004 Sensors and Effectors

Cell Signalling Biology Michael J.
Berridge r Module 4 r Sensors and Effectors 4 r1
Module 4
Sensors and Effectors

Synopsis
Signalling pathways regulate cellular processes by acting through sensors to stimulate the
downstream effectors that are responsible for controlling different cellular processes. In some cases,
these effectors might be relatively simple, consisting of a single downstream effector system, whereas
there are more complicated effectors made up of multiple components such as those driving
processes such as membrane and protein trafficking, endocytosis, exocytosis, phagocytosis, motor
proteins, gene transcription, gene silencing and actin remodelling.
Sensors Ca2 + sensors

Intracellular sensors detect the signals coming from the For the Ca2 + signalling pathway, there are a number
different signalling pathways (Module 2: Figure cell sig- of Ca2 + sensors, such as troponin C (TnC), calmodulin
nalling pathways) and use the information to stimulate the (CaM), annexins, S100 proteins and the synaptotagmins.
effectors that bring about different cellular processes.
There are many different sensors:
Calmodulin (CaM)
2+ Calmodulin (CaM) is one of the major Ca2 + sensors in
The Ca signalling system employs a range of different
cells. It contains four helixloophelix folding motifs con-
Ca2+ sensors:
taining EF-hand Ca2 + -binding sites (Module 4: Figure
Calmodulin (CaM)
EF-hand motif). These sites are arranged into two lobes
Troponin C (TnC)
(the N- and C-terminal lobes) at either end of the mo-
Neuronal Ca2+ sensor (NCS) proteins
lecule separated by a flexible linker (Module 4: Figure CaM
Ca2+ -binding proteins (CaBPs)
structure). Each lobe has two EF-hands with sites I and II
Calcium and integrin binding protein 1 (CIB1)
in the N-terminal domain and III and IV in the C-terminal
S100 proteins
domain. The latter two sites have a Ca2 + affinity that is
Annexins
10-fold higher than for sites I and II. When Ca2 + binds
Synaptotagmins
to these lobes, the molecule undergoes a pronounced con-
The regulatory subunit of protein kinase A (PKA) binds formational change that exposes a hydrophobic pocket and
the second messenger cyclic AMP [Module 2: Figure increases its affinity for its various targets that have char-
protein kinase A (PKA)]. acteristic CaM-binding domains. In effect, the N- and C-
In the case of protein kinase C (PKC), which responds terminal lobes wrap themselves around the CaM-binding
to both diacylglycerol (DAG) and Ca2 + , the enzyme is domains of their effector proteins to induce the conforma-
both the sensor and the effector (Module 2: Figure PKC tional changes responsible for altering the activity of many
structure and activation). different signalling components:
The sensors for cyclic GMP are located on its two main
effectors: cyclic GMP-dependent protein kinase (cGK) Opening of intermediate-conductance (IK) and small-
and the cyclic nucleotide-gated channel (CNGC) (Mod- conductance (SK) K + channels (Module 3: Figure
ule 3: Figure cyclic nucleotide-gated channels). IK/SK channel opening)
The many sensors, which function in protein-lipid inter- CaV 1.2 L-type channel (Module 3: Figure CaV 1.2 L-
actions, have lipid-binding domains capable of sensing type channel).
different lipid messengers (Module 6: Figure modular Ca2+ /calmodulin-dependent protein kinases (CaMKs)
lipid-binding domains). Myosin light chain kinase (MLCK)
Phosphorylase kinase
Activation of NO synthase (Module 2: Figure NO syn-
thase mechanism)
Green text indicates links to content within this module; blue text Neuromodulin
indicates links to content in other modules. Neurogranin
Please cite as Berridge, M.J. (2014) Cell Signalling Biology; Ras guanine nucleotide release-inducing factors
doi:10.1042/csb0001004 (RasGRFs)

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2014 Portland Press Limited www.cellsignallingbiology.org
Licensed copy. Copying is not permitted, except with prior permission and as allowed by law.
Cell Signalling Biology Michael J. Berridge r Module 4 r Sensors and Effectors 4 r2
Module 4: Figure EF-hand motif
Structural organization of the EF-hand.

The orientations of the two helices that make up the EF-hand resemble a finger and a thumb as illustrated on the right. The small loop connecting these
two helices contains aspartate and glutamate residues that provide the carbonyl-group oxygen atoms to co-ordinate the Ca2 + ion. Reproduced, with
permission, from the Annual Review of Biochemistry, Volume 45 c 1976 by Annual Reviews; http://www.annualreviews.org; see Kretsinger 1976.
Human vacuolar protein sorting 34 (hVps34) palmitoylated cysteine residues, has the unusual property
of being able to bind to CaM at resting concentrations of
EF-hand Ca2 + . In response to a local elevation in Ca2 + , the CaM
The EF-hand is one of the major Ca2 + -binding regions is released from neuromodulin and diffuses away to carry
found on many of the Ca2+ sensors. It takes its name from out its signalling functions. The IQ domain on neuromod-
the fact that the binding site is located between an E and ulin that binds CaM has a Ser-41 that is phosphorylated
an F helix that are aligned like a thumb and a finger (Mod- by protein kinase C (PKC) and this blocks CaM binding
ule 4: Figure EF-hand motif). A small loop of 12 amino and thus represents another way in which the level of CaM
acids, which connects the two helices, contains aspartate might be regulated.
and glutamate side chains that have carbonyl groups that
provide the oxygen atoms to co-ordinate the Ca2 + . The Neurogranin
number of EF-hands on proteins can vary. In the case of the Neurogranin is a neural-specific calmodulin (CaM)-
classical sensors such as calmodulin (CaM) and troponin binding protein that functions primarily in the postsyn-
C (TnC), there are four EF-hands. aptic region. It has some sequence homology to neur-
The Stromal interaction molecule (STIM), which func- omodulin particularly within the IQ domain that binds
tions in the mechanism of store-operated channel (SOC) CaM that also has a phosphorylation site for protein kinase
activation to sense the Ca2 + content of the endoplasmic C (PKC). Neurogranin may function to regulate CaM
reticulum (ER) has a modified EF-hand with an affin- levels in postsynaptic regions just as neuromodulin does in
ity matched to the higher concentrations of Ca2 + located presynaptic regions. In response to an increase in Ca2 + , the
within the lumen of the ER (Module 3: Figure SOC sig- neurogranin/CaM complex will be disrupted and the re-
nalling components). leased CaM will be free to carry out its many postsynaptic
functions (Module 10: Figure neuronal gene transcription).
Neuromodulin
Neuromodulin, which is also known as GAP43, B-50 or Troponin C (TnC)
P-57, is a neural-specific calmodulin (CaM)-binding pro- Troponin C (TnC) resembles calmodulin in that it has
tein that is located mainly in the presynaptic region. It two pairs of Ca2 + -binding EF-hands. It has a specific
is found at concentrations resembling that of CaM and function during excitation-contraction (E-C) coupling in
may thus play a role in regulating the free level of CaM. skeletal muscle and in cardiac muscle cells. Its function
Neuromodulin, which is tethered to the membrane by two has been worked out in some detail in the case of skeletal

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Module 4: Figure CaM structure
The EF-hand Ca2 + -binding domain.

These ribbon diagrams illustrate the structure of calmodulin in the absence (left) and presence (red balls in the centre and right-hand panels) of
Ca2 + . When Ca2 + binds, calmodulin (CaM) undergoes a remarkable change in conformation that enables it to wrap around parts of proteins (shown
in green on the right) to alter their properties. Such an action is evident in the Ca2 + -dependent opening of K + channels (Module 3: Figure IK/SK
channel opening). Reproduced, with permission, from the Annual Review of Pharmacology and Toxicology, Volume 41 2001 c by Annual Reviews;
http://www.annualreviews.org; see Hook and Means 2001.
muscle (Module 7: Figure skeletal muscle E-C coupling). of ion channels, membrane trafficking, receptor modu-
The Ca2 + released from the sarcoplasmic reticulum (SR) lation, gene transcription and cell survival. There are 14
acts through TnC to stimulate contraction. TnC is one members of the NCS family, which are typical EF-hand
of the components of a regulatory complex located on Ca2 + -binding proteins (Module 2: Table Ca2+ signalling
actin that determines its ability to interact with myosin. toolkit). They possess four EF-hands (Module 4: Figure
One of these proteins is tropomyosin, which has a long EF-hand motif), but only three of them bind Ca2 + . In the
rod-like structure made up of two chains arranged in a case of recoverin, only two of the EF-hands are functional.
coiled-coil that lies on the rim of the groove of the actin While most of the NCS proteins are fairly widely distrib-
helix (Module 7: Figure skeletal muscle structure). These uted throughout the neuronal population, some have a
tropomyosin rods are separated at 40 nm intervals (cor- limited expression, such as hippocalcin, which is restricted
responding to seven actin subunits) by a complex of reg- mainly to the hippocampal pyramidal neurons. Hippocal-
ulatory troponin proteins that include troponin I (TnI), cin may contribute to the Ca2 + -dependent activation of
troponin T (TnT) and TnC. The TnI binds to both actin endocytosis during the process of long-term depression
and TnC, whereas TnT interacts with tropomyosin and (LTD).
TnC. This troponin complex regulates the position of the Many of the NCS proteins are multifunctional in that
tropomyosin rods relative to the groove of the actin helix they can control different downstream effectors. For ex-
in a Ca2 + -dependent manner. In the resting muscle, the ample, KChIP3/DREAM/calsenilin has three quite dis-
tropomyosin is displaced out of the groove to provide a tinct functions. Likewise, NCS-1 also interacts with a
physical barrier (steric hindrance) preventing the myosin number of downstream effectors. Most of the effectors
heads from interacting with the actin subunits. During E- controlled by the NCS proteins are located on membranes,
C coupling, Ca2 + binds to the EF-hands on TnC to induce and there is some variability concerning the way they loc-
a conformational change in the troponin complex that is ate these targets. A characteristic feature of many NCS
transmitted to tropomyosin, causing it to move a small proteins is that they have an N-terminal myristoyl group,
distance (approximately 1.5 nm) towards the groove, and which functions to attach them to membranes. In some
this then permits the myosin heads to interact with actin cases, there is a Ca2+ -myristoyl switch that enables the
to begin the contractile process. protein to associate with membranes in a reversible man-
ner, depending on the level of intracellular Ca2 + . For some
Neuronal Ca2 + sensor (NCS) proteins of the NCS proteins, such as NCS-1 and K + -channel-
The neuronal Ca2 + sensor (NCS) proteins are mainly interacting protein 1 (KChIP1), the myristoyl group is
found in neurons, where they were first discovered, but exposed in the absence of a Ca2 + signal, which means
some are also found to function in other cell types. They that they are already associated with the membrane and
have multiple functions in cells, including the regulation can thus respond rapidly to brief Ca2 + transients. On

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the other hand, the proteins that use the Ca2 + -myristoyl erties by enhancing the current flow and by lowering the
switch have slower response times and are thus tuned to rate of recovery. The channel will then remain open for
respond to slower global changes in intracellular Ca2 + . longer and will thus act to reduce excitability. In the case
The association of NCS proteins with membranes is fairly of KChIP2, which is expressed in heart cells, transgenic
specific and is mainly confined to interactions with the mice that lack this channel display ventricular tachycar-
plasma membrane or membranes of the trans-Golgi net- dia, thus emphasizing the importance of the KChIPs in
work, where they play a variety of roles as outlined in the regulating membrane excitability. This may have relevance
following descriptions of individual NCS proteins: for epilepsy, because patients suffering from this condition
have reduced levels of KChIP/DREAM/calsenilin.
Neuronal Ca2 + sensor-1 (NCS-1) KChIP is an unusual protein in that it has multiple func-
Neuronal Ca2 + sensor-1 (NCS-1) is widely expressed in tions. Not only does it regulate K + channel activity but
neurons. It has an N-terminal myristoyl group, which at- it also functions as a Ca2 + -sensitive transcription factor
taches it to membranes even in the absence of Ca2 + , which (DREAM) and as a regulator of the presenilins, and was
means that NCS-1 can respond rapidly to Ca2 + transi- thus called calsenilin. To avoid confusion, it has been re-
ents. It has been implicated in the control of different cel- ferred to here as KChIP/DREAM/calsenilin.
lular processes. One action is to modulate ion channels,
and particularly members of the voltage-operated chan- KChIP/DREAM/calsenilin
nels (VOCs) such as the P/Q-, N- and L-type channels. It KChIP/DREAM/calsenilin is a multifunctional neuronal
can inhibit the internalization of dopamine D2 receptors Ca2 + sensor (NCS) protein that was discovered through
by interacting with both the receptor and the G protein- three independent studies to regulate quite different pro-
coupled receptor kinase 2 (GRK2). There is evidence that cesses, and for each one it was given a specific name:
NCS-1 can have an effect on exocytosis, which may de-
pend on its ability to activate the type III PtdIns 4-kinase It was found to be one of the K+ channel auxiliary
(PtdIns 4-K) III. The formation of PtdIns4P is one of the subunits and was called K + -channel-interacting protein
steps in the PtdIns4,5P2 regulation of exocytosis. NCS-1 (KChIP) (Module 3: Figure K+ channel domains).
also functions in Golgi to plasma membrane transfer at It was also found to be a Ca2 + -sensitive transcrip-
the trans-Golgi network (TGN) (See step 3 in Module 4: tion factor, and was called downstream regulatory
Figure membrane and protein trafficking). element antagonistic modulator (DREAM). DREAM
NCS-1 has been implicated in inositol 1,4,5-trisphos- binds constitutively to its promoter, and this transcrip-
phate receptor (InsP3 R) modulation where it acts to pro- tional activity is inhibited by Ca2 + , which acts to re-
mote the release of Ca2 + from the endoplasmic reticulum move DREAM (Mechanism 3 in Module 4: Figure tran-
(Module 3: Figure InsP3 R regulation). scription factor activation). In the absence of Ca2 + ,
NCS-1 has been found to associate with interleukin-1 DREAM binds to its promoter sites, where it functions
receptor accessory protein-like (IL1RAPL) protein, which as a transcriptional repressor. This gene repression is re-
is mutated in X-linked mental retardation. There is an up- moved when Ca2 + binds to the EF-hands on DREAM
regulation of NCS-1 in the prefrontal cortex in patients to reduce its affinity for DNA.
with schizophrenia and bipolar disorders. It was found to regulate the presenilins, and was
thus called calsenilin. The KChIP3/DREAM/calsenilin
Guanylyl cyclase-activating proteins (GCAPs) binds to the -secretase complex responsible for the
Expression of the guanylyl cyclase-activating proteins processing of the -amyloid precursor protein (APP)
(GCAPs) is restricted to the retina, where they play an resulting in the formation of the -amyloids respons-
important role in light adaptation during phototransduc- ible for Alzheimers disease (AD) (Module 12: Figure
tion (Step 11 in Module 10: Figure phototransduction). In APP processing).
the light, the level of Ca2 + declines and this allows the
GCAPs to stimulate guanylyl cyclase to restore the level Having these three names has led to some confusion
of cyclic GMP. regarding its terminology and here I have referred to this
protein as KChIP/DREAM/calsenilin to emphasize that
K + -channel-interacting proteins (KChIPs) it is the same protein with three very different functions.
There are four members of the K + -channel-interacting
proteins (KChIPs), which were first identified as regulat- Recoverin
ors of the Kv 4 voltage-dependent K+ (Kv ) channels re- Recoverin is located in the retina, where it functions to
sponsible for the A-type K + current in neurons. These prevent inactivation of phototransduction by inhibiting
K + channels have six membrane-spanning regions, with the rhodopsin kinase that phosphorylates rhodopsin (Step
the N- and C-termini facing the cytoplasm (Module 3: 2 in Module 10: Figure phototransduction).
Figure K+ channel domains). The KChIPs bind to the N-
terminal region, where they have two main effects. Firstly, Ca2 + -myristoyl switch
they control the trafficking of K + channels to the plasma For some of the neuronal Ca2 + sensor (NCS) proteins
membrane. In the absence of KChIP, the channel remains [hippocalcin, neurocalcin , visinin-like protein (VILIP)-
in the Golgi. Secondly, binding of KChIP to channels in the 1 and VILIP-3], the position of the myristoyl group is
plasma membrane can markedly influence channel prop- sensitive to Ca2 + , and this provides a mechanism for

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the proteins to translocate to cell membranes in a Ca2 + - In cardiac cells, CIB1 functions to anchor calcineurin
dependent manner. Under resting conditions, the myris- (CaN) to the sarcolemma where it responds to the Ca2 +
toyl group is tucked away in a hydrophobic pocket. The signals that activate the cardiac NFAT shuttle respons-
binding of Ca2 + induces a conformational change that ex- ible for the onset of cardiac hypertrophy.
poses the myristoyl group, which then inserts itself into CIB1 has been shown to interact with presenilin 2 and
membranes and pulls the protein on to the membrane sur- could thus contribute to the onset of Alzheimers disease
face. (AD).
Activity of the IIb/3 , which functions in integrin sig-
Ca2 + -binding proteins (CaBPs) nalling in blood platelets and megakaryocytes, is regu-
The Ca2 + -binding proteins (CaBPs) are a small group of lated by CIB1.
EF-hand proteins that are related to calmodulin (CaM)
and the neuronal Ca2+ sensor (NCS) proteins. As is evid-
Annexins
ent from Module 2: Table Ca2+ signalling toolkit, the
The annexins are a large group of Ca2 + sensors that are also
CaBPs are found predominantly in the brain and ret-
capable of binding to membrane phospholipids. There are
ina. Caldendrin was the first member of this family to be
12 annexin subfamilies (Module 2: Table Ca2+ signalling
characterized. There are two splice variants of caldendrin,
toolkit). The annexins respond to an increase in Ca2 + by
known as long CaBP1 (L-CaBP1) and short CaBP1 (S-
translocating to the membranes, both the plasma mem-
CaBP1). CaBP-1 has attracted interest because it seems
brane and internal membranes. Annexin structure is dom-
to function as an ion channel modulator with effects on
inated by the annexin core, which is the region responsible
both P/Q voltage-operated channels and on the inositol
for the translocation to membrane surfaces.
1,4,5-trisphosphate receptor (InsP3 R). Neurons also have
When annexins bind to membranes, they can have a
two proteins called calneuron-1 and calneuron-2, which
number of effects, such as the organization and attachment
are closely related to caldendrin. The calneurons seem to
of the cytoskeleton, regulation of membrane trafficking
act by controlling the PtdIns 4-KIII that functions in the
including exocytosis, endocytosis and membrane transfer
PtdIns4P signalling cassette to regulate vesicle trafficking
between intracellular compartments, linking membranes
during the Golgi to plasma membrane transfer of proteins.
together and the regulation of ion fluxes. One of the prob-
lems has been to link these various effects, many of which
Calcium and integrin binding protein 1 (CIB1) have been uncovered in artificial membrane systems or
Calcium and integrin-binding protein 1 (CIB1), which in cultured cells, to specific cellular control mechanisms.
is also known as calmyrin and kinase-interacting protein However, various transgenic approaches have begun to re-
(KIP), is a 22 kDa EF-hand protein that resembles calm- veal a number of cellular approaches that will be revealed
odulin and several other calcium-binding proteins. As its in the description of individual annexins.
name implies, CIB1 was originally identified through its
ability to bind to the cytoplasmic tail of platelet integrin
IIb. It was found subsequently to be widely distributed Annexin structure
and is able to bind to a number of other targets. CIB1 has The central feature of annexin structure is the annexin
four EF-hand motifs, but only two of these can bind Ca2 + . core, which is made up of four annexin regions, each of
CIB1 can bind to membranes through an N-terminal myr- which has 70 amino acids with a large number of carbonyl
istoyl group. and carboxy groups that make up the Ca2 + -binding re-
The binding of Ca2 + to the EF hand motifs on CIB1 gions (Module 4: Figure annexin structure). The affinity
induces a conformational change that enables this effector for Ca2 + is in the low-micromolar range, and this sens-
to modulate the activity of its various target protein: itivity can be altered following tyrosine phosphorylation
of the N-terminal region. When Ca2 + binds to this site,
CIB1 responds to an elevation of Ca2 + by activating it induces a conformational change that exposes a con-
p21-activated kinases (PAK1) to bring about the phos- vex surface where the Ca2 + can form salt bridges with
phorylation and inactivation of gelsolin that contrib- the membrane phospholipids. The molecular structure of
utes to actin polymerization (Module 2: Figure Rac sig- this Ca2 + -bound form is shown in panel b in Module 4:
nalling). Figure annexin molecular structures. It clearly illustrates
Apoptosis signal-regulating kinase 1 (ASK1), which is the convex region that attaches to the membrane and the
a component of the mitogen-activated protein kinase concave region where attachments to other proteins are
(MAPK) signalling toolkit (Module 2: Table MAPK made. Another consequence of Ca2 + binding is that the
signalling toolkit) where it acts to stimulate both the N-terminal region swings away and becomes accessible to
JNK signalling and p38 signalling pathways, is inhib- phosphorylation by serine/threonine and tyrosine kinases,
ited through its association with CIB1. In response to an which not only alters Ca2 + sensitivity, but also can make
elevation of Ca2 + , this inhibition is removed resulting these proteins susceptible to proteases.
in an activation of the ASK1-JNK and ASK1-p38 sig- The function of annexins is dependent on interac-
nalling pathways. CIB1 may thus act as a Ca2 + -sensitive tions with other proteins. For example, annexins 1 and
antagonist of stress-induced signalling events. 2 can interact with members of the S100A family to form
CIB1 has been implicated in the control of spermato- symmetrical heteromeric complexes that have the potential
genesis. to bind together different membrane surfaces (see panel B

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Module 4: Figure annexin structure
Annexins N C
Annexin A6 N C
A B
S100A10
2+
Ca
Actin
Annexin structure and membrane association.

The annexins have four annexin repeats (lilac), which makes up the annexin core domain. Each repeat has a Ca2 + -binding domain. Annexin A6
is unusual in that the core domain is duplicated, which has resulted from the fusion of annexins A5 and A10. When annexins bind Ca2 + (see A),
a conformational change occurs to expose a hydrophobic concave surface that attaches to membranes through a salt bridge formed by Ca2 +
interacting with the carbonyl and carboxy groups of the annexins and the negative charges on the phospholipid head groups in the membrane. The
conformational change can also displace the N-terminal region (green) to open up the concave surface, which can then interact with other proteins
such as actin. Annexin can also interact with S100A10 to form a symmetrical tetrameric structure capable of bringing together two membranes (see
B).
Module 4: Figure annexin molecular structures
Molecular structure of different annexins.

Panels ad illustrate the molecular structure of annexins in their Ca2 + -bound forms, which associate with membranes depicted as the green triangles.
a. This panel illustrates the Ca2 + ions (blue) attaching the convex surface of the core domain to the membrane. b. The protein core illustrating how the
four annexin repeats (coloured differently) are composed of five -helices. c. The symmetrical heteromeric complex composed of a central S100A10
dimer (blue) connected to two annexin A2 core domains attached to different membranes. A schematic diagram of this arrangement is shown in
Module 4: Figure annexin structure. d. Structural organization of annexin A6, which has duplicate core domains connected by a flexible linker that
enables each half to assume different orientations. The bound Ca2 + is shown in blue. Reproduced by permission from Macmillan Publishers Ltd: Nat.
Rev. Mol. Cell Biol. Gerke, V., Creutz, C.E. and Moss, S.E. (2005) Annexins: linking Ca2 + signalling to membrane dynamics. 6:449461. Copyright
(2005); http://www.nature.com/nrm/; see Gerke et al. 2005.

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in Module 4: Figure annexin structure and panel c in Mod- Annexin A7

ule 4: Figure annexin molecular structures). Annexin A7 was originally identified in adrenal medulla
cells, where it appeared to be associated with chromaffin
Annexin A1 granules. Subsequently, it was found to have an effect on
Annexin A1 has all the hallmarks of a typical annexin. release of Ca2 + from internal stores by both the inositol
It is activated by Ca2 + (see panel A in Module 4: Fig- 1,4,5-trisphosphate receptor (InsP3 R) and the ryanodine
ure annexin structure). The N-terminal tail can be phos- receptor (RYR). In the case of the latter, it is of interest that
phorylated by tyrosine kinases to alter its Ca2 + -sensitivity. annexin A7 associates with sorcin, which is known to act as
Like annexin A2, it can segregate lipids within the plasma a negative regulator of the coupling of L-type Ca2 + chan-
membrane, and there appears to be a particularly high af- nels and RYR2s in heart cells. If annexin A7 contributes
finity for the PtdIns4,5P2 to form raft-like domains rich in to such a negative regulation of Ca2 + release mechanisms,
this lipid. Since annexin A1 can also bind actin, it is thought it might explain the phenotypes of some of the transgenic
to provide attachment points where the cytoskeleton links animal experiments. Astrocytic Ca2 + waves from annexin
to the plasma membrane. A7 / mice were found to have higher velocities, con-
Annexin A1 can bind to S100A10 and S100A11 to form sistent with an increased propensity to release Ca2 + . The
heteromeric complexes, as shown in panel B in Module 4: increased sensitivity may also explain why the astrocytes
Figure annexin structure. displayed an increased rate of proliferation and were also
more prone to cancer. Indeed, it is considered that annexin
A7 could function as a tumour promoter gene.
Annexin A2
Annexin A2 behaves much as annexin A1 with regard to
Annexin A11
its association with the plasma membrane and its ability to
Annexin A11 may play an important role in the traffick-
bind to PtdIns4,5P2 , and to provide membrane attachment
ing and insertion of vesicles. One role occurs during the
points for the cytoskeleton. This annexin also associates
process of cytokinesis, when the daughter cell separates
with actin at the comet tails that propel endocytic vesicles
into two at the time of cell division (Module 9: Figure cy-
away from the plasma membrane.
tokinesis). It appears to enter the nucleus at prophase and
The complex formed between annexin A2 and S100A10
locates to the midbody, where it is in position to contrib-
has also been implicated in the control of plasma membrane
ute to vesicle trafficking during cytokinesis. Annexin 11
Cl channels, the TRPV5 and TRPV6 channels and the
may also play a role in the trafficking of vesicles during
TWIK-like, acid-sensitive K+ channel-1 (TASK1).
COPII-mediated transport from ER to Golgi (Module 4:
Annexin A2 has a nuclear export signal (NES) and is nor-
Figure COPII-coated vesicles).
mally excluded from the nucleus. Following phosphoryla-
Annexin A11 binds to S100A6.
tion of tyrosine residues on the N-terminal region, it can
translocate into the nucleus. Since it has been shown to Annexin A13
bind to RNA, it could play a role in RNA transport. Annexin A13 is unusual in that it can associate with mem-
branes through an N-terminal myristoylation in a Ca2 + -
Annexin A4 independent manner.
Annexin A4 has been implicated in the control of Cl
channels in the plasma membrane. S100 proteins
S100 proteins are the largest subgroup of the EF-hand
Annexin A5 Ca2 + -binding protein family. There are about 20 S100
An unusual feature of annexin A5 is that the binding of proteins that are very divergent with regard to their dis-
Ca2 + allows a tryptophan residue to insert itself into the tribution, function and Ca2 + -binding properties. An in-
membrane to interact with the hydrocarbon chains. This teresting aspect of their function is that they can operate
propensity to intercalate with the hydrophobic region of both intra- and extra-cellularly. When functioning within
the membrane is enhanced further at low pH when the pro- the cell, they can have multiple effects on cytoskeletal dy-
tein integrates into the membrane such that it can function namics, gene transcription, Ca2 + homoeostasis, cell pro-
as a Ca2 + channel in biophysical experiments. However, liferation and differentiation. With regard to their extracel-
there is little evidence for such a channel function in cells lular function, some of the S100 proteins (e.g. S100B and
under normal conditions. S100A12) are secreted and can act as extracellular ligands.
Like annexin A2, annexin A5 can also enter the nucleus One of their targets is the receptor for advanced glycation
following its tyrosine phosphorylation. end-product (RAGE).
They have attracted considerable attention because there
Annexin A6 are a number of disorders, such as cancer, inflammation,
Annexin A6 is an unusual annexin in that it contains two cardiomyopathy and neurodegeneration, that have been
core domains that arose from the fusion of annexins A5 linked to deregulation of these proteins.
and A10 (Module 4: Figure annexin structure). The two One of the curious features of the S100 protein family is
Ca2 + -binding core domains are connected by a flexible that many of the genes are clustered in a small region (1q21)
linker that enables it to bind different membrane regions on human chromosome 1 (Module 4: Figure S100 phylo-
(panel d in Module 4: Figure annexin molecular structures). genetic tree). This suggests that the S100 family expanded

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Module 4: Figure S100 phylogenetic tree
Human
S100A1 chromosome 1 1700
S100A10
S100B S100A11
S100P
Trichohyalin
S100A10 1600
S100Z Profilaggrin
S100A6 C1orf10
p 1500
S100A3
S100A5
S100A2
S100A4
S100A13 400 S100A9
S100A14 S100A12
S100A8
S100A9 1q21
300 S100A7
S100A12
S100A11 kb S100A6
S100A5
S100A7 q 200 S100A4
S100A3
Trichohyalin S100A2
C1orf10 100 S100A13
S100A1 S100A14
Profilaggrin
S100A8 0
Phylogenetic tree and clustering of many S100 genes on human chromosome 1.

The phylogenetic tree shown on the left illustrates the relationships between the S100 protein family, which may have grown through gene duplication.
The gene map on the right shows how many of the genes (i.e. those highlighted in orange on the phylogenetic tree) are clustered in the 1q21 region
of human chromosome 1. The nomenclature of the S100 proteins, summarized by Heizmann et al. 2003, is based on this gene clustering (Module 2:
Table Ca2+ signalling toolkit). Redrawn from Handbook of Cell Signaling, Volume 2 (R.A. Bradshaw and E.A. Dennis, eds), Heizmann, C.W., Schafer,
B.W. and Fritz, G., The family of S100 cell signalling proteins, pages 8793. Copyright (2003), with permission from Elsevier; see Heizmann et al. 2003.
by gene duplication. The link between S100 proteins and a regulator of cardiac contractility. It is up-regulated during
cancer emerged from the observation that tumours arise compensated hypertrophy, but reduced during cardiomy-
from deletions or rearrangements within this chromosome opathy. When overexpressed in cardiac cells, it markedly
region. increases the amplitude of the Ca2 + transients, apparently
Members of the S100 proteins contain two EF-hand by increasing the uptake of Ca2 + into the SR through some
motifs (Module 4: Figure EF-hand motif). The C-terminal action on the sarco/endo-plasmic reticulum Ca2+ -ATPase
motif is similar to the canonical Ca2 + -binding site found (SERCA) pump.
on other EF-hand proteins, whereas the N-terminal motif
has a slightly different structure that is characteristic of
the S100 proteins and has been referred to as a pseudo- S100A2
EF-hand. The S100 proteins usually function as homo- S100A2 is increased in various tumours, and the expression
or hetero-dimers. In response to an elevation of Ca2 + , the level has proved to have considerable diagnostic value in
dimers usually bind four Ca2 + ions, which induce the con- assessing the severity of laryngeal squamous carcinoma.
formational changes that expose an internal hydrophobic
region responsible for activating its downstream effectors.
Some of the S100 proteins can also bind Zn2 + and Cu2 + . S100A4
For example, S100A3 has a much higher affinity for Zn2 + The expression of S100A4 has been associated with cancer,
compared with that for Ca2 + . where it seems to play a role in promoting metastasis. One
The S100 proteins have been implicated in a very large proposal is that it may be released from cells to act as an
number of cellular processes, and it has proved difficult to extracellular ligand to activate tumour angiogenesis.
clearly identify their precise function in specific cell types.
The following descriptions of some of the individual mem- S100A6
bers illustrate how this large family has been implicated in This S100 protein is also known as calcyclin. Like many
many different cellular control mechanisms: other members of the S100 family, S100A6 is often up-
regulated in tumours, and especially in those showing
S100A1 high metastatic potential. Its expression is also increased
S100A1 is preferentially expressed in cardiac cells, where during neurodegeneration in both Alzheimers disease and
it is located near the sarcoplasmic reticulum (SR) and the in amyotrophic lateral sclerosis (ALS). In the case of the
contractile filaments. There are indications that it might be former, it is markedly increased in the astrocytes, especially

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in the regions surrounding the amyloid plaques (Module Module 4: Figure S100A6 in AD neocortex
4: Figure S100A6 in AD neocortex). These astrocytes also
express large amounts of S100B.
S100A8
S100A8 appears to have a role in inflammation. It is also
thought to control wound healing by reorganizing the ker-
atin cytoskeleton in the epidermis.
S100A9
S100A9 has a similar mode of action as S100A8 in inflam-
mation and wound healing.
S100A10
S100A10 is somewhat unusual in that it is constitutively
active even at low Ca2 + levels. It can thus bind to its
targets independently of binding Ca2 + . Its action is intim-
ately connected with that of annexin A1 and annexin A2.
It forms a symmetrical heteromeric complex with these
annexins (Module 4: Figure annexin structure).
S100B
S100B is located mainly in the brain astrocytes where it
has both intra- and extracellular functions. It acts within
the cell to modulate microtubule assembly and it also reg-
ulates the cell cycle by interacting with p53. S100B is also
released to the extracellular space where it functions much
like a cytokine to stimulate neurite outgrowth. However, Comparison of S100A6 levels in the neocortex of normal and
at high concentrations it stops having this neurotrophic Alzheimers disease-affected brain.
function and begins to activate apoptosis. Various neuro- There is a large increase in the histochemical labelling of S100A6 in
Alzheimers disease (AD)-affected neocortex (B) compared with that
degenerative diseases, such as Alzheimers disease, Downs of normal subjects (A). Reproduced from Biochim. Biophys. Acta, Vol.
syndrome and amyotrophic lateral sclerosis (ALS), are as- 1742, Broom, A., Pochet, R., Authelet, M., Pradier, L., Borghgraef, P.,
sociated with an increased expression of S100B. The ap- Van Leuven, F., Heizmann, C.W. and Brion, J.-P., Astrocytic calcium/zinc
binding protein S100A6 over expression in Alzheimers disease and in
optosis may arise from an inflammatory response, because PS1/APP transgenic mice models, pages 161168. Copyright (2004),
high levels of S100B are known to increase the production with permission from Elsevier; see Broom et al. 2004.
of nitric oxide (NO) and reactive oxygen species (ROS).
S100B is thought to act through the receptor for ad-
vanced glycation end-products (RAGE), which is a mem-
ber of the immunoglobulin (Ig) superfamily of receptors. aptotagmins are embedded in the vesicle, whereas others
An S100B tetramer binds to the V domain of the RAGE are found in the plasma membrane. For example, synaptot-
receptor and this complex then interacts with another agmins I and II are embedded in the vesicle with their two
S100B/RAGE complex to form the functional dimer that C2 domains facing the cytosol, where they bind to Ca2 +
then triggers cell signalling. and undergo a conformational change that helps to trigger
There also is strong evidence to suggest that S100B can Ca2+ -dependent exocytosis. On the other hand, synaptot-
inhibit the activity of the tumour suppressor p53. S100B agmin VII is embedded in the plasma membrane.
interacts with both the p53 oligomerization domain and
the C-terminal domain that is phosphorylated by protein Effectors
kinase C (PKC). S100B can thus reduce p53 binding to Effector is a rather general term used to describe the
DNA and its transcriptional activity. A Ca2 + -dependent cellular process responsible for carrying out the actions
activation of p53 may thus suppress the activity of p53, of signalling pathways. Information provided by the sig-
which will contribute to cancer progression. Such an ac- nalling systems instructs the effectors to control a variety
tion is consistent with the observation that S100B is over- of cellular processes. Some of these effectors are single en-
expressed in many melanomas, astrocytomas and gliomas. tities, such as an ion channel or an enzyme regulating a
Antibodies against S100B have been used for tumour typ- metabolic process. However, there are more complex ef-
ing and the diagnosis of melanoma. fector mechanisms that often are the targets of multiple
signalling pathways. These effectors are then responsible
Synaptotagmins for controlling a variety of cellular processes:
Synaptotagmins are a family of Ca2 + -binding proteins that
function as Ca2 + sensors to control exocytosis (Module 4: Exocytosis
Figure Ca2+ -induced membrane fusion). Some of the syn- Phagocytosis

C
Gene transcription Ca2 + /calmodulin-dependent protein kinase I (CaMKI)

Ciliary beating This ubiquitous enzyme is located in the cytosol. Its activ-
Actin remodelling ation requires both Ca2 + /calmodulin (CaM) binding and
phosphorylation of its activation loop on Thr-177 (Module
4: Figure activation of CaMKs).
Ca2 + effectors
Ca2 + -sensitive cellular processes in cells depend upon a Ca2 + /calmodulin-dependent protein kinase II
wide range of Ca2 + effectors: (CaMKII)
Ca2 + /calmodulin-dependent protein kinase II (CaMKII)
Ca2+ -sensitive K+ channels is a highly versatile enzyme that can phosphorylate a range
Ca2+ -sensitive Cl channels (CLCAs) of substrates. One of its proposed functions is to operate as
Ca2+ /calmodulin-dependent protein kinases (CaMKs) a spike frequency detector to decode frequency-modulated
Calcineurin (CaN) (FM) Ca2 + signals (Module 6: Figure encoding oscillatory
Ca2 + -sensitive Ras-GAPs such as Ca2 + -promoted information). It also has a central role as a molecular switch
Ras inactivator (CAPRI) and RASAL (Ras GTPase- in learning and memory.
activating-like) (Module 2: Figure Ras signalling) The eight to twelve subunits that make up the holoen-
Phosphorylase kinase zyme are encoded by four separate genes: , , and ).
Myosin light chain kinase (MLCK) Structural analysis reveals that the subunits are organized
in two-stacked hexameric rings (Module 4: Figure struc-
ture of CaMKII). The CaMKIIs expressed in different cells
Ca2 + /calmodulin-dependent protein kinases (CaMKs) contain different proportions of these four isoforms. For
The Ca2 + sensor calmodulin (CaM) activates a family of example, the majority of brain CaMKII is present as the
Ca2 + /CaM-dependent protein kinases (CaMKs) (Module / isoforms in a ratio of 3:1, whereas the predominant
4: Figure structure of CaMKs). Some of these CaMKs isoform in the heart is CaMKII, which exists as differ-
are dedicated kinases in that they have a single sub- ent spliced variants (e.g. B and C ). The CaMKIIB vari-
strate, such as phosphorylase kinase and myosin light ant contains a nuclear localization signal and is found in
chain kinase (MLCK). There are other multifunctional en- both the cytoplasm and nucleus, whereas the CaMKIIC
zymes such as Ca2+ /calmodulin-dependent protein kinase variant is confined to the cytoplasm. All of the isoforms
I (CaMKI), Ca2+ /calmodulin-dependent protein kinase II are alternatively spliced to give up to 30 spliced versions.
(CaMKII) and Ca2+ /calmodulin-dependent protein kinase These subtle variations in the structure of the different iso-
IV (CaMKIV) that phosphorylate a wide range of sub- forms are probably responsible for substrate targeting and
strates. CaMKI and CAMKIV are monomeric and share subcellular localization. The catalytic headgroups that are
a similar activation process. Ca2 + uses CaM as a sensor arranged close to each other in the multimeric complex
to activate these enzymes. Activation of CaMKI and are activated by Ca2 + in a series of steps, as illustrated in
CaMKIV depends upon a sequence of events begin- Module 4: Figure CaMKII activation:
ning with the binding of Ca2 + /CaM, which is then
followed by phosphorylation of their activation loop 1. Ca2 + binds to calmodulin (CaM) to form the bilobed
by a Ca2+ /calmodulin-dependent protein kinase kinase Ca2 + /CaM structure.
(CaMKK). These enzymes are thus organized into cas- 2. The bilobed Ca2 + /CaM wraps around the Ca2 + /CaM-
cades during which information is transferred to down- binding domains (blue) so that the catalytic site (C)
stream targets. CAMKII, which is a multimer of eight to moves away from the autoinhibitory domain (red) and
twelve subunits, has a different activation mechanism that is now free to carry out two types of phosphorylation
depends solely on the binding of Ca2 + /CaM. However, reactions (Steps 3 and 4).
this isoform has a complex autophosphorylation mechan- 3. In one type of phosphorylation reaction, the catalytic
ism that gives it unique properties to function both as a domain (C) phosphorylates substrates of the down-
frequency detector and as a long-term storage of informa- stream effectors controlled by CaMKII. These sub-
tion. strates have a substrate domain (red) that resembles
the autoinhibitory domain.
4. The other type of phosphorylation reaction is a
Ca2 + /calmodulin-dependent protein kinase kinase kinase/kinase reaction in that the catalytic site (C) can
(CaMKK) also phosphorylate Thr-286 in the autoinhibitory do-
This enzyme is located in both the cytoplasm and nucleus. main (red) of a neighbouring subunit. In order for this
It functions as part of a phosphorylation cascade to ac- intermolecular phosphorylation to occur, two neigh-
tivate either Ca2 + /calmodulin-dependent protein kinase I bouring subunits have to be activated by each bind-
(CaMKI) or IV (CaMKIV) (Module 4: Figure activation ing a Ca2 + /CaM, which then enables one to act as a
of CaMKs). kinase and the other as a substrate. In effect, there is an
CaMKK may also function to phosphorylate other sub- internal kinase cascade that operates between contigu-
strates such as AMP-activated protein kinase (AMPK) as ous subunits. The phosphorylation of Thr-286 opens
part of the AMP signalling pathway (Module 2: Figure up the molecule to unveil new binding sites for CaM,
AMPK control of metabolism). and this can lead to the phenomenon of CaM trapping,

C
Module 4: Figure structure of CaMKs
2+
Ca -calmodulin-dependent protein kinases (CaMKs)
A C CaMKK Autoinhibitory domain
A C CaMKK Ca 2+/CaM-binding domain
A C CaMK I ATP-binding site (A)

Association Catalytic domain (C)
domain
A C CaMK II
A C CaMK IV
CaMK II
A C CaMK IV
C
A
A
CaMK I
C
A A C
A C
A
A
C
C
The domain structure and organization of Ca2 + /calmodulin-dependent protein kinases (CaMKs).
The Ca2 + /calmodulin-dependent protein kinases (CaMKs) share a number of similar domains. They all have an ATP-binding site (A), a catalytic
domain (C) and an autoinhibitory domain (red) that overlaps the Ca2 + /CaM (calmodulin)-binding domain (blue). Most of the CaMKs function as
monomers, as shown for CaMKI at the bottom of the figure. In the resting state, the catalytic site (C) bends around to interact with the autoinhibitory
domain (red). CaMKII is a multimeric enzyme, and half of the complex is illustrated on the right, where six monomers connect together through their
C-terminal association domains to create a wheel-like structure (Module 4: Figure structure of CaMKII).
Module 4: Figure activation of CaMKs
CaM
Ca 2+
CaM.Ca
CaMKK CaMKI or CaMKIV
A C A C
C C
A A
P C
A
P
Substrate
Ca2 + activation of Ca2 + /calmodulin-dependent protein kinases I and IV (CaMKI/CaMKIV).

Ca2 + /CaM (calmodulin)-dependent protein kinase I (CaMKI) and IV (CaMKIV) are described together because they share a similar activation
mechanism. The action of Ca2 + begins with it binding to CaM to form the Ca2 + /CaM complex, which then has two separate actions. Firstly, it
binds to the Ca2 + /CaM-binding site of CaMK kinase (CaMKK), resulting in a conformational change that leads to the activation of the catalytic
domain. In its second action, Ca2 + /CaM binds to CaMKI or CaMKIV, causing a similar conformational change that then enables the activated CaMKK
to phosphorylate a threonine residue on the activation loops of CaMKI (Thr-177) or CaMKIV (Thr-196), which are then able to phosphorylate their
substrates.

C
Module 4: Figure structure of CaMKII
Three-dimensional structure of Ca2 + /calmodulin-dependent protein kinase II (CaMKII).

The domain structure at the bottom illustrates the different structural regions of one of the Ca2 + /calmodulin-dependent protein kinase (CaMKII)
monomers that make up the dodecameric structure. The structure of the multimeric holoenzyme was determined by reconstructing electron micro-
scopic images. It has a barrel-shaped structure made up of the association domains forming the inner core, with the catalytic domains protruding
as foot structures from either side. In effect, there will be a ring of six catalytic domains on either side of the molecule that are located close enough
for the intermolecular interactions necessary for enzyme activation (Module 4: Figure CaMKII activation). The stain model-based reconstructions
were reproduced from Kolodziej, S.J., Hudmon, A., Waxham, M.N. and Stoops, J.K. (2000) Three-dimensional reconstructions of calcium/calmodulin-
dependent (CaM) kinase IIa and truncated CaM kinase IIa reveal a unique organisation for its structural core and functional domains. J. Biol. Chem.
275:1435414359, with permission from the American Society for Biochemistry and Molecular Biology; see Kolodziej et al. 2000.
which becomes apparent during the recovery phase CamKII has multiple functions:
when Ca2 + is removed.
It mediates the action of Ca2 + in triggering chromo-
5. When Ca2 + returns to its resting level, CaM comes
some separation at anaphase (Module 9: Figure chro-
off those subunits that have not been phosphorylated,
mosome separation).
and these subunits return to their resting configura-
It carries out the action of Ca2 + in stimulating PtdIns 3-
tions. However, those that have been phosphorylated
kinase during phagosome maturation (Module 4: Figure
on Thr-286 remain in an active state. There are two
phagosome maturation).
types of autonomous activity: those molecules that have
It enhances inositol 1,4,5-trisphosphate (InsP3) forma-
not trapped CaM, whereas others have a trapped CaM
tion by inhibiting the inositol polyphosphate 5-phos-
(Steps 6 and 7 respectively).
phatase.
6 and 7. The phosphorylation of Thr-286 thus creates
CaMKII functions as a molecular switch in learning and
Ca2 + /CaM-independent states that remain competent
memory (Module 10: Figure Ca2+ control of LTD and
to phosphorylate downstream substrates even though
LTP).
Ca2 + has returned to its resting level. It is this
Phosphorylation of phosphodiesterase 1B (PDE1B) by
autonomous activity that gives the enzyme such in-
CaMKII results in a decrease in its sensitivity to Ca2 +
teresting properties. This autonomous activity implies
activation.
that CaMKII has a memory, and this long-lasting
CaMKII is one of the enzymes that phosphorylates
activity has been implicated as the molecular switch in
phospholamban (PLN) to control the pumping activ-
learning and memory (Module 10: Figure Ca2+ control
ity of the sarco/endo-plasmic reticulum Ca2 + -ATPase
of LTD and LTP).
2a (SERCA2a) pump (Module 5: Figure phospholam-
ban mode of action).
CaMKII phosphorylates Syn-GAP, which is one of
The fact that the holoenzyme has 12 subunits all capable the GTPase-activating proteins (GAPs) responsible for
of being switched into an autonomous state means that the switching off Ras signalling (Module 2: Figure Ras sig-
enzyme is capable of counting. Indeed, a role for CaMKII nalling).
in frequency decoding may play a key role in the process CaMKII stimulates cytosolic phospholipase A2
of encoding and decoding of Ca2+ oscillations. (PLA2), which is phosphorylated on Ser-515.

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CaMKII associates with the N-methyl-d-aspartate important in regulating the process of glycogenolysis both
(NMDA) receptor and can alter its activity by phos- in skeletal muscle (Module 7: Figure skeletal muscle E-C
phorylating sites on both NR2A and NR2B subunits coupling) and in liver cells (Module 7: Figure glycogen-
(Module 3: Figure NMDA receptor). olysis and gluconeogenesis).
CaMKII activates the transcription factor cyclic AMP
response element-binding protein (CREB) to modulate Myosin light chain kinase (MLCK)
the circadian clock (Module 6: Figure circadian clock Myosin light chain kinase (MLCK) is a Ca2 + -sensitive
input-output signals). enzyme that functions to phosphorylate the myosin light
In parietal cells, CaMKII phosphorylates the Ca2 + - chain that regulates the activity of myosin II (NMII)
sensitive phosphoprotein of 28 kDa (CSPP-28) during found in both smooth muscle and a variety of non-muscle
the onset of acid secretion (Module 7: Figure HCl se- cells:
cretion).
Control of smooth muscle contraction (Module 7: Fig-
Ca2 + /calmodulin-dependent protein kinase IV ure smooth muscle cell E-C coupling).
(CaMKIV) Control of cytokinesis during cell division (Module 9:
Ca2 + /calmodulin-dependent protein kinase IV Figure cytokinesis).
(CaMKIV) has a somewhat limited tissue distribu- Control of endothelial permeability (Module 7: Figure
tion in that it is located mainly in the nucleus. CaMKIV endothelial cell contraction).
is inactive until CaMK kinase (CaMKK) phosphorylates
a single threonine residue (Thr-196) on its activation loop Calcineurin (CaN)
(Module 4: Figure activation of CaMKs). This activation Calcineurin (CaN), which is also known as protein phos-
process is reversed by protein phosphatase 2A (PP2A), phatase 2B (PP2B), is a member of the phosphopro-
which is constitutively active and is normally found
tein phosphatase (PPP) family of serine/threonine pro-
closely associated with CaMKIV. There is some indication tein phosphatases (Module 5: Table serine/threonine phos-
that Ca2 + can dissociate this complex, thus prolonging phatase classification). The function of calcineurin is in-
the active phosphorylated form of CaMKIV. hibited by the immunosuppressant drugs cyclosporin A
The CaMKIV located in the nucleus has a number of (CsA) and FK506, which act through the immunophilins.
functions: Calcineurin functions as a heterodimeric complex com-
CaMKIV phosphorylates the transcription factor cyclic posed of a catalytic A subunit (CaNA), a regulatory B
AMP response element-binding protein (CREB) (Mod- subunit (CaNB) and calmodulin (CaM). Both the B sub-
ule 4: Figure CREB activation). Such a mechanism op- unit and calmodulin confer the Ca2 + -sensitivity of the en-
erates during the control of neuronal gene transcription zyme. There are three isomers of the A subunit (CaNA,
(Module 10: Figure neuronal gene transcription). CaNA and CaNA). Whereas CaNA and CaNA are
CaMKIV contributes to the differentiation of skeletal expressed in many different cells, CaNA is restricted to
muscle. It phosphorylates histone deacetylase (HDAC), the testis and certain regions of the brain. An alteration in
which is then exported from the nucleus in association the CaNA has been linked to schizophrenia.
with the 14-3-3 protein, thus terminating the deacetyla- CaN is activated by Ca2 + through a two-stage process
tion of chromatin as occurs during the activation of (Module 4: Figure calcineurin):
MyoD (Module 4: Figure MyoD and muscle differenti-
1. As the cytosolic Ca2 + level rises, Ca2 + binds to the two
ation).
low-affinity sites on CaNB to induce a conformational
CaMKIV plays a prominent role in activating the tran-
change in CaNA, resulting in the exposure of the CaM-
scription factor myocyte-enhancer factor 2 (MEF2)
binding domain (CaM-BD) site.
(Module 4: Figure MEF2 activation).
2. In the next step, Ca2 + activates CaM, which wraps
The transcriptional repressor methyl-CpG-binding
around the CaM-BD site to induce a further conform-
protein 2 (MeCP2) is phosphorylated by CaMKIV dur-
ational change that pulls the autoinhibitory domain
ing the activation of neural genes such as that encoding
(AID) away to free up the catalytic site so that it can be-
brain-derived neurotrophic factor (BDNF) (Module 4:
gin to hydrolyse its phosphorylated substrates such as
Figure MeCP2 activation).
nuclear factor of activated T cells (NFAT) and protein
Cell depolarization produces Ca2 + signals that activ-
phosphatase 1 (PP1).
ate CAMKIV to regulate the alternative splicing of ion
channels through a CAMKIV-responsive RNA element In cardiac cells, CaN is anchored to the sarcolemma by
(CaRRE). binding to calcium and integrin-binding protein 1 (CIB1).
During cardiac hypertrophy, there is an increased expres-
Phosphorylase kinase sion of CIB1 and this may contribute to the way CaN
Phosphorylase kinase is a Ca2 + -sensitive enzyme that is triggers the cardiac NFAT shuttle (Module 12: Figure hy-
regulated by a resident calmodulin (CaM). The cyclic AMP pertrophy signalling mechanisms).
signalling pathway can phosphorylate this enzyme and this The Ca2 + -dependent activation of CaN then acts by
can enhance the activity of the enzyme by increasing its dephosphorylating a number of key signalling compon-
sensitivity to Ca2 + . Phosphorylase kinase is particularly ents:

C
Module 4: Figure CaMKII activation
CaM
C
CaMK II
A
C
A A C Ca 2+
A
A
C
A
A
C 2
C
C
A
2+
Ca /CaM
C
C
A
C A C
AA
AA
A A
CC
C
C
C
Substrate C
A
C
A A
P A C
A
A
C
C
A
C A
3
C
AA
4
C
C
C
C
C
A
A
C
P C AA
C A
A
6
C
AA
A
5
A
CC
A
C
C
C
C
P
C
P
A
7 Substrate
C
C
A
AA C
A
CaM trapped
A
CaM
CC
C
C
Ca2 + activation of Ca2 + /calmodulin-dependent protein kinase II (CaMKII).

The activation of Ca2 + /calmodulin-dependent protein kinase II (CaMKII) by Ca2 + (half-maximal at a Ca2 + concentration of 0.51 M) depends upon
a sequential series of events that gives rise to a number of CaMKII activation states. See the text for details of Steps 17.
One of the major targets is the transcription factor The number of calcineurin molecules in cells can vary
NFAT (Module 4: Figure NFAT activation). enormously: 5000 in lymphocytes, but 200 000 in hippo-
CaN acts together with PP1 during synaptic plasticity campal and cardiac cells.
in neurons (Module 10: Figure Ca2+ control of LTD
and LTP). It plays a central role in the mechanism of Immunophilins
Ca2 + -induced synaptic plasticity where it acts to erase The immunophilins are a family of proteins that modulate a
memories (Module 10: Figure Ca2+ -induced synaptic number of signalling components. They were first defined
plasticity). through their ability to bind to immunosuppressive drugs
CaN controls Ca2 + -dependent gene transcription in such as cyclosporin A (CsA) and FK506. The major im-
glucagon-secreting -cells (Module 7: Figure -cell sig- munophilins are cyclophilin A and the FK506-binding
nalling). proteins (FKBPs).
CaN dephosphorylates the transducer of regulated
CREB (TORC), thus enabling it to enter the nucleus Cyclophilin A
to co-operate with CREB to switch on transcription Cyclophilin A is the protein that binds cyclosporin A to
(Module 4: Figure CREB activation). regulate the activity of calcineurin (CaN). This inhibition

C
Module 4: Figure calcineurin
CaNB
1 CaNB
-BD
CaNB-BD
CaNA CaM-BD CaNA
CaM-BD
Catalytic Catalytic
AID AID site
site
Cytosolic
2+
Ca
CaNA
Ca Catalytic
NB
-BD site
Ca
M-
2+ P
BD
CaM 2 Ca /CaM
P
AID
Phosphorylated
substrates
( e.g. NFAT, PP1)
Calcineurin (CaN) is a Ca2 + /calmodulin-sensitive serine/threonine protein phosphatase.

Shown on the top is the catalytic calcineurin A (CaNA) subunit, which has the catalytic site, a calcineurin B-binding domain (CaNB-BD), a calmodulin
(CaM)-binding domain (CaM-BD) and an autoinhibitory domain (AID) that inhibits the catalytic site in the inactive state. CaNB is an EF-hand Ca2 + -
binding protein that has a similar structure to CaM and is bound to the enzyme under resting conditions. The stimulatory action of Ca2 + is carried out
by two Ca2 + sensors, CaNB and CaM, which act by removing the inhibitory effect of AID that occurs in two steps, as described in the text.
of CaN is particularly evident in the inhibition of nuc- Cyclosporin A (CsA)

lear factor of activated T cells (NFAT) activation in T cells FK506
(Module 9: Figure T cell Ca2+ signalling), which is the Downs syndrome critical region 1 (DSCR1)
basis of decreasing the rejection of transplanted organs by Calcineurin homologous protein (CHP)
the immunosuppressant drug cyclosporin A (CsA). CsA is Cain/Cabin
also able to inhibit the cardiac gene transcription respons- Carabin
ible for hypertrophy.
Cyclosporin A (CsA)
FK506-binding proteins (FKBPs) Cyclosporin A (CsA) is a potent immunosuppressant drug
The FK506-binding proteins (FKBPs) were first recog- that acts to inhibit a family of cyclophilins. CsA binds to
nized through their ability to bind the immunosuppress- one of the cyclophilins, and this CsA/cyclophilin complex
ant drugs FK506 and rapamycin. There are approximately then binds to the active site of calcineurin (CaN) to block
eight family members in mammals, with most attention enzymatic activity. By inhibiting the activation of nuclear
being focused on FKBP12 and FKBP12.6. These members factor of activated T cells (NFAT) in T cells (Module 9:
of the immunophilin family have peptidylpropyl cistrans Figure T cell Ca2+ signalling), CsA is capable of reducing
isomerase (PPIase) activity. the rejection of transplanted organs. CsA is also able to
One of the functions of FKBP12 is to regulate the activ- inhibit the cardiac gene transcription responsible for hy-
ity of the target of rapamycin (TOR) (Module 9: Figure pertrophy.
target of rapamycin signalling). Another important func- CsA is also a potent inhibitor of apoptosis. It binds
tion of the FKBPs is to regulate the Ca2 + release channels. to the cyclophilin-D (CyP-D), which is a component of
In the case of muscle cells, FKBP12 (calstabin1) regulates the mitochondrial permeability transition pore (mPTP)
the ryanodine receptor 1 (RYR1), whereas FKBP12.6 (cal- (Module 5: Figure ER/mitochondrial shuttle).
stabin2) modulates the activity of ryanodine receptor 2
(RYR2) in cardiac cells. FK506
Like cyclosporin A, FK506 is a potent immunosuppress-
Calcineurin inhibitors ant drug capable of reducing the rejection of transplanted
The activity of calcineurin (CaN) is sensitive to a number organs. It acts by binding to the FK506-binding protein
of inhibitors, both endogenous and exogenous: (FKBP), and this FK506/FKBP complex then binds to the

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active site of calcineurin (CaN) to block enzymatic activity. trated in Module 4: Figure membrane and protein traffick-
FK506 has been shown to reverse Ca2+ -dependent neuro- ing:
degeneration by preventing the memory loss in a mouse
model of Alzheimers disease (AD) (Module 12: Figure 1. Endoplasmic reticulum/Golgi transport mechanisms
amyloids and Ca2+ signalling). describe the two-way protein transport pathways that
operate between the ER and the Golgi. The coat pro-
Downs syndrome critical region 1 (DSCR1) tein complex II (COPII) vesicles carry cargo from the
The Downs syndrome critical region 1 (DSCR1) gene is ER to the Golgi, whereas the COPI vesicles return
found in the critical region of chromosome 21 that is amp- certain ER proteins from the Golgi back to the ER.
lified by trisomy. There are approximately 230 supernu- 2. Golgi protein sorting and packaging.
mary genes on this extra region of DNA. DSCR1, which is 3. Golgi-to-plasma membrane transfer
also known as the regulator of calcineurin 1 (RCAN1) or The trans-Golgi network (TGN) is a major protein
the modulator calcineurin interacting protein 1 (MCIP1), sorting organelle functioning to direct newly synthes-
is thus one of the candidate genes that may be respons- ized proteins either to the plasma membrane or to
ible for Downs syndrome. DSCR1 is part of a family that various endosomal compartments. Control of vesicle
includes ZAKI-4 and DSCR1L2 (DSCR1-like 2), which formation at the TGN depends on Ca2 + regulation
also act to inhibit calcineurin (CaN). These inhibitors are of the PtdIns 4-KIII that forms the PtdIns4P neces-
strongly expressed in the brain, and DSCR1 and ZAKI-4 sary for this trafficking process. In addition, it can also
are also found in the heart and skeletal muscle. receive cargo back from the endosomes. This bidirec-
One of the interesting features of DSCR1 is that its tional transfer between the TGN and endosomes is
expression is induced by Ca2 + acting through a cal- particularly important for sorting and directing lyso-
cineurin (CaN)/nuclear factor of activated T cells (NFAT)- somal hydrolases towards the lysosome. These hydro-
dependent mechanism, thus representing a negative- lases are carried on the cation-independent mannose 6-
feedback loop to limit the activity of CaN (Module 4: phosphate receptor (CI-MPR), which is then returned
Figure NFAT control of Ca2+ signalling toolkit). to the TGN by the early endosome to trans-Golgi net-
work (TGN) trafficking pathway (Module 4: Figure
Calcineurin homologous protein (CHP) endosome budding TGN).
Calcineurin (CaN) homologous protein (CHP) shares 4. Exocytosis
some homology with CaN regulatory B subunit (CaNB) 5. Endocytosis and the transport of vesicles to the early
and competes with the latter to inhibit the activity of CaN. endosome by myosin motors such as myosin VI.
6. Endosome vesicle fusion to early endosomes
7. Early endosome protein sorting and intraluminal ves-
Cain/Cabin
icle formation.
Cain/Cabin are non-competitive inhibitors of calcineurin.
The endocytic vesicles that bud off from the plasma
Cain/Cabin can reduce the cardiac gene transcription re-
membrane fuse with the early endosome to deliver a
sponsible for cardiac hypertrophy. It can act as a repressor
number of different proteins. These proteins have to
to inhibit the activity of the transcription factor MEF2
be sorted and then packaged for transport to a number
(Module 4: Figure MEF2 activation)
of other cellular locations.
8. Early endosome to plasma membrane trafficking
Carabin 9. Early endosome to trans-Golgi network (TGN) traf-
Carabin has 446 amino acid residues and has a pu- ficking
tative Ras/Rab GAP domain at the N-terminus and 10. Early endosome maturation to lysosomes
a calcineurin-binding domain at the C-terminus. It is As the early endosome begins to accumulate int-
strongly expressed in spleen and peripheral blood lymph- ralumenal vesicles it matures into a multivesicular en-
ocytes. During T cell receptor (TCR) signalling, there is dosome (MVE). At this stage, there is a large accumu-
a marked up-regulation of carabin that may thus exert a lation of PtdIns3,4,5P2 , which is a phosphoinositide
negative-feedback loop to inhibit T cell signalling (Mod- lipid signalling molecule (Module 2: Figure localized
ule 9: Figure T cell Ca2+ signalling). Carabin also inhibits inositol lipid signalling) that activates the TRPML1
Ras signalling through its Ras GTPase activity and may channels to create the local domains of Ca2 + that trig-
thus provide a cross-talk mechanism between the Ras and gers the fusion events to form the lysosomes.
Ca2 + signalling pathways. 11. There is an alkaline phosphatase (ALP) pathway re-
sponsible for transporting ALP from the Golgi to the
endosomallysosomal complex. The ALP and another
Membrane and protein trafficking cargo protein Vam3 have di-leucine sorting signals that
Membranes and their protein components are constantly direct them into adaptor protein-3 (AP-3)-coated ves-
being turned over through a mechanism that has multiple icles. The AP-3 is associated with hVps41 that helps
components and pathways. Most emphasis will be focused to transport the vesicles to the endosomallysosomal
on the proteins that are synthesized on the endoplasmic complex. The SORT1 trafficking protein may use a
reticulum and then begin their journey through the cell similar pathway to transport proteins from the Golgi
through a number of pathways some of which are illus- to the lysosomes.

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Module 4: Figure membrane and protein trafficking
TFR EGFR
EGFR
Exocytosis
Exocytosis Endocytosis
5
4
Recycling 6
Early
endosome
endosome
Actin 8
7
Trans-Golgi
network (TGN)
Golgi 3 Intralumen
CI-MPR Lysosomal hydrolases endosomal
9 vesicle
2 Early 10
endosome
hVps41 (MVE)
Microtubule
AP-3
ERGIC
COPI
COPII 1
Endoplasmic
ALP pathway 11
reticulum
Lysosome
Nucleus
Membrane and protein trafficking pathways.

The turnover of membrane components and proteins depends on a constant flux between the plasma membrane and an array of intracellular
organelles such as the endoplasmic reticulum, Golgi, various endosomal compartments and the lysosome. The lipid signalling pathways responsible
for controlling these trafficking processes is described in Module 2: Figure localized inositol lipid signalling. The major pathways that connect these
different organelles are described in the text. CI-MPR, cation-independent mannose 6-phosphate receptor.
There are two hVps41 SNPs that may increase the In this section, we will consider the two-way transport
susceptibility of developing Parkinsons disease. between the ER and the Golgi that is orchestrated by coat
protein complex I and II (COPI and COPII). COPII-me-
diated transport from ER to Golgi is responsible for the
Endoplasmic reticulum/Golgi transport anterograde transport system (Module 4: Figure COPII
mechanisms coated vesicles), whereas COPI-mediated transport from
The first step in membrane and protein trafficking is the Golgi to ER takes care of the retrograde transport of cer-
transfer of proteins from the ER to the Golgi (see step 1 in tain proteins that are returned to the ER (Module 4: Figure
Module 4: Figure membrane and protein trafficking). The COPI-coated vesicle).
Golgi is a highly dynamic organelle that processes large
amounts of protein that not only is being exported to the COPII-mediated transport from ER to Golgi
plasma membrane, but is also constantly being exchanged The anterograde transport of newly synthesized proteins
with the ER and the endosomal system. To carry out these from the ER to the ERGolgi intermediate compart-
dynamic Golgi functions, it is essential that this organelle ment (ERGIC) is carried out by coat protein complex II
maintains its characteristic morphology. The PtdIns4 sig- (COPII) through the following sequence of events (Mod-
nalling cassette (Module 2: Figure localized inositol lipid ule 4: Figure COPII-coated vesicles):
signalling) seems to play an important role in orchestrat-
ing both the morphology and function of the Golgi. The 1. The ER exit sites (ERES), which are specialized to
PtdIns4P binds to proteins such as oxysterol-binding pro- communicate with the Golgi, have the secretory 12
tein (OSBP), phosphatidylinositol-Four-P AdaPtor Pro- (Sec12) type II transmembrane protein that functions
tein (FAPP) and the ceramide transfer protein (CERT). as a guanine nucleotide-exchange factor (GEF) for the
CERT functions in the generation and function of cer- small GTPase Sar-1. When cytoplasmic Sar-1 interacts
amide and sphingosine 1-phosphate (S1P) (see Step 2 in with Sec12, it exchanges its GDP for GTP, which then
Module 2: Figure sphingomyelin signalling). In addition, induces a conformational change causing the protru-
GOLPH3 binds to PtdIns4P to provide an anchor, which sion of an N-terminal amphipathic -helix that attaches
is linked to myosin 18A and then to actin to provide a Sar-1 to the membrane.
tensile force that stretches out the membrane stacks to 2. The membrane-bound Sar-1.GTP complex then re-
maintain the characteristic shape of the Golgi. cruits components of the COPII complex beginning

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with the Sec23/Sec24 dimer. The Sec23 has two func- final approach may be facilitated by the golgin protein
tions. First, it is a GTPase-activating protein (GAP) p115, which attaches to Rab1 on the Golgi membrane.
that will come into play later when the COPII coat 9. Once the SNAREs begin to interact with each other,
is removed (see step 6 below). Secondly, it functions they drive vesicle fusion thus enabling the ER cargo
to attach the vesicle to the Golgi surface by binding proteins to reach the Golgi
to the tethering complex trafficking protein particle 1
(TRAPP1). The Sec24 is responsible for capturing the COPI-mediated transport from Golgi to ER
cargo that is to be transported to the Golgi. The COPI retrieval pathway functions to return those
3. The Sec13/Sec31 heteromeric complex then attaches to ER-resident proteins that escaped to the Golgi through
the Sec23/Sec24 dimer to complete the formation of the the COPII-mediated transport from ER to Golgi (Module
COPII complex. As these COPII complexes accumu- 4: Figure COPII-coated vesicles). This retrieval pathway
late, they induce a localized curvature of the membrane (see step 2 in Module 4: Figure membrane and protein traf-
that then matures into a bud. SNARE proteins, which ficking) is somewhat more complex than the anterograde
will be used for vesicle fusion to the Golgi, are also pathway because it can originate from multiple locations
incorporated into the maturing vesicle. within the Golgi. ER-resident proteins that move along
4. The mechanism of bud scission is still not fully un- the Golgi as it matures can be removed at all levels and
derstood. There are indications that the hydrolysis of moved backwards to be returned to the Golgi. This ret-
phosphatidylcholine (PC) by phospholipase D (PLD) rograde transport to the ER, which depends on the coat
to form phosphatidic acid (PA) may play a role in de- protein complex I (COPI), occurs through the follow-
forming the membrane during bud scission. PA may ing sequence of events (Module 4: Figure COPI-coated
also be formed from diacylglycerol (DAG) through vesicles):
the activity of DAG kinase (DAGK). Both DAG and
PA are cone-shaped lipids that can bring about negative 1. The initial step in this retrograde pathway depends on
curvature of the membrane that can facilitate scission of the activation of the small GTPase Rab1b, which then
the vesicle. The BFA-induced ADP-ribosylation sub- recruits the golgin protein p115.
strate (BARS), whose activity seems to depend on PA, 2. The Rab1b/p115 complex helps to recruit the Golgi-
has also been implicated in the process of cutting off specific brefeldin A-resistant factor 1 (GBF1), which
the vesicle. is a guanine nucleotide-exchange factor (GEF) for
5. Once the vesicle has been released from the ER, it is the small monomeric G protein ADP-ribosylation
carried along microtubules towards the Golgi by the factor 1 (Arf1) (Module 2: Table monomeric G protein
dynein motor. It is the p50 component of dynactin, toolkit). The Arf signalling pathway has an import-
which is a dynein adaptor (Module 4: Figure dynein), ant role in controlling key events that occur during
that uses the golgin bicaudal D and Rab6 to attach the coat formation, actin polymerization and Golgi ves-
motor to the COPII vesicle. icle budding (Module 2: Figure Arf signalling).
6. As the COPII-coated vesicle approaches the ERGolgi 3. When cytoplasmic Arf1.GDP interacts with GBF1,
intermediate compartment (ERGIC) membrane sys- it exchanges its GDP for GTP, which then induces a
tem, it begins to shed its COPII coat. Part of this shed- conformational change causing the protrusion of an
ding process seems to be driven by the inactivation of N-terminal amphipathic -helix capped by a myris-
the Sar-1.GTP complex when the GTP is hydrolysed to toyl group that attaches Arf1.GTP to the membrane
GDP through a process facilitated by the GAP activity that sets the stage for assembling the COPI coat.
of Sec23 (see step 2 above) (Module 4: Figure COPII 4. The membrane-bound Arf1.GTP complex then be-
coated vesicles). gins to assemble components of the COPI complex
7. The Sec23 has an additional function of attaching the beginning with the p23/p24 heterodimer and this is
vesicle to the TRAPP1 tethering complex, which con- then followed by the large COPI complex that con-
tains six main subunits. It is the Bet3 subunit that is sists of multiple subunits organized into two groups:
responsible for binding to the Sec23 subunit on the F-COP (, , , and ) and B-COP (, and ). It is
vesicle. The TRAPP1 is also a guanine nucleotide- the -COP and the -COP that bind to the Arf1.GTP.
exchange factor (GEF) for Rab1 and thus converts At this stage, the cargo marked out for transport also
inactive Rab1.GDP into active Rab1.GTP that func- associates with this large COPI coat.
tions in vesicle tethering. Sec31 can bind the penta-EF- 5. As these COPI complexes begin to accumulate, they
hand protein apoptosis-linked gene 2 (ALG-2), which induce a localized curvature of the membrane that
also interacts with Annexin-11 (ANX11). Alg-2 and then matures into a bud. SNARE proteins, which will
ANX11 are Ca2 + -binding proteins that may carry out be used for vesicle fusion to the Golgi, are also in-
a Ca2 + -dependent regulatory step to modify the mem- corporated into the maturing vesicle. It is during this
brane events related to either vesicle formation or the budding stage that the ArfGAP1 and the two closely
later function of COPII vesicles to the Golgi. To what related ArfGAPs 2 and 3 (ArfGAP2/3) associate with
extent Ca2 + plays a role in the regulation of this ER- the developing bud through separate mechanisms. Ar-
to-Golgi transport system remains to be determined. fGAP1 recognizes the curvature of the bud using
8. The next step is for the SNARE complexes on the two an ArfGAP1 lipid sensory (ALPS) motif, which is
membranes to begin to interact with each other. This normally unstructured, but when it detects a curved

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Module 4: Figure COPII-coated vesicles
ERGIC
9 Rab1 Rab1
Rab1 _
GTP
GDP TRAPP1
Cargo
8 7 Sec23
p115
SNARE
2+
Ca
Dynein
Sec13/Sec31
Microtubule
Sec23 Sec24 Alg-2
ANX11
+ 5
Sar-1 GDP
3
GTP GDP
2 +
1 Sec23 Sec24 Alg-2
Sec23 Sec24
ANX11 4
Sar-1 GTP Sar-1 GTP Sar-1 GTP
Sec12 BARS
DAGK PLD
DAG PA PC
Cargo
Endoplasmic reticulum SNARE
Cargo transport from the ER to the Golgi using COPII-coated vesicles.

The small GTPase Sar-1 initiates the transport process by bringing together the COPII coat made up of the Sec23/Sec24 and Sec13/Sec31 complexes
that bind to the cargo. The COPII, perhaps helped by the Ca2 + -binding proteins apoptosis-linked gene 2 (ALG-2) and annexin-11 (ANX11), then
deforms the membrane to form a bud that is cut off and is transported along microtubules towards the ERGolgi intermediate compartment (ERGIC)
using the dynein motor. The COPII coat is removed allowing the vesicle to be captured by tethers such as trafficking protein particle 1 (TRAPP1),
which enables the SNARE proteins to engage with each other and to fuse with the early Golgi compartment. Note that there is a change in scale
between steps 1 and 4 and the subsequent steps in the sequence. See the text for further details.
membrane it forms an amphipathic -helix enabling 9. The next step is for the SNARE complexes on the two
the molecule to bind to the developing vesicle. membranes to begin to interact with each other.
The mechanism of bud scission is still not fully 10. This SNARE interaction finally drives vesicle fusion
understood. There are indications that the hydro- thus enabling the Golgi proteins to reach the ER.
lysis of phosphatidylcholine (PC) by phospholipase
D (PLD) to form phosphatidic acid (PA) may play Golgi sorting and protein packaging
a role. PA may also be formed from diacylglycerol The Golgi consists of a series of flattened cisternal mem-
(DAG) through the activity of DAG kinase (DAGK). branes, which are stacked on top of each other (Module
Both DAG and PA are cone-shaped lipids that can 4: Figure membrane and protein trafficking). The Golgi
bring about negative curvature of the membrane that is polarized with the cis-face exchanging proteins and lip-
can facilitate scission of the vesicle. The BFA-induced ids with the endoplasmic reticulum while the trans-face
ADP-ribosylation substrate (BARS), whose activity sends secretory proteins to the plasma membrane and also
seems to depend on PA, has also been implicated in communicates with the endosomal system.
the process of cutting off the vesicle. This stack like organization appears to be held together
6. Once the vesicle has been released from the Golgi, it by interactions with the cytoskeleton and also through the
is carried along actin filaments towards the ER by a action of the coiled-coil proteins of the golgin family.
kinesin-2 motor.
7. As the COPI-coated vesicle approaches the ER, it Golgins
begins to shed its COPI coat. Part of this shedding The Golgins are a family of coiled-coil proteins that op-
process seems to be driven by the inactivation of the erate within the Golgi during the process of Golgi sorting
Arf1.GTP complex when the GTP is hydrolysed to and protein packaging. The golgins help to maintain the
GDP through a process facilitated by the GAP activ- structural organization of the Golgi and may also func-
ities of both ArfGAP1 and ArfGAP2/3. tion as membrane tethers during vesicle transfer between
8. The initial contact between the vesicle and the ER different vesicle compartments. The golgins function as di-
depends on a tethering complex called syntaxin 18, mers held together by their coiled-coil regions, and some
which may also act to draw in the SNARE proteins of the family members have interaction domains that en-
necessary for subsequent membrane fusion. able them to interact with various small GTPases such as

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Module 4: Figure COPI-coated vesicle
p23/p24 Cargo SNARE

ERGIC
DAG PA PC
Rab1b Rab1b GBF1 5 BARS PLD
GTP GTP Arf1 GTP Arf1 GTP DAGK
ArfGAP
2/3
3
2 4

1
p115
Kinesin-2
ArfGAP1
GBF1 F-COP 6
ArfGAP
Rab1b 2/3
Arf1 GDP B-COP

8
10 9
Syntaxin 18
Endoplasmic
reticulum SNARE
Retrograde cargo transport from the Golgi to the ER using COPI-coated vesicles.
The small GTPase Rab1b initiates the transport process by activating Arf1 that then acts to bring together the p23/p24 dimer, the COPI coat made up
of multiple subunits and the cargo protein that is to be returned to the ER. The COPI then helps to deform the membrane to form a bud that is then
cut off and transported along actin filaments towards the ER using kinesin-2 motors. The COPI coat components are removed allowing the vesicle to
be captured by tethers such as syntaxin 18, which then enables the SNARE proteins to engage with each other and to fuse with the ER. Note that
there is a change in scale between steps 1 and 4 and the subsequent steps in the sequence. See the text for further details.
Rab1, ADP-ribosylation factor (Arf) and Arf-like (ARL) 97 and golgin-245 attach to ARL1 through their GRIP
(Module 2: Table monomeric G protein toolkit) that appear domains.
to control their interaction with the Golgi membranes. The 5. Arf-associated golgins, such as GMAP-210, are re-
following are some of the main golgin family members: cruited to membranes through the small GTPase AD-
P-ribosylation factor (Arf).
1. Transmembrane golgins such as Giantin, Golgin-84

and CCAAT-displacement protein (CDP) alternatively
Golgi to plasma membrane transfer
The trans-Golgi network (TGN) is a major protein-sorting
spliced product (CASP) are attached to membranes
organelle functioning to direct newly synthesized proteins
through a C-terminal transmembrane domain.
either to the plasma membrane or to various endosomal
2. Adaptor-associated golgins are attached to mem-
compartments (see step 3 in Module 4: Figure membrane
branes through the Golgi reassembly stacking protein
and protein trafficking). The formation of vesicles at the
(GRASP) adaptors, which associate with membranes
TGN appears to be regulated by a local Ca2 + signal that
through an N-terminal myristoyl group. For example,
stimulates the PtdIns4 signalling cassette (Module 2: Fig-
GM130 is attached to GRASP65, whereas Golgin-45 is
ure localized inositol lipid signalling). A key component
attached to GRASP55.
of this Golgi lipid signalling system is PtdIns 4-KIII. At
3. Rab-associated golgins are recruited to membranes
resting levels of Ca2 + , this lipid kinase is kept inactive
through the small Rab GTPases. Rab1 recruits p115
when bound to the Ca2 + -sensing proteins calneuron-1 or
during COPI-mediated transport from Golgi to
calneuron-2. In response to a local pulse of Ca2 + , the in-
ER (Module 4: Figure COPI-coated vesicle). The
hibitory calneurons are replaced by neuronal Ca2+ sensor
Rab1/p115 complex also functions as a tether on the
1 (NCS-1) that stimulates PtdIns 4-KIII to begin to pro-
Golgi surface during COPII-mediated transport from
duce the PtdIns4P necessary for vesicle formation.
ER to Golgi (Module 4: Figure COPII-coated vesicles).
Rab6 binds to the golgin Bicaudal D, which functions
in vesicle transport between the Golgi and the ER, to Exocytosis
attach the dynein motor complex to the vesicle (Mod- Many aspects of cell communication depend upon the
ule 4: Figure dynein) for its transfer from the ER to the process of exocytosis to release signalling molecules such
Golgi membranes. as hormones and neurotransmitters. These signalling mo-
4. ARL-associated golgins are recruited to membranes lecules are stored in membrane vesicles that are released
through the Arf-like (ARL) small GTPase. The golgin- during cellcell communication. This regulated release

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of stored vesicles occurs through a process of Ca2 + - kiss-and-run fusion has been described in chromaffin cells
dependent exocytosis. There are two exocytotic mech- (Module 7: Figure chromaffin cell exocytosis).
anisms: the classical exocytotic/endocytotic cycle and a
briefer kiss-and-run vesicle fusion mechanism. In both
cases, the problem is to understand how an elevation in Exocytosis triggered by Ca2 + entry through
Ca2 + can trigger the initial event of membrane fusion. voltage-operated channels (VOCs)
Since there is a natural reluctance for membranes to fuse The most extensively studied form of exocytosis is the re-
with each other, special exocytotic machinery is used to lease of synaptic vesicles at neuronal presynaptic endings,
force the two membranes together so that fusion occurs. which depends upon Ca2+ -dependent exocytosis triggered
There are two types of Ca2 + -dependent exocytosis by the entry of Ca2 + through the CaV 2 family of N-type,
(Module 4: Figure Ca2+ -dependent exocytosis): P/Q-type and R-type channels. A remarkable aspect of
this process is its rapid kinetics. The phenomenal compu-
Exocytosis triggered by Ca2+ entry through voltage-op- tational ability of the brain depends upon neurons being
erated channels (VOCs) able to communicate with each other in less than 2 ms
Exocytosis triggered by Ca2+ release from internal (Module 10: Figure kinetics of neurotransmission). Dur-
stores ing this process of synaptic transmission, the arrival of
Delivery of AMPARs to the postsynaptic membrane, an action potential at the synaptic ending can trigger the
which plays an important role in Ca2+ -dependent synaptic release of neurotransmitter in less than 200 s. The organ-
plasticity (Module 10: Figure Ca2+ -induced synaptic plas- ization of the exocytotic machinery appears to be specially
ticity), is also carried out by exocytosis, but how this is designed to achieve these high reaction rates.
controlled remains to be determined. Exocytosis is part of an orderly vesicle cycle (Module
4: Figure vesicle cycle). Vesicles move from a reserve pool
Exocytotic mechanisms to dock with the membrane, during which the exocytotic
Membrane vesicles lying close to the plasma membrane are machinery is assembled and primed to respond to the final
primed to fuse with the plasma membrane in response to event of Ca2 + -induced exocytosis. The key to achieving
a pulse of Ca2 + . The neuronal Ca2+ sensor-1 (NCS-1) can such rapid responses is therefore to have the exocytotic
enhance exocytosis and this may depend upon its ability to machinery assembled and primed prior to the arrival of
facilitate the priming step. NCS-1 activates the PtdIns 4-k- the Ca2 + signal, which then functions just to trigger the
inase (PtdIns 4-K), which contributes to the PtdIns4,5P2 final step of membrane fusion.
regulation of exocytosis. Once the vesicles are primed,
membrane fusion is triggered by a brief pulse of Ca2 + .
Exocytosis triggered by Ca2 + release from internal
When fusion occurs, the contents of the vesicles are free
stores
to diffuse out through the fusion pore. Just how much
Some cells seem to be capable of stimulating exocytosis
of the content is released depends upon the subsequent
by releasing Ca2 + from internal stores (Module 4: Figure
events. In the case of the classical exocytotic/endocytotic
Ca2+ -dependent exocytosis). The concentration of Ca2 +
cycle, all of the contents are released. On the other hand,
within the microdomains that form around the opening
the kiss-and-run vesicle fusion mechanism is much briefer
of internal release channels, such as the inositol 1,4,5-tri-
and can be repeated a number of times, thus enabling the
sphosphate receptors (InsP3 Rs) and ryanodine receptors
same vesicle to function repeatedly.
(RYRs), is very high and thus will be capable of triggering
the exocytotic process. There are a number of examples
Exocytotic/endocytotic cycle
of vesicle release being triggered by release of Ca2 + from
The classical exocytotic/endocytotic cycle (Module 4: Fig-
internal stores:
ure vesicle cycle) depends upon sequential processes of
docking, priming, exocytosis and endocytosis. During
this process, the vesicle fuses with the plasma membrane Astrocytes use InsP3 -induced release of Ca2 + to trigger
to release all of its contents, and this is then followed the exocytosis of vesicles containing glutamate as part of
by the membrane of the empty vesicle being taken up the astrocyte-neuronal communication system (Module
again through the process of endocytosis. The scaffold- 7: Figure astrocyte tripartite synapse).
ing protein intersectin may play an important role in co- Mossy fibre presynaptic Ca2+ release.
ordinating the processes of exocytosis and endocytosis, Cerebellar basket cell presynaptic Ca2+ release.
Hypothalamic neuronal presynaptic Ca2+ release.
Kiss-and-run vesicle fusion Neocortical glutamatergic presynaptic Ca2+ release.
As its name implies, the kiss-and-run vesicle fusion pro- Release of luteinising hormone (LH) and follicle-
cess depends upon individual vesicles fusing repeatedly stimulating hormone (FSH) from gonadotrophs (see
with the membrane, during which process they release a step 5 in Module 10: Figure gonadotroph regulation).
small proportion of their contents. This mechanism is par- Release of thyroid-stimulating hormone (TSH) from the
ticularly evident in the small synaptic endings found in thyrotrophs is triggered by Ca2 + released from the in-
the brain, which have 2030 synaptic vesicles that can be ternal store (Module 10: Figure thyrotroph regulation).
re-used repeatedly to give transient pulses of neurotrans- Release of 5-HT from type III presynaptic cells during
mitter during synaptic transmission. Another example of taste reception (Module 10: Figure taste receptors).

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Module 4: Figure Ca2 + -dependent exocytosis
A. VOC
Exocytosis
**
Calcium triggered by
Vesicle entry calcium entry
Calcium
microdomain
B.
Exocytosis
Vesicle * triggered by
calcium release
Calcium
RYR or release
InsP R
3
Endoplasmic
reticulum
Two types of Ca2 + -dependent exocytosis.

A. Exocytosis triggered by Ca2 + entry through voltage-operated channels (VOCs). B. Exocytosis triggered by Ca2 + released through ryanodine
receptors (RYRs or inositol 1,4,5-trisphosphate receptors (InsP3Rs from the endoplasmic reticulum. The asterisks indicate the local microdomains of
Ca2 + responsible for triggering membrane fusion.
Module 4: Figure vesicle cycle
Neurotransmitter
2+
Ca
2. Priming
3.Fusion
4.Endocytosis
ATP
1. Docking
Reserve pool
of vesicles
The exocytotic vesicle cycle.

1. Docking. A vesicle held within the reserve pool of vesicles embedded in a cytoskeletal matrix moves towards the membrane and uses its vesicle
soluble N-ethylmaleimide-sensitive fusion protein-attachment protein receptors (v-SNAREs) to dock with the target SNAREs (t-SNAREs) in the plasma
membrane. 2. Priming. The vesicle is primed through an ATP-dependent process that prepares the exocytotic machinery for fusion. 3. Fusion. Upon
membrane depolarization, Ca2 + entering through a voltage-operated channel (VOC) triggers membrane fusion: the process of exocytosis (Module
4: Figure Ca2+ -induced membrane fusion). 4. Endocytosis. The membrane of the empty vesicle is retrieved through a process of endocytosis.

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Module 4: Figure Ca2 + -induced membrane fusion
Depolarization
Ca2+
Plasma membrane proteins
Vesicle proteins
VOC Syntaxin
Synaptobrevin
SNAP-25 Synaptotagmin VII Synaptotagmin I/II
Exocytotic machinery: a hypothetical mechanism for Ca2 + -induced membrane fusion.

The major proteins that function in exocytosis are the vesicle soluble N-ethylmaleimide-sensitive fusion protein-attachment protein receptor (v-SNARE)
synaptobrevin and the target SNAREs (t-SNAREs) syntaxin and 25 kDa synaptosome-associated protein (SNAP-25). These three SNAREs interact
with each other in the docked state (left) to form a ternary complex. This complex may be prevented from interacting further by regulatory proteins
such as the Ca2 + -sensitive proteins synaptotagmin I/II. A single voltage-operated channel (VOC), a member of the CaV2 family of N-type, P/Q-type
and R-type channels, is anchored to syntaxin or SNAP-25 and opens in response to depolarization to introduce a pulse of trigger Ca2 + that acts
through the synaptotagmins to enable the SNAREs to drive the two membranes to fuse with each other. These synaptotagmins have two adjacent C2
domains (C2A and C2B), which have Ca2 + -binding pockets with the low affinities (40 M to 1 mM) necessary to respond to the high levels of Ca2 +
found in the microdomains surrounding the vesicles. The site where the SNAREs interact with the CaV2 family of VOCs is shown in Module 3: Figure
CaV 2 channel family.
Exocytotic machinery allel arrays (Module 4: Figure Ca2+ -induced membrane

The exocytotic machinery is made up of many differ- fusion). The current models of exocytosis consider that
ent components that are distributed between the plasma the SNARE proteins zipper up with each other, thereby
membrane and the synaptic vesicle. The latter are encrus- inducing fusion by driving the two membranes together.
ted by a large number of proteins that include those that Mutation in the gene encoding PICALM, which func-
function in exocytosis such as synaptobrevin, and syn- tions in the trafficking of synaptobrevin, has been linked
aptotagmin (Module 10: Figure synaptic vesicle). Mem- to familial Alzheimers disease (FAD).
brane fusion is driven by an interaction between these The rapidity of the fusion process described in the
vesicle proteins and the plasma membrane proteins (Mod- section on exocytosis triggered by Ca2+ entry through
ule 4: Figure Ca2+ -induced membrane fusion). Some of voltage-operated channels (VOCs) can be accounted for
the key players in this membrane fusion mechanism are by the fact that the fusion proteins may begin the zipper-
the soluble N-ethylmaleimide-sensitive fusion protein- ing processes during the docking/priming events, and are
attachment protein receptors (SNAREs), composed of the thus poised for completion when given the appropriate
vesicle SNAREs (v-SNAREs) and the target SNAREs (t- signal through the process of Ca2+ -dependent exocytosis.
SNAREs) located on the plasma membrane (Module 4:
Figure Ca2+ -induced membrane fusion):
Synaptobrevin [also known as vesicle-associated mem- Ca2 + -dependent exocytosis

brane protein 2 (VAMP2)] is a v-SNARE. The process of membrane fusion during exocytosis in
Syntaxin 1a and 25 kDa synaptosome-associated protein neurons is driven by an influx of Ca2 + through the
(SNAP-25) are examples of t-SNAREs. CaV 2 family of N-type, P/Q-type and R-type channels.
A characteristic feature of these voltage-operated channels
These three SNAREs are weakly homologous proteins, (VOCs) is that they possess a binding site that tightly
especially with regard to a SNARE motif consisting of anchors them to the exocytotic machinery (Module 3:
a coiled-coil domain, that bind to each other with high Figure CaV 2 channel family), and they are thus posi-
affinity through hydrophobic interactions to form par- tioned to provide the rapid, high-intensity pulse of Ca2 +

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necessary to trigger membrane fusion (Module 4: Figure synaptic vesicle recycling. The uptake of integral mem-
Ca2+ -induced membrane fusion). In many endocrine cells, brane proteins, such as the transferrin receptor (TFR),
activation of L-type Ca2 + channels produces the global el- occurs through various stages (Module 4: Figure endo-
evation of Ca2 + necessary to trigger exocytosis. Just how cytosis):
this Ca2 + triggers fusion is still somewhat of a mystery.
The synapse has a number of Ca2 + sensors that may play Cargo selection by sorting proteins
different functions, as they seem to be sensitive to different Membrane invagination and scission
Ca2 + concentrations. The zipper model implies that, once Vesicle transport
the exocytotic machinery has been primed, it is preven- Coat removal
ted from going to completion by inhibitory mechanisms
that are removed by the pulse of Ca2 + . It therefore seems Cargo selection by sorting protein
reasonable to imagine that the Ca2 + -sensitive synaptotag- The initial step of cargo sorting depends on the assembly
min family of proteins might mediate this inhibition. Dif- of various sorting proteins that recognize the cargo. The
ferent synaptotagmins seem to play a role in exocytosis. sorting proteins function as adaptors to connect cargo pro-
Synaptotagmins I and II are integral membrane proteins teins to the clathrin coat during the process of endocytosis
anchored in the vesicle through membrane-spanning re- (Module 4: Figure endocytosis). In order to carry out this
gions. On the other hand, synaptotagmin VII is located in adaptor function, the sorting proteins such as the adaptor
the plasma membrane. These synaptotagmins are ideally proteins (APs) and the clathrin-associated sorting proteins
suited to regulate fusion in that they can bind to syn- (CLASPs) have to bind multiple partners. For example ad-
taxin and their C2 domains can bind to phospholipids in aptor protein 2 (AP2) associates with the membrane by
a Ca2 + -dependent manner. These Ca2 + -sensitive C2 do- binding to PtdIns4,5P2 , formed by the PtsIns4P 5-kinase
mains, which are separated from each other by a flexible I (PIPKI), and to the sorting signals located on the cyto-
linker, contain a -barrel and bind multiple Ca2 + ions plasmic domain of the cargo proteins (Module 4: Figure
during which there is a conformational switch that might cargo sorting signals). These sorting proteins then form a
activate the exocytotic machinery. One possibility is that molecular platform that binds clathrin, which is an essen-
the conformational change in synaptotagmin relieves the tial feature of the coated vesicles. The cytoplasmic domain
inhibition on the exocytotic machinery, thus enabling fu- of the cargo proteins has the sorting signals that enable
sion to occur (Module 4: Figure Ca2+ -induced membrane them to be recognized by the sorting proteins. Many of
fusion). The plasma membrane and vesicular synaptotag- these sorting signals are short amino acid sequences that are
mins appear to have different affinities for Ca2 + : those on found on cargo proteins that are taken up constitutively.
the plasma membrane are high-affinity sensors involved in However, the endocytosis of some proteins is regulated
slow exocytosis, whereas those on the vesicle have low- through a post-translational modification such as the ubi-
affinity sensors capable of fast Ca2 + -dependent exocyt- quitination that occurs during the Cbl down-regulation
osis. of cell signalling components (Module 1: Figure receptor
Another protein called piccolo/aczonin, which has C- down-regulation).
terminal C2A and C2B domains, may be a low-affinity The adaptor protein (AP) family are particularly im-
Ca2 + sensor that functions as a regulator when Ca2 + ac- portant sorting proteins, with AP2 playing a major role
cumulates during repetitive activity. in endocytosis. In addition to AP2, there are a number of
clathrin-associated sorting proteins (CLASPs) that func-
tion in cargo recognition during endocytosis.
Endocytosis
Adaptor protein (AP)
Cells take up a wide range of molecules through a num-
The adaptor protein family has three members: AP1, AP2
ber of mechanisms such as clathrin-mediated endocytosis
and AP3. Most attention has focussed on adaptor protein
(CME), caveolin-mediated endocytosis, clathrin/caveolin-
2 (AP2), which has a primary role to play in clathrin-
independent endocytosis and macropinocytosis (Module
mediated endocytosis (CME) (Module 4: Figure endocyt-
4: Figure membrane and protein trafficking). The endo-
osis). AP2 consists of four subunits (, 2, 2 and 2) that
cytic vesicles, which carry molecules away from the plasma
form a heterotetrameric complex that has a large trunk
membrane, are directed towards the early endosome and
and two appendage domains located on flexible linkers
the subsequent process of endosome vesicle fusion to early
that come from the - and 2-subunits (Module 4: Figure
endosomes, enabling the molecules taken up from the
cargo sorting signals). The trunk region attaches AP2 to the
plasma membrane to enter the intracellular protein traf-
cargo and the membrane, whereas the appendages bind to
ficking system (Module 4: Figure membrane and protein
various accessory proteins that contribute to forming the
trafficking). A number of signalling mechanisms function
molecular layer that coats the vesicle.
in the control of endocytosis.
The YXX sorting signal, which is located on pro-
teins such as the transferrin receptor (TFR), CD-M6PR,
Clathrin-mediated endocytosis (CME) LAMP1, LRP1, PAR1, P2X4 receptor and the 2-subunit
Most attention has focused on clathrin-mediated endo- of the GABAA receptor, is recognized by a region on the
cytosis (CME), which has multiple functions, such as 2-subunit of AP2. The latter also binds to phosphatidyl
down-regulation of surface receptors, nutrient uptake and 4,5-bisphosphate (PtdIns4,5P2 ), which also functions as an

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Module 4: Figure endocytosis
TFR Cargo sorting
PtdIns4P P PIPKI PtdIns4,5P2 P P P P

P P P P
P Invagination
Actin plume
P P
P P
AAK1 P P P
P P
AP2
Scission Dynamin ring
Dynamin P CaN Actin plume

Clathrin
Amphiphysin P
DYRK1A
Protein Auxilin P
P
phosphatase SJ1
P
Hsc70 P
P
P
P
P
P Coat removal
P
P
P
P
P
P
SJ1
P
P P
P
P
PtdIns4P
P P
P
P P
Myosin VI
Vesicle transport
Control of clathrin-mediated endocytosis.

The uptake of plasma membrane proteins by the process of endocytosis is regulated by two main mechanisms. First, the adaptor protein 2 (AP2)
is phosphorylated by adaptor-associated kinase 1 (AAK1), which helps it to bind to cargo proteins such as the transferrin receptor (TFR). Secondly,
the PtdIns4P 5-kinase (PIPKI) phosphorylates PtdIns4P to PtdIns4,5P2 , which contributes to the binding of cargo. As the AP2TFR complexes
aggregate, they begin to bind clathrin and the membrane starts to invaginate. Various proteins, which bind to the neck region, are responsible for the
process of scission during which the vesicle is budded off into the cytoplasm. Dephosphorylation of AP2 and PtdIns4,5P2 by protein phosphatases
and synaptojanin-1 (SJ1) respectively result in disassembly of the coat components that are re-used for further rounds of endocytosis.
adaptor to glue AP2 to the membrane. A separate group The receptor down-regulation of G protein-coupled re-
of cargo proteins such as CD4, CD3, LIMP2 and Nef ceptors (GPCRs) is carried out by -arrestins that behave
have the sorting signal [DE]XXXL[LI], which recognizes like CLASPs (Module 1: Figure homologous desensitiza-
the 2-subunit. tion). When the arrestins bind to the hyperphosphorylated
GPCRs, they reveal calthrin- and AP2-binding motifs that
guide the complex into the coated pits ready for internal-
Clathrin-associated sorting proteins (CLASPs) ization (Module 4: Figure cargo sorting signals).
The clathrin-associated sorting proteins (CLASPs) func-
tion as adaptors to select cargo during endocytosis (Mod- Membrane invagination and scission
ule 4: Figure cargo sorting signals). Some of these CLASPs, One sorting protein, such as the adaptor protein 2 (AP2),
such as disabled 2 (DAB2), autosomal recessive hypercho- have trapped and concentrated cargo proteins, clathrin be-
lesterolemia (ARH) and Numb, have a PTB domain, which gins to coat the macromolecular complexes and the mem-
recognizes the [FY]XNPX[YF] sorting signal found on brane invaginates to form a concentric coated bud (Mod-
cargo proteins such as the LDL receptor, LRP1, LRP2 ule 4: Figure scission of endocytic vesicles). The clathrin,
(megalin), P-selectin and 1A integrin1 and 2. These which consists of two subunits, an elongated heavy chain
CLASPs contribute to the coat by binding to clathrin and and a light chain, polymerize to form triskelia. These clath-
AP2. rin triskelia, which have three legs radiating out from a
The epsins and the epidermal growth factor re- hub, interact with each other to form a web that coats the
ceptor substrate 15 (EPS15) contribute to endocytosis vesicular bulb. This bulb is then cut off through a scission
by functioning as sorting proteins to detect ubiquitin- process that is not fully understood, but some of the main
ated cargo. They have ubiquitin-interacting motifs (UIMs) players have been identified. A key event appears to be the
that bind to the ubiquitinated cargo such as the epidermal formation of a macromolecular spiral that wraps around
growth factor receptor (EGFR) as occurs during the Cbl the neck of the vesicular bud. The main components of this
down-regulation of cell signalling components (Module 1: spiral are SNX9 and the large GTPase dynamin. The PX
Figure receptor down-regulation). Epsin 1 and EPS15 can domain on SNX9 binds to PtdIns4,5P2 , which is present
recognize the polyubiquitination of Lys-63 on the EGFR. at high levels, to induce a conformation change resulting

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Module 4: Figure cargo sorting signals
TFR
CD-M6PR
PAR1 LDL receptor
LAMP1 CD4 LRP1 EGFR
P2X LIMP2 LRP2 MHC Class I
GABA (2) CD3 P-selectin Delta
Cargo 1 integrin Nef 1 integrin EnaC
proteins GPCRs
PtdIns4,5P 2
Sorting Ubiquitin P
YXX [DE]XXXL[LI] [FY]XNPX[YF]
signals
2 2 ARH Arrestins
Epsins
DAB2
Sorting Adaptor 2 2 Numb
EPS15
proteins protein 2 Myosin VI
(AP2)
Clathrin
Cargo recognition by sorting proteins.

A wide range of cargo proteins located in the plasma membrane have specific sorting signals that are recognized by various sorting proteins. These
sorting proteins have a variety of motifs that recognize both the sorting signals on the cargo proteins as well as clathrin. These sorting proteins also
have lipid-binding motifs enabling them to bind to phosphatidyl 4,5-bisphosphate (PtdIns4,5P2 ). The ability of the sorting proteins to connect cargo
proteins to the clathrin coat is an essential part of the process of endocytosis (Module 4: Figure endocytosis).
in its oligomerization and this then provides a platform vesicles). Myosin 1E is a plus-end motor that will pull the
to draw in other proteins. For example, dynamin binds to dynamin ring towards the plasma membrane, whereas the
both SNX9 and to PtdIns4,5P2 to form part of the spiral. minus-end motor myosin VI will pull the vesicle in the
The SNX9/dynamin spiral provides a platform to opposite direction towards the cytoplasm.
assemble actin remodelling proteins such as cortactin,
N-WASP and Arp2/3 responsible for the nucleation of Dynamin
actin filaments (Module 4: Figure scission of endocytic Dynamin is a large GTPase that functions in clathrin-
vesicles). In addition, the SNX9 also provides another con- mediated endocytosis (CME) (Module 4: Figure scission of
nection to actin by binding to myosin 1E. Another mo- endocytic vesicles). At the time of scission, dynamin is part
lecular motor (myosin VI) is connected to the vesicular of a macromolecular complex containing amphiphysin, en-
region of the bulb by binding to both the sorting pro- dophilin, epsin, Eps15, synaptojanin, syndapin, N-WASP,
tein DAB2 and to PtdIns4,5P2 . As the clathrin-coated pit cortactin, mammalian actin-binding 1 (mAbp1), inter-
matures, the phospholipid composition of the membrane sectin and profilin. One of its functions is to provide a
begins to change to prepare the vesicle for its transfer to the protein scaffold that assembles many of the proteins re-
endocytic system (Module 2: Figure localized inositol lipid quired for scission. One of its scaffolding functions is to
signalling). Firstly, the PtdIns4,5P2 is dephosphorylated to link the neck of the pit to the actin cytoskeleton. It can reg-
PtdIns4P, which is then phosphorylated to PtdIns3,4P2 by ulate F-actin dynamics by binding to accessory proteins
PI3KC2. This PtdIns3,4P2 is then dephosphorylated by such as cortactin, mammalian actin-binding 1 (mAbp1),
inositol polyphosphate 4-phosphatase type II (INPP4B) intersectin and profilin. At the time of scission, dynamin
to form PtdIns3P, which is the characteristic inositol lipid hydrolyses GTP and this induces a conformational change
found in endosomes. of the spiral that stretches out the neck resulting in release
As the various scission molecules bind, the neck begins of the coated vesicle.
to thin and is then severed (scission) through a process Dynamin is part of a dynamin superfamily of large
that seems to depend on the GTP-dependent action of dy- GTPases, such as the dynamin-like proteins, Mx pro-
namin. In addition, the two motor proteins might help to teins, OPA1, Mitofusins and GBP/atlastin-related pro-
pull the vesicle into the cytoplasm through their interac- teins, which all seem to function in either membrane tu-
tion with actin (Module 4: Figure scission of endocytic bulation or scission.

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Amphiphysins its cofactor auxilin. The auxilin is recruited by a lipid sig-

There are two genes encoding the amphiphysins: am- nal, which appears to be the PtdIns4P that is formed when
phiphysin 1 expressed in the brain and amphiphysin 2, PtdIns4,5P2 is hydrolysed by synaptojanin 1. Cells ex-
which has a wider distribution and has numerous spliced press two auxilins, brain-specific auxilin 1 and the more
isoforms. They have an N-terminal Bar domain that en- ubiquitous auxilin 2, which is also known as cyclin G-
ables two molecules to dimerize to form a positively- associated kinase (GAK). Auxilin is rapidly recruited just
charged concave surface that enables the dimers to interact before scission occurs and enables the 70 kDa heat shock
with membranes. The C-terminal region has a SH3 do- protein (Hsc70) to uncoat the membrane through an ATP-
main that enables it to interact with its binding partners dependent process. The energy derived from ATP hydro-
such as dynamin and synaptojanin 1 (SJ1) during the late lysis enables Hsc70 to drive disassembly of the clathrin
stages of endocytosis. Amphiphysin is found in a 1:1 stoi- coat to liberate the individual triskelions (Module 4: Fig-
chiometry with dynamin and may function to facilitate the ure scission of endocytic vesicles).
recruitment of dynamin to form the spiral responsible for
scission (Module 4: Figure scission of endocytic vesicles).
Control of endocytosis
The ability of amphiphysins to associate with dynamin
The process of endocytosis is regulated at a number of dif-
and the cell membrane is controlled by a phosphoryla-
ferent stages (Module 4: Figure endocytosis). During the
tion/dephosphorylation system (Module 4: Figure endo-
initial step of cargo selection by sorting proteins, the sort-
cytosis). Phosphorylation by the dual-specificity tyrosine
ing protein adaptor protein 2 (AP2) is phosphorylated by
phosphorylation regulated kinase 1A (DYRK1A) inhibits
adaptor-associated kinase 1 (AAK1), which helps it to bind
binding, whereas dephosphorylation by calcineurin (CaN)
to cargo proteins such as the transferrin receptor (TFR).
removes this inhibition to allow amphiphysins to particip-
Association with the membrane also depends on the en-
ate in endocytosis.
zyme PtdIns4P 5-kinase (PIPKI) that phosphorylates
Amphiphysins have also been implicated in the forma-
PtdIns4P to PtdIns4,5P2 , which contributes to the bind-
tion of the T-tubules found in muscle cells.
ing of cargo. As the AP2/TFR complexes aggregate, they
begin to bind clathrin and this coincides with membrane
Endophilin
invagination.
The endophilin family has three members: endophilin
These processes are then reversed during the later stages
1 (SH3p4), endophilin 2 (SH3p8) and endophilin 3
of scission and coat removal. The large GTPase protein
(SH3p13). Endophilin 1 is particularly abundant in nerve
dynamin helps to assemble the actin fibres and plays a
terminals in the brain, whereas endophylin 2 is the main
critical role in the scission process. The action of dynamin
isoform in non-neuronal cells. Endophilin has a structure
and its various endocytic accessory proteins such as am-
resembling that of the amphiphysins in that it has an N-
phiphysin and synaptojanin 1 (SJ1) is regulated by a phos-
terminal BAR domain and a C-terminal SH3 domain. The
phorylation/dephosphorylation cycle (Module 4: Figure
latter binds to the proline-rich regions of dynamin and
scission of endocytic vesicles). Phosphorylation of these
synaptojanin. One of the primary functions of endophilin
proteins by the dual-specificity tyrosine-phosphorylation
is to recruit synaptojanin, which plays a major role in coat
regulated kinase 1A (DYRK1A) and by the neuronal cyc-
removal following scission (Module 4: Figure scission of
lin-dependent kinase 5 (CDK5) prevents them from being
endocytic vesicles).
recruited into the macromolecular complexes that drive
In non-neuronal cells, endophilin 2 plays a major role
scission and subsequent coat removal (Module 4: Figure
in the Cbl down-regulation of cell signalling components
scission of endocytic vesicles). These inhibitory phosphate
during the endocytosis of various tyrosine kinase-coupled
groups are removed by the Ca2 + -sensitive phosphatase
receptors such the Trk receptors and EGF receptors. In
calcineurin (CaN) and this can account for the Ca2 + -
these examples, Cbl functions to recruit both endophilin
sensitivity of endocytosis, particularly in the case of syn-
and Cbl-interacting protein of 85 kDa (CIN85) to con-
aptic vesicle retrieval at synaptic endings during the events
trol receptor internalization (Module 1: Figure receptor
related to Ca2+ and synaptic plasticity (Module 10: Figure
down-regulation). Phosphorylation of endophilin by Rho
Ca2+ -induced synaptic plasticity).
kinase inhibits endocytosis.
After the coated vesicle has been formed, the coat is re-
moved using various mechanisms. First, the auxilin/Hsc70
Vesicle transport
system removes clathrin (Module 4: Figure scission of en-
Following scission of the bud, the coated vesicles enters
docytic vesicles). Secondly, the removal of sorting pro-
the cytoplasm where the various coat proteins are removed
teins such as AP2 follows the hydrolysis of PtdIns4,5P2 to
and the endosomal vesicles move towards the early endo-
PtdIns4P by synaptojanin 1 (SJ1) (Module 4: Figure endo-
some where it fuses to deliver its membrane cargo (see step
cytosis). The coat components are then re-used for further
5 in Module 4: Figure membrane and protein trafficking).
rounds of endocytosis.
Coat removal
When the coated vesicles enter the cytoplasm and begin to Dual specificity tyrosine-phosphorylation regulated
move towards the early endosomes, the various coat pro- kinase 1A (DYRK1A).
teins are removed (Module 4: Figure endocytosis). This The gene DYRK1A encodes the dual-specificity tyrosine-
uncoating process depends on proteins such as Hsc70 and phosphorylation regulated kinase 1A (DYRK1A), which

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Module 4: Figure scission of endocytic vesicles
Endophilin
N-WASP Cytosolic
Arp2/3 2+
Ca
Actin CaM
Cortactin
GTP Myosin
1E Amphiphysin
GDP
Dynamin-SNX9 P
Dynamin spiral SJ1
P
SJ1
Clathrin SNX9 DYRK1A
P
ADP
Dynamin
Hsc70 SJ1
K P
ATP GA
ilin/ Amphiphysin
Aux P
P PtdIns4,5P2
DAB2
P PtdIns4P Myosin
VI
P Clathrin-coated pit
AP2
Endocytic vesicle scission and removal of the clathrin coat.

Once the clathrin-coated pit has formed, the neck is cut off to release the vesicle into the cytoplasm. The sequence of events responsible for scission
remains to be established. This Figure attempts to illustrate the structural organization of some of the major components responsible for scission.
Once the vesicle is formed, the clathrin coat is removed by an ATP-dependent process activated by Hsc70. See the text for further details.
is an orthologue of Drosophila minibrain kinase (MNB). are activated by proteins on the particle cell surface. En-
This kinase not only functions in early brain development, gagement of the Fc receptors results in the formation of
but it continues to operate in the adult brain. DYRK1A is pseudopodia that rise up to engulf the particle. On the
a serine/threonine protein kinase that has multiple func- other hand, particles coated with complement C3 recept-
tions both in the nucleus and in the cytoplasm. The bipart- ors simply sink into the cell.
ite nuclear targeting sequence enables it to operate in both The process of Fc-mediated phagocytosis depends
locations. The DYRK1A located in the nucleus is respons- upon the formation of pseudopodia that engulf the particle.
ible for phosphorylating the transcription factor NFAT to The PtdIns4,5P2 regulation of phagocytosis brings about
promote its export from the nucleus (Module 4: Figure the actin remodelling necessary to form the pseudopodia.
NFAT control of Ca2+ signalling toolkit). DYRK1A also In addition, there is a process of focal exocytosis, during
phosphorylates various endocytic accessory proteins that which membrane vesicles are added to the growing tips of
function during membrane invagination and scission dur- the pseudopodia. The large GTPase protein dynamin-2 ap-
ing the process of endocytosis (Module 4: Figure scission pears to play a role in regulating this process of exocytosis.
of endocytic vesicles). For example, it phosphorylates the This exocytosis is revealed by an increase in capacitance
proline-rich domains (PRDs) of dynamin, amphiphysin that precedes the rapid decrease when the particle is finally
and synaptojanin 1 (SJ1). The phosphorylation of these internalized.
proteins seems to inhibit their recruitment at endocytic
sites. Dephosphorylation of these proteins by calcineurin
(CaN) activates the recruitment and assembly of these en- Phagosome maturation
docytic accessory proteins. When a pathogenic organism has been engulfed within the
DYRK1A is also located on the Down syndrome crit- phagosome, a complex cascade of events drives a matur-
ical region 1 (DSCR1) on human chromosome 21, where ation process whereby the phagosome is converted into
trisomy occurs resulting in an elevation of this protein that a phagolysosome (Module 4: Figure phagosome matura-
could contribute to the Downs syndrome phenotype. tion). During phagosome maturation, there is a dramatic
change both in the composition of the surrounding mem-
brane and in the contents of the phagosome brought
Phagocytosis about primarily by an orderly fusion of vesicles from
The process of phagocytosis, which is particularly evident the endocytic pathway. A large number of signalling mo-
in haematopoietic cells such as macrophages, is a special- lecules and accessory proteins collaborate in this matura-
ized form of endocytosis. During phagocytosis, the cell tion process, and the precise sequence of events remains
engulfs large particles such as bacteria through two main to be worked out. The process begins when Fc recept-
mechanisms determined by the nature of the receptors that ors (FcRs) on the cell surface recognize an opsonized

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micro-organism to initiate phagocytosis to form a pha- Endosome vesicle fusion to early

gosome. After the phagosome has formed, one of the endosomes
earliest events to occur is the activation of the Class III Endocytic vesicles coming from the plasma membrane
PtdIns 3-kinase (PtdIns 3-K), which converts PtdIns into travel inwards to fuse with the early endosome through
PtdIns3P that builds up rapidly on the surface of the pha- an intracellular exocytotic mechanism (Step 6 in Module
gosome (Module 4: Figure PtdIns3P formation in pha- 4: Figure membrane and protein trafficking). The fusion
gosomes). The PtdIns3P appears within minutes and per- machinery is controlled by the Rab signalling mechanism
sists for at least 30 min. This PtdIns3P then has a crucial (Module 2: Figure Rab signalling). The GTP-binding pro-
role in recruiting endocytic vesicles (EVs) by binding to tein Rab5, which is a member of the Rab family of mono-
proteins such as Rab5 and the early endosome antigen meric GTP-binding proteins (G proteins) (Module 2: Table
1 (EEA1). EEA1 contains a FYVE motif that specific- monomeric G protein toolkit). Like other Rabs, Rab5 ex-
ally recognizes PtdIns3P (Module 6: Figure modular lip- ists in two forms. It is either soluble in the cytoplasm where
id-binding domains). Once the endosome is docked near it is associated with GDP-dissociation inhibitor (GDI) or
the phagosome, a family of membrane-tethered coiled-coil it is associated with the membranes of vesicles or intra-
proteins, resembling those of the exocytotic machinery cellular organelles. This association with the membrane is
[e.g. soluble N-ethylmaleimide-sensitive fusion protein- assisted by the prenylated Rab acceptor 1 (PRA1), which
attachment protein receptor (SNARE) and synaptosome- acts as a GDI displacement factor. Rab5 recruits various
associated protein (SNAP)], are used to drive fusion of the tethering components and effectors that orchestrate the
endocytic vesicle with the phagosome (Module 4: Figure close apposition of the membranes necessary for SNARE
phagosome maturation). A phagolysosome forms when proteins to induce membrane fusion (Module 4: Figure en-
the late endosome/lysosome vesicles fuse with the phago- dosome vesicle fusion). This assembly of a fusion complex
some. depends on the Rab5-dependent recruitment and activa-
Just how the PtdIns 3-K on the phagosome is activ- tion of the Class III PtdIns 3-kinase called hVps34 to pro-
ated remains to be established, but there appears to be duce a local accumulation of PtdIns3P. Three of the Rab5
an important role for Ca2 + that is generated through effectors [early endosome antigen 1 (EEA1), rabenosyn 5
an interaction between the phospholipase D (PLD) sig- and rabankyrin 5] not only bind Rab5, but they also have
nalling pathway (Module 2: Figure PLD signalling) and FYVE domains that enable them to bind PtdIns3P. The
the sphingomyelin signalling pathway (Module 2: Figure EEA1 has N- and C-terminal Rab5-binding sites that may
sphingomyelin signalling). Just how the FcR activates help to tether the incoming vesicles to the early endosome.
PLD1 is unknown, but it is likely to depend upon some The Rab5 and its associated effector proteins interact
of the known activators such as RhoA, ADP-ribosylation with the SNAREs to position them such that they can
factor (Arf) or protein kinase C (PKC). Activation of interact with each other to bring about membrane fu-
PLD1 located on either the sorting endosome (SE) or the sion. The EEA1 and rabenosyn associate with the target-
endoplasmic reticulum (ER) produces phosphatidic acid SNAREs syntaxin-6 and syntaxin-13 on the early en-
(PA), which then activates sphingosine kinase (SPHK) to dosome whereas the v-SNARE VAMP4 associates with
convert sphingosine into sphingosine 1-phosphate (S1P). Rabex-5 and rabankyrin-5. The N-ethylmaleimide sens-
The latter then releases Ca2 + from internal stores using itive factor (NSF), the soluble attachment protein (-
channels that remain to be identified. The pulses of Ca2 + SNAP) and hVPS45 are accessory factors that contribute
appear to have two roles: either they act through CaMKII to the priming of the SNARE complexes for fusion to
to stimulate the PtdIns 3-K, or they may also trigger the en- occur.
dosome/phagosome fusion events (Module 4: Figure pha-
gosome maturation).
One of the interesting aspects of phagosome maturation
is the way that it is blocked by certain pathogens: Early endosome protein sorting and
intraluminal vesicle formation
Mycobacterium tuberculosis, which causes tuberculosis, The early endosome is the initial clearing house for pro-
is able to survive within the phagosomes of the host teins that it receives following the fusion of endocytic ves-
by switching off the signalling processes responsible for icles and the first task is to sort them out so that they can
converting it into a phagolysosome. M. tuberculosis pro- be sent to different locations (Step 7 in Module 4: Figure
duces lipoarabinomannan (LAM), which then acts to membrane and protein trafficking). One sorting mechan-
inhibit both the PLD1 and the SPHK that generate the ism deals with proteins such as the EGFR that are destined
Ca2 + signals responsible for driving various aspects of to be degraded by the lysosome (Module 1: Figure receptor
the maturation process (Module 4: Figure phagosome down-regulation). The first step in the degradation path-
maturation). way is for the EGFRs to be corralled within an intralu-
The bacterium Chlamydia trachomatis, which causes minal endosomal vesicle (Module 4: Figure intraluminal
chlamydial diseases, is also able to inhibit the process of endosomal vesicle formation). An endosomal sorting com-
phagosome maturation. plex required for transport (ESCRT) complex functions to
The bacterium Listeria monocytogenes, which causes lis- sort proteins and to form the intraluminal vesicles. There
teriosis, uses the bacterial virulence factor listeriolysin are four ESCRT complexes (ESCRT0ESCRTIII) that
O (LLO) to escape from the phagosome. cooperate with each other to sort cargo into a specific

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Module 4: Figure PtdIns3P formation in phagosomes
Rapid accumulation of PtdIns3P in the early phagosome following ingestion of IgG-opsonized red blood cells.
The appearance of PtdIns3P was detected using green fluorescent protein (GFP) fused to two FYVE domains that binds to this lipid. The progress
of two engulfed particles (black arrows in the interference contrast image A) illustrates the time course of the PtdIns3P response. At the beginning of
the fluorescence recording, the bottom particle is already brightly labelled (B), but this begins to wane 6 min later (C). The particle at the top had just
been engulfed in B and shows no evidence of PtdIns3P, but this is clearly evident 6 min later, and is still present 37 min later (D). Reproduced from
Vieira et al. 2002, with permission from the Biochemical Society.
Module 4: Figure phagosome maturation
Mycobacterium
tuberculosis
LAM Opsonized
microorganism
FcRI
_ _
PLD1
+
RhoA Phagosome
PtdIns4,5P 2
+ Arf
PA PC PKC
SPHK SE or ER EV
? SNARE
+ b5
S1P Ra 1 SNAP
2+
EEA
Sphingosine Ca PtdIns
+
b5
Late endosome/ Ra
A1
lysosome EE 3P
+ + In
s- PtdIns 3-KIII
d
Pt CaMKII
Phagolysosome
Phagosome maturation.
This working hypothesis summarizes some of the major signalling events that are thought to regulate the orderly conversion of a phagosome into a
phagolysosome. See the text for further details.

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Module 4: Figure endosome vesicle fusion
Endosomal vesicle
TFR EGFR
GDI
GDI
VAMP4
Rab5
Ra b5
b5 Ra 5
in-
yr
Rab5 nk
-SNAP ba
Ra
NSF Rabex-5 45
hVPS
EEA1 Rabenosyn-5
hVPS34 Rab5 Rab5 Rab5
Rab5
PtdIns3P Syntaxin-13
Syntaxin-6
PRA1
PtdIns
Early endosome
Endocytic vesicle fusion to early endosomes.

Endocytic vesicles that have budded off the plasma membrane travel to the early endosome where they fuse through a mechanism resembling the
exocytosis seen at the plasma membrane. This exocytotic mechanism depends on a number of components whose activity is orchestrated by Rab5
and the local formation of phosphatidyl 3-phosphate (PtdIns3P), which functions to recruit proteins that regulate the fusion process.
membrane region where an inward deformation of the then dissociated through an ATP-dependent process that
membrane buds off to form the intraluminal vesicle. is driven by the ATPase Vps4p.
Just how these complexes interact with each other re-
mains to be worked out, and one hypothesis is that they
operate as a conveyer belt to both sort the cargo and to
form the bud. As for many other endosomal functions, Early endosome to plasma membrane
the local formation of PtdIns3P by the Class III PtdIns trafficking
3-kinase called hVps34 plays a role in initiating the sort- The early endosome can rapidly recycle certain membrane
ing process by the ESCRT-0 complex. The hepatocyte proteins, such as the transferrin receptor (TFR), back to the
growth factor-regulated tyrosine kinase substrate (HRS) plasma membrane through two pathways (Step 8 in Mod-
has a FYVE domain that targets it to the PtsIns3P. The HRS ule 4: Figure membrane and protein trafficking). A rapid
then binds to the signal transducing adaptor molecule 1 recycling pathway transports TFR back to the membrane
(STAM1) that recognizes the ubiquitin (UB) moiety on the directly, whereas in the slower pathway the TFR passes ini-
cytoplamic tail of EGFR that marks it out for this degrad- tially to the recycling endosome before being transferred
ative pathway. ESCRTI and ESCRTII have proteins with to the plasma membrane. In both cases, the initial sorting
ubiquitin-binding domains, such as tumour susceptibility of the cargo is carried out by various members of the large
gene 101 (TSG101) and VPS36p that feed cargo to the fi- family of sorting nexins (SNXs). Assembly of the SNX4
nal step that depends on components of ESCRTIII that complexes depend on activation of the Class III PtdIns
form the bud. The initial deformation of the membrane 3-kinase called hVps34 to produce a local accumulation
seems to depend on lysobisphosphatidic acid (LBPA) and of PtdIns3P (Module 4: Figure early endosome budding).
the LBPA-binding protein ALIX [apoptosis-linked gene Once the TFR has been sorted, the cytoskeletal-associated
2 (ALG-2)-interacting protein X]. Before the vesicle is recycling or transport (CART) complex, which consists of
formed, ubiquitin hydrolases remove the ubiquitin group actinin-4, brain-expressed RING finger protein (BERP)
that is re-used for further cycles of receptor internalization and myosin V, directs the vesicle to the plasma membrane.
and degradation. In mammals, this deubiquitinization is The molecular motor myosin V and Rab4 are responsible
carried out by deubiquitinating enzymes (DUBs) such as for moving the vesicle along the actin filaments.
associated molecule with the Src homology 3 (SH3) do- Similar processes are responsible for sorting cargo such
main of STAM (AMSH) and ubiquitin-specific protease Y as TFR for its transfer to the recycling endosome. As for
(UBPY). the fast recycling mechanism described above, sorting de-
Once the vesicle and its cargo have been internalized, the pends on SNX4. The Lemur tyrosine kinase 2 (LMTK2)
function of the ESCRT complexes is complete and they are appears to have a role in controlling this sorting process.

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Module 4: Figure intraluminal vesicle formation
UB
ESCRT-0 ESCRT-I ESCRT-II ESCRT-III
VPS36p AMSH
hVPS34
HRS TSG101
STAM1
UB UB UBPY
AL
UB IX
AL
IX
EGFR
PtdIns3P LBPA
PtdIns
Early
endosome
Intralumen
endosomal
vesicle
Protein sorting and formation of intraluminal vesicles in early endosomes.

Proteins destined for degradation are sorted into intraluminal endosomal vesicles. The sequence of events is orchestrated by an endosomal sorting
complex required for transport (ESCRT) complexes (0III). The main ESCRT components are illustrated on the basis of the conveyer belt hypothesis
that proposes that the ESCRT complexes act in sequence to sort and transport the proteins into the internal vesicle.
The vesicles that budd off from the end of the tubules are The last sequence of this retrieval process is vesicle
transported along actin using myosin VI and Rab4. excision when the tips of the tubules bud off vesicles.
The scaffolding protein Eps15p homology (EH) domain-
containing protein 1 (EDH1), which has an ATPase do-
main, may function to pinch off the end of the tubule.
The phosphofurin acid cluster sorting protein 1 (PACS1),
Early endosome to trans -Golgi network which binds acidic clusters on cargo proteins such as CI-
(TGN) trafficking MRP and furin, has also been implicated in the endosome
Certain proteins, such as the cation-independent mannose to TGN trafficking process.
6-phosphate receptor (CI-MPR) move between the trans-
Golgi network (TGN) and the early endosome (see step
3 in Module 4: Figure membrane and protein trafficking). Phosphofurin acid cluster sorting protein 1
(PACS1)
Once CI-MPR has released its cargo of lysosomal hydro-
The phosphofurin acid cluster sorting protein 1 (PACS1)
lases to the early endosome, it is returned to the LGN
has been implicated in early endosome to trans-Golgi net-
through a specific trafficking pathway (Module 4: Fig-
work (TGN) trafficking. It plays a role in the trafficking of
ure endosome budding to TGN). The term retromer has
both furin and mannose-6-phosphate receptor by connect-
been used to describe the retrieval complex that sorts cargo
ing their acidic-cluster-containing cytoplasmic domains to
and orchestrates the tubulation and subsequent budding to
the adaptor-protein complex-1 (AP-1).
form the vesicles that returns cargo to the TGN. Activation
A related phosphofurin acid cluster sorting protein 2
of the Class III PtdIns 3-kinase called hVps34 produces a
(PACS2) may contribute to the stability of mitochondri-
local accumulation of PtdIns3P that contributes to the as-
a-associated ER membranes (MAMs).
sembly of various members of the family of sorting nexins
(SNXs). The BAR domains of SNX1 and SNX4 dimerize
to form the concave structure that facilitate formation of Sorting nexins (SNXs)
the tubules where sorting occurs through a cargo selec- The family of sorting nexins (SNXs) are characterized
tion complex consisting of VPS26, VPS29 and VPS35. The by having a PX domain, which enables them to bind to
VPS35 binds to the C-terminal tail of CI-MPR, which has lipid messengers such as phosphatidylinositol 3-phosphate
a phosphoserine motif that was placed there by the sorting (PtdIns3P) during certain events that occur during mem-
retromer at the TGN in order to direct the CI-MPR to- brane and protein trafficking. Some members of the fam-
wards the early endosome. The removal of this phosphate ily also possess BAR domains that dimerize with adjacent
by VPS29 enables the CI-MPR to cycle back to the TGN. domains to form a concave structure that binds to the

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Module 4: Figure early endosome budding
To plasma
hVPS34
membrane TFR SNX4
Rab4
Myosin V Actinin-4 BERP

Early
endosome
Cytoskeletel-associated recycling or transport (CART) complex
Actin
PtdIns
3P
Myosin VI PtdIns
hVPS34
Rab4
TFR SNX4
To recycling
endosome
Early endosome budding.

One of the functions of the early endosome is to recycle various membrane proteins such as the transferrin receptor (TFR) back to the plasma
membrane. These proteins are either returned directly to the plasma membrane or they are recycled via the recycling endosome (See Module 4:
Figure membrane and protein trafficking). In both cases, the proteins are sorted at tubular extension before being budded off into vesicles that are
carried away by various motor proteins.
Module 4: Figure endosome budding to TGN
Early endosome
CI-MPR
Actin
PtdIns
Myosin
EDHI
To TGN
Rab4
s3P
PtdIn
hVPS34
EDHI
P 1
9 NX
VPS2 VPS35 SNX
4 S
6
VPS2
P
Early endosome budding and transport to TGN.

The early endosome receives vesicles from the Golgi carrying proteins such as the cation-independent mannose 6-phosphate receptor (CI-MPR),
which is a carrier of various hydrolases. This CI-MPR carrier is then returned to the trans-Golgi network (TGN) once it has been sorted and transferred
into vesicles by the mechanism illustrated in this figure.

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surface of membrane tubules as occurs during early endo- Once cargo reaches the vicinity of its final destination, it
some to plasma membrane trafficking (Module 4: Figure is often transferred to the actin-dependent myosin motors
early endosome budding) or during the early endosome to for the final transfer to the plasma membrane.
trans-Golgi network (TGN) trafficking (Module 4: Figure
endosome budding to TGN). Myosin
A large myosin superfamily is responsible for different
forms of cell motility. These myosins emerged early in
Early endosome maturation to evolution and are widely distributed throughout the euk-
lysosomes aryotes. Some myosins, such as myosin VIII and myosin
During the process of early endosome protein sorting and IX are found only in plants. Most of the myosins move to-
intraluminal vesicle formation proteins are sorted into dif- wards the plus-end of actin with the exception of myosin
ferent groups that are then sent off to different locations. VI, which is a minus-end directed motor. The conventional
Some proteins are recycled by being sent back to either myosin II family operates in muscle cells to bring about
the plasma membrane or to the recycling endosome (see large scale cellular contractions, whereas the unconven-
Step 8 in Module 4: Figure membrane and protein traf- tional myosin motor proteins, which are exemplified by
ficking). Others, such as the cation-independent mannose myosin Va, transport a great variety of cargoes (synaptic
6-phosphate receptor (CI-MPR) are sent back to the Golgi vesicles, secretory granules, melanosomes, InsP3 -sensitive
(see Step 9 in Module 4: Figure membrane and protein Ca2 + stores and mRNAprotein complexes) along actin
trafficking ). Finally, proteins such as the EGFR that are tracks to different intracellular destinations.
destined to be degraded by lysosomes (Module 1: Fig- Despite their multiple functions, all of the myosins have
ure receptor down-regulation) are isolated into intralu- a highly conserved N-terminal motor domain, whereas the
minal endosomal vesicle (Module 4: Figure intraluminal C-terminal region is highly divergent and enables the my-
endosomal vesicle formation). As the internal vesicles ac- osin motors to interact with many different cargo proteins.
cumulate, the early endosome gradually matures into a
multivesicular endosome (MVE) and eventually end up Myosin I
as lysosomes (see Step 10 in Module 4: Figure membrane Myosin IE plays a role in the process of membrane inva-
and protein trafficking). The PtdIns3,5P2 signalling cas- gination and scission during the endocytosis of clathrin-
sette (Module 2: Figure PIKfyve activation) plays an im- coated vesicles (Module 4: Figure scission of endocytic
port role in this late endosome-lysosome transformation. vesicles).
Sortilin 1 (SORT1) Myosin II

Sortilin 1 (SORT1) is a transmembrane protein that binds The myosin II subfamily consists of two main types: the
a number of molecules to direct their transport within the conventional type II myosins and the non-muscle myosin
trans-Golgi network (TGN). It is a type-1 receptor protein II (NMII). All of the type II myosins have the same ba-
that consists of a large luminal domain, a single transmem- sic structure. Two myosin heavy chains form dimers that
brane domain and a short C-terminal cytoplasmic domain are held together by their -helical coiled-coil N-terminal
that resembles that on the mannose 6-phosphate receptor globular heads that have the ATPase catalytic region that
(CI-MPR). SORT1 can traffick proteins from the Golgi converts the energy from ATP hydrolysis into mechan-
apparatus either to the plasma membrane or in the oppos- ical force. There are also two myosin light chains (MLCs)
ite direction towards the lysosomes. located between this head domain and the long coiled-coil
It is a multifunctional receptor capable of transporting tail. Each myosin II dimer aggregates to form myosin fil-
many different proteins such as low-density lipoprotein aments, with the head regions lined up precisely opposite
(LDL), neurotensin, progranulin (PGRN), proBDNF and the actin filaments. In skeletal muscle, each filament con-
proNGF. Its role in transporting LDL has implications for tains approximately 200 myosin molecules that lie in the
hypercholesterolaemia and atherosclerotic lesion forma- middle of the sarcomere between the actin fibres (Mod-
tion. ule 7: Figure skeletal muscle structure). By contrast, the
SORT1 can operate independently of CI-MPR by func- non-muscle filaments are smaller comprising approxim-
tioning as a clearance receptor that directs proteins from ately 1220 myosin molecules.
the cell surface through the Golgi apparatus to the lyso- The main difference between the type II myosins lies
somes (step 11 in Module 4: Figure membrane and protein in their mode of activation. The conventional type II
trafficking). For example, progranulin (PGRN) may be myosins, which function in skeletal and cardiac muscle,
metabolized by this SORT1-clearance pathway. are controlled by Ca2 + acting on troponin C (TnC) as
described in the process of excitation-contraction (E-C)
coupling in skeletal muscle cells (Module 7: Figure skeletal
muscle structure). In the case of smooth muscle type II
Motor proteins myosins and the non-muscle type II myosins, the primary
The kinesin or dynein motors, which travel along micro- regulation is exerted by phosphorylation of the 20 kDa
tubules, are responsible for long-range transport from the MLC. This phosphorylation can occur either through ac-
nucleus to the cell periphery and back, such as the ante- tivation of myosin light chain kinase (MLCK) or through
and retro-grade process of axonal transport in neurons. a smooth muscle Rho/Rho kinase signalling pathway that

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inhibits a myosin phosphatase (MYPT) as illustrated by are connected to the coiled-coil regions via an -helical 24
smooth muscle cell excitation-contraction coupling (Mod- nm lever arm that has six IQ motifs that bind to calmodulin
ule 7: Figure smooth muscle cell E-C coupling). In the case (CaM). These long lever arms enable MyoV to take 36 nm
of the non-muscle myosin II motors, phosphorylation of steps, which is approximately equal to the pseudo-repeat
the MLCKs can also be carried out by a number of other distance of the actin helix. This arrangement enables the
kinases including citron kinase, leucine zipper interact- motor to move straight along the actin without having to
ing kinase (ZIPK) and myotonic dystrophy kinase-related spiral around the helical actin filament. The long coiled-
CDC42-binding kinase (MRCK). coil forms the region where the two molecules of MyoV
There are three non-muscle type II myosin heavy chains: are tied together to form the functional motile unit. The
NMHCIIA, NMHCIIB and NMHCIIC coded for by the C-terminal region contains the globular domains that bind
Myh9, Myh10 and Myh14 genes respectively. Like the con- to different cargo proteins and it is this region that mainly
ventional myosin II, these non-muscle myosins also have defines the three Myo5 motors.
two MLCs that regulate their function. These non-muscle MyoV exists in two states. A compact inactive state
myosins (NMIIA, NMIIB and NMIIC) differ in their kin- where the two cargo-binding globular tail domains fold
etic properties and particularly with regard to their duty over to interact with the motor head domains (see panel B
ratio, which is defined by the time that the myosin head in Module 4: Figure myosin motors). In this inactive state,
remains attached to actin during the course of a typical one of the motor heads can bind to actin so that the motor
contraction cycle. NMIIA has the shortest duty ratio in is positioned on the actin track waiting for the activation
that it has the highest rate of ATP hydrolysis and thus signal, which converts the folded molecule into its exten-
moves over actin at the highest rate. By contrast, NMIIB ded open state capable of transporting cargo. Just how this
has the longest duty ratio and maintains tension on the activation is triggered is somewhat of a mystery and there
actin filaments for longer periods enabling it to contrib- appear to be two mechanisms. First, there are indications
ute to the tonic contraction of smooth muscle cells. These that activation is triggered by Ca2 + acting on the resident
non-muscle myosins have multiple functions in cells such calmodulin (CaM) molecules. The concentration of Ca2 +
as cell migration, adhesion and mitosis: appears to be critical because very high levels will displace
some of the CaMs resulting in the lever arms becoming
In endothelial cells, the endothelial regulation of para- too inflexible to participate in the motile cycle. Secondly,
cellular permeability depends on non-muscle myosin II the presence of cargo binding to the C-terminal globular
providing the contractile force to opens up the paracel- domains may pull the latter away from the motor domains
lular pathway (Module 7: Figure regulation of paracel- allowing the molecule to extend out into its active state.
lular permeability).
Non-muscle myosin II functions in neutrophil chemo-
taxis during actin assembly, pseudopod formation and Myosin Va
uropod contraction. At the front of the cell, inactive Myosin Va (MyoVa) is a multifunctional motor protein
non-muscle myosin IIA (NMIIA) helps to form the capable of transporting a number of different cargoes.
cables that attach to the integrin receptors. At the back One of the functions of MyoVa is to transport melano-
of the cell, Rho stimulates the NMIIA to contract the somes into the dendrites of melanocytes (Module 7: Figure
uropod actin network to propel the cell forward (Mod- melanogenesis). The C-terminal globular domains bind to
ule 11: Figure neutrophil chemotactic signalling). the adaptor protein melanophilin, which is also known
The actin fibres attached to integrin receptors are sta- as synaptotagmin-like protein homologue lacking C2 do-
bilized by binding to non-muscle myosin II filaments main a (Slac2-a). The melanophilin is attached through
(Module 6: Figure integrin signalling). the GTPase Rab27a to the melanosome (see panel C in
The activation of contraction during the process of cy- Module 4: Figure myosin motors). The MyoVa then trans-
tokinesis depends on phosphorylation of the myosin ports the melanosomes down the dendrites towards the
light chains (MLCs) of myosin II filaments (Module 9: periphery where they are transferred across to the kerat-
Figure cytokinesis). inocytes (see steps 8 and 9 in Module 7: Figure melanogen-
Clot retraction, which is the final stage in blood platelet esis). Myo5a also functions to transport secretory granules
activation (step f in Module 11: Figure thrombus forma- in both chromaffin cells and insulin-secreting cells. The
tion), may be driven by contraction of non-muscle my- interaction between this motor and the granules is car-
osin II filaments (Module 11: Figure platelet activation). ried out by Rab27a and the myosin- and Rab-interacting
protein (Myrip), which is also known as synaptotagmin-
Myosin V like protein homologue lacking C2 domain c (Slac2-c) (see
There are three myosin V genes (MYO5A, MYO5B and panel C in Module 4: Figure myosin motors). Both Slac2-
MYO5C) that are widely expressed, particularly in the c/MyRIP and synaptotagmin-like protein 4-a play a role
nervous system. These three motor proteins have similar together with Rab27 in the control of amylase release from
motor domains, but variable C-terminal domains that re- rat parotid acinar cells.
cognize different cargo proteins. The N-terminal region During the process of early endosome to plasma mem-
of Myosin Va (MyoVa) has the motor domain that binds brane trafficking, myosin Va transports recycling vesicles
to actin and is responsible for motility (see panel A in from the early endosome to the plasma membrane (Mod-
Module 4: Figure myosin motors). The motor domains ule 4: Figure early endosome budding).

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Module 4: Figure myosin motors
A Melanophilin B Open
Myrip Compact
Rab11-FIP2 inactive state
Ca 2+ activation active state
_ +
C C _
C-terminal
globular domain +
Actin
monomers Cargo activation
_ +
Coiled-coil
2+
regions C
Ca
Secretory
Melanosome vesicle Synaptic
vesicle
Lever Rab27a Rab27a Rab11

arm
24nm
CaM Motor Melanophilin Myrip FIP2
domain
N Actin N
MyoVa MyoVa MyoVb
_
36nm +
Myosin V motor structure and function.

The myosin V family exemplifies the activity of many of the unconventional myosin motors. A: myosin V functions as a dimer with the two heavy chains
joined together through a coiled coil region. The N-terminal motor domains are attached to actin and the C-terminal globular domains are linked to
various cargoes by different adaptors. Six calmodulins (CaMs) are attached to each of the 24 nm lever arms. B: activation of myosin V is driven either
by Ca2 + or the binding of cargo. C. The Myosin V family members bind to different cargoes using a variety of adaptors (melanophilin, myrip and FIP2 )
and Rab GTPases (Rab11 and Rab27a).
When the secretory and synaptic vesicles leave the actin Myosin VI has been implicated in a number of vesicu-
filaments and begin to approach the plasma membrane in lar transport mechanisms during protein trafficking where
preparation for exocytosis, the myosin Va may contribute it functions in both exocytosis and endocytosis. The C-
to the docking process by the molecular motor interacting terminal tail has binding domains enabling it to associate
with syntaxin-1 on the plasma membrane. with various vesicle components such as the phosphol-
Myosin Va plays an important role in transporting en- ipid PtdIns4,5P2 and Disabled-2 (Dab2) that enables it to
doplasmic reticulum vesicles containing InsP3 receptors bind clathrin-coated vesicles. With regard to the latter, it
along the dendrites to their location in the dendritic spines. functions in the membrane invagination and scission of
Another function of myosin Va is to transport messenger clathrin-coated vesicles and then helps to transport these
RNA ribonucleoprotein that have mRNA-binding pro- vesicles from the cell surface towards the early endosome
teins. (Module 4: Figure scission of endocytic vesicles).
Mutation of the MYO5a has been linked to Griscelli Various other vesicle proteins can bind to myosin VI,
syndrome (GS). such as glucose-transporter binding protein (GIPC) and
FIP2 (also known as optineurin).
Myosin Vb Myosin VI has a role in hair cells where it is located at
Myosin Vb (MyoVb) is highly enriched in neuronal syn- the base of the stereocilia and transports components that
aptic spines where it functions to transport endosomes car- are essential for the structural integrity of the mechano-
rying AMPARs to the exocytotic sites (Module 10: Figure sensitive mechanisms. In stereocilia, the myosin VI moves
Ca2+ -induced synaptic plasticity). toward the base and this helps to maintain stability by
The myosin Vb also plays a role in non-clathrin- increasing the internal tension.
dependent endocytosis that is driven by Rab8A. Mutations in FIP2/optineurin have been linked to
Mutations in myosin Vb has been linked to microvillus some forms of glaucoma and amyotrophic lateral sclerosis
inclusion disease. (ALS).
Myosin VI Kinesin
Myosin VI is unusual in that it moves backwards towards The superfamily of kinesin motors transports a great vari-
the minus-end of actin filaments. Just how the myosin IV ety of intracellular targets along microtubules. The kinesin
moves along the actin is still debated. motor domain uses the energy of ATP to provide the force

C
to drag cargos through the cytoplasm. The large kinesin Module 4: Table kinesin superfamily
superfamily, which contains 45 KIF genes, has been di- The superfamily of kinesin motors.
vided into three separate families that are defined on the Kinesin superfamily
N-terminal kinesins The N-kinesins superfamily (39
basis of the location of kinesin motor domain (Module 4: (N-kinesins) KIF genes) have the globular
Table kinesin superfamily). The motor domain is located motor domain located at the
at the N-terminus of the 12 N-kinesins (kinesin 112), in N-terminus and usually
transport cargo towards the
the middle of the molecule for the M-kinesins (kinesin-13) plus end of microtubules
and at the C-terminus in the C-kinesins (kinesin-14). Each (Module 4: Figure kinesin
of the 14 kinesin families has variable numbers of motors, cargo transport in neurons)
Kinesin-1 KIF5 plays a major role in
which are referred to as KIFs. transporting cargo in neurons
The structure of the KIFs has three main regions: the (Module 4: Figure kinesin
globular motor and cargo-binding domains are connected cargo transport in neurons)
KIF5A
through variable stalk regions. These linker regions often KIF5B
form coiled-coil segments when subunits dimerize to form
either homo- or hetero-dimers (Module 4: Figure kinesin KIF5C
Kinesin-2
motor structure). The globular motor domains of all of KIF3A
the KIFs display a high degree of homology. It is this KIF3B
region that has the specific microtubule-binding domain KIF3C
KIF17
and the ATP-binding domain. Most variability is found in Kinesin-3
the cargo-binding domain responsible for interacting with KIF1A
the different cargos that are transported through the cell. KIF1B, KIF1B
KIF1C
Considering this variability, it is difficult to generalize and KIF13A
each kinesin has to be considered separately. KIF13B
Kinesin uses its two heads to progress along the surface Kinesin-4
KIF4A
of the microtubule with the two heads alternating with KIF4B
each other as they bind and then detach from the tubulin KIF21A
dimers (Module 4: Figure kinesin and dynein motor mech- KIF21B
Kinesin-5
anisms). The motor moves in steps of approximately 8 nm KIF11
as the heads take turns to move over the tubulin surface. Kinesin-6
ATP is used to drive each attachment/detachment cycle. KIF20
KIF23
The ability of these motors to transport cargo around the Kinesin-7
cell is controlled by a number of kinesin motor regulatory KIF10
mechanisms. Kinesin-8
KIF18
KIF19
Kinesin-1 Kinesin-9
Kinesin-10
Kinesin-1 is a typical N-kinesin and has three family mem-
KIF22
bers (KIF5A, KIF5B and KIF5C) (Module 4: Table kinesin Kinesin-11
superfamily). The KIF5 motors, which function as dimers, Kinesin-12
KIF12
have the typical N-terminal globular motor domain that
KIF15
is connected via a stalk to the C-terminal cargo-binding Middle kinesins (M-kinesins) The M-kinesins superfamily (3
domain (Module 4: Figure kinesin motor structure). The KIF genes) usually transport
cargo towards the plus end
cargo-binding domain associates with two kinesin light
of microtubules
chains (KLCs) to form a fan-like structure responsible for Kinesin-13
binding a number of different cargos that attach to either KIF2A
KIF2B
the KLCs or the cargo-binding domains, often using spe-
KIF2C
cific scaffolding proteins. The processivity of kinesin-1 is C-terminal kinesins
enhanced by the acetylation of the -tubulin subunits. (C-kinesins)
Kinesin-14
The KIF5 motors are particularly active in carrying dif-
KIFC1
ferent cargos down neuronal axons and dendrites (Module KIFC2
4: Figure kinesin cargo transport in neurons). For example, KIFC3
the cargo-binding domain uses glutamate receptor-inter-
acting protein 1 (GRIP1) to bind to AMPARs that are
transported down the dendrites. The cargo-binding do-
main of KIF5 also functions to transport a large oligo- this complex code for proteins, such as activity-regulated
meric complex of proteins and mRNAs into the dend- cytoskeletal-associated protein (ARC) and the -subunit
rites. This ribonucleoprotein (RNP) particle has approx- of CAMKII that function within the postsynaptic spines
imately 40 proteins, such as fragile X syndrome protein (Module 10: Figure Ca2+ -dependent synaptic plasticity).
1 (FXRP1), mStaufen and cofactors for mRNP localiza- On the other hand, the KLCs use the JIPs to interact with
tion in dendrites such as PUR and PUR. The FMRP1 the -amyloid precursor protein (APP) and apolipopro-
links the particle to KIF5. The mRNAs that are attached to tein E receptor 2 (APOER2) that is moved down the axons.

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Module 4: Figure kinesin motor structure
Motor Cargo-binding
domain Stalk Hinge domain
Kinesin-6
Kinesin-1 KLC
KIF5
Kinesin-7
KIF3A KIF10
KAP
KIF3B
Kinesin-2 Kinesin-8
KIF17
Kinesin-10
KIF22
Kinesin-3
KIF1
Kinesin-12
Kinesin-4
KIF4 Kinesin-13
KIF2
Kinesin-5 Kinesin-14
KIF11 KIFC
Structure of kinesin motors.

The kinesin superfamily of motor proteins, which has 45 KIF genes, has been divided into 14 families. The structure of some of the KIFs illustrates
their three main regions: globular motor domain (orange), cargo-binding domain (green) and a stalk region (black). Many of the motors form dimers
through coiled-coil segments of these stalk regions. Some of these stalks are interrupted by hinge regions that enables the cargo-binding domain
to bend around to interact with the motor domain to set up an autoinhibitory interaction in the absence of any cargo. See the text for further details.
Information for this Figure was taken from Box 1 in Verhey and Hammond (2009).
Kinesin-2 Kinesin-2 transports vesicles during COPI-mediated

Kinesin-2 is an N-kinesin, which has four family mem- transport from Golgi to ER (Module 4: Figure COPI
bers (KIF3AC and KIF17) (Module 4: Table kinesin su- coated vesicle).
perfamily). KIF3A can form heterodimers with KIF3B
and KIF3C, which then form heterotrimeric complexes Fodrin
when their N-terminal region interacts with kinesin Fodrin, which is also known as non-erythroid spectrin,
superfamily-associated protein 3 (KAP3) (Module 4: Fig- consists of - and -subunits that bind together to form
ure kinesin motor structure). One of the functions of this filaments that bind to actin at both ends to create a network
KIF3A/KIF3B/KAP3 complex is to transport large ves- just beneath the plasma membrane. The -fodrin is cleaved
icles (90150 nM) that are associated with fodrin (Module by caspases during apoptosis.
4: Figure kinesin cargo transport in neurons). KIF3A also In adipocytes, fodrin might play a role in the transloca-
transports the partitioning protein 3 (PAR3), which is part tion and fusion of the GLUT4 storage vesicles (GSVs). The
of the polarity complex (PAR3/PAR6/atypical PKC) that GSVs have VAMP2 that interacts with syntaxin 4 to trig-
helps to establish which neurites will grow into axons. ger membrane fusion. The cortical fodrinactin network
The KIF17 motor transports vesicles containing NM- may play a role in moving GSVs to the plasma membrane.
DARs down dendritic microtubules at a rate of approxim-
ately 0.75 m/s towards the postsynaptic spines (Module Kinesin-3
4: Figure kinesin cargo transport in neurons). The attach- Kinesin-3 is a N-kinesin that has a number of family mem-
ment between the KIF17 motor domain and the NR2B bers (KIF1A, KIF1B and KIF1B, KIF1C, KIF13A and
subunit of the NMDAR is carried out by a trimeric scaf- KIF13B) (Module 4: Table kinesin superfamily). These
folding complex containing the Munc18-interacting pro- kinesin-3 motors can function either as monomers, as
tein (MINT1), calcium/calmodulin-dependent serine pro- for KIF1A and the two alternatively spliced KIF1B and
tein kinase (CASK) (LIN-2) and the vertebrate LIN-7 KIF1B, or as homodimers (Module 4: Figure kinesin mo-
homologue (VELIS), which is also known as mammalian tor structure).
LIN-7 protein (MALS). These proteins have PDZ domains In neurons, KIF1A and KIF1B transport organ-
that enable Velis/MALS to bind to the NR2B subunit and elles containing synaptic vesicle precursors (Module 4:
MINT1 to attach the whole complex to KIF17. Figure kinesin cargo transport in neurons), such as

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synaptotagmin, synaptophysin and Rab3A. On the other Kinesin-13

hand, the KIF1B isoform can transport mitochondria Kinesin-13 is an M-kinesin, which has three members
down axons. KIF2A, KIF2B and KIF2C (Module 4: Table kinesin su-
A point mutation in the ATP-binding site of the mo- perfamily), and is unusual because the motor domain is
tor domain of KIF1B has been linked to a hereditary located in the middle of the molecule (Module 4: Figure
peripheral neuropathy called Charcot-Marie-Tooth dis- kinesin motor structure). While it is not capable of direc-
ease (CMT) type 2A. ted movement, it is capable of destabilizing microtubules.
KIF13A is used to transport vesicles containing the Kinesin-13 is strongly expressed in the developing brain
mannose 6-phosphate receptor (M6PR), which recognizes where it has an important role in controlling microtubule
the sorting signal located on adaptor protein 1 (AP-1). dynamics within the growth cones.
AP-1 functions as an adaptor/scaffold that binds to both
clathrin and to the motor protein KIF13A to transport Kinesin-14
coated vesicles from the trans-Golgi network to the early Kinesin-14 is a C-kinesin, which has three members
endosome (see step 3 in Module 4: Figure membrane and KIFC1, KIFC2 and KIFC3 (Module 4: Table kinesin su-
protein trafficking). AP-1 consists of four adaptin sub- perfamily), and is unusual because the motor domain is
units (1, , 1 and 1) and it is the 1-adaptin subunit located at the C-terminus (Module 4: Figure kinesin motor
that binds to the KIF13A subunit. structure). It has the ability of moving along and destabil-
izing microtubules.
Kinesin-4
Kinesin-4 is an N-kinesin that has a number of family Kinesin motor regulation
members (KIF4A, KIF4B, KIF21A and KIF21B). The two The regulation of kinesin motor activity depends on a
KIF4 members carry the cargo poly(ADP-ribose) poly- number of mechanisms that can operate during the course
merase 1 (PARP1). CaMKII acts to release PARP1 from of a typical transport cycle. This cycle has three main pro-
KIF4 and the PARP1 then enters the nucleus to control cesses: it begins with the selection of cargo and attachment
transcription and DNA repair. to the microtubule, processivity (co-ordinated movement
along the microtubule) and ends with motor detachment.
Kinesin-5 The selection of cargo and attachment to the microtu-
Kinesin-5 is a typical N-kinesin and has a single mem- bule is an important site of control and varies somewhat
ber KIF11 (Module 4: Table kinesin superfamily). KIF11 between the different motors. Many of the motors, such as
forms homodimers that then interact with each other to kinesin-1, kinesin-2 (KIF17) and kinesin-7 (KIF10), exist
form homotetramers that line up with their globular do- in an inactive folded state where the C-terminal cargo-
mains facing in opposite directions (Module 4: Figure kin- binding domain bends over to interact with the motor
esin motor structure). KIF11 is activated by phosphoryla- domain thereby blocking the nucleotide pocket and thus
tion of its C-terminal cargo-binding domain by cyclin- inhibits the binding and hydrolysis of the ATP required
dependent kinase 1 (CDK1). for movement. Various mechanisms are used to relieve
this autoinhibitory state. In the case of kinesin-1, autoin-
hibition is relieved by binding two proteins: fasciculation
Kinesin-6
and elongation protein-1 (FEZ1, also known as zygin1),
The kinesin-6 family of motor proteins has two members:
which binds to the C-terminal cargo-binding domain and
KIF20A [also known as Rab6 kinesin or mitotic kinesin-
Jun N-terminal kinase (JNK)-interacting protein 1 (JIP1),
like protein 2 (MKLP2)] and KIF23 [also known as mitotic
which attaches to the kinesin light chains (KLCs) (Module
kinesin-like protein 1 (MKLP1)].
4: Figure kinesin cargo transport in neurons). The disrup-
ted in schizophrenia 1 (DISC1) protein is also known to
Kinesin-7 interact with FEZ1. Another way of activating the motors
Kinesin-7 is a typical N-kinesin and has a single mem- is through phosphorylation as occurs for kinesin-5 and
ber KIF10 (Module 4: Table kinesin superfamily), which is kinesin-7. Following phosphorylation, these motor pro-
also known as centromere-associated protein E (CENPE). teins unfold to a more extended state thus enabling them
Like some other kinesins, KIF10 is autoinhibited when the to interact and move along the microtubules.
C-terminal cargo-binding domain bends over to interact Once motors attach themselves to the microtubule their
with the motor domain. This inactivation is reversed fol- rate of movement tends to depend on the state of the mi-
lowing phosphorylation of the cargo-binding domain by crotubules that can be modified in different ways usually in
various kinases such as CDK1/cyclin B or by monopolar the form of a post-translational modification that not only
spindle protein 1 (MPS1). The latter is located on the kin- provide a mechanism for regulating the rate of movement,
etochore where KIF10 functions during mitosis. but may also mark out microtubules to direct kinesins to
carry cargo to specific cellular destinations. For example,
Kinesin-8 acetylation of the -tubulin subunits can enhance the pro-
Kinesin-8, which is a typical N-kinesin that has two mem- cessivity of kinesin-1, whereas detyrosination of the same
bers KIF18 and KIF19 (Module 4: Table kinesin superfam- subunits has the effect of directing kinesin-1 to transport
ily), has a unique ability to both walk along and depoly- cargo to specific destinations. Polyglutamylation of tu-
merize microtubules. bulin subunits seems to enhance the ability of KIF1 to

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Module 4: Figure kinesin cargo transport in neurons
APP APOER2
Miro
JIP1 JIP1 Milton Synaptic vesicle
precursors
Fodrin
KIF1A
KIF5 KIF5 KIF5 KIF3 KIF1B KIF1B
_
Microtubule +
Dendrites Axonal transport of presynaptic components
Axon
Dendritic transport of postsynaptic components
AMPAR NMDARs mRNA

GluR2 RNP
GRIP1 NR2B FMRP1 particle
VELIS
KIF5 CASK
KIF17
MINT1 KIF5
+ _
Microtubule
Transport of cargo by kinesin motors in neurons.

In the case of the KIF5 motors, the C-terminal cargo-binding domain (green) carries cargo (AMPARs) down the dendrites whereas cargo that is
attached to the KLCs (yellow), such as the amyloid precursor protein (APP) and apolipoprotein E receptor 2 (APOER2) moves down the axons. This
Figure is based on the information shown in Figure 3 in Hirokawa and Takemura (2005).
transport synaptic vesicle precursors (Module 4: Figure Dynein

kinesin cargo transport in neurons). Dynein is a motor protein that carries cargo along micro-
Finally, there are a number of mechanisms that terminate tubules in the minus-end direction. It has important func-
processivity by enhancing the detachment of the kinesin tions during membrane and protein trafficking as it moves
motors. One is a physical mechanism whereby the vari- cargo from the cell periphery towards the cell centre. This
ous microtubule-associated proteins (MAPs), such as tau, is particularly evident in neurons where dynein transports
may act to block motors such as kinesin-1. Displacement various components from the synapses back to the cell
of the motors may also be controlled by phosphorylation. body. Dynein also moves proteins down the axoneme of
The JIP1 scaffolding protein, which attaches cargo to KIF5 cilia. There are a large number of dynein heavy chains, but
(Module 4: Figure kinesin cargo transport in neurons), is only two of these function as motors to transport mater-
also an activator of the MAP kinase signalling pathway ials around the cell. Intraflagellar transport (IFT) dynein
that is known to dissociate JIP1 from its motor thus ter- transports cargo down the axoneme whereas cytoplasmic
minating transport. A similar action of the MAP kinase dynein transports cargo such as vesicles, proteins, mRNA
signalling may disrupt cargo transport by the kinesin-2 and also functions during mitosis. The large dynein com-
motor proteins that operate in cilia and flagella. plex can be divided into separate functional components
The Ca2 + signalling system may also play a role in reg- (Module 4: Figure dynein):
ulating the interaction between cargo and kinesin motors.
For example, phosphorylation of the C-terminal cargo- Dynein catalytic motor protein has the catalytic com-
binding domain region of the Kinesin-2 family member ponent that converts the energy of ATP to move dynein
KIF17 releases the NMDAR cargo vesicles that are car- Dynein non-catalytic subunits link the dynein motor to
ried down the microtubules to the dendrites. In this way, various adaptors
local elevations of Ca2 + in the presynaptic region will Dynein adaptors link dynein to various cargos
serve to recruit NMDARs as part of the process of syn-
aptic remodelling responsible for learning and memory. Dynein catalytic motor protein
The Ca2 + -sensitive mitochondrial Rho-GTPase (MIRO) The dynein catalytic motor protein consists of a single
responds to local elevations of Ca2 + by releasing mito- protein, a large catalytic heavy chain that has three main
chondria (Module 5: Figure mitochondrial motility). regions: a motor domain, linker domain and an N-terminal

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Module 4: Figure kinesin and dynein motor mechanisms and AAA5, forms a stalk that has the microtubule-binding
domain that attaches the heavy chain to the tubulin sub-
units. The chain that emerges from AAA1 forms the linker
domain that connects to the coiled-coil region where the
N-terminal tails of the two heavy chains dimerize with
each other. The dynein non-catalytic subunits are bound
to this N-terminal tail.
Dynein non-catalytic motor subunits

A number of subunits are associated with the coiled-coil
region of the N-terminal tail (see the green box in Mod-
ule 4: Figure dynein). The light intermediate chain (LIC)
and the dynein intermediate chain (IC) bind directly to
the heavy chain, whereas the smaller dynein light chain 7
(LC7), dynein light chain 8 (LC8) and T-complex testis-
specific protein 1 (TCTEX1) are all attached to IC. These
subunits function by interacting with various dynein ad-
aptor proteins, as described below.
Dynein adaptors
There are a number of adaptors (see pink boxes in Module
4: Figure dynein). The most important adaptor is dynein
activator (dynactin), which is made up of 11 subunits. The
interaction between dynein intermediate chain (IC) and
dynactin is particularly important in controlling the mo-
tor function of dynein. A major component of dynactin is
the p150 subunit that links to the dynein catalytic motor
protein complex through the IC. At its other end, p150 has
a cytoskeleton-associated protein glycine-rich (CAP-Gly)
domain that can bind to tubulin. The interaction between
tubulin and p150 is facilitated by the latter interacting
with two plus-end-binding proteins called end-binding 1
(EB1) and CAP-Gly domain-containing linker protein 170
(CLIP170). A short filament made up from actin-related
protein 1 (ARP1) plays an important role in binding to
some cargos especially those that are membrane-bound.
This ARP1 filament of dynactin binds to the filamentous
protein III spectrin that is often found on the surface
Motile mechanisms of kinesin and dynein motors
of many membranes including those of the Golgi. At the
The proposed motile mechanism for kinesin and dynein motors. A. For
kinesin, one head is always firmly attached whereas the othe head de- pointed end of the ARP1 filament, there is a complex of
taches and then moves forward to attach to tubulin 8 nM away. Once it proteins consisting of actin-related protein 11 (ARP11),
is bound the trailing head can detach to repeat the sequence. Reprinted
p62, p25 and p27. The barbed end of the ARP1 filament
from Curr. Opin. Cell Biol., 21, Gennerich, A. and Vale, R.D., Walking the
walk: how kinesin and dynein coordinate their steps, 5967, c 2009, with has a number of proteins. There is an actin-capping protein,
permission from Elsevier. a p24 subunit and a p50 tetramer, which is also known as
dynamitin. The p50 interacts with other dynein adaptors,
such as Bicaudal D1 and Rod-ZW10-Zwilch (RZZ). Bi-
caudal D was originally discovered in Drosophila where
tail where the non-catalytic subunits are bound (Mod- it functions to transport mRNA during pattern formation
ule 4: Figure dynein). The C-terminal motor domain has in early development. The two mammalian homologues
two main regions: the ATP-binding modules (16) and (Bicaudal D1 and Bicaudal D2) belong to the golgin fam-
the microtubule-binding domain located at the end of a ily and appear to function in vesicle transport between the
short stalk. These heavy chains belong to the superfam- Golgi and the ER. The GTPase Rab6 binds to Bicaudal D
ily of ATPase associated with various cellular activities and thus provides a mechanism to attach the dynein motor
(AAA + ATPase), but is somewhat unusual in that its six complex to the vesicle for the COPII-mediated transport
AAA domains are all part of the same polypeptide chain. from ER to Golgi (Module 4: Figure COPII-coated ves-
These six AAA domains form a catalytic ring responsible icles). Bicaudal D1 may also interact with LC8.
for binding and hydrolysing ATP. Hydrolysis of ATP by In addition to the large dynactin complex, there are
AAA1 and AAA3 are mainly responsible for providing some other adaptors that can attach to the dynein complex.
the energy to drive the motor along the tubulin subunits Lissencephaly (LIS1), which interacts with either nuclear
of the microtubule. A long loop, which connects AAA4 distribution protein (NUDE) or the closely related

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Module 4: Figure dynein
Kinetocore III spectrin CARGO

ZW10
ARP1 filament
CENPF
Actin-capping protein
6b
Ra
N
N
6b
Ra
TCTEX1
NUDEL p24
NUDE
ARP11, p25,
DISC1 p27, p62
LC8 p50
LIS1 LIS1 LIC IC Rab6
Linker LC7
Dynein adaptors domain
ATP Bicaudal D1
C 1 C ADP
1
6 2 6 RZZ
2 p150
DYNEIN 5 ATP
3 5 3
4 DYNACTIN
4 ADP
Stalk
CAP-Gly
domain CLIP170
Microtubule-binding
domain EB1
Tubulin subunits
_
+
Microtubule
Structure and function of dynein.

The dynein motor complex consists of the large catalytic dynein heavy chain (thick black line) that has six ATP-binding modules (16), a microtubule-
binding domain located at the end of a short stalk and an N-terminal region that forms a coiled-coil with a neighbouring chain. Attached to this
N-terminal region of the heavy chain, there are a number of dynein non-catalytic subunits (shown in green). A number of dynein adaptors (shown in
the pink boxes) provide links to various cargoes. The main adaptor is the dynactin complex (shown in yellow). See text for further details.
NUDE-like (NUDEL), is attached to the kinetocore last process, DISC1 may function by binding to the
by interacting with centromere protein F (CENPF) and actin-binding protein fasciculation and elongation pro-
ZW10 (Module 4: Figure dynein). LIS1 has the unique tein zeta-1 (FEZ-1), which seems to function in kinesin
ability to bind to the AAA1 domain and this may function motor regulation.
to regulate ATPase activity and hence motor activity. The The hydrolysis of cyclic AMP by PDE4B is inhibited by
centrosomal proteins NUDE and NUDEL also interact DISC1. The antidepressant Rolipram inhibits PDE4B.
with disrupted in schizophrenia 1 (DISC1) as part of a DISC1 may also function to regulate gene transcription
complex that functions in mitosis, neuronal migration and through different mechanisms. It can interact with tran-
microtubule organization during brain development. scription factors such as ATF4 and ATF5. DISC1 can
The human disease lissencephaly may be caused by also interact with glycogen synthase kinase 3 (GSK3),
mutations in the gene that encodes lissencephaly 1 (LIS1). which is part of the PtdIns 3-kinase signalling pathway
(Module 2: Figure PtdIns 3-kinase signalling). Modific-
Disrupted in schizophrenia 1 (DISC1) ation of GSK3 activity will then influence the opera-
The gene for disrupted in schizophrenia 1 (DISC1) confers tion of the canonical Wnt/-catenin pathway that con-
an increased risk for various mental illnesses, including bi- trols gene transcription through activation of -Catenin
polar disorder and schizophrenia. DISC1 is one of the most (Module 2: Figure Wnt canonical pathway).
highly associated susceptibility genes for schizophrenia. A
chromosomal translocation between chromosome 11 and
chromosome 1 (where DISC1 is located) results in the
truncation of DISC1 and the subsequent loss of function Gene transcription
seems to be responsible for disrupting both brain struc- A major function of many signalling pathways is to activ-
ture and function. These widespread changes can be ex- ate gene transcription. This regulation of gene expression
plained by the fact that DISC1 has many binding partners operates throughout the life history of a typical cell. Devel-
responsible for carrying out multiple physiological pro- opmentally regulated transcription factors begin to func-
cesses. Some of these processes are shown in Module 12: tion early in development to define developmental axes,
Figure schizophrenia: and they contribute to cellular differentiation to form spe-
cialized cells. An important component of differentiation
DISC1 is part of the NUDEL/LIS1 complex that func- is signalsome expression, during which each cell type ex-
tions as one of the dynein adaptors for the dynein mo- presses the specific signalling components that are neces-
tor protein (Module 4: Figure dynein) to carry out sary to meet its functional requirements (Module 8: Fig-
processes such as mitosis, neuronal migration, neur- ure signalsome expression). Once cells have differentiated,
ite formation and axon elongation. With regard to the transcription plays an important role in maintaining both

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the stability of the differentiated state and its signalling Mitochondrial transcription factor (Tfam)
pathways. An important aspect of this phenotypic stabil- Myocyte enhancer factor-2 (MEF2)
ity is the way in which the signalling and transcriptional Myc
systems co-operate to create a quality assessment system MyoD
that ensures signalsome stability. Nuclear factor of activated T cells (NFAT)
All of these processes require making transcripts of in- Nuclear factor B (NF-B)
dividual genes, which is carried out by RNA polymerase Nuclear factor erythroid 2 related factor 2 (NRF-2)
II (pol II). Gene regulation depends upon careful con- Nuclear transcription factor Y (NF-Y)
trol of polymerase II by a host of transcription factors Nuclear response factor-1 (NRF-1)
and transcriptional co-regulators. The way in which tran- p53
scription factors communicate with pol II is carried out Parkin-interacting substrate (PARIS)
by a mediator complex, which can exert a significant role Peroxisome-proliferator-activated receptors (PPARs)
in controlling whether or not specific genes are tran- Peroxisome-proliferator-activated receptor (PPAR)
scribed. While most attention will focus on the transcrip- coactivator-1 (PGC-1)
tion factors, it is clear that co-regulators such as the co- Pituitary-specific transcription factor (Pit-1)
activators and co-repressors also play a significant role by Single-minded 1(Sim1)
recruiting chromatin remodelling enzymes such as histone Smads
acetyltransferases (HATs), histone deacetylases (HDACs) Specificity protein 1 (Sp1)
and protein methylases to form the large macromolecu- Sterol regulatory element-binding proteins (SREBPs)
lar signalling complexes called transcriptosomes that reg- Signal transducers and activators of transcription
ulate transcription. There also is an important relationship (STATs)
between ubiquitin signalling and gene transcription that Serum response factor (SRF)
operates at many different levels. Transcription initiation factor IB (TIF-IB)
Most attention will be focused on the transcription Vitamin D receptor (VDR)
factor activation mechanisms used by signalling pathways X-Box binding protein 1 (XBP-1)
to control gene expression.
Transcription factors Transcriptional co-regulators

There is a bewildering variety of transcription factors that The action of transcription factors often depends on co-
can either function as activators or repressors of gene tran- regulators such as the transcriptional co-activators and
scription. These activators and repressors do not function transcriptional co-repressors. These additional compon-
in isolation, but are usually part of a multi-protein tran- ents facilitate the activity of the transcriptional activators
scriptosome made up of transcriptional co-regulators and and repressors in different ways. In many cases, they func-
associated factors. tion by recruiting chromatin remodelling proteins that
A transcription factor classification has been introduced alter the way transcription factors access gene promoter
to provide a framework for understanding the diverse sites.
functions of the following transcription factors:
Activating protein 1 (AP-1) (Fos/Jun) Transcriptional co-activators

Activating transcription factor 6 (ATF6) Co-activators, which function to enhance gene expression,
APP intracellular domain (AICD) usually act by binding to transcriptional activators. Many
-Catenin of the coactivators function by regulating the transcrip-
BCL6 tion of genes that control cellular differentiation, migra-
CCAAT/enhancer-binding protein (C/EBP) tion, and proliferation. Some of these coactivators are his-
CSL (CBF-1, Suppressor of Hairless, Lag-1) tone acetyltransferases (HATs) that add acyl groups to the
Cyclic AMP response element-binding protein (CREB) lysine groups on histones resulting in an increase in the ac-
Downstream regulatory element antagonistic modu- cessibility of DNA to transcription factors. The following
lator (DREAM) are examples of such co-activators:
E2F family of transcription factors
E twenty-six (ETS)
Forkhead box O (FOXO) CREB binding protein (CBP)
Glucocosteroid receptor (GR) Myocardin family
Hepatocyte nuclear factor 4 (HNF4) p300
Hypoxia-inducible factor (HIF) Peroxisome-proliferator-activated receptor (PPAR)
Interferon-regulatory factors (IFNs) coactivator-1 (PGC-1)
Kruppel-like factors (KLFs) p300/CBP association factor (PCAF)
LBP1 family of transcription factors p53-binding protein 1 (53BP1) is a p53 co-activator
c-Maf whose binding is facilitated by p53 methylation on
Methyl-CpG-binding protein 2 (MeCP2) K370me2.
Microphthalamia transcription factor (MITF) Tat interactive protein 60 (TIP60)

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CREB binding protein (CBP) gions (CH1-3); the CH3 domain enables p300/CBP to
CREB binding protein (CBP) and the closely related pro- bind to PCAF, p53, MyoD, E2F and c-fos.
tein p300 are histone acetyltransferase (HAT) paralogues Once p300/CBP is recruited to the transcriptional ac-
that arose through gene duplication. Since they share a tivator it can acetylate various proteins responsible for
similar structure and function, they are often referred to driving transcription. In most cases, p300/CBP acet-
as p300/CBP. Details of how these two proteins function ylates histones to relax chromatin structure to facilitate
is described in the section on p300. gene transcription. In addition, it can also acetylate tran-
Mutations in CBP are responsible for Rubinstein-Taybi scription factors such as p53. The following examples
syndrome (RTS). illustrate the action of p300/CBP in controlling gene
transcription:
Myocardin family
The myocardin family consists of closely related transcrip-
Phosphorylation of Ser-133 on cyclic AMP response
tional coactivators such as myocardin itself, which is loc-
element-binding protein (CREB) results in the recruit-
ated mainly in the nucleus, myocardin-related transcrip-
ment of p300/CBP (Module 4: Figure CREB activation).
tion factor-A (MRTF-A) and myocardin-related transcrip-
Activation of CREB has a central role in controlling
tion factor-B (MRTF-B). These three coactivators have a
neuronal gene transcription (Module 10: Figure neur-
similar structure consisting of an N-terminal RPEL do-
onal gene transcription).
main, which enables MRTF-A and MRTF-B to bind to
During muscle differentiation, activated MyoD recruits
actin. The RPEL domain of myocardin cannot bind to
p300 to induce histone acetylation (Module 4: Figure
actin. In the middle of these myocardin family molecules
MyoD and muscle differentiation).
there is a basic ( + ), a glutamine-rich (Q), a SAP (SAF-
A p53 acetylation reaction activates the transcriptional
A/B, Acinus, and PIAS) and an LZ domain with a tran-
activity of p53 (Module 4: Figure p53 function).
scriptional activation domain (TAD) domain at the C-
Activation of transcription by FOXO is facilitated by
terminal region. The association between SRF and these
p300 (Module 4: Figure FOXO control mechanisms).
myocardin family members is mediated by the basic ( + )
Activation of the transcription factor MEF2 depends
and glutamine-rich (Q) regions.
on the recruitment of p300 (Module 4: Figure MEF2
Myocardin activation).
Myocardin is a potent coactivator that binds to serum re- Acetylation of peroxisome-proliferator-activated re-
sponse factor (SRF) that controls the expression of cyto- ceptor (PPAR) coactivator-1 (PGC-1), which is one
skeletal and contractile proteins. It is located mainly in the of the main transcriptional regulators of uncoupling
nucleus and acts to control cardiac development (Module protein 1 (UCP-1) expression during differentiation of
8: Figure cardiac development) and smooth muscle cells brown fat cells, depends on p300 (Module 8: brown fat
(Module 8: Figure smooth muscle cell differentiation). cell differentiation).
Myocardin-related transcription factor-A (MRTF-A) Mutations in CBP are responsible for Rubinstein-Taybi
Myocardin-related transcription factor-A (MRTF-A), syndrome (RTS)
which is also known as MAL, MKL-1 and BSAC, is
strongly expressed in mesenchymal, epithelial and muscle
cells during embryogenesis. The MRTF-A plays an im- p300/CBP association factor (PCAF)
portant role in mediating the link between actin dynamics p300/CBP association factor (PCAF) is a transcriptional
and gene transcription where it acts as a transcriptional coactivator that associates with p300 and CBP. It is a
coactivator of the serum response factor (SRF) (Module 4: histone acetyltransferase (HAT) that acetylates proteins
Figure actin dynamics and gene transcription). (Module 1: Figure protein acetylation). PCAF activity is
regulated by acetylation either through autoacetylation
Myocardin-related transcription factor-B (MRTF-B)
or by p300. One of the important functions of PCAF is
Myocardin-related transcription factor-B (MRTF-B) is
to acetylate and activate transcription initiation factor IB
also known as MKL-2. During embryogenesis, MRTF-B
(TIF-IB) that regulates the activity of RNA polymerase I
functions in the branchial arch arteries and in the develop-
(Pol I).
ing nervous system.
p300 General control of amino-acid synthesis (GCN5)

The transcriptional co-activator p300 and the closely re- General control of amino-acid synthesis (GCN5), which
lated CREB binding protein (CBP) are histone acetyltrans- is also known as lysine acetyltransferase 2A (KAT2A), is
ferase (HAT) paralogues that arose through gene duplic- a typical histone acetylase (HAT). One of its activities is
ation. Since they share a similar structure and function, to acetylate and inhbit peroxisome-proliferator-activated
they are often referred to as p300/CBP. The structure of receptor (PPAR) coactivator-1 (PGC-1) that reg-
p300/CBP is dominated by a central HAT domain that is ulates genes that contribute to ATP generation, such as
flanked by other protein interaction domains. There is an those that function in fatty acid oxidation, glycolysis and
N-terminal KIX domain that promotes interactions with mitochondrial biogenesis (see Step 4 in Module 2: Figure
CREB and MYB. There are three cysteine/histidine re- AMPK control of metabolism).

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Tat interactive protein 60 (TIP60) switching and hence its name. SIN3 lacks DNA bind-
Tat interactive protein 60 (TIP60), which is also known ing activity and is drawn into transcription complexes by
as lysine acetyltransferase 5 (KAT5), is a typical histone interacting with various repressors such as Mad that si-
acetyltransferase (HAT) that acetylates proteins (Module lences genes normally controlled by the proto-oncogene
1: Figure protein acetylation). TIP60 is inactivated by pro- Myc (Module 4: Figure Myc as a gene activator). The tran-
teosomal degradation following its ubiquitinylation by the scription factor myocyte enhancer factor-2 (MEF2) is also
ubiquitin ligase Mdm2. regulated by SIN3 (Module 4: Figure MEF2 activation).
One of the functions of TIP60 is to acetylate and ac- SIN3 carries out its co-repressor activities through its tar-
tivate various components of the DNA damage repair geting and scaffolding functions. For example, it has a PAH
mechanism (Module 9: Figure G1 checkpoint signalling). domain that binds to the SIN3 interaction domain (SID)
It contributes to the arrest of growth by acetylating p53 on located on repressors such as Mad. With regard to its scaf-
lysine-120 (Module 4: Figure p53 domains). By acetylating folding function, SIN3 assembles a large number of pro-
the histones H2A and H4, it opens up the chromatin for the teins. The core SIN3/HDAC complex contains histone
various repair processes. For example, it acetylates ataxia deacetylases (HDAC1 and 2), retinoblastoma-associated
telangiectasia mutated (ATM), which is a key component proteins 46 and 48 (RbAp46/48), SIN-associated pro-
of the repair mechanism (Module 9: Figure G1 checkpoint tein 18 and 30 (SAP18 and SAP30) and SDS3. The
signalling). HDACs function to deacetylate histones that tightens the
TIP60 can also function to control autophagy by activ- histoneDNA interaction to prevent transcription. The
ating ULK1 (Module 11: Figure autophagy). RbAp46/48 proteins provide a bridge to the nucleosomes
by binding to histones H4 and H2A. The SAP proteins
Transcriptional co-repressors have a stabilizing role particularly with regard to the in-
Transcriptional co-repressors, which function to decrease teraction between SNI3 and HDAC1. The SDS3 pro-
gene expression, usually act by binding to transcriptional tein stabilizes the complex by binding both SIN3 and the
repressors such as Mad, some members of the E2F family HDACs.
of transcription factors (E2F4E2F7), methyl-CpG-bind- Other enzymes such as the DNA and histone
ing protein 2 (MeCP2) and CSL (CBF-1, Suppressor of methylases can be added to this SIN3/HDAC core com-
Hairless, Lag-1). One of their actions is to recruit histone plex to expand its chromatin remodelling function. One
deacetylases (HDACs) that remove acyl groups from the such protein methylation enzyme is ERG-associated pro-
lysine groups on histones resulting in the DNA being less tein with SET domain (ESET) is a histone H3-specific
accessible to transcriptional activators. There are a number methyltransferase. Another example is ALL-1, which is a
of such co-repressors: histone 3 lysine 4 (H3K4) methyl transferase.
C-terminal binding protein (CtBP) Mediator complex

Nuclear receptor co-repressor (NCoR) The mediator complex consist of a collection of proteins
Ligand-dependent co-repressor (LCOR) that play a role in initiating gene transcription, which con-
Silencing mediator of retinoic and thyroid hormone re- sists of a group of initiation factors (e.g. TFIIB etc.) that
ceptor (SMRT) are associated with RNA polymerase II (pol II) at the core
Switch independent 3 (SIN3) promoter. The gap between the transcription factors loc-
Transcriptional intermediary factor 1 (TIF1) ated on their specific promoters and this initiation complex
C-terminal binding protein (CtBP) is bridged by this mediator complex as illustrated for the
C-terminal binding protein (CtBP) is a transcriptional co- regulation of peroxisome-proliferator-activated receptor
repressor that is recruited to target promoter sites by bind- (PPAR) action (Module 4: Figure PPAR activation).
ing to PxDLS motifs on various repressors. NADH, which The terminology of the large number of proteins making
is part of the NAD signalling pathway, plays a role in di- up this complex is confusing, because some of the names
merization of CtBP monomers. It was originally iden- refer to the different complexes whereas others refer to
tified through its interaction with the adenovirus A1A specific proteins. Examples of the former are the MED,
oncoprotein. CtBP functions as a bridging molecule by thyroid hormone receptor-associated complex (TRAP),
recruiting various histone deacetylases (HDACs) and his- vitamin D receptor interacting protein (DRIP) complex,
tone methyltransferases. The two CtBP members (CtBP1 ARC, CRSP and PC2. As far as the individual proteins
and CtBP2) are closely homologous. One difference is within these complexes are concerned, there are Mediator
that CtBP2 lacks the Lys-428 sumolyation residue found (MED1-31) proteins and the mediator-associated kinases
on CtBP1. Translocation of CtBP from the cytoplasm in such as CDK8 and CDK11. Cyclin 7 usually associates
to the nucleus is controlled by sumoylation. with CDK8 when it binds to the mediator complex to
One of the functions of CtBP is to contribute to the phosphorylate various components of the initiator com-
transcriptional repression of the E-cadherin gene, which is plex.
one of the classical cadherins.
Transcription factor classification
Switch independent 3 (SIN3) Transcription factors (TFs) can be classified on the basis of
The switch independent (SIN3) co-repressor was first their functional characteristics and their mode of activation
identified in yeast where it functioned in mating-type (Module 4: Figure transcription factor classification). Most

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Module 4: Figure transcription factor classification
TRANSCRIPTION FACTORS
CONSTITUTIVE REGULATORY
Sp1
CCAAT
Nf1
DEVELOPMENTALLY- SIGNAL
REGULATED DEPENDENT
GATA
HNFs
MyoD
Myf1 NUCLEAR CELL SURFACE
Myf5 INTERNAL
RECEPTORS SIGNALS RECEPTOR-DEPENDENT
Myf6
Hox
GR SREBP
ER ATF6
PR p53
TR HIF RESIDENT SHUTTLE LATENT
RARs
NUCLEAR NUCLEAR CYTOPLASMIC
RXRs
PPARs FACTORS FACTORS FACTORS
VDR CREB
DREAM STATs
E2F1 SMADs
ETS FOXO
ATMs NFB
SRF Catenins
FOS-JUN NFAT
MEF2 Tubby
Myc GLI1
MeCP2 MITF
Classification of transcription factors based on their function and mode of activation.

Some transcription factors (TFs) are always present in the nucleus and are constitutively active. The regulatory transcription factors fall into two groups.
The developmentally regulated TFs are often cell-type-specific, such as those that regulate muscle differentiation (MyoD and Myf5) and those that
are signal-dependent. The latter can be subdivided further on the basis of whether they are regulated by steroid hormones, internal signals or by
cell-surface receptors. The cell-surface receptor-dependent TFs can be divided further into those that are resident within the nucleus, those that can
shuttle between the nucleus and the cytoplasm, and the latent cytoplasmic TFs that migrate into the nucleus upon receptor activation. Modified with
permission from Brivanlou, A.H. and Darnell, Jr, J.E. (2002) Signal transduction and the control of gene expression. Science 295:813818. Copyright
(2002) American Association for the Advancement of Science; see Brivanlou and Darnell 2002.
attention will be focused here on the signal-dependent 6 (ATF6) and sterol-regulatory-element-binding pro-
transcription factors, with special attention on how their teins (SREBPs), which are then released and impor-
transcriptional activity is regulated. The nuclear receptors ted into the nucleus (Module 2: Figure ER stress sig-
belong to a superfamily containing approximately 48 hu- nalling).
man transcription factors, which are located mainly in the 2. Receptors on the cell surface generate cytosolic signals
nucleus, where they are activated by steroids or by other that activate latent transcription factors in the cyto-
lipids such as fatty acids. Internal signal-dependent tran- plasm, which are then imported into the nucleus. Some
scription factors are activated by signals generated within receptor-dependent cytosolic signals enter the nucleus
the cell. Many of these are activated by the endoplasmic to activate different types of transcription factors.
reticulum (ER) stress signalling pathway (Module 2: Fig- 3. Nuclear signals can inactivate repressors such as
ure ER stress signalling). Most of the transcription factors downstream regulatory element antagonistic mod-
described so far are the cell-surface receptor-dependent ulator (DREAM), Forkhead box O (FOXO) and
transcription factors that are activated following the stim- methyl-CpG-binding protein 2 (MeCP2). Once re-
ulation of cell-surface receptors. These receptors activate moved from the DNA, some of these such as DREAM
cell signalling pathways that then stimulate either resident and FOXO are then exported from the nucleus.
nuclear factors or latent cytoplasmic factors that are then 4. Nuclear signals activate or inhibit latent transcription
induced to enter the nucleus to initiate transcription. factors that are already attached to DNA.
5. Nuclear signals activate latent transcription factors in
Transcription factor activation mechanisms the nucleoplasm that then bind to DNA.
Cells employ a great variety of activation mechanisms to
control the transcription factors that regulate gene tran-
scription. As illustrated in Module 4: Figure transcription Gene silencing
factor activation, the signalling mechanisms that control When considering transcription factor activation mechan-
transcription can act both in the cytoplasm and within the isms, it is appropriate to include the process of gene silen-
nucleus: cing that can occur through different mechanisms. First,
the expression of gene mRNA transcripts can be regu-
1. One of the functions of the endoplasmic reticulum lated by microRNAs. Secondly, the genes themselves can
stress signalling mechanisms is to activate latent tran- be switched off for long periods. Such gene silencing is re-
scription factors such as activating transcription factor sponsible for the process of imprinting. One of the major

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Module 4: Figure transcription factor activation
Latent
Endoplasmic transcription
reticulum factor
Cytosolic Cytosolic
2 signals signals Activated
transcription
factor
1
Export
Import
Nuclear
signals
5
3 4
SREBP STATs NFB DREAM CREB FOS-JUN

ATF6 SMADs Catenins FOXO ETS Myc
GLI1 NFAT MeCP2 MEF2
HIF MITF
Location and mechanism of transcription factor activation.

Transcription factors are activated or inhibited by a variety of cell signalling pathways that act either within the cytosol or within the nucleus (see the
text for details of the different mechanisms).
mechanisms of gene silencing depends upon the epigenetic normally imprinted with paternal silencing, any mutation
mechanism of DNA methylation, which is a global phe- in the functional maternal copy of the gene will result in
nomenon in that the methylation occurs throughout the disease. In contrast, there is no effect from mutations in
genome. DNA methyltransferases, such as DNA methyl- the silenced paternal copy. Relatively few of the approx-
transferase 1 (DNMT1), carry out this methylation, which imately 85 human imprinted genes have been associated
occurs on cytosine (C) residues that are followed by guan- with a human disease:
ine residues (CpGs). There are a large number of such
CpG dinucleotides distributed throughout the DNA se- Prader-Willi syndrome (PWS),
quence, which is thus marked when the cytosine residues Angelman syndrome (AS)
are methylated. Of particular interest are those methyl- Mental retardation
CpGs that occur within 5 regulatory regions where they BeckwithWiedemann syndrome (BWS)
are responsible for gene silencing as they bring about a RussellSilver (SRS)
localized alteration in chromatin structure that shuts genes
down for prolonged periods.
The alteration in chromatin structure depends upon a Ubiquitin signalling and gene transcription
number of proteins that have methyl-CpG-binding do- The ubiquitin signalling system, which is characterized by
mains that attach to the CpG islands and recruit various the reversible ubiquitination of cell signalling components
transcriptional repressors, such as methyl-CpG-binding (see top panel in Module 1: Figure protein ubiquitination),
protein 2 (MeCP2) and MBD14 to silence transcriptional is particularly important in regulating gene transcription
activity. Much attention has focused on MeCP2 because at a number of levels as illustrated by the following ex-
mutations in the MECP2 gene are responsible for Rett amples:
syndrome. MeCP2 may also play an important role in re-
pressing the expression of neuroligin 1 (NLGN 1) and this The activity of the transcription factor p53 is regulated
may play a significant role in mediating the effect of inflam- by its p53 ubiquitination and degradation (Module 4:
mation in Alzheimers disease (AD) (see Step 6 in Module Figure p53 function).
12: Figure Inflammation and Alzheimers disease). The ubiquitin system responsible for Myc degradation
is carefully controlled to regulate the turnover of this
Imprinting transcription factor (Module 4: Figure Myc as a gene
Gene silencing is responsible for the process of imprint- activator).
ing whereby only the paternal or maternal copy of certain The FOXO4 transcription factor is activated by mon-
genes are expressed. Such imprinting has interesting con- oubiquitination (Module 4: Figure FOXO control
sequences with regard to genetic diseases. For a gene that is mechanisms)

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Developmentally regulated transcription factors (HDAC), which deacetylates chromatin to inhibit tran-
The developmentally regulated transcription factors (TFs) scription.
are often cell-type-specific and function to control the dif- 5. The inhibitor of DNA binding (Id) protein , which is
ferentiation of the many different cell types found in the a dominant-negative inhibitory protein, binds to E2A
body (Module 7: Table cell inventory). The myogenic reg- that promotes differentiation by binding to MyoD as a
ulatory factors (MRFs) that function in the development heterodimer.
of skeletal muscle are examples of such developmentally All of these mechanisms ensure that MyoD is prevented
regulated TFs. One of these factors is MyoD, which is re- from switching on the differentiation programme while
sponsible for activating a large number of muscle-specific the myoblasts continue to proliferate. These inhibitory
genes during the differentiation of skeletal muscle. processes are reversed when the myoblasts stop grow-
ing and MyoD begins to activate the differentiation
Myogenic regulatory factors (MRFs) programme by stimulating the expression of muscle-
The myogenic regulatory factors (MRFs) play a key role specific genes (lower box in Module 4: Figure MyoD
early in skeletal muscle myogenesis (Module 8: Figure and muscle differentiation):
skeletal muscle myogenesis). They belong to a family of 6. During differentiation, there is an induction of cell cycle
basic helixloophelix (bHLH) transcription factors: inhibitors such as p21, which inactivate CDK4, thus
removing its inhibitory action on MyoD.
MyoD (also known as Myf3) 7. The Rb protein, which is dephosphorylated when cells
Myf5 stop proliferating, is able to bind to MyoD to facilitate
Myogenin (also known as Myf1) its activation.
MRF4 also known as Myf6/herculin 8. Activation of Ca2+ /calmodulin-dependent protein
The way in which such myogenic factors function is kinase IV (CaMKIV) phosphorylates HDAC, which
typified by MyoD. is then exported from the nucleus in association with
14-3-3 protein, thus terminating the deacetylation of
chromatin.
MyoD
9. The activated MyoD associated with MEF2 now binds
Like the other myogenic regulatory factors, MyoD is ex-
the histone acetyltransferases (HATs) p300/CBP and
pressed exclusively in skeletal muscle, where it functions
PCAF, which then facilitates transcription by remod-
to activate a large number of muscle-specific genes (Mod-
elling chromatin by histone acetylation.
ule 4: Figure MyoD and muscle differentiation). MyoD
10. The inhibitory Id proteins leave E2A, which is then
has two important domains, a DNA-binding region and
able to associate with MyoD to form an active het-
the basic helixloophelix (bHLH). The latter enables it
erodimer capable of stimulating the transcription of
to form heterodimers with regulators such as E2A. One
muscle-specific genes.
of the important functions of MyoD is its ability to re-
model chromatin by forming complexes with acetylating
Paired box (Pax)
enzymes such as p300 and the p300/cyclic AMP response
A family of paired box (Pax) transcription factors func-
element-binding protein (CREB)-binding protein (CBP)-
tion during development to orchestrate the development of
associated protein (PCAF).
specific tissues and organs. For example, Pax3 and Pax7 are
MyoD acts at a critical phase during the prolifera-
activated early during skeletal muscle myogenesis where
tion-differentiation switch. It is therefore not surprising
they control the expression of myogenic regulatory factors
to find that there are interactions between MyoD and pro-
(MRFs) such as MyoD (Module 8: Figure skeletal muscle
teins of the cell cycle machinery.
myogenesis).
The way in which the myoblasts are maintained in a pro-
Pax expression also plays an important role in maintain-
liferative state and then switched into the process of differ-
ing stem cell progenitor cell populations:
entiation are summarized in Module 4: Figure MyoD and
muscle differentiation. The inhibitory mechanisms that Pax3 functions in the self-maintenance of melanocyte
operate on MyoD during proliferation are shown in the stem cells. It functions again during the process of
upper panel: melanogensis (see step 3 in Module 7: Figure melanogen-
esis). Mutations in Pax3 have been linked to Waarden-
1. The cyclin-dependent kinase 4 (CDK4), which is activ-
burg syndrome 3 (WS3).
ated early during cell proliferation (Module 9: Figure
Pax7 functions in the self-maintenance of satellite cells,
cell cycle signalling mechanisms), inhibits the activity
which are skeletal muscle stem cells (Module 8: Figure
of MyoD.
Satellite cell function).
2. The cyclin D/CDK4 complex hyperphosphorylates,
and thus inactivates, the Rb protein, which is prevented The Pax family has been divided into four subfamilies
from stimulating MyoD (see lower panel) (Module 4: (Module 4: Table Pax transcription factors).
Figure MyoD and muscle differentiation).
3. MyoD is inhibited by activating protein 1 (AP-1), the Sex-determining region Y (SRY)-box (SOX)
Jun/Fos heterodimer. transcription factors
4. The coactivator myocyte enhancer factor-2 (MEF2) The sex-determining region Y (SRY)-box (SOX) functions
binds to MyoD and draws in histone deacetylase to control the differentiation of different cell types:

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Module 4: Figure MyoD and muscle differentiation
14-3-3
P P
HDAC
P
1 P Rb 2 3
CDK4 _ P Jun 14-3-3
P
Cyclin D Fos
5 MyoD
P P
MEF2
E2A Id E-box HDAC
HDAC
Ac
Ac 4
Ac
Ac Ac
Deacetylation
Proliferation
8
Differentiation CaMKIV
_
CDK4
p21 7
6
Cyclin D
Rb -Actin
10 Muscle creatine
E2A MyoD
p300/ kinase (MCK)
CBP Troponin I
E-box
Id 7 integrin
PCAF
Ac Ac Ac Ac Ac Ac Ac Ac Ac
9 Muscle-specific genes
Acetylation
The role of MyoD in skeletal muscle differentiation.

MyoD is one of the myogenic regulatory factors (MRFs) that control muscle differentiation. MyoD binds to the E-box, which is a DNA motif (CANNTG)
found on the promoters of many muscle-specific genes. MyoD is expressed in myoblasts early in muscle development when mesodermal cells acquire
a myogenic lineage. Since these myocytes continue to proliferate early in development, differentiation is kept in abeyance by various mechanisms
that inhibit the activity of this potent myogenic factor. (See the text for details of the mechanisms in the two panels.)
Module 4: Table Pax transcription factors The specific transcription factor Sox9 and its two targets
Paired box (Pax) transcription factors. Sox5 and Sox6 controls the conversion of mesenchymal
Paired box (Pax) Developmental expression stem cells (MSCs) to chondrocytes.
transcription factors in tissues/organs
Subfamily 1
Pax3 CNS, craniofacial tissue, trunk
neural crest (peripheral
nervous system, Nuclear receptors
melanocytes, endocrine The nuclear receptors belong to a lipid-activatable super-
glands, connective tissue), family that contains approximately 48 human transcrip-
skeletal muscle somites
Pax7 CNS, craniofacial tissue, tion factors. The nuclear receptor toolkit reveals the pres-
skeletal muscle somites ence of a variety of transcription factors, coactivators and
Subfamily 2 repressors (Module 4: Table nuclear receptor toolkit). In
Pax4 Pancreas, gut
Pax6 CNS, pancreas, gut, nose eye many cases, the nuclear receptors act to inhibit gene tran-
Subfamily 3 scription and this effect depends on their association with
Pax 2 CNS, kidney, ear various co-repressors such as silencing mediator for retin-
Pax5 CNS, kidney, thyroid
Pax8 CNS, B-lymphocytes oid and thyroid hormone receptor (SMRT) and nuclear re-
Subfamily 4 ceptor co-repressor (N-CoR). These co-repressors act by
Pax1 Skeleton, thymus, parathyroid recruiting chromatin remodelling complexes containing
Pax9 Skeleton, thymus, craniofacial
tissue, teeth histone deacetylases (HDACs). These nuclear receptors
The paired box (Pax), which are divided into four subfamilies, function to control many different cellular processes:
control the development of many tissues and organs. Information
contained in this table was taken from Table 1 in Buckinham and Peroxisome-proliferator-activated receptors (PPARs)
Relaix (2007). are particularly important in regulating energy metabol-
ism by controlling the expression of many of the meta-
In embryonic stem cells (ES), Sox2 functions together bolic components that regulate lipid and carbohydrate
with Oct4 and Nanog to maintain pluripotency. metabolism.
In melanocytes Sox10 contributes to the expression Liver X receptors (LXRs) function in the control of
of the transcription of the microphthalamia-associated lipid metabolism.
transcription factor (MITF) to control melanogenesis Thyroid hormone receptor (TR) mediates the action of
(Module 7: Figure melanogenesis). thyroid hormone in many different cell types.

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Module 4: Table nuclear receptor toolkit CCAAT/enhancer-binding protein (C/EBP)

The toolkit for the nuclear factor superfamily of transcription factors. A family of transcription factors (C/EBP, C/EBP,
Nuclear receptor superfamily Comments C/EBP and C/EBP) are expressed in preadipocytes
Transcription factors
Oestrogen receptor- (ER) ER controls expression of where they function in the differentiation of white fat
enzymes that function in cells (Module 8: Figure white fat cell differentiation). They
adipogenesis and appear very early during differentiation and function to
mitochondrial biogenesis
(Module 5: Figure switch on the peroxisome-proliferator-activated receptors
mitochondrial biogenesis) (PPARs) responsible for the expression of adipose genes.
Oestrogen receptor- (ER)
Hepatocyte nuclear factor 4 This signalling pathway Glucocorticoid receptor (GR)
(HNF4) operates to regulate insulin
biosynthesis (Module 7: The glucocorticoid receptor (GR) belongs to the nuclear
Figure -cell signalling) receptor family (Module 4: Table nuclear receptor toolkit).
Glucocorticoid receptor (GR) See Module 10: Figure The inactive receptor is present in the cytoplasm in an oli-
corticotroph regulation
Liver X factor (LXR) gomeric complex together with Hsp90, FKBP52 and p23.
Thyroid hormone receptors See Module 8: Figure brown fat Upon binding its ligands cortisol or corticosterone it dis-
(TRs) cell differentiation and sociates from this complex, forms a dimer and translocates
Module 10: Figure thyrotroph
regulation into the nucleus where it binds to the glucocorticoid re-
TR Also known as NR1A1 sponse element (GRE) on the promoter of glucocorticoid
TR Also known as NR1A2 genes. The transcriptional activity of the GR is often fa-
Retinoid X receptors (RARs)
RAR Also known as NR1B1 cilitated by transcriptional coactivator complexes such as
RAR Also known as NR1B2 CREB binding protein (CBP), p300, PCAF, and SRC1
RAR Also known as NR1B3 (steroid receptor coactivator-1).
Vitamin D receptor (VDR) Functions in PTH secretion
(Module 7: Figure PTH Once GR has bound to GRE, it can either activate or
secretion) repress genes and this can depend on the phosphorylation
Peroxisome-proliferator- of GR on specific sites. For example, phosphorylation of
activated receptors
(PPARs) GR on serine-211 by cyclin-dependent kinase 5 (CDK5)
PPAR Also known as NR1C1 results in activation of the Hdac2 gene and the resulting
PPAR Also known as NR1C2; contains increase in HDAC2 may contribute to the neurodegener-
two isoforms created by gene
splicing ation seen in Alzheimers disease.
PPAR1 Expressed in liver, muscle, The glucocorticoid receptor (GR) exerts negative-
macrophages, colon feedback control in corticotrophs (see step in 7 in Module
PPAR2 Expressed mainly in fat cells
PPAR Also known as NR1C3 10: Figure corticotroph regulation).
Nuclear receptor cofactors Activation of the GR with glucocorticoids such as pred-
Androgen-associated protein nisone, dexamethasone and hydrocortisone has been used
(ARA70)
Glucocorticoid receptor to treat asthma.
interacting protein 1 (GRIP1)
Cyclic AMP response Liver X receptor (LXR)
element-binding protein One of the functions of the liver X receptors (LXRs),
(CREB)-binding protein
(CBP)/p300 which come in two isoforms LXR and LXR, is to con-
PPAR-binding protein (PBP) trol the expression of various components that function
PPAR coactivator-1 (PGC-1) in lipid metabolism such as apolipoprotein E (ApoE) and
PGC-1 Contributes to action of PPAR
in liver ABCA1.
PGC-1
PPAR coactivator-2 (PGC-2) Thyroid hormone receptor (TR)
Steroid receptor coactivator-1 Contributes to the activity of the The thyroid hormone receptor TR is a typical example of
(STC-1) VDR (Module 7: Figure
vitamin D receptor activation) a nuclear receptor (Module 4: Figure transcription factor
Steroid receptor coactivator-2 classification). There are various isoforms: TR-1, TR-
(STC-2) 1 and TR-2. The TR mediates the action of thyroid
Receptor-interacting protein Acts with PGC-1 to induce
140 (RIP140) uncoupling protein 1 (UCP-1) hormone in a number of cells:
in brown fat cells
Nuclear receptor co-repressors The TR-2 is restricted to the nervous system and an-
Silencing mediator for retinoid Binds to PPAR terior pituitary where it functions as part of a negative-
and thyroid hormone receptor
feedback loop enabling T3 to inhibit both the expres-
(SMRT)
Nuclear receptor co-repressor Binds to PPAR and PPAR sion of thyrotropin-releasing hormone (TRH) by the
(N-CoR) hypothalamic neurons and the expression of thyroid-s-
timulating hormone (TSH) by the thyrotrophs (Module
10: Figure thyrotroph regulation).
The TR controls the expression of uncoupling protein-1
The glucocorticoid receptor (GR) exerts negative feed- (UCP-1), which contributes to the differentiation of
back control in corticotrophs (see step in 7 in Module brown fat cells (Module 8: brown fat cell differenti-
10: Figure corticotroph regulation) ation).

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Retinoid X receptor (RXR) Apolipoprotein E (ApoE)

Retinoid X receptors (RXRs), also known as retinoic Apolipoprotein E (ApoE) is the major apolipoprotein in
acid receptors are members of the nuclear receptor family the brain where it functions to distribute lipids between
(Module 4: Table nuclear receptor toolkit) and are widely the glial cells and neurons. ApoE is synthesized by the as-
distributed in many different cell types. There are three trocytes and microglia and once it is released, the ABCA1
RXRs, RXR, RXR and RXR, that normally function transporter functions in its lipidation. The APOE gene en-
by forming heterodimers together with other nuclear re- codes three isoforms ApoE2, ApoE3 and ApoE4. One of
ceptors such as liver X receptor (LXR), peroxisome-pro- the functions of ApoE is to prevent the build-up of the
liferator-activated receptors (PPAR), thyroid hormone re- -amyloids by enhancing their hydrolysis and by regu-
ceptor (TR) and the vitamin D receptor (VDR) (Module lating the processing of the amyloid precursor protein
7: Figure vitamin D receptor activation). (APP) (see step 7 in Module 12: Figure amyloid cascade
The cancer drug bexarotene acts by stimulating RXR hypothesis).
and may thus increase the activity of the vitamin D receptor A polymorphism in the ApoE4 isoform increases the
(VDR) (Module 7: Figure vitamin D receptor activation). risk of developing Alzheimers disease (AD).
Such an action might explain the reported beneficial effects
on both Parkinsons disease and Alzheimers disease, but Estrogen-related receptor (ERR)
these findings are somewhat controversial. Estrogen-related receptor (ERR) belongs to the nuclear
receptor superfamily but is an orphan receptor in that there
is no known agonist. One of the functions of EER is act
Nuclear receptor co-repressor (N-CoR) together with PGC-1 to regulate some of the compon-
The nuclear receptor co-repressor 1 (N-CoR1) and the ents required for fatty acid oxidation during mitochondrial
closely related silencing mediator for retinoid and thyroid biogenesis and maintenance (Module 5: Figure mitochon-
hormone receptor (SMRT), which is also known as N- drial biogenesis).
CoR2, are co-repressors of the nuclear receptors. They
are large multidomain proteins that associate with the Hepatocyte nuclear factor 4 (HNF4)
nuclear receptors to repress gene transcription. N-CoR1 Hepatocyte nuclear factor 4 (HNF4) belongs to the
and SMRT have deacetylase activation domains (DADs) nuclear receptor superfamily (Module 4: Table nuclear
that enable them to associate with histone deacetylase 3 receptor toolkit). It usually functions as a homodimer
(HDAC3) to enhance gene transcription. One of the func- and can bind a number of coactivators such as GRIP1,
tions of the N-CoR1HDAC3 complex is to help control p300/CBP, DRIP205 and PGC-1. Like many other nuc-
the circadian clock molecular mechanisms. lear receptors, HNF4 can bind a lipid and, in this case, it
is linoleic acid. It is not clear whether such ligand binding
influences the activity of this transcription factor.
Chromatin remodelling HNF4 is strongly expressed in the liver where it func-
In its condensed form, DNA is wrapped around histones tions to control expression of the glycolytic genes. The
to form nucleosomes and other higher-order compact activity of HNF4 is regulated by the AMP signalling
chromatin structures. Remodelling of chromatin opens pathway (Module 2: Figure AMPK control of metabol-
up this compact structure to enable gene transcription, ism). When the level of the metabolic messenger AMP is
DNA replication and DNA repair to occur. This chro- high the AMP-activated protein kinase (AMPK) represses
matin remodelling is carried out by a number of differ- gene transcription by phosphorylating and inactivating the
ent types of chromatin modifying mechanisms. There are hepatocyte nuclear factor 4 (HNF4). This signalling
enzyme-based complexes that modify histones through pathway operates to regulate insulin biosynthesis (Mod-
protein acetylation, methylation, phosphorylation or ubi- ule 7: Figure -cell signalling).
quitinylation. There also are a number of ATP-dependent Mutations in HNF4 have been linked to diabetes.
chromatin-remodelling complexes that use the energy of
ATP to restructure the nucleosome to open up the DNA Peroxisome-proliferator-activated receptors (PPARs)
and make it accessible for the transcriptional machinery. The peroxisome-proliferator-activated receptors (PPARs)
Examples of these remodelling complexes are SWI/SNF, are members of the nuclear receptor superfamily (Module
ISWI, NuRD/Mi-2/CHD, INO80 and SWR1. 4: Table nuclear receptor toolkit). They play a particularly
There also is a chromodomain helicase DNA-binding important role in controlling both lipid and glucose ho-
(CHD) family that plays an important role in regulating moeostasis. The PPARs always function as heterodimers
gene transcription by acting as a repressor. One of these bound to the retinoid X receptor (RXR). This heterodimer
is CHD8 that binds directly to -catenin and suppresses then binds to a peroxisome-proliferator-response element
signalling through the canonical Wnt/-catenin pathway (PPRE), which has a six-nucleotide motif (AGATCA).
(Module 2: Figure Wnt canonical pathway). The CHD8, There usually are two PPRE elements located close to-
which appears to act by binding to histone H1, is expressed gether to provide binding sites for the two components
mainly during embryonic development. Such an action of the PPAR/RXR dimer. The PPAR is activated by lipid
may explain why mutations in the CHD9 gene have been moieties such as free fatty acids or by eicosanoids, whereas
linked to autism spectrum disorders (ASDs). the RXR element can be activated by 9-cis-retinoic acid. It

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is not necessary for both components to bind their ligands aroyl CoA desaturase 1 (SCD-1) and oxidized low-density
for the dimer to be active. lipoprotein (oxLDL) receptor 1, all of which contribute to
The PPAR group of transcription factors has three an increase in the fat content of white fat cells (Module
members with different cellular distributions and func- 7: Figure white fat cell metabolism). In addition, there
tions: also is an increase in phosphoenolpyruvate carboxykinase
(PEPCK), glycerol kinase and aquaporin 7 (a glycerol
PPAR transporter), all of which enhance the recycling of FFAs
PPAR within the fat cells.
PPAR The activation of PPAR is also responsible for driving
PPAR differentiation of white fat cells (Module 8: Figure white
Peroxisome-proliferator-activated receptor (PPAR) is fat cell differentiation). While this process is normally con-
mainly expressed in liver, heart, kidney proximal tubule fined to the period of development, strong activation of
cells and enterocytes at the tips of the villi. All of these cells fibroblast-like preadipocytes in adults can result in the
are characterized by having high levels of mitochondrial differentiation of new white fat cells and this may be par-
and peroxisome -oxidation. Like other members of the ticularly important during the onset of obesity.
family, PPAR is stimulated by free fatty acids, and its
activity can also be modulated by insulin released from the PPAR
insulin-secreting -cell and glucocorticoids released from Peroxisome-proliferator-activated receptor (PPAR) is
the adrenal cortex. expressed ubiquitously and is particularly abundant in
The activity of PPAR is markedly enhanced by the per- the nervous system, skeletal muscle, cardiac cells, pla-
oxisome-proliferator-activated receptor (PPAR) co- centa and large intestine. Like the other PPAR isoforms,
activator-1 (PGC-1), which is particularly important dur- it plays an important role in controlling energy metabol-
ing the transcriptional cascade that up-regulates gluconeo- ism, but its action is subtly different. It seems to play a
genesis in liver cells (Module 7: Figure liver cell signalling). particular role in the metabolism of fatty acids and in the
enzymes that function in adaptive thermogenesis. With
PPAR regard to the latter, it appears to act antagonistically to
Peroxisome-proliferator-activated receptor (PPAR) is PPAR by promoting the burning of fat rather than its
mainly expressed in white and brown fat cells, but is also storage, a property that is attracting considerable attention
found in other tissues such as the brain, intestinal mucosa, as a possible therapeutic target for controlling obesity and
liver and heart. While its primary role is as a physiological diabetes.
lipid sensor in white fat cells, it also has been implicated in
a number of pathophysiological processes, such as athero- Peroxisome-proliferator-activated receptor (PPAR)
sclerosis, kidney oedema and tumours. coactivator-1 (PGC-1)
Like other nuclear receptors, PPAR is inhibited by the The peroxisome-proliferator-activated receptor
co-repressors nuclear receptor co-repressor 1 (N-CoR1) (PPAR) coactivator-1 (PGC-1) is the founder mem-
and the closely related silencing mediator for retinoid ber of a family that also contains PGC-1 and PGC-1
and thyroid hormone receptor (SMRT) (Module 4: Figure related coactivator (PRC). They all functions to control
PPAR activation). PPAR is also inhibited by FOXO1. major metabolic functions in cells by acting as transcrip-
The sirtuins also play a key role in inactivation by de- tional co-activators to strongly potentiate the activity of
acylating both PPAR and FOXO1. many of the nuclear receptors (NRs) (Module 4: Table
The activation of PPAR is driven by a number of nuclear receptor toolkit) such as the PPAR transcription
factors that are all related to a build-up of excessive nu- factors (PPAR and PPAR), thyroid hormone receptor,
trients. Of particular importance is the positive-feedback retinoid receptors, hepatic nuclear factor-4 (HNF-4),
loop whereby an increase in free fatty acids (FFAs) stim- liver X receptor (LXR), and the estrogen-related receptor
ulates the white fat cells to increase their propensity to (ERR). In addition, PGC-1 can bind to other
store fat (Module 7: Figure metabolic energy network). types of transcription factors such as FOXO1, SREBP1
The increased exposure to FFAs activates PPAR directly. and myocyte enhancer factor-2 (MEF-2). The PGC-1
Insulin also plays a role in that it phosphorylates FOXO1 performs its co-activation function by binding to these
to reduce its inhibitory effect and it strongly promotes the multiple transcriptional partners to provide a platform
expression of the co-activator PGC-1 (Module 4: Fig- to bring in other regulatory factors such as p300/CBP,
ure PGC-1 gene activation). PPAR is activated further nuclear respiratory factor1 (NRF-1) and -2 (NRF-2) and
following its acetylation by p300/CBP, which is drawn the Mediator complex. In addition to functioning as a
into the complex by binding to the N-terminal transcrip- transcriptional coactivator, it can also act to increase the
tional activation domain of PGC-1 (Module 4: Figure expression of transcription factors such as ERR.
PPAR activation). This activation of PPAR sets up a The PGC-l family members are strongly expressed in
feed-forward mechanism by increasing the expression of cells that have a high oxidative capacity and are rich in
a number of genes that promote lipogenesis. Some of the mitochondria such as muscle (heart and skeletal muscle),
genes that are activated include adipocyte lipid-binding brown fat cells, brain and kidney. Expression PGC-1
protein (aP2), fatty acid transport protein 1 (FATP1), a seems to be driven by stimuli such as cold temperat-
fatty acid translocase (FAT) also known as FAT/CD36, ste- ures, exercise and nutritional status that will require cells

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Module 4: Figure PPAR activation
Insulin
PI 3-K
Ac P
FOXO1
+ + PGC-1
PKB Fatty
acids
SIRT2 p300/ Multiple
CBP Mediator
PPAR-sensitive
p300/ genes
SMRT + CBP
aP2
NCoR SIRT1 Ac PGC-1
PPAR Cd36
FOXO1 PPAR X activation PPAR pol II
FATP1
Aquaporin 7
PPRE PPRE SCD1
PPAR Glycerol kinase
+ + inactivation TFII
+
NAD
Adipogensis
Calorie
restriction
PPAR activation.
The peroxisome-proliferator-activated receptor (PPAR) is a regulator of lipid metabolism in cells such as the white and brown fat cells. It binds to
the PPRE promotor site and is inactivated by corepressors such as NcoR and SMRT and is also inhibited by FOXO1. In response to insulin and an
elevation of fatty acids, PPAR is activated and begins to stimulate transcription of multiple genes associated with the activation of adipogenesis.
to enhance mitochondrial energy production (Module 4: in enhancing thermogenesis by switching on genes re-
Figure PGC-1 gene activation). A number of signalling sponsible for increasing fuel uptake, mitochondrial ox-
pathways are used to translate these external stimuli into idation and the expression of the uncoupling protein-1
the activation of different transcription factors that con- (UCP-1) to ensure that this oxidation is converted into
trol PGC-1 gene expression. In muscle, the elevation heat.
in Ca2 + associated with exercise stimulates calcineurin In dopaminergic neurons, PGC-1 stimulates the ex-
(CaN) that dephosphorylates MEF2. In addition, Ca2 + pression of the transcription factor nuclear response
stimulates Ca2+ /calmodulin-dependent protein kinase IV factor-1 (NRF-1), which is responsible for maintain-
(CaMKIV) that acts to phosphorylate HDAC and CREB. ing the levels of both antioxidants and detoxifying en-
The activity of PGC-1 is inhibited following its acet- zymes that protect against the deleterious effects of ROS
ylation by the general control of amino-acid synthesis (Module 12: Figure signalling pathways in Parkinsons
(GCN5). This inactivation is reversed by the sirtuin SIRT1 disease).
that deacetylates PGC-1. This activation by SIRT1 plays
an important role in the maintenance of energy metabol-
Parkin interacting substrate (PARIS)
ism and antioxidant defences (Module 12: Figure ageing
Parkin-interacting substrate (PARIS) is a transcriptional
mechanisms) and has been implicated in the process of
repressor that controls the peroxisome-proliferator-activ-
ageing.
ated receptor (PPAR) coactivator-1 (PGC-1) that
The multiple actions of PGC-1 contribute to the oper-
has a number of important metabolic functions. The level
ation of the metabolic energy network by coactivation of
of PARIS is regulated by the ubiquitin E3 ligase Parkin,
genes that function in both lipid and glucose metabolism,
which is one of the genes known to be mutated in Par-
as indicated by the following examples:
kinsons disease (Module 12: Figure signalling pathways in
Parkinsons disease).
PGC-1 is rapidly induced in liver cells during fasting
or following stimulation with glucagon, and then acts
as a cofactor with PPAR to increase the expression of Nuclear transcription factor Y (NF-Y)
a number of the components that function in gluconeo- Nuclear transcription factor Y (NF-Y), which is com-
genesis (Module 7: Figure liver cell signalling). posed of three subunits (NF-YA, NF-YB and NF-YC)
In brown fat cells, PGC-1 plays a central role in the to form a heterotrimeric transcription factor that binds to
differentiation of brown fat cells by inducing the ex- the CCAAT sequence in the regulatory regions of many
pression of uncoupling protein-1 (UCP-1) (Module 8: different genes. The NF-YA binds to DNA, whereas the
Figure brown fat cell differentiation). It also plays a role B and C subunits have histone-fold motifs.

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Module 4: Figure PGC-1 gene activation
Exercise Insulin Cell Cold Glucagon

stress
Adrenergic
receptor
14-3-3
P P
HDAC
Ca 2+ PIP 3 ROS Cyclic
AMP
14-3-3
+
+ + +
CREB
P P P
P
HDAC MEF2 FOXO
Glucneogenesis
PKA
Thermogenesis
CaMKIV CaN ERK1/2 ERK1/2
CaMKIV Mitochondrial
biogenesis
HDAC
FOXO PGC-1 Fatty acid
MEF2 CBP P P
oxidation
TTGTTTAC CRE CREB PGC-1
Electron transport
and
Oxidative
phosphorylation
Activation of the PGC-1 gene.

The PGC-1 gene is activated by a number of external stimuli that act through various signalling pathways to regulate the activity of three main
transcription factors: MEF2, FOXO and CREB. The expressed PGC-1 functions by binding to and coactivating many other genes that regulate
cellular energy metabolism.
Many of these NF-Y target genes express proteins that signalling pathways in Parkinsons disease).The NRF-1
regulate a number of different cellular processes: binds to an antioxidant response element (ARE) located
in the promoter region of these antioxidant and detoxify-
The cell cycle [e.g. topoisomerase II alpha (topo II),
ing genes. Mutation of the Parkin gene, which controls the
cyclins (cyclin A and cyclin B1) and cdc25B and cdc25C,
expression of PGC-1, regulates this signalling pathway,
p21]
resulting in a decrease in the formation of antioxidants and
Protein degradation (proteasome proteins)
is thus consistent with the calcium and ROS hypothesis of
The biological clock through control of the BMAL1
Parkinsons disease.
regulatory loop.
The NRF-1 also acts to increase the expression of the
The checkpoint function of p53 that prevents mitotic
mitochondrial transcription factor (Tfam) that has a vital
cell death may depend on an interaction between p21,
role in regulating the replication and transcription of mito-
the transcription factor NF-Y and the Polo-like kinases
chondrial DNA during mitochondrial biogenesis (Module
1 (Plk1).
5: Figure mitochondrial biogenesis).
NP-Y controls the T-cell leukaemia homeobox 3 (TLX3
also known as Hox11L2) transcription factor that reg-
ulates the development of the visceral nervous system Nuclear factor erythroid 2 related factor 2 (NRF-2)
and the noradrenergic neurons found in the brainstem The nuclear factor erythroid 2 related factor 2 (NRF-2)
that control the respiratory and cardiovascular systems. is a stress-sensing transcription factor that responds to
reactive oxygen species (ROS) by enhancing the cells anti-
NP-Y may also regulate human diseases by regulating oxidant defences. The transcriptional activity of NRF-2 is
expression of proteins such as laminin-1 (muscular dys- regulated by a number of mechanisms that determines its
trophy) and transforming growth factor type II receptor nuclear import/export balance and its degradation (Mod-
(TRII) (cancer). ule 4: Figure NRF-2 antioxidant function).
NRF-2 is continuously formed and enters the nucleus
Nuclear respiratory factor-1 (NRF-1) to maintain the expression of the antioxidants. In the ab-
Nuclear response factor-1 (NRF-1) is a transcription factor sence of cell stress and ROS, some of the NRF-2 binds
that functions to co-ordinate the induction of genes that to Kelch-like ECH-associated protein 1 (Keap1), which
encode a number of detoxifying enzymes that protect represses NRF2 activity and is associated with the ubi-
against oxidative stress. The expression of NRF-1 is con- quitin ligase Cullin 3 that ubiquitinates NRF-2 resulting
trolled by peroxisome-proliferator-activated receptor in its degradation by the proteasome (Module 4: Figure
(PPAR) coactivator-1 (PGC-1) (Module 12: Figure NRF-2 antioxidant function). When ROS levels rise in

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response to cell stress, the Keap1 is oxidized and is no loop, because the NRF-2 functions to activate the expres-
longer capable of binding NRF-2, which means that more sion of p62.
of the NRF-2 can enter the nucleus to boost antioxid- The C-terminal region has an ubiquitin-association do-
ant formation. This nuclear entry of NRF-2 can also be main (UBA) that enables p62 to form aggregates of ubi-
enhanced following its phosphorylation by a number of quitinylated proteins that are then removed from the cyto-
protein kinases such as the MAP kinases (ERK1/2, JNK plasm by autophagy (Module 11: Figure autophagy) and
and p38) and protein kinase C (PKC). NRF-2 can also proteasomal degradation.
be phosphorylated by PERK following endoplasmic re- There are indications that a decrease in the expression
ticulum (ER) stress signalling (Module 2: Figure ER stress and cytosolic levels of p62 may contribute to various
signalling). In addition, there are two important regulators neurodegenerative diseases and particularly those associ-
of NRF-2: p62 and DJ-1, which seem to act by inhibiting ated with the accumulation of misfolded protein aggregates
the interaction between NRF-2 and Keap1 and thus re- as seen in Alzheimers disease (AD) and Parkinsons dis-
ducing the degradation of NRF-2, which then enters the ease (PD). In addition, a decline in p62 activity will also
nucleus (Module 4: Figure p62 function). result in a decrease in NRF-2 activity and in the NF-B
NRF-2 binds to the antioxidant response element signalling pathway that could contribute to oxidative stress
(ARE) to enhance the expression of a large number of and inflammation respectively.
antioxidant and detoxifying enzymes. This transcriptional
activity of NRF-2 depends on its binding to MafG, which Internal signal-dependent transcription factors
is one of the small musculo-aponeurotic fibrosarcoma There are a number of transcription factors that are stim-
Mafs. Transcription through the ARE site can be en- ulated by signals generated from within the cell. Classical
hanced by valproate, which inhibits histone deacetylase examples are activating transcription factor 6 (ATF6) and
(HDAC) indicating that histone acetylation has an im- p53, which are generated by various forms of cell stress.
portant role in maintaining cellular antioxidant levels. In Other examples include sterol regulatory element-bind-
addition, the transcriptional activity of NRF-2 is also fa- ing proteins (SREBPs), which function in sterol sensing
cilitated by interacting with a number of other factors and cholesterol biosynthesis, and hypoxia-inducible factor
such as AP-1, ATF-1, PPAR and RAR. This NRF-2 (HIF), which is an oxygen-sensitive transcription factor.
redox regulator controls the expression of a large num-
ber of proteins such as the enzyme glutamate cysteine Activating transcription factor/cyclic AMP response
ligase (GCL) that synthesizes the redox buffer gluta- element binding protein (ATF/CREB) family
thione (GSH), glutathione S-transferase, haemoxygenase The activating transcription factor/cyclic AMP response
1 (HO1), NAD(P)H quinone oxidase 1 (NQO1), per- element binding protein (ATF/CREB) family are members
oxyredoxins and thioredoxin (TRX). of a leucine zipper family of transcription factors (ATF1-
The export of NRF-2 from the nucleus is regulated by 7), which have the consensus binding site cAMP responsive
glycogen synthase kinase-3 (GSK-3) (Module 4: Figure element (CRE). Both ATF4 and ATF6 play prominent
NRF-2 antioxidant function). The GSK-3 activates this roles in the endoplasmic reticulum (ER) stress signalling
export either by phosphorylating NRF-2 directly or by (Module 2: Figure ER stress signalling).
acting indirectly by stimulating the tyrosine kinase Fyn.
Inhibition of GSK-3 by lithium can markedly enhance Activating transcription factor 4 (ATF4)
the expression of NRF-2. Activating transcription factor 4 (ATF4) is a member of
There is considerable evidence to suggest that a decline the activating transcription factor/cyclic AMP response
in NRF-2 activity may contribute to numerous diseases element binding protein (ATF/CREB) family of leucine
such as cancer, Alzheimers disease (AD), Parkinsons dis- zipper transcription factors. ATF4 is induced by endo-
ease (PD) (Module 12: Figure signalling pathways in Par- plasmic reticulum (ER) stress signalling (Module 2: Figure
kinsons disease), amyotrophic lateral sclerosis, chronic ER stress signalling). ATF4 is a universal stress-responsive
pulmonary obstructive disease, and various inflammatory gene that seems to have a protective role by regulating
disorders. cellular adaptation to adverse conditions. However, it is
A decrease in the activity of NRF-2 is also an important also capable of inducing the transcription of factors such
component of the ROS hypothesis of ageing. as CHOP, PUMA and Noxa that promote cell death.
p62
The p62 protein, which is also known as sequestosome-1, Activating transcription factor 6 (ATF6)
has a number of different functions. Its domain structure Activating transcription factor 6 (ATF6) is part of a tran-
has all the hallmarks of a scaffolding protein capable of scriptional pathway that is activated by the endoplasmic
interacting with different cellular functions and signalling reticulum (ER) stress signalling pathway. ATF6 normally
systems (Module 4: Figure p62 function). One of its im- resides in the ER/sarcoplasmic reticulum (SR) membrane
portant functions is to regulate the stability of the nuclear through a single transmembrane domain located in the
factor erythroid 2 related factor 2 (NRF-2) by inhibiting centre of the molecule. Upon Ca2 + depletion, the cytoso-
its interaction with Keap1, which directs NRF-2 to the lic N-terminal domain is released by proteolysis to enter
protein degradation pathway (Module 4: Figure NRF-2 the nucleus, where it interacts with the ER stress re-
antioxidant function). This interaction sets up a feedback sponse element of the C/EBP (CCAAT/enhancer-binding

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Module 4: Figure NRF-2 antioxidant function
Cell stress
HOS Keap1 Proteasome

ROS
Protein
NRF-2 Ub Ub degradation
+ Ub Ub
HS Keap1
Cullen 3
NRF-2 HS Keap1
NRF-2
ERK1/2
JNK DJ-1 Lithium
p38
p62
PERK
PKC
-
AP-1 PPAR
ATF1 RAR GSK-3
Valproate
- p62
P Glutathione S-transferase
HDAC Hemoxygenase-1 (HO-1)
MafG NRF-2
Glutamate-cysteine lygase
MARE ARE
Thioredoxin
- Peroxyredoxin
NRF-2 antioxidant function.

The nuclear factor erythroid 2 related factor 2 (NRF-2) is a stress-sensing transcription factor that responds to reactive oxygen species (ROS). In the
absence of cell stress and ROS, NRF-2 binds to Kelch-like ECH-associated protein 1 (Keap1), which is associated with the ubiquitin ligase Cullin 3
that ubiquitinylates NRF-2 resulting in its degradation by the proteasome. When ROS levels rise, the Keap1 is oxidized and no longer binds to NRF-2,
which then enters the nucleus where it binds to the antioxidant response element (ARE) to enhance the expression of a large number of antioxidant
and detoxifying enzymes.
Module 4: Figure p62 function
Protein
degradation
Proteasome
Cullen 3
NRF-2 HS Keap1
Protein Ub Ub
Ub Ub
+
p62
HS Keap1
NRF-2
- p62
DJ-1
p62
NRF-2
ARE
p62 function.
p62 is a multifunctional protein. Its expression is controlled by nuclear factor erythroid 2 related factor 2 (NRF-2) and it then feeds back to enhance
the stability of NRF-2 by inhibiting Keap1, which directs it towards proteasomal degradation (Module 4: Figure NRF-2 antioxidant function).

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protein)-homologous protein 10 (CHOP) gene (Module to the signalling pathways that determine its activation.
2: Figure ER stress signalling). This complex activates Under normal conditions, its level is kept low through
various stress-response proteins, such as 78 kDa glucose- p53 ubiquitination and degradation. This rapid turnover is
regulated protein (GRP78)/immunoglobulin heavy-chain- prevented by genotoxic stimuli that act through a variety of
binding protein (BiP). As part of this stress response, post-translational modifications, such as p53 phosphoryla-
there is a compensatory increase in the expression of tion, p53 acetylation and p53 SUMOylation, to stabilize
sarco/endo-plasmic reticulum Ca2+ -ATPase 2 (SERCA2), its level and to promote its accumulation in the nucleus
which plays an important role in signalsome stability of resulting in p53-induced cell cycle arrest and p53-induced
the Ca2 + signalling system. apoptosis. Since the major functions of p53 are to regulate
the cell cycle and to promote apoptosis, there is a major
Specificity protein 1 (Sp1) role for p53 in tumour suppression. The importance of
The specificity protein 1 (Sp1) is a ubiquitous zinc finger its role in tumour suppression is evident by the fact that
transcription factor that regulates the expression of a large there is a strong relationship between alterations in p53
number of proteins that function in cell growth, differ- and cancer.
entiation, immune responses, regulation and antioxidant
defences. With regard to the latter, Sp1 appears to con- p53 domain structure and function
trol the expression of the antioxidant protein DJ-1, which The p53 protein contains a number of domains that de-
is often mutated in Parkinsons disease (PD) (Module 12: termine its function as a regulator of gene transcription
Figure signalling pathways in Parkinsons disease). (Module 4: Figure p53 domains). Beginning at the N-
One of its actions is to remodel chromatin by binding terminal region, there is a transactivation domain (TAD)
p300 and histone deacetylases (HDACs). that interacts with a number of cofactors including other
Sp1 plays a role in regulating the transcription of the transcription factors, mouse double minute-2 (MDM2)
type 1 inositol 1,4,5-trisphosphate (InsP3 ) receptor in re- ubiquitin ligase responsible for p53 ubiquitination and de-
sponse to the action of tumour necrosis factor (TNF) gradation, and acetyltransferases such as cyclic AMP re-
during inflammation in Alzheimers disease (AD) (Module sponse element-binding protein (CREB)-binding protein
12: Figure inflammation in Alzheimers disease). (CBP)/p300. Next, there is a proline-rich Src homology
3 (SH3)-like domain, which enables p53 to bind Sin3 to
Sterol regulatory element-binding proteins (SREBPs) protect it from degradation. The middle of the molecule
The sterol regulatory element-binding proteins (SREBPs) has a DNA-binding domain (DBD). Many of the muta-
function in sterol sensing and cholesterol biosynthesis. tions in p53 that result in the onset of cancer are located
SREBPs are integral membrane proteins located in the in the DBD region that interacts with DNA (Module 4:
endoplasmic reticulum (ER). The N-terminal region is Figure p53/DNA complex). The C-terminal end contains
a latent transcriptional regulator, which is cleaved by a nuclear localization signals (NLSs) and nuclear export sig-
sterol-regulated system of proteases (Module 2: Figure nals (NESs) responsible for the translocation of p53 into
sterol sensing). Once released into the cytosol, these tran- and out of the nucleus. There is a tetramerization (TET)
scription factors dimerize through a basic loop and are domain that enables the p53 monomers to oligomerize into
imported into the nucleus. the tetramers that function in gene transcription. Finally,
AMP-activated protein kinase (AMPK) can inhibit the there is a C-terminal regulatory domain (REG), which
activity of SREBP (Module 2: Figure AMPK control of contains a large number of basic amino acids.
metabolism). Under normal conditions, the activity of p53 is some-
what benign; it is kept in a quiescent state by virtue of the
p53 persistent p53 ubiquitination and degradation processes,
The transcription factor p53 is part of a family of similar which is controlled by its negative regulator mouse double
proteins consisting of p53 itself, p63 and p73. Most atten- minute-2 (MDM2) (Module 4: Figure p53 function). How-
tion has been focused on p53, which is often referred to as ever, in response to a variety of stress stimuli, it is rapidly
the guardian of the genome as it occupies a pivotal pos- activated to begin the processes of p53-induced cell cycle
ition right at the centre of the processes that regulate the arrest and p53-induced apoptosis. There is a close relation-
cell cycle (Module 9: Figure cell cycle network). Its par- ship between these p53 functions and microRNAs in this
ticular function is to maintain DNA integrity, especially regulation of cell cycle arrest and apoptosis. The former
in the face of stress stimuli such as oncogene activation, seems to take precedence over the latter, such that the cell
UV light and ionizing radiation. In healthy individuals, stops proliferating to allow the repair mechanism to re-
p53 is continually being produced, but its level is kept store normal function. If this repair process fails, the cell
low as it is rapidly degraded. In response to various forms then induces apoptosis. The activation of p53 to begin the
of DNA damage, p53 is activated to begin its protective processes of cell cycle arrest and apoptosis is driven by
role by contributing to the checkpoint signalling system. multisite post-translational modifications carried out by a
One of the components of this checkpoint signalling is the number of processes, including p53 phosphorylation, p53
acetylation of p53 by TIP60 (Module 9: Figure G1 check- acetylation, p53 methylation and p53 sumoylation (Mod-
point signalling). The p53 domain structure and function ule 4: Figure p53 domains). The modifications are located
is characteristic of other transcription factors, with regions primarily at the two ends of the molecule. One of the
specialized to bind DNA and other regions that respond functions of these modifications is to pull p53 away from

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the MDM2 that is normally responsible for degrading it non-receptor protein tyrosine kinase Abl, which contrib-
through p53 ubiquitination and degradation. utes to the process of DNA repair (Module 1: Figure Abl
The list below provides details of the enzymes shown in signalling).
Module 4: Figure p53 domains:
Ataxia telangiectasia mutated (ATM) p53 ubiquitination and degradation

Ataxia telangiectasia mutated (ATM) and Rad3-related Degradation of p53 is carried out by various ubiquitin
(ATR) ligases such as mouse double minute-2 (MDM2), Pirh2
Cyclin-dependent kinase (CDK) and COP1. MDM2 is classified as an oncogene because
Casein kinase I (CKI) it causes tumours when overexpressed in cells. The action
COP9 signalsome-associated kinase complex (CSN-K) of MDM2 is facilitated by a related protein called, Mdmx,
DNA-dependent protein kinase (DNAPK) which binds to MDM2 to form heterodimers. MDM2 has
Extracellular-signal-regulated kinase (ERK); see Mod- dual functions: it is responsible for both the nuclear export
ule 2: Figure ERK signalling of p53, and its polyubiquitination and degradation by the
Glycogen synthase kinase-3 (GSK-3) 26S proteasome (Module 4: Figure p53 function). MDM2
c-Jun N-terminal kinase (JNK); see Module 2: Figure also facilitates the nuclear export of p53 by entering the
JNK signalling nucleus, where it carries out the mono-ubiquitination of
p38: part of a mitogen-activated protein kinase (MAPK) p53 to expose the nuclear export signal (NES) resulting in
signalling pathway; see Module 2: Figure MAPK sig- its translocation into the cytoplasm, where it is polyubi-
nalling quitinated prior to its degradation by the 26S proteasome.
Protein kinase C (PKC) The ubiquitination of p53, which is an example of the re-
Double-stranded RNA-activated protein kinase (PKR) lationship between ubiquitin signalling and gene transcrip-
TATA-box-binding protein-associated factor 1 (TAF1) tion, is a dynamic process because a ubiquitin-specific pro-
tease Usp7, which is also known as herpes virus-associated
Ataxia telangiectasia mutated (ATM) ubiquitin-specific protease (HAUSP), is a p53-binding
This is an example of a sensor kinase that functions in the protein that functions to de-ubiquitinate p53. The activ-
checkpoint signalling response of cells to DNA damage ity of Usp7/HAUSP is regulated by the actin-modulating
induced by either ionizing radiation or UV. In the case of protein supervillin. The Usp7/HAUSP also plays an im-
the latter, ATM is sensitive to a specific wavelength of UV portant role in stabilizing MDM2, which can be autoubi-
light, which has been separated into UVA (UVAI, 340 quitinated leading to its degradation. The Usp7/HAUSP
400 nm, and UVAII, 320340 nm), UVB (280320 nm) stabilizes the level of MDM2 thus helping it to prevent the
and UVC (180280 nm). ATM responds to UVA light. In increases in p53 that would lead to cell cycle arrest.
addition to phosphorylating Ser-15 of p53 directly, ATM The negative regulator MDM2 and p53 are connected
also acts on other components. It can activate other kinases, together by a negative-feedback loop whereby p53 induces
such as checkpoint kinase 2 (CHK2) (Module 9: Figure G1 the transcription of MDM2, whereas the latter acts to in-
checkpoint signalling). It can also phosphorylate mouse hibit p53 action by inducing its degradation. This feedback
double minute-2 (MDM2), which results in inhibition of mechanism has to be modified in order for stressful stimuli
MDM2 and thus a corresponding increase in the level of to increase the activity of p53. This feedback loop operat-
p53. ing between p53 and MDM2 can be adjusted by a number
This mutation of ATM is responsible for the human of mechanisms:
genetic disorder ataxia-telangiectasia (AT) syndrome.
One of the main mechanisms depends upon p53 phos-
Ataxia telangiectasia mutated (ATM) and Rad3-related
phorylation.
(ATR)
The tumour suppressor alternative reading frame (ARF)
The Ataxia telangiectasia mutated and Rad3-related (ATR)
stabilizes p53 by binding to MDM2 in the nucleus, and
is a sensor kinase that functions in S and G2 /M checkpoint
thus prevents p53 from leaving the nucleus to be de-
signalling to single-stranded DNA (Module 9: Figure S/G2
graded in the cytoplasm.
phase checkpoint signalling).
p53 activity is prolonged through Abl inhibition of
mouse double minute-2 (MDM2) (Module 1: Figure Abl
DNA-dependent protein kinase (DNA-PK) signalling).
DNA-dependent protein kinase (DNA-PK) is a sensor
kinase that plays an important role in the G1 checkpoint
signalling to DNA double-strand breaks (DSBs), which is Mdmx
an important response of cells to DNA damage induced Mdmx functions together with mouse double minute-2
by either ionizing radiation or UV (Module 9: Figure G1 (MDM2) to regulate p53 ubiquitination and degradation.
checkpoint signalling). It is one of the kinases that is ac- Mdmx has some sequence homology with MDM2, but
tivated at the site DNA strand breaks that occur following unlike the latter, it lacks E3 ligase activity. Mdmx forms
DNA damage. One of its actions is to phosphorylate p53 heterodimers with MDM2 and may contribute to the ac-
on Ser-15 and Ser-37 (Module 4: Figure p53 domains). In tion of MDM2 in inhibiting p53. There is some indication
response to DNA damage, DNA-PK also activates the that Mdmx binds to the transactivation domain of p53.

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Module 4: Figure p53 domains
ERK p300
CK1 p38 CDK
ATR TIP60
GSK-3
ATM
DNA-PK JNK PCAF p38
PKR
CHK1/2 MDM2
FACT-
p38 CSN-K Ck2
ATR p300
DNA-PK
PKC
p38
TAF1 p300
ERK2
SUMO-1
JNK
P P P P P P P P P P A P P P A P A Nd A P A S P
S S S T S S S S T T K S T T K S K K K S K K S
6 9 15 18 20 33 37 46 55 81 120 149 150 155 305 315 320 370 373 376 382 386 392
TAD SH3 DBD NLS TET REG
NES
The domain structure and post-translational modification sites of the transcription factor p53.
The major domains of p53 consist of an N-terminal transactivation domain (TAD), followed by a Src homology 3 (SH3)-like domain. In the middle of
the molecule there is a large DNA-binding domain (DBD). The C-terminal region has a nuclear localization signal (NLS), a tetramerization domain
(TET) and a regulatory (REG) domain. There are a large number of post-translational sites that are modified by phosphorylation (P), acetylation (A),
sumoylation (S) and neddylation (Nd) by a large number of signalling pathways. (See the text for a description of the different enzymes.)
Module 4: Figure p53/DNA complex
A ribbon drawing of p53 complexed to DNA.

This figure illustrates the proposed interaction between p53 (shown on the left) and DNA in its vertical orientation on the right. Many of frequently
mutated residues of p53 are located on the surface that interacts with DNA. Reproduced with permission from Cho, Y., Gorina, S., Jeffrey, P.D.
and Pavletich, N.P. (1994) Crystal structure of a p53 tumor suppressorDNA complex: understanding tumorigenic mutations. Science 265:346355.
Copyright (1994) American Association for the Advancement of Science; http://www.sciencemag.org/; see Cho et al. 1994.
p53 phosphorylation and allows its level to rise such that it can begin to induce
Phosphorylation of p53 plays a key role in its transcrip- gene transcription (Module 4: Figure p53 function). There
tional activation. Many of the phosphorylation sites are are at least 16 sites on p53 that are phosphorylated by a
located in the same N-terminal region where ubiquitin lig- large variety of serine/threonine protein kinases (Module
ase MDM2 is bound. Phosphorylation of these sites blocks 4: Figure p53 domains). Certain sites are phosphorylated
this binding of MDM2 and thus prevents p53 degradation by a single kinase; for example, Ser-6, Ser-9 and Thr-18

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Module 4: Figure p53 function
Ionizing Cell
radiation stress
PARC
JNK
+
ATM + P p38 p53
Proteasome
Chk1/2
Supervillin
+
+
+
+
P
U
P P _ U
NES
p53
U
p53
USP U p53
+
HAU 7/ P P p53
SP
Strap
P
MDM2
DNA-PK
ARF
MDM2
DNA ARF P P
P P P
MDM2
damage
P NES
P53
p53
MDM2 U
HDAC
P P P
P Bax, Bcl-L,FAS1,
Apoptosis
Strap
JMY FASL, IGF-BP3

p53
p300 A
A Cyclin D, p21,GADD45, Cell cycle
TGF, 14-3-3, EGFR arrest
The activation and function of p53.

The transcriptional activity of p53 is regulated by a complex set of processes that control its stability, activation and translocation. The transcriptional
activity of p53 is normally kept at a low level either by its binding to various proteins such as p53-associated parkin-like cytoplasmic protein (PARC),
which effectively acts as a buffer, or by its rapid turnover. The ubiquitin ligase mouse double minute-2 (MDM2) is responsible for the degradation and
for the nuclear export of p53. The activity of MDM2 is inhibited by alternative reading frame (ARF), which acts to stabilize p53. A variety of stressful
stimuli such as DNA damage and ionizing radiation stimulate the protein kinases and acetylases responsible for the post-translational modifications
(Module 4: Figure p53 domains) that activate p53. Once activated, it accumulates in the nucleus where it binds to the p53-responsive element found
on a large number of genes, many of which function to control cell cycle arrest or apoptosis. One of the genes switched on by p53 codes for MDM2,
thus setting up a negative-feedback loop to curb the activity of p53.
are specific for casein kinase I (CKI). However, for some from the nucleus and its degradation (Module 4: Figure
of the other sites, there is considerable redundancy in that p53 function).
they can be phosphorylated by a number of kinases, as The cofactor function of p300 is augmented by junction-
is the case for Ser-15. It seems that p53 integrates a large mediating and regulatory protein (JMY). The interaction
number of input signals, and the sum of the modifications of p300 and JMY is facilitated by a scaffolding protein
then determines the specificity and the magnitude of its called serine/threonine kinase receptor associated pro-
transcriptional activity. There also are indications that the tein (STRAP), which has a tandem series of tetratri-
degree of p53 phosphorylation fluctuates during the course copeptide repeats that function in proteinprotein inter-
of the cell cycle, which is in keeping with the process of actions. The scaffolding function is regulated by its phos-
p53-induced cell cycle arrest. phorylation by ataxia telangiectasia mutated (ATM), which
enables STRAP to enter the nucleus, where it binds to the
p53 acetylation p300/JMY complex to acetylate p53. In addition to acet-
Acetylation is an important post-translational modifica- ylating p53, p300 will also acetylate histones to open up
tion that regulates the transcriptional activation of p53. the chromatin for transcription to occur.
The histone acetyltransferases (HATs) p300 and CREB
binding protein CBP (p300/CBP), p300/CBP-associated p53 methylation
factor (PCAF) and Tat-interactive protein 60 (TIP60) func- The transcriptional activity of p53 is regulated by pro-
tion to acetylate specific lysine residues located in the C- tein methylation. The Lys-370 site undergoes both mono-
terminal region of p53 (Module 4: Figure p53 domains). methylation (K370me1) carried out by Smyd-2 and di-
This acetylation enhances the stability of p53, thus con- methylation (K370me2) through an unknown methyl-
tributing to its function in gene transcription. Conversely, transferase. The K370me1 represses transcriptional activ-
the deacylation of p53 by histone deacetylases (HDACs), ity whereas the K370me2 enhances transcription by
such as the sirtuin Sirt1, may initiate the process of de- promoting association of p53 with the transcriptional
gradation, because some of the deacylated residues appear co-activator p53-binding protein 1 (53BP1). This activa-
to be those used for the mono-ubiquitination by mouse tion step is reversed by histone lysine-specific demethylase
double minute-2 (MDM2) that results in the export of p53 (LSD1).

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p53 sumoylation that controls a key step in microRNA biogenesis (Module

The transcriptional activity of p53 can be modulated by 4: Figure microRNA biogenesis). These three miRs then
sumoylation. This is a post-translational modification that help to switch off the cell cycle.
depends on the formation of an isopeptide bond between
the ubiquitin-like protein SUMO1 and the -amino group Transcription-dependent mechanism.
of Lys-386 on p53. The effect of this sumoylation is still The primary action of p53 is to activate the transcription
not clear, but recent evidence seems to indicate that it may of many of the key components of apoptosis (Module 4:
repress the transcriptional activity of p53. Figure p53 function). It acts by up-regulating components
of both the extrinsic pathway (e.g. Fas) and the intrinsic
p53-induced cell cycle arrest pathway (e.g. Bax, Bid, Apaf-1, Puma and Noxa). Altern-
One of the functions of p53 is to arrest the cell cycle atively, p53 suppresses some of the anti-apoptotic genes
to enable damaged DNA to be repaired. This arrest is such as Bcl-2, and Bcl-XL . In addition, it can also pro-
achieved by stimulating the transcription of many of the mote the expression of phosphatase and tensin homologue
cell cycle signalling components, such as p21, growth- deleted on chromosome 10 (PTEN), which thus will re-
arrest and DNA-damage-inducible protein 45 (GADD45), duce the cell survival signalling mechanisms controlled by
Wip1, proliferating-cell nuclear antigen (PCNA), cyclin the PtdIns 3-kinase signalling pathway (Module 2: Figure
D1, cyclin G, transforming growth factor (TGF) and PtdIns 3-kinase signalling).
14-3-3 (Module 4: Figure p53 function). One of the main
mechanisms used by p53 is to activate the G1 arrest by Transcription-independent mechanism.
activating p21, which is a potent cyclin-dependent kinase The transcription-independent mechanism depends upon
(CDK) inhibitor (Module 9: Figure proliferation signalling an effect of p53 at the mitochondrion, where it binds to the
network). p21 inhibits the CDK2 that activates transcrip- anti-apoptotic proteins Bcl-2, Bcl-XL and Mcl-1, thereby
tion of the E2F-regulated genes that are required for the contributing to the activation of the pro-apoptotic pro-
onset of DNA replication (Module 9: Figure cell cycle sig- teins Bax and Bak. The manganese superoxide dismutase
nalling mechanisms). Another important checkpoint con- (MnSOD) might be another target for p53 in the mito-
trol mechanism is regulated by a protein called GADD45a, chondria.
which is another of the proteins up-regulated by p53 act-
ing together with another tumour suppressor Wilms tu-
mour suppressor (WT1). GADD45a acts at the G2 /M p53 in tumour suppression
checkpoint by dissociating the cyclin B/CDK1 complex p53 is one of the main tumour suppressors that functions
by binding to CDK1 (Module 9: Figure proliferation sig- by preventing the emergence of cancer cells. Under normal
nalling network). circumstances, this function of p53 is held in abeyance and
is only called into action by various sensor kinases, such
p53-induced apoptosis as ataxia telangiectasia mutated (ATM) and DNA-depend-
One of the main tumour suppressor functions of p53 is ent protein kinase (DNAPK), which detect DNA damage
to induce the apoptosis of damaged cells (Module 9: Fig- and begin to phosphorylate p53 (Module 4: Figure p53
ure proliferation signalling network). An increase in tum- function). The activated p53 then begins its suppression
origenesis occurs when this pro-apoptotic function is in- of tumour formation through the twin-track approach of
activated. p53 promotes apoptosis through transcription- p53-induced cell cycle arrest and p53-induced apoptosis.
dependent and transcription-independent mechanisms. If this function of p53 as a negative regulator of cell growth
is reduced either by mutation or by its binding to various
p53 function and microRNAs oncogenic viruses, such as simian virus 40 (SV40) large T
There is a close relationship between the functions of p53 antigen, adenovirus E1B 55 kDa protein and human papil-
and microRNAs (Module 4: Figure microRNAs and p53 lomavirus E6 (HPV E6), the causative link between p53
function). Translation of the TP53 gene transcript to form and cancer emerges.
p53 is regulated by miR-125b and by miR-380-5p. Once A role for p53 in cancer may depend in part on its in-
formed, p53 phosphorylation by various stimuli, such as teraction with the Ca2 + -binding protein S100B. Elevated
ionizing radiation, cell stress and DNA damage, results in levels of Ca2 + will increase the S100B-dependent inhibi-
the activation of p53 to bring about p53-induced cell cycle tion of p53, and this could contribute to the progression
arrest and p53-induced apoptosis. Regulation of these pro- of tumours. Such an interaction is another example of the
cesses by p53 is carried out by two main transcriptional relationship between Ca2+ signalling and cancer.
mechanisms. First, p53 increases the transcription of com- A germline mutation that results in a single copy of the
ponents that control the cell cycle (e.g. p21 and GADD45) TP53 gene that codes for p53 is the cause of Li-Fraumeni
and apoptosis (e.g. Bax, FAS1 and FASL) (Module 9: Fig- syndrome.
ure proliferation signalling network). Secondly, p53 regu-
lates the transcription of a number of microRNAs (miRs),
such as the miR-34 family, that can contribute to the regu- p63
lation of both cell cycle arrest and apoptosis. In some cases The p53 tumour suppressor family consists of three mem-
(e.g. miR-16-1, miR-143 and miR-145), p53 not only ac- bers: p53, p63 and p73. The primary function of p63 seems
tivates the initial transcription but it also enhances Drosha to be related to maintenance and development of stem cells.

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Module 4: Figure microRNAs and p53 function
P miR-16-1
p53
P Drosha miR-143
p53
p53RE miR-16-1
miR-143 miR-145
miR-145
P P
p53 p53 miR-34
Ionizing p53RE miR-34
Cell cycle
+ +
radiation
arrest
Cell
stress
p53
p21 Apoptosis
P GADD45
miR-125b p53
p53RE p21,GADD45
miR-380-5p Bax
FAS1
P
FASL
p53
p53RE Bax, FAS1,FASL
TP53
MicroRNAs and p53 function.

There are intimate relationships between the functions of microRNAs (miRs) and p53. The expression of p53 is regulated by miR-125b and miR-380-5p,
whereas activated p53 can control the transcription of a number of miRs that contribute to regulation of cell cycle arrest and apoptosis.
p73 role in hypoxia-inducible factor (HIF) activation when

The p53 tumour suppressor family consists of three mem- cells are subjected to low O2 tensions.
bers: p53, p63 and p73. The function of p73, which re- The transcription factor NF-B plays a role in link-
sembles that of p53, is complicated in that it exists as mul- ing innate immunity and inflammation to the hypoxic re-
tiple isoforms. The TAp73 isoform promotes apoptosis sponse by increasing expression of HIF-1 (Module 4:
and this might be related to its ability to inhibit tumorigen- Figure NF-B activation and function).
esis. TAp73 can also promote the expression of TWIK-1,
which is also known to inhibit tumour growth.
Hypoxia-inducible factor (HIF) structure

Hypoxia-inducible factor (HIF) There are a number of hypoxia-inducible factors (HIFs),
Hypoxia-inducible factor (HIF) functions in one of the which belong to the basic helixloophelix (bHLH) group
O2 -sensing responses that cells employ when facing pro- of proteins. Most attention has focused on HIF-1 and
longed hypoxia. This is particularly important in the HIF-1, which interact with each other to form the het-
growth of tumours where new cells become starved of erodimer, which is the active form of this transcription
O2 as they divide and move away from the existing vascu- factor. In addition, there is HIF-2, which seems to act
lature. These new cells respond to the lack of O2 by sending like HIF-1, but acts on different target genes. Finally,
out signals to activate angiogenesis, which creates the new there is HIF-3, which seems to function as an inhib-
blood vessels required to oxygenate the growing tumour. itor of HIF-1. The N-terminal region of HIF-1 has the
An important component of this angiogenic response is bHLH motif responsible for binding to HIF-1 to form
the activation of HIF that then increases the expression of the functional heterodimer (Module 4: Figure HIF struc-
a large number of components that function in one way ture). The N-terminal transactivation domain (N-TAD)
or another in the regulation of cell proliferation (Mod- contains the two proline residues that are hydroxylated by
ule 4: Figure HIF functions). For example, one of these the prolyl hydroxylase domain (PHD2) protein to pro-
components is vascular endothelial growth factor A (VE- duce the binding sites for the von HippelLindau (VHL)
GF-A), which promotes the angiogenic response. HIF also protein. The C-terminal transactivation domain (C-TAD)
regulates the expression of chemokines, such as CXCL12 has the Asp-803 residue that is hydroxylated by the factor
and its receptor CXCR4 that contributes to a relation- inhibiting HIF (FIH) to displace the p300 that facilitates
ship between chemokines and cancer. Hypoxia-inducible the transcription of HIF target genes. These hydroxyla-
factor (HIF) structure reveals that this transcription factor tion events play a critical role in hypoxia-inducible factor
is hydroxylated by O2 -sensitive enzymes that play a key (HIF) activation under hypoxic conditions.

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Module 4: Figure HIF functions
TRANSCRIPTIONAL REGULATION
CELL PROLIFERATION
Cyclin G2; IGF-2; IGFBP1; DEC1; DEC2; ETS-1; NUR77
IGFBP2; IGFBP3; WAF1;
TGF-: TGF- pH REGULATION
Carbonic anhydrase 9
CELL SURVIVAL
ADM; EPO; NOS2; VEGF REGULATION of HIF-1
p35srj
APOPTOSIS
NIP3; NIX; RTP801 DRUG RESISTENCE
MDR1
CELL ADHESION
MIC2 IRON METABOLISM
Ceruloplasmin; Transferrin;
MOTILITY
HIF-1 Transferrin receptor
AMF/GPI; C-MET; LRP1
AMINO ACID METABOLISM
VASCULAR TONE Transglutaminase 2
1B - Adrenergic receptor; ADM;
Et1; Haem oxygenase-1; NOS2 NUCLEOTIDE METABOLISM
Adenylate kinase 3
CYTOSKELETAL STRUCTURE Ecto-5-nucleotidase
KRT14; KTR18; KRT19; VIM
GLUCOSE METABOLISM
EXTRACELLULAR MATRIX Hk1; Hk2; AMF/GPI; ENO1; GLUT4;
CATHD; Collagen V; Fn1; MMP2; GAPDH; LDHA; PFKBF3; PFKL;
PGK1; PKM; TPI
PAI1; UPAR; Prolyl-4-hydroxylase
Hypoxia-inducible factor 1 (HIF-1) activates the transcription of multiple genes.

There are a large number of genes that are regulated by hypoxia-inducible factor 1 (HIF-1). Some of these target genes function in the regulatory
networks that control cell proliferation, survival and apoptosis. (Redrawn from Figure 3 from Semenza 2003.)
Module 4: Figure HIF structure
N-TAD C-TAD
HIF-1 HLH PAS
Pro 402 Pro 564 Asp803
OH OH OH
VHL
Prolyl Factor inhibiting

hydroxylase (PHD2) HIF (FIH)
VHL p300
O2 O2
The structure and activation of hypoxia-inducible factor 1 (HIF-1).

The O2-sensitive hypoxia-inducible factor 1 (HIF-1) has a basic helixloophelix (HLH) domain that enables it to form a heterodimer with the
O2-insensitive HIF-1 subunit to form the functional transcription factor. The activity of HIF-1 depends on the hydroxylation of proline residues at
402 and 564 and an asparagine residue at 803. The proline residues are hydroxylated by a prolyl hydroxylase (PHD2), whereas a factor inhibiting HIF
(FIH) hydroxylates the asparagine residue. These hydroxy groups provide binding sites for the von HippelLindau (VHL) protein that directs HIF-1
to the proteasomal degradation pathway and for p300 that regulates its transcriptional activity. See Module 4: Figure HIF activation for details of how
O2 regulates the activity of HIF-1.

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Module 4: Figure HIF activation
HIF-1 HIF1
Normal oxygen Low oxygen

tension tension (hypoxia)
PHD2
O2 1
FIH 4
Proteasome
HIF-1
HIF1
HIF-1 OH
OH OH
3 HIF-1 OH
2
OH OH
E3 ubiquitin VHL
ligase complex
P
5 P
ERK1/2
SP1
P 6 P P 7 VEGF-A Angiogenesis
HIF-1
HIF1
AP1 CXCL12 Cancer

Jun Fos CXCR4
mestasasis
-RCGTG-
Activation of the hypoxia-inducible factor (HIF).

1. At the normal oxygen tension of blood, the hypoxia-inducible factor 1 (HIF-1) isoform is constantly being degraded by a process that is driven
by O2, which activates the O2-sensitive prolyl hydroxylase domain (PHD2) protein and factor inhibiting HIF-1 (FIH). These two proteins hydroxylate
HIF-1. 2. Two of these hydroxy groups provide binding sites for the von HippelLindau (VHL) protein, which is a component of the E3 ubiquitin ligase
complex. 3. Ubiquitinated HIF-1 is degraded by the proteasome. 4. At low O2 tensions, PHD2 and FIH are inactive. 5. HIF-1 is free to associated
with HIF-1 to form a heterodimer. 6. The heterodimer translocates into the nucleus to initiate the transcription of genes such as vascular endothelial
growth factor A (VEGF-A) that function in angiogenesis.
Hypoxia-inducible factor (HIF) activation 6. For many of these target genes, such as the vascular
The activation of hypoxia-inducible factor (HIF) during endothelial growth factor A (VEGF-A) gene, there are
hypoxia depends upon two O2 -sensitive hydroxylases that additional binding sites for transcription factors such
control both the stability and transcriptional activity of the as activating protein (AP-1) and specificity protein 1
HIF-1 subunit. At normal oxygen tensions, the HIF- (SP1).
1 subunit is unstable and is constantly being degraded 7. The HIF target genes include VEGF-A that controls
through the following sequence of events (Module 4: Fig- angiogenesis and the chemokine CXCL12 and its re-
ure HIF activation): ceptor CXCR4 that contribute to the relationship
between chemokines and cancer metastasis.
1. The O2 activates two hydroxylases. A prolyl hy-
droxylase domain (PHD2) protein hydroxylates Pro-
402 and Pro-564 in the N-terminal transactivation do- Cell-surface receptor-dependent
main (N-TAD) (Module 4: Figure HIF structure). transcription factors
2. These two hydroxylations provide a binding site for the A variety of mechanisms are used to activate receptor-
von HippelLindau (VHL) protein, which is a com- dependent transcription factors (Module 4: Figure tran-
ponent of the E3 ubiquitin ligase complex (Module 1: scription factor activation). Receptors on the cell surface
Figure ubiquitin-proteasome system), which ubiquit- employ various signalling pathways to stimulate transcrip-
inates HIF-1 and thus marks it out for destruction. tion factors located either in the cytosol or in the nucleus.
3. The ubiquitinated HIF-1 is then degraded by the pro- In the case of some transcription factors, activation de-
teasome (Module 4: Figure HIF activation). Under nor- pends upon their expression, as is the case for the Myc
mal oxygen tensions, therefore, the HIF-1 is unstable family.
and thus unable to activate transcription.
4. At low oxygen tensions, the hydroxylation enzymes Activation of transcription by cytosolic signals
are inactive and the HIF-1 is stabilized and now can There are a large number of transcription factors that lie
bind to HIF-1 to form the active heterodimer. dormant within the cytoplasm until they are activated by
5. The activated HIF-1 translocates into the nucleus a variety of signalling pathways (Mechanism 2 in Module
where it binds to the hypoxia response element (HRE), 4: Figure transcription factor activation):
which contains the core sequence -RCGTG-, on the
promoter region of the large number of HIF target APP intracellular domain (AICD)
genes. -Catenin

C
Interferon-regulatory factors (IFNs) These Mafs bind to Maf-recognition elements (MAREs)

LBP1 family of transcription factors of target genes that have many functions. They have been
Notch intracellular domain (NICD) implicated in the control of interleukin-4 (IL-4) and also
Signal transducers and activators of transcription interleukin-21 (IL-21). The latter is important in driving
(STATs) the final stages of B-cell differentiation in the lymph node
Nuclear factor of activated T cells (NFAT) (Module 8: Figure B cell maturation signalling). MafA ac-
Nuclear factor B (NF-B) tivates the insulin gene promoter in -cells. MafG com-
Smads bines with the nuclear factor erythroid 2 related factor 2
(NRF-2) to control the expression of antioxidant enzymes
APP intracellular domain (AICD) (Module 4: Figure NRF-2 antioxidant function).
The APP intracellular domain (AICD) is formed when The oncogenic v-Maf, which was identified in the gen-
mutant APP is hydrolysed to form amyloid 42 (A42 ) ome of the acute transforming avian retrovirus AS42, in-
(see steps 4 and 5 in Module 12: Figure amyloid cascade duces musculoaponeurotic fibrosarcoma (Maf).
hypothesis). The AICD then functions as a transcription
factor that may play a role in controlling the expression Signal transducers and activators of
transcription (STATs)
of some of the Ca2 + signalling components such as the
The Janus kinase (JAK)/signal transducer and activator
SERCA pump and the ryanodine receptor (RYR). This
of transcription (STAT) signalling pathway is another ex-
link between amyloid processing and the remodelling of
ample of transcriptional activation by cytosolic signals
the Ca2 + signalling system may be a significant step in the
(Module 2: Figure JAK/STAT function). Following re-
progression of Alzheimers disease (AD).
ceptor activation, the JAKs are activated to phosphorylate
tyrosine residues on the STATs, which then dimerize be-
-Catenin as a transcription factor fore migrating into the nucleus to activate transcription.
-Catenin is the transcription factor that is activated by Mutations in STAT3 have been linked to hyper-IgE syn-
the Wnt signalling pathways. Under resting conditions, drome (HIES).
the cytosolic level of -catenin is kept low by proteasomal
degradation. Following Wnt activation, the -catenin de- Notch intracellular domain (NICD)
gradation complex is inhibited allowing -catenin to ac- The Notch intracellular domain (NICD) is the cytoplas-
cumulate and to enter the nucleus, where it induces the mic domain of the Notch protein that functions in the
transcription of the Wnt genes responsible for regulating Notch signalling pathway. When Notch is hydrolysed by
development and cell proliferation (Module 2: Figure Wnt the -secretase complex, NICD is released into the cyto-
canonical pathway). plasm and then diffuses into the nucleus where it induces
the transcription of multiple Notch target genes (steps 4
Interferon-regulatory factors (IFRs) and 5 in Module 2: Figure Notch signalling).
The interferon-regulatory factors (IFRs) are a family of
transcription factors that contribute to the function of Nuclear factor of activated T cells (NFAT)
the type I interferons (IFNs) [(interferon- (IFN-) and The nuclear factors of activated T cells (NFATs) are a
interferon- (IFN-)]. There are at least nine members of family of cytosolic transcription factors (NFAT1NFAT4)
the family. These IRFs are activated when cells respond to that function not only during early development, but also
IFNs and also during the induction of the type I IFNs. The in the subsequent response of cells to external signals that
latter is illustrated by the role of IRF3 and IRF7 during activate processes such as cell proliferation, neural control
the response of cells to viral infections (Module 2: Figure of differentiation and cardiac hypertrophy. Nuclear factor
viral recognition). of activated T cells (NFAT) structure reveals the many
features that are required for NFATs to respond to cytoso-
LBP1 family of transcription factors lic signals and to bind to DNA. Nuclear factor of activ-
The leader-binding protein-1 (LBP1) family has two genes ated T cells (NFAT) activation depends upon cytosolic
that give rise to different splice variants. One gene gives Ca2 + signals that activate calcineurin (CaN) to dephos-
rise to LBP1a and LBP1b, whereas the other gene produces phorylate NFAT, thereby enabling it to enter the nucleus.
LBP1c and LBP1d. These different isoforms can form both Nuclear factor of activated T cells (NFAT) function is
homo- and heterodimers. The LBC1c variant, which is also very diverse. It operates during early development and
known as CP2 and LSF, has been implicated in the onset of during the subsequent process of differentiation into spe-
Alzheimers disease because it may interact with the APP cialized cells. NFAT also continues to function in fully
intracellular domain (AICD) to regulate gene transcription differentiated cells, where it controls adaptation and main-
(Module 12: APP processing). tenance of the differentiated state.
c-Maf Nuclear factor of activated T cells (NFAT) structure

c-Maf is considered to be a proto-oncogene. It belongs The four nuclear factor of activated T cells (NFAT) family
to the family of small musculoaponeurotic fibrosarcoma members share similar structural domains (Module 4: Fig-
Mafs, which are basic region leucine zipper domain tran- ure NFAT structure). The Rel-homology region (RHR)
scription factors consisting of MafF, MafG and MafK. is very similar to that found in the nuclear factor B

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Module 4: Figure NFAT structure
Phosphorylation sites
1 928
RHR-N RHR-C
Rel homology
region (RHR)
Calcineurin binding domain (B)
KTS
Third Ser-Pro sequence (SP-3)
Nuclear localization signal (NLS)
Second Ser-Pro sequence (SP-2)
First Ser-Pro sequence (SP-1)
Serine rich region (SRR-1)
Calcineurin binding domain (A)
Activation domain
The structural domains of nuclear factor of activated T cells (NFAT).

The various domains located along the length of the nuclear factor of activated T cells (NFAT) molecule perform different functions during its activation
of transcription. Starting with the N-terminal region, there is an activation domain. The interaction between NFAT and calcineurin depends on two
calcineurin-binding domains (A and B, shown in blue). Calcineurin then removes the 13 phosphate groups (red dots) that are clustered in four
regions: a serine-rich region (SRR-1), two Ser-Pro sequences (SP-2 and SP-3) and one at a KTS site. Removal of these phosphates unveils a nuclear
localization signal (NLS) that enables NFAT to enter the nucleus. Once in the nucleus, NFAT binds to DNA through the Rel-homology region (RHR),
which has two parts: an N-terminal specificity domain (RYR-N), which binds to DNA, and a C-terminal dimerization domain (RHR-C), which binds to
other transcription factors such as activating protein 1 (AP-1) (Module 4: Figure NFAT/AP-1/DNA complex). Modified, with permission, from Hogan,
P.G., Chen, L., Nardone, J. and Rao, A. (2003) Transcriptional regulation by calcium, calcineurin, and NFAT. Genes Dev. 17:22052232. Copyright
(2003) Cold Spring Harbor Laboratory Press; see Hogan et al. 2003.
(NF-B)/Rel family. It is responsible for DNA binding. lear/cytoplasmic shuttle. The control of this shuttle de-
While NFAT can bind to DNA as a homodimer, the inter- pends upon the balance between Ca2 + stimulating the
action with DNA is weak. Under normal circumstances, its import process and the action of various kinases such
DNA-binding is facilitated by binding to other transcrip- as glycogen synthase kinase-3 (GSK-3) and mitogen-
tion factors, such as activating protein 1 (AP-1) (Fos/Jun), activated protein kinase (MAPK) p38 (Module 2: Fig-
c-Maf or GATA4, to form a high-affinity DNA-binding ure MAPK signalling) that stimulate export back into the
complex (Module 4: Figure NFAT/AP-1/DNA complex). cytosol. The activation sequence that begins with an elev-
The formation of such complexes greatly expands the ver- ation of cytosolic Ca2 + is illustrated in Module 4: Figure
satility of the NFAT transcriptional system and also serves NFAT activation.
to enhance its integrative capacity by providing additional
inputs from other signalling pathways (Module 4: Figure 1. Ca2 + binds to calmodulin (CaM), which then interacts
NFAT activation). with calcineurin (CaN).
2. The activated CaN then removes the multiple phos-
phates located on NFAT to unveil a nuclear localization
Nuclear factor of activated T cells (NFAT) activation signal (NLS) that promotes import into the nucleus,
Nuclear factor of activated T cells (NFAT) is an example where it can have two actions.
of a transcription factor that is activated in the cytoplasm 3. The dephosphorylated NFAT can interact with other
before being imported into the nucleus (Mechanism 2 in transcription factors such as activating protein 1 (AP-1)
Module 4: Figure transcription factor activation). Activa- (Fos/Jun), and the ternary Fos/Jun/NFAT complex
tion of NFAT is controlled by an increase in the concentra- then binds to DNA sites on the promoters of NFAT
tion of cytosolic Ca2 + , which then stimulates the enzyme target genes.
calcineurin to dephosphorylate NFAT (Module 4: Figure 4. The other action of NFAT, which is confined to
NFAT activation). It is this dephosphorylated NFAT that NFAT2, is to bind to the myocyte enhancer factor-2
is imported into the nucleus as part of an NFAT nuc- (MEF2), where it helps to recruit the p300 coactivator.

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Module 4: Figure NFAT/AP-1/DNA complex
Structure of the nuclear factor of activated T cells (NFAT)/activating protein 1 (AP-1)/DNA complex.
The N-terminal region of nuclear factor of activated T cells (NFAT) is shown in yellow, whereas the C-terminal region that interacts with Fos (red)
and Jun (blue) is shown in green. Reproduced by permission from Macmillan Publishers Ltd: Nature, Chen, L., Glover, N.M., Hogan, P.G., Rao, A.
and Harrison, S.A. (1998) Structure of the DNA-binding domains from NFAT, Fos and Jun bound specifically to DNA. 392:4248. Copyright (1998);
http://www.nature.com; see Chen et al. 1998.
5. The recruitment of p300 contributes to chromatin re- location process is examined at different frequencies of
modelling and gene transcription by histone acetyla- Ca2 + signalling (Module 6: Figure NFAT nuclear trans-
tion. location). This NFAT shuttle may thus represent a mech-
6. The inactivation of NFAT depends upon its export anism whereby cells can decode oscillatory information
from the nucleus following its rephosphorylation by through integrative tracking (Module 6: Figure decod-
various NFAT kinases. ing oscillatory information). In general, low-frequency
7. One of these kinases is glycogen synthase kinase-3 high-amplitude transients are ineffective, whereas more
(GSK-3), which appears to initiate the rephosphoryla- frequent transients are very effective. This is evident in
tion cascade by phosphorylating sites at the N-terminal skeletal muscle, where stimulation at 1 Hz had little effect,
serine-rich region (SRR-1) (Module 4: Figure NFAT whereas stimulation at 10 Hz resulted in a gradual trans-
structure). The nuclear activity of NFAT is prolonged fer of NFAT from the cytosol to the nucleus (Module 8:
by the PtdIns 3-kinase signalling cassette, which uses Figure nuclear import of NFAT).
protein kinase B (PKB) to phosphorylate GSK-3.
8. NFAT is also phosphorylated by p38, one of the MAPK Nuclear factor of activated T cells (NFAT) function
signalling pathways. Nuclear factor of activated T cells (NFAT) has a multitude
of functions, since it is a transcription factor that oper-
The Ca2 + -dependent translocation of NFAT from the ates throughout the life of an animal. One of its earliest
cytoplasm into the nucleus can be visualized by express- developmental functions is in axis formation, as has been
ing a green fluorescent protein (GFP)/NFAT construct shown for dorsoventral specification. It is employed again
in baby hamster kidney (BHK) cells (Module 4: Figure during differentiation, where it is particularly significant in
NFAT translocation). controlling the expression of tissue-specific genes (Module
The time that NFAT resides within the nucleus is crit- 4: Figure NFAT activation). As part of this differentiation
ically dependent on the balance between the rates of im- process, it has a special role in signalsome expression when
port and export, which can vary considerably between the signalling pathways characteristic of each cell type is
cell types. The other characteristic feature of this NFAT being established. Finally, NFAT continues to function in
shuttle is that it is a dynamic process whose equilibrium the maintenance of fully differentiated cells and in their
is very dependent on the temporal properties of the Ca2 + adaptation to external stimuli. An example of the latter is
signal. This becomes particularly evident when the trans- signalsome stability, where the transcription of genes such

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Module 4: Figure NFAT translocation
The Ca2 + -dependent translocation of nuclear factor of activated T cells (NFAT) from the cytoplasm into the nucleus.
When a green fluorescent protein (GFP)/nuclear factor of activated T cells (NFAT) construct is expressed in baby hamster kidney (BHK) cells, it is
located in the cytoplasm at rest (see panel A,i). Upon an increase in Ca2 + as shown in the lower panels of A, the GFP/NFAT moves from the cytoplasm
into the nucleus as shown in panel A,iv. The time course of these changes is shown in B. Note how the increase in Ca2 + induces a fall in the cytosolic
level of GFP/NFAT, with a corresponding increase in the nucleus. Reproduced by permission from Macmillan Publishers Ltd: EMBO J., Tomida, T.,
Hirose, K., Takizawa, A., Shibasaki, F. and Iino, M. (2003) NFAT functions as a working memory of Ca2 + signals in decoding Ca2 + oscillation. EMBO
J., 22:38253832. Copyright (2003); http://www.nature.com/emboj; see Tomida et al. (2003).
as NFAT may contribute to a feedback mechanism that en- There appears to be a link between Downs syndrome
sures stability of the differentiation state of cells, including and NFAT function. Downs syndrome may result from
their signalling pathways (Module 4: Figure NFAT control a dysregulation of NFAT resulting from an increased ex-
of Ca2+ signalling toolkit). pression of two of the proteins that function to regulate the
NFAT thus plays a role in the regulation development, NFAT shuttle (Module 4: Figure NFAT control of Ca2+
proliferation and cellular adaptation to changing circum- signalling toolkit).
stances:
Nuclear factor B (NF-B)
Remodelling the cardiac signalsome during compensat- The nuclear factor B (NF-B) is a multifunctional tran-
ory hypertrophy (Module 12: Figure hypertrophy sig- scription factor that is used to regulate a large number
nalling mechanisms). of processes, such as inflammation, cell proliferation and
Regulating the biosynthesis of glucagon (Module 7: Fig- apoptosis (Module 4: Figure NF-B activation and func-
ure -cell signalling). tion). NF-B belongs to the group of transcription factors
T cell activation is critically dependent on the activation that lie latent in the cytoplasm and then translocate into the
of NFAT (Module 9: Figure T cell Ca2+ signalling). nucleus upon activation (mechanism 2 in Module 4: Figure
The formation of slow-twitch skeletal muscle during the transcription factor activation). The way in which NF-B
neural control of differentiation, where it contributes is activated is described in the section on the nuclear factor
to gene activation by the transcription factor myocyte B (NF-B) signalling pathway (Module 2: Figure NF-B
enhancer factor-2 (MEF2). activation).
Development of the vasculature, which depends upon
cross-talk between the developing vessels and the sup- Single-minded 1(Sim1)
porting tissues, requires the activation of NFAT. A hypothalamic transcription factor located in neurons
-Cell secretagogues that elevate Ca2 + maintain the that function in the neural network for the control of
supply of insulin by stimulating the proliferation of food intake and body weight (Module 7: Figure control
-cells through the NFAT pathway (Module 7: -cell of food intake). Sim1 is strongly expressed in the para-
signalling). ventricular nuclei (PVN) which are innervated by the
NFAT regulates the expression of many components POMC/CART neurons located in the arcuate nucleus
of the Ca2 + signalling system, such as endothelin, (ARC). These POMC/CART neurons release -MSH,
transient-receptor potential-like channel 3 (TRPC3), in- which acts on the melatonin receptor MC4R responsible
ositol 1,4,5-trisphosphate receptor 1 (InsP3 R1), NFAT2 for stimulating Sim1. Haploinsufficiency of the Sim1 gene
and Downs syndrome critical region 1 (DSCR1) (Mod- causes hyperphagic obesity indicating that the activation of
ule 4: Figure NFAT control of Ca2+ signalling toolkit). this transcription factor functions in a signalling pathway

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Module 4: Figure NFAT activation
Cytosolic
CaM PP
2+ P NFAT
Ca P
2
1
CaN NFAT MKK3
shuttle
PKB
6 MKK6
Import Export
GSK3
7 8
p38
GENE CELL TYPE
AP1
Endothelin Endothelium
Cox2
NFAT Glucagon Pancreatic islet
Jun Fos BNP Heart
3 4 IL-2; IL-3; IL-4; Lymphocyte
Il-5; GM-CSF;
IFN-; FasL.
T Carabin
p300 FA
N Myf-5; MYC I; Skeletal muscle
AT
P
SERCA2a;
NF
MEF2 TRPC3.
Jun Fos Ac Ac Ac Ac Ac
aP2 Adipocyte
IP3 receptor 1 Hippocampal
Acetylation
neuron
5 Calcitonin Osteoclasts
receptor
The Ca2 + -dependent activation of nuclear factor of activated T cells (NFAT).

An increase in the concentration of cytosolic Ca2 + initiates a series of events that culminate in the transcriptional activation of a large number of
genes. [See the text for details of the Ca2 + -dependent activation of nuclear factor of activated T cells (NFAT).]
Module 4: Figure NF-B activation and function
Growth Bacterial Receptor Oxidative

Cytokines factors products ligands Viruses stress
TNF- PDGF LPS HIV
CD40 Ozone
IL-1 EGF HTLV-1
IL-12 Exotoxin N-CAM
EBV
RECEPTORS
AND TRANSDUCERS
NF-B p50 p65
Hypoxic response
NF-B-sensitive genes
Inflammation
HIF-1a
TNF-, Rantes, IL-1 Recruitment of

inflammatory cells
IL-8, MCP-1, ICAM
Prostaglandin & NO
p50 p65 COX2, iNOS production
B
c-Myc, cyclin-D Proliferation
TRAF1/2, C-IAP, XIAP Apoptosis

CD40, Ig light chain
Immune responses
Activation and function of the transcription factor nuclear factor B (NF-B).

A number of different stimuli acting through various receptors and transducing mechanisms activate nuclear factor B (NF-B), which then enters the
nucleus, where it binds to B promoter elements to induce the expression of many genes that function in many different cellular processes. Adapted
from Handbook of Cell Signaling, Vol. 3, (Bradshaw, R.A. and Dennis, E.A., eds), Westwick, J.K., Schwamborn, K. and Mercurio, F., NFB: a key
integrator of cell signalling, pp. 107114. Copyright (2003), with permission from Elsevier; see Westwick et al. 2003.

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that carries information to the satiety centres to terminate Regulating the biosynthesis of glucagon (Module 7: Fig-
feeding. ure -cell signalling).
CREB is particularly important in activation of neur-
Smads onal gene transcription responsible for learning and
In the case of the Smad signalling pathway, the activa- memory (Module 10: Figure neuronal gene transcrip-
tion process is relatively simple. Smads are activated dir- tion), the expression of clock genes (Module 6: Fig-
ectly through serine/threonine phosphorylation by the ure circadian clock input-output signals), the expression
receptor-associated kinases (Module 2: Figure TGF-R of brain-derived neutrophic factor (BDNF) (Module
activation). The activated Smads then diffuse into the nuc- 4: Figure MeCP2 activation) and to activate the genes
leus to activate transcription (Module 2: Figure Smad that defines the phenotype of fast-spiking interneurons
signalling). (Module 12: Figure schizophrenia).
The CREB/TORC transcriptional duo play a critical
role in energy metabolism by controlling the expres-
Activation of transcription by nuclear signals
sion of the peroxisome-proliferator-activated receptor
Many of the signalling pathways in the cytosol are capable
(PPAR) coactivator-1 (PGC-1), which acts together
of entering the nucleus, where they operate to activate dif-
with the PPAR transcription factors to control the ex-
ferent transcription factors (Mechanisms 35 in Module 4:
pression of components that function in liver cell gluc-
Figure transcription factor activation). There are transcrip-
oneogenesis (Module 7: Figure liver cell signalling).
tional activators and repressors that can shuttle between
CREB controls the transcription of the aquaporin
the nucleus and cytoplasm, and whose activity is altered
(AQP) channels AQP2 and AQP3 during vasopress-
by these nuclear signals:
in-induced antidiuresis (Module 7: Figure collecting
Activating protein 1 (AP-1) (Fos/Jun) duct function).
Cyclic AMP response element-binding protein (CREB) CREB is one of the major transcription factors that is
Downstream regulatory element antagonistic modu- activated during the process of melanogenesis (step 3 in
lator (DREAM) Module 7: Figure melanogenesis).
E twenty-six (ETS) NMDA receptor hypofunction in schizophrenia is
E2F family of transcription factors caused by a decrease in the Ca2 + -dependent phos-
Forkhead box O (FOXO) phorylation and activation of CREB that is responsible
Methyl-CpG-binding protein 2 (MECP2) for maintaining the phenotypic stability of inhibitory
Myocyte enhancer factor-2 (MEF2) interneurons in the brain (Module 12: Figure schizo-
Serum response factor (SRF) phrenia).
Cyclic AMP response element-binding protein (CREB)

The cyclic AMP response element-binding protein B cell lymphoma 6 (BCL-6)
(CREB) is a ubiquitous multifunction transcription factor. B cell lymphoma 6 (BCL-6) is a member of the BTB/POZ
CREB belongs to a family of transcription factors that have zinc-finger family of transcription factors. It functions as a
a series of leucine residues that function as a leucine zipper repressor to control the very rapid proliferation of centro-
to form both homo- and hetero-dimers. CREB is activ- blasts during B-cell differentiation in the lymph node
ated by a number of signalling pathways (Module 4: Fig- (Module 8: Figure germinal centre). This repressor role
ure CREB activation). CREB activation is also dependent can have different outcomes depending on the ability of
on Ca2 + signalling, which has two main actions. Firstly, it BCL-6 to recruit different co-repressor complexes. For
activates the nuclear Ca2+ /calmodulin-dependent protein example, BCL-6co-repressor (BCoR)/nuclear-receptor
kinase IV (CaMKIV), which is one of the kinases that co-repressor (NCoR)/silencing mediator for retinoid and
phosphorylate CREB. Secondly, it activates calcineurin thyroid receptor (SMRT) complex seem to play a role in
(CaN), which dephosphorylates the transducer of regu- suppressing the genes that control growth and apoptosis.
lated CREB (TORC) thus enabling it to enter the nucleus On the other hand, recruitment of the nucleosome remod-
to co-operate with CREB to switch on transcription. elling and deacetylase (Mi-2-NuRD) complex switches off
The phosphorylation of CREB enables it to bind the the genes that control the differentiation of the germinal
transcriptional co-activator proteins p300 and CBP, which centre B-cells.
are histone acetyltransferases (HATs) that acetylates his-
tones thereby remodelling the chromatin so that transcrip-
tion can proceed.
CREB functions in the control of many different cellular Pituitary-specific transcription factor (Pit-1)
processes: Pituitary-specific transcription factor (Pit-1) controls the
development of some of the anterior pituitary endocrine
The survival and proliferation of -cells is regulated cells such as the somatotrophs, lactotrophs and thyro-
by glucose and by various hormones such as glucagon- trophs. In the case of the somatotrophs, Pit-1 functions to
like peptide-1 (GLP-1), which appear to act synergist- control the expression of growth hormone (GH) and per-
ically to activate the CREB co-activators called TORCs haps also the growth hormone-releasing hormone receptor
(Module 7: Figure -cell signalling). (GHRH-R) (Module 10: Figure somatotroph regulation).

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Module 4: Figure CREB activation
Cyclic AMP
Ca2+
+
P P P
ERK1/2
Cyclic AMP P TORC2 PKA
+
CaN SIK2
+
+
TORC2
P
PKA ERK1/2 CaMKIV
P
Ser-133 P P
CREB
CREB
TORC2
P P
CREB
CRE
p300/CBP
Ac Ac Ac Ac Ac Ac Ac Ac
PCAF
Acetylation
Activation of the cyclic AMP response element-binding protein (CREB).

Cyclic AMP response element-binding protein (CREB) is phosphorylated by a number of signalling pathways that employ different kinases to
phosphorylate the critical residue Ser-133, which is essential for its transcriptional activity. In addition to activating CREB within the nucleus, some
of these signalling pathways also regulate the activity of cofactors called transducers of regulated CREB (TORCs). Under resting conditions, TORC
is phosphorylated and resides in the cytoplasm, but upon dephosphorylation, it enters the nucleus, where it acts together with CREB to promote
transcription. TORC phosphorylation depends upon a Ca2 + -dependent dephosphorylation mediated by calcineurin (CaN) and the cyclic AMP/protein
kinase A (PKA)-dependent inhibition of the salt-inducible kinase 2 (SIK2) that phosphorylates TORC.
Forkhead box O (FOXO) be dephosphorylated, enabling it to return to the

This family of forkhead transcription factors has been re- nucleus.
named as the forkhead box O (FOXO1, FOXO3a and 5. Once it has been dephosphorylated, the FOXO is free
FOXO4) factors. These resident nuclear factors, which to re-enter the nucleus to begin gene transcription. Dur-
are constitutively active, are examples of shuttle nuclear ing stress, the FOXO4 isoform is monoubiquitinated
factors (Module 4: Figure transcription factor classifica- and this facilitates its entry into the nucleus. The ubi-
tion) that are regulated within the nucleus. The FOXO quitin group may also facilitate its interaction with vari-
factors have a DNA-binding domain and their movement ous transcriptional co-activators such as p300 (Module
across the nuclear pores is mediated by a nuclear local- 4: Figure FOXO control mechanisms).
ization signal (NLS) and a nuclear export signal (NES). 6. Such stress-induced activation is reversed by the ubi-
The PtdIns 3-kinase signalling cassette (Module 2: Fig- quitin-specific protease Usp7/HAUSP. Removal of
ure PtdIns 3-kinase signalling) is responsible for inhibit- ubiquitin enables FOXO4 to return to the cytoplasm.
ing the transcriptional activity of FOXO, as illustrated in
the sequence of events shown in Module 4: Figure FOXO
control mechanisms: The function of FOXO is linked to decisions related to
cell proliferation/quiescence. Cells that are quiescent, i.e. at
1. External signals operating through the PtdIns 3-kinase G0 , have various mechanisms for suppressing the cell cycle,
signalling cassette generate the lipid second messenger and FOXO may play a role in this because it is known to
PtdIns3,4,5P3, which then activates protein kinase B code for components such as the cyclin-dependent kinase
(PKB) (Module 2: Figure PtdIns 3-kinase signalling). (CDK) inhibitor p27 and the retinoblastoma (Rb) fam-
2. Activated PKB enters the nucleus and phosphorylates ily protein p130, which play a role inhibiting the cell
FOXO on threonine and serine residues, some of which cycle (Module 9: Figure cell cycle signalling mechanisms).
lie within the NLS. FOXO3a regulates the transcription of manganese super-
3. The phosphorylated FOXO is recognized by 14-3-3 oxide dismutase (MnSOD), which contributes to redox
protein, or by the exportin Crm1, which facilitate its signalling in apoptosis by providing greater protection
export into the cytoplasm. against reactive oxygen species (ROS)-induced apoptosis.
4. When the PtdIns 3-kinase signalling system is switched FOXO can regulate the expression of Bim, which is a
off, the phosphorylated FOXO in the cytoplasm can pro-apoptotic factor that contributes to the intrinsic

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Module 4: Figure FOXO control mechanisms
Stimulus
PtdIns 4,5P2 PI 3-K PtdIns3,4,5P 3 PTEN PtdIns 4,5P 2

FOXO P
1
4 P PKB
5
FOXO P 14-3-3
Ubiquitin
FOXO P
TTGTTTAC P 14-3-3
3
FOXO P 14-3-3
P
FOXO
2
PKB
Usp7/
HAUSP
p27 Inhibit cell
6
p130 proliferation
p300 FOXO
Bim Proapoptotic factor
TTGTTTAC
MnSOD Inhibit redox
Acetylation signalling
Control of forkhead box O (FOXO) proteins by the PtdIns 3-kinase signalling cassette.
The forkhead box O (FOXO) family are typical shuttle nuclear transcription factors that bind to a specific promoter sequence to activate the transcription
of genes such as p27, p130, Bim and manganese superoxide dismutase (MnSOD). Like many other transcription factors, FOXO can bind p300,
thereby acetylating histone to make DNA more accessible to transcription. The transcriptional activity of FOXO is inhibited by phosphorylation through
the PtdIns 3-kinase signalling cassette, resulting in export from the nucleus into the cytoplasm. (See the text for details of the signalling pathway.)
pathway of apoptosis (Module 11: Figure Bcl-2 family These ETS transcription factors are made up of a fam-
functions). ily of so-called ternary transcription factors that form a
The inactivation of FOXO by the PtdIns 3-kinase sig- complex with DNA and with SRF. Typical members are
nalling cassette thus contributes to the sequence of mo- ETS-1, ETS-2, the ETS-like transcription factor-1 (Elk-
lecular events that control the cell cycle and apoptosis 1), the SRF accessory protein-1 (Sap-1) and Net. These
when cells are stimulated to proliferate (Module 4: Figure ETS transcription factors have four main domains They
FOXO control mechanisms). The ability of phosphatase all have a winged helixturnhelix DNA binding domain
and tensin homologue deleted on chromosome 10 (PTEN) (ETS domain), which recognizes ETS-binding sites (EBS)
to function as a tumour suppressor may well depend upon that contain a conserved CGAA/T sequence. Some of the
its ability to reduce the level of PtdIns3,4,5P3 , thus re- ETS family also has a Pointed (PNT) domain responsible
moving the inhibitory effect on PKB activation to allow for interactions with other proteins such as SRF. There is
FOXO to express the cell cycle regulators that prevent also an activation domain (AD) which is phosphorylated
proliferation. The onset of tumorigenesis in renal and pro- by the MAPK signalling pathway. The Elk-1 transcription
state carcinoma cells that have a defect in PTEN may thus factor regulates a variety of cellular processes:
result from an uncontrolled elevation of PtdIns3,4,5P3 and
inhibition of FOXO activity.
Phosphorylation of Elk-1 plays an important role in

E twenty-six (ETS) reversing the differentiation of smooth muscle when
The E twenty-six (ETS) domain transcription factors are cells are switched back into a proliferative state.
typical resident nuclear factors (Module 4: Figure tran- Elk-1 plays a role in the transcriptional events that occur
scription factor classification) that are activated primar- in medium spiny neurons during drug addiction (Mod-
ily through the mitogen-activated protein kinase (MAPK) ule 10: Figure medium spiny neuron signalling).
signalling pathway. They act together with the serum re- KLF4 plays a role in regulating the differentiation of
sponse factor (SRF) to activate many of the early response smooth muscle cells (Module 8: Figure smooth muscle
genes, such as Fos (Module 4: Figure ETS activation). cell differentiation).

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Module 4: Figure ETS activation
Cytoplasm
P
ERK1/2
P
Nucleus
P
Elk-1(active)
AD
P
p300 PNT
Fos
ETS SRF
EBS CArG Box
Acetylation
AD
Elk-1(inactive)
HDAC PNT
ETS SRF
EBS CArG Box
Deacetylation
Gene activation through co-operation of the E twenty-six (ETS) and serum response factor (SRF) transcription factors.
A ternary complex is formed through DNA, the serum response factor (SRF) bound to the CArG box and the E twenty-six (ETS) transcription factors
such as Elk-1, which bind to the ETS-binding site (EBS) on DNA and to SRF through their Pointed (PNT) domains. In the inactive state, as shown
at the bottom, ETS functions as a repressor by recruiting inhibitors such as the histone deacetylases (HDACs), which deacylate histone to increase
chromatin condensation. A major activator of transcription is the extracellular-signal-regulated kinase (ERK) pathway of the mitogen-activated protein
kinase (MAPK) signalling pathway, with the effector ERK1/2 phosphorylating sites on the activation domain (AD). Just how this phosphorylation
induces transcription is unclear, but there does appear to be an activation of histone acetyltransferases (HATs), such as p300, to increase histone
acetylation to decondense the chromatin.
CSL (CBF-1, Suppressor of Hairless, Lag-1) ferentiation switch (Module 8: Figure proliferation-differ-
CSL (CBF-1, Suppressor of Hairless, Lag-1) is a tran- entiation switch) and may thus be important in neuronal
scriptional repressor that functions in the Notch signalling differentiation. In immature neurons, expression of the Ids
pathway (Module 2: Figure Notch signalling). It functions suppresses a number of the differentiation genes. During
to repress Notch target genes by providing a framework neuronal maturation, MeCP2 represses the expression of
to recruit co-repressors such as SMRT, SHARP, SKIP and the Ids and this unmasks the neuronal differentiation genes
CIR. such as Neurod1.
MeCP2 is normally located in the nucleus associated
with the methyl-CpG islands through its methyl-CpG-
Methyl-CpG-binding protein 2 (MeCP2)
binding domain. It also has a transcriptional repression do-
Methyl-CpG-binding protein 2 (MeCP2) is a transcrip-
main (TRD) and two nuclear localization signals (NLSs).
tional repressor that functions in gene silencing (Module 4:
The activity of MeCP2 is sensitive to various signalling
Figure MeCP2 activation). It has the methyl-CpG-binding
pathways as illustrated in the following sequence of events
domain that enables it to associate with those methyl-CpG
(Module 4: Figure MeCP2 activation):
islands that are found in the promoter regions of many
genes.
MeCP2 is found in a number of cell types, but is mainly 1. During neural activity, membrane depolarization (V)
expressed in brain where it appears in postmitotic neur- activates voltage-operated channels (VOCs) that intro-
ons where it functions as a regulator of neuronal gene duce Ca2 + into the cell.
expression. Levels of MeCP2 are low during early devel- 2. Ca2 + acts through Ca2+ /calmodulin-dependent pro-
opment but then increase progressively as neurons ma- tein kinase IV (CaMKIV) to phosphorylate the MeCP2
ture preceding the onset of synapse formation. Microar- that is bound to mCpG islands causing it to leave the
ray analysis has begun to identify some of the MeCP2- DNA. In addition, CaMKIV also phosphorylates the
regulated genes, which include brain-derived neurotrophic transcription factor CREB as part of the gene activation
factor (BDNF), insulin-like growth factor binding protein mechanism.
3 (IGFBP3), the ubiquitin ligase UBE3A, -aminobutyric 3. MeCP2 is a multifunctional protein that interacts
acid (GABA) receptor and the inhibitor of DNA binding with a number of other proteins such as DNA
(Id) proteins. The latter function in the proliferation-dif- methyl transferases 1 (DNMT1) and the transcriptional

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co-repressors switch independent (SIN3) that recruits 4: Figure transcription factor classification), it is activated
histone deacetylase (HDAC) to function in chromatin by a sequence of Ca2 + -sensitive steps within the nucleus
remodelling. Histone deacetylation by HDAC inhib- (Module 4: Figure MEF2 activation):
its gene transcription. Once MeCP2 is removed, these
remodelling complexes also leave the DNA, preparing 1. In the absence of Ca2 + signals, MEF2 acts as a
the way for the onset of gene transcription. repressor to inhibit transcription by associating with
4. An important aspect of this onset of gene transcrip- various negative regulators. It binds the class II his-
tion is the activation of CREB by phosphorylation of tone deacetylases (HDAC4, HDAC5, HDAC7 and
Ser-133 by CaMKIV or by the cyclic AMP signalling HDAC9), which induce chromatin condensation and
pathway. inhibit transcription by deacetylating the N-terminal
5. CREB binds to the CRE element and helps to build tails of histones.
up a transcriptional complex that includes chromatin 2. An increase in nuclear Ca2 + binds to calmodulin
remodelling components such as p300 that acetylate the (CaM) within the nucleus.
histones to open up the chromatin so that transcription 3. The Ca2 + /CaM activates the resident nuclear
can proceed. Ca2+ /calmodulin-dependent protein kinase IV
6. One of the functions of MeCP2 is to regulate the gene (CaMKIV).
coding for brain-derived neurotrophic factor (BDNF), 4. The Ca2 + /CaM/CaMK IV complex phosphorylates
which is essential for neural development especially the class II HDACs on two conserved serine residues.
the changes in synaptic plasticity associated with learn- 5. The phosphorylated HDAC is recognized by 14-3-3
ing and memory such as long-term potentiation (LTP). protein, which removes it from MEF2 and assists its
MeCP2 also regulates the expression of neuroligin 1, export from the nucleus into the cytosol.
which plays an important role in synaptic plasticity by 6. In some cells, such as T cells, CaM can have a more dir-
forming and stabilizing the contact between pre- and ect role in activating MEF2 when it is bound through
post-synaptic endings (Module 10: Figure postsynaptic Cabin1 to the class I HDACs (HDAC1 and HDAC2)
density). through the co-repressor switch independent (SIN3).
The Ca2 + /CaM binds to Cabin1 to release both the
Mutations in the MECP2 gene that codes for MeCP2, Cabin1/SIN3 complex and the class I HDACs.
which is the primary cause of Rett syndrome, have also 7. Once the HDACs have been removed (Steps 5 and
been found in a number of other neurological disorders 6), the MEF2 is able to bind activators such as NFAT,
such as autism, Angelman syndrome (AS), neonatal en- CREB and p300. The CaMK IV facilitates this process
cephalopathy and X-linked mental retardation. Microglial by phosphorylating CREB.
inflammation in Alzheimers disease results in MeCP2 ac- 8. The calcineurin (CaN) that is associated with NFAT
tivation and a decrease in the expression of neuroligin 1, helps to activate MEF2 by removing an inhibitory
which may play a role in the loss of memory associated phosphate.
with this neurodegenerative disorder (see step 6 in Mod- 9. The recruitment of p300 acetylates the histones to fa-
ule 12: Figure Inflammation and Alzheimers disease). cilitate transcription by opening up the chromatin.
10. Activation of the MEF2 promotor results in an in-
Myocyte enhancer factor-2 (MEF2) crease in the transcription of PGC-1 and many other
The myocyte enhancer factor-2 (MEF2) plays a role in genes responsible for multiple processes such as mito-
cell proliferation and differentiation. As its name implies, chondrial biogenesis and fatty acid oxidation.
the MEF2 family, which belongs to the MADS family of MEF2 functions in the regulation of a number of pro-
transcription factors that includes serum response factor cesses, many of which are related to morphogenesis:
(SRF), was originally discovered in muscle cells, but is now
known to regulate important decisions in cell proliferation The formation of slow-twitch skeletal muscle during the
and differentiation in many other cell types. The family neural control of differentiation.
consists of four members (MEF2A, MEF2B, MEF2C and Remodelling the cardiac signalsome during the onset of
MEF2D), all encoded by separate genes. The N-terminal hypertrophy (Module 12: Figure hypertrophy signalling
region has a MADS domain responsible for dimerization mechanisms).
and for DNA binding. Next to the MADS domain there Smooth muscle cell proliferation.
is a MEF2-specific domain that enables it to bind to a
number of other transcription factors and cofactors, such Activating protein 1 (AP-1) (Fos/Jun)
as MEF2 itself, to form a homodimer. The function of The activating protein 1 (AP-1) family has an important
MEF2 is very much defined by this ability to associate with function in regulating cell proliferation. It is a heterodimer
other factors, some of which are activators, such as MyoD formed by the association between members of the Jun
(Module 4: Figure MyoD and muscle differentiation) and family (c-Jun, Jun B and Jun D) and the Fos family (c-Fos,
the nuclear factor of activated T cells (NFAT) (Module Fos B, Fra-1 and Fra-2). These two genes are classical ex-
4: Figure NFAT activation), whereas others are inhibitory amples of immediate early genes that are activated rapidly
[e.g. the histone deacetylases (HDACs) and Cabin1]. following growth factor stimulation. This Fos/Jun dimer
The function of MEF2 is particularly sensitive to Ca2 + is held together by a leucine zipper (Module 4: Figure SRF
signalling. Since MEF2 is a resident nuclear factor (Module and AP-1).

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Module 4: Figure MeCP2 activation
Activation of methyl-CpG-binding protein 2 (MeCP2).

Many genes that contain methyl CpG (mCpG) islands are silenced by MeCP2 that recruits histone deacetylases (HDACs) and DNA methyl transferases
1 (DNMT1) to alter chromatin structures through histone deacylation and DNA methylation respectively. An increase in Ca2 + leads to phosphorylation
of MeCP2 that leaves the DNA enabling gene transcription to proceed through transcription factors such as CREB. One of the genes controlled by
this mechanism is brain-derived neurotrophic factor (BDNF) that functions in synaptic plasticity.
Module 4: Figure MEF2 activation
Function of nuclear Ca2 + in myocyte enhancer factor-2 (MEF2) activation.

There are a number of ways in which an increase in the level of Ca2 + within the nucleus can influence the conversion of MEF2 from a repressor into
an activator of transcription. (See the text for the sequence of Ca2 + -activated events.)
The activation of AP-1 (Fos/Jun) has two compon- form the dimer that then goes on to activate transcription
ents. Firstly, growth factors and other stimuli can induce of a set of genes that have a 12-O-tetradecanoylphorbol
a rapid transcription of both Fos and Jun family mem- 13-acetate (also called phorbol 12-myristate 13-acetate)
bers. Fos transcription is activated by the serum response (TPA)-responsive element (TRE). Secondly, the tran-
factor (SRF), whereas Jun is controlled by a combination of scriptional activity of AP-1 (Fos/Jun) is regulated by
Jun and activating transcription factor (ATF). Once these both phosphorylation and by redox signalling. The c-Jun
two transcription factors are formed, they zipper up to N-terminal kinase (JNK) pathway plays a major role by

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Module 4: Figure SRF and AP-1
P
Immediate early genes
P
ERK1/2
P
ATF
P JUN c-Jun
GSK-3
P
?TRE Casein kinase II
Elk-1
SRF c-Fos c-Fos c-JUN
SRE SOH P
P
P JNK
Ref-1
P
c-Fos c-JUN
SH
TRE
Activation of serum response factor (SRF) and activating protein-1 (AP-1).

One of the earliest consequences of growth factor activity is to stimulate the transcription of the transcription factors c-Fos and c-Jun. The latter
then forms a dimer through a leucine zipper and binds to the 12-O-tetradecanoylphorbol 13-acetate (also called phorbol 12-myristate 13-acetate)
(TPA)-responsive element (TRE). The interaction with TRE is regulated both by phosphorylation and by the oxidation state of a highly conserved
cysteine residue located in the DNA-binding domain. A redox factor 1 (Ref-1) controls the redox state of a cysteine residue in the DNA-binding domain.
phosphorylating Jun (Module 2: Figure JNK signalling). (MEF2) family. The 57-amino-acid MADS-box enables
Transcription can also be inhibited by glycogen syn- these transcription factors to dimerize and bind to DNA
thase kinase-3 (GSK-3) and casein kinase II (CK2), which to regulate approximately 160 genes that control processes
phosphorylate sites on the DNA-binding domain. A link such as cell growth, migration and muscle cell differenti-
between redox signalling and gene transcription has also ation. SRF may also regulate survival through its control
been identified for AP-1 (Fos/Jun) (Module 4: Figure SRF of the anti-apoptotic Bcl-2 gene. The activity of SRF is
and AP-1). Binding to DNA is very dependent on the very dependent on coactivators, particularly members of
oxidation state of a critical cysteine residue located in the myocardin family.
the DNA-binding domain. The nucleus contains a redox SRF is stimulated by growth factors and plays an im-
factor 1 (Ref-1), which functions to control transcription portant role by activating the transcription of c-Fos, which
by reducing this cysteine residue. is a component of activating protein 1 (AP-1) (Fos/Jun).
The binding of AP-1 (Fos/Jun) can also support the The SRF, which is associated with p62TCF, binds to the
binding of other transcription factors to form larger tran- serum response element (SRE) of the c-Jun promoter. The
scriptional complexes. An example is the interaction of extracellular-signal-regulated kinase (ERK) pathway plays
AP-1 with nuclear factor erythroid 2 related factor 2 an important role in stimulating transcription by phos-
(NRF-2) (Module 4: Figure NRF-2 antioxidant func- phorylating the ETS-like transcription factor-1 (Elk-1)
tion) and nuclear factor of activated T cells (NFAT) (p62TCF), which is a cofactor of SRF (Module 4: Figure
(Module 4: Figure NFAT activation). The organization SRF and AP-1).
of the NFAT/AP-1 complex is shown in Module 4: Figure One of the functions of SRF is to participate in the
NFAT/AP-1/DNA complex. AP-1 also contributes to the action of either myocardin or the E twenty-six (ETS) tran-
transcriptional activity of hypoxia-inducible factor (HIF) scription factors (Module 4: Figure ETS activation), as oc-
(Module 4: Figure HIF activation). curs during the proliferation/differentiation switch dur-
ing the differentiation of smooth muscle (Module 8: Fig-
Serum response factor (SRF) ure smooth muscle cell differentiation). In the presence of
Serum response factor (SRF) belongs to a small family the transcriptional coactivator myocardin, the SRF stimu-
of MADS (MCM1, Agamous, Deficiens, SRF) transcrip- lates the genes responsible for smooth muscle cell differ-
tion factors that include the myocyte enhancer factor-2 entiation. SRF is also activated by the myocardin-related

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transcription factors (MRTFs), which provide a critical role in the control of cell proliferation (Module 9: Figure
link between actin dynamics and gene transcription (Mod- G1 proliferative signalling). Myc structure reveals that it
ule 4: Figure actin dynamics and gene transcription). SRF is a member of the basic helixloophelix leucine zipper
regulates the expression of a large number of genes and (bHLH-Zip) group of transcription factors. Since Myc
many of these encode proteins that function in actin form- has a short half-life (approximately 20 min), its level in
ation and in the coupling of actin to the integrins. quiescent cells is low. When cells are stimulated to grow,
SRF also plays a role in the differentiation of cardiac Myc formation is markedly increased by growth factor
cells (Module 8: Figure cardiac development) and in the signalling pathways that activate Myc transcription. Myc
regulation of miR-133a to control Ca2+ signalling and car- action depends upon it binding to another bHLH-Zip pro-
diac hypertrophy (Module 12: Figure miRNA and cardiac tein called Max to form a Myc/Max heterodimer, which
hypertrophy). then activates a large number of Myc targets. Most of these
targets function in the control of cell proliferation. Given
Transcription initiation factor IB (TIF-IB) this central role of Myc in the regulation of cell prolifera-
Cell growth requires protein synthesis, which in turn de- tion, it is not surprising to find a strong link between Myc
pends on ribosomal RNA (rRNA) transcription and ribo- dysregulation and cancer development.
some formation. Transcription of rRNA is carried out by
RNA polymerase I (Pol I), which is controlled by vari- Myc structure
ous transcription factors such as transcription initiation The Myc family of transcription factors is composed of
factor IB (TIF-IB), which is a multimeric protein complex four members (c-Myc, N-Myc, L-Myc and S-Myc). Most
containing TATA-binding protein (TBP) and four TBP- attention has focused on the first three members, which
associated factors (TAFs) that are specific for polymerase have been strongly implicated in the genesis of human
I transcription. One of these TAFs is TAF1 68, which is cancers. Myc belongs to the basic helixloophelix leucine
regulated by reversible acetylation. When it is acetylated zipper (bHLH-Zip) group of transcription factors (Mod-
by PCAF, there is an increase in its DNA-binding activ- ule 4: Figure Myc structure).
ity and this increases Pol I transcription. This activation is Myc formation
reversed following deacylation of TAF1 68. The formation of Myc is dependent on the action of growth
factors that act to rapidly stimulate its transcription. Myc
X-Box binding protein 1 (XBP-1) is thus a typical immediate early gene in that it is rapidly
The X-Box binding protein 1 (XBP-1) acts to regulate switched on when cells are stimulated to grow and prolif-
the transcription of genes that function in both immune erate. Myc has a short half-life (20 min), and its function
responses and in the endoplasmic reticulum (ER) stress in the cell is determined by its rate of formation. It is the
signalling pathway (for details see step 3 in Module 2: increase in Myc concentration that enables it to bind to its
Figure ER stress signalling). partner Max to form the active dimers that are respons-
ible for Myc action to stimulate transcription. The level of
Kruppel-like factors (KLFs) Myc is determined by a balance between Myc formation
The Kruppel-like factors (KLFs), which take their name and Myc degradation.
from the Drosophila Kruppel protein, function in a num- The Myc promoter contains binding sites for the nuc-
ber of cellular processes such as proliferation, differenti- lear factor of activated T cells c1 (NFATc1) isoform of the
ation and survival. The C-terminus has three zinc fingers Ca2 + -sensitive transcription factor NFAT, suggesting that
(Cys2 His2) that are separated from each other by a highly the Ca2 + signalling pathway may play a role in stimulat-
conserved H/C link. ing the expression of Myc. This activation pathway may
In the case of the differentiation of white fat cells, KLF2 be relevant to Myc dysregulation and cancer development,
inhibits transcription of the Pparg gene in the preadipo- because large amounts of NFATc1 are expressed in pan-
cytes but is replaced by stimulatory KFL5 and KFL15 creatic cancer cells.
isoforms during the process of differentiation (Module 8:
Figure white fat cell differentiation). KLF4 plays a role Myc degradation
in regulating the differentiation of smooth muscle cells The stability of Myc is regulated by various cell sig-
(Module 8: Figure smooth muscle cell differentiation). nalling pathways. For example, the mitogen-activated pro-
tein kinase (MAPK) pathway acts to stabilize Myc by
Activation of transcription factors by regulating phosphorylating Ser-62 (Module 4: Figure Myc as a gene
their expression activator). On the other hand, glycogen synthase kinase-3
The activity of some transcription factors is determined by (GSK-3) phosphorylates Thr-58, which destabilizes Myc
adjusting their intracellular concentration. In the case of by initiating its degradation through the ubiquitin-protea-
Myc, this is achieved through a growth factor-dependent some system. The PtdIns 3-kinase signalling pathway can
increase in expression. prevent this degradation by inhibiting the phosphoryla-
tion of Ser-58 by GSK-3.
Myc A prolyl isomerase (PIN1) recognizes the phos-
Myc was identified as a component of the myelocyto- phorylated Thr-58 and this then leads to isomerization
matosis transforming virus (v-Myc) and was subsequently of Pro-59, which in turn allows the protein phosphatase
found to be a normal human gene, which plays a central 2A (PP2A) to remove the stabilizing phosphate on Ser-58.

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Module 4: Figure Myc structure
TIP48/49 P300
P-TEFb TIP60 Miz1
TRRAP TRRAP Max
FBW7 SKP2 SKP2
TAD I II III HLH ZIP c-Myc
NLS
TAD I II III HLH ZIP N-Myc
TAD I II III HLH ZIP L-Myc
NLS
HLH ZIP Max
SID NLS
HLH Mad1, Mad3,
ZIP
Mad4 and Mxi1
HLH ZIP Mnt
Zn finger
PDZ Zn fingers 1-12 Myc binding 13 Miz1
Structure of the Myc family and their associated proteins Max, Mad, Mnt and Miz.
The three main Myc proteins are shown at the top. The C-terminal region contains the basic helixloophelix (HLH) and the associated leucine zipper
(ZIP) domains. The N-terminal region contains a transactivation domain (TAD) and the conserved Myc-boxes (IIII), which play an important role in
binding to various transcriptional coactivators (denoted by the black bars shown above the c-Myc structure). The HLH-ZIP domains of Myc bind to
similar domains on its partner Max. Max also binds to the Mads, Mxi1 and Mnt, which also have HLH-ZIP domains. Miz1 is a zinc-finger protein that
also binds to Myc through a Myc-binding domain that does not depend on the HLH-ZIP domain. A nuclear localization signal (NLS) is located on
c-Myc, Max and Mad1.
The phosphate that remains at Thr-58 is then recognized the E-box of those genes that function in cell growth or the
by the SCF ubiquitin ligases Fbw7 and Skp2 (SCFFbw7 and cell cycle (Module 4: Figure Myc as a gene activator). When
SCFSkp2 ), which label it for degradation by the protea- cells are either quiescent (G0 ) or differentiated, these genes
some. The ubiquitin-specific protease Usp28 can stabilize are silenced by the binding of dimers formed between Max
Myc by removing the ubiquitin groups that target it for and its other partners, the transcriptional repressors such
degradation by the proteasome. as Mad or Mnt. These repressors act by recruiting the
transcriptional co-repressor switch independent (SIN3),
Myc action which assembles a chromatin remodelling complex that
Myc appears to be a master gene in that it can regulate up contains histone deacetylases that remove acetyl groups
to 15% of all genes. The action of Myc is complex in that from the histones. When the level of Myc rises, it displaces
it can function by either activating or repressing genes, the repressors (Mads and Mnt) by binding to Max to form
depending on its various binding partners. In general, it the Myc/Max activation complex.
activates those genes that promote the cell cycle, such as the The repressor action of the Myc/Max complex is
cyclins D1 and D2 , and cyclin dependent kinase 4 (CDK4) achieved by interfering with the activation of genes that are
(Module 4: Figure Myc as a gene activator), whereas it normally induced by the transcription factor Miz1 (Mod-
represses those genes that inhibit the cell cycle, such as ule 4: Figure Myc as a gene repressor). Some of these genes
the various CDK inhibitors p27, p21 and p15 (Module 4: code for the CDK inhibitors such as p15 and p21, which
Figure Myc as a gene repressor). function to inhibit events during the G1 phase of the cell
The transcriptional activity of Myc depends upon it cycle (Module 9: Figure cell cycle signalling mechanisms).
forming a dimer by combining with another basic helix In the case of the p21 gene, which is activated by p53 (Mod-
loophelix zipper (bHLH-Zip) protein, Max (Module 4: ule 4: Figure p53 function), the Myc/Max dimer inactivates
Figure Myc structure), to form a Myc/Max heterodimer the Miz1 bound to the initiator (INR) site (Module 4: Fig-
that functions to regulate a large number of gene targets ure Myc as a gene repressor). The transforming growth
that control both cell growth and the cell cycle. When Myc factor (TGF-) inhibition of cell proliferation depends
functions in gene activation, the Myc/Max dimer binds to upon the Smad signalling pathway that uses the activated

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Smad1/4 complex to bind to the serum response factor numerous positive- and negative-feedback loops that en-
(SRF) site on the promoter of the p15 gene (Module 4: hances the robustness of this regulatory mechanism. So
Figure Myc as a gene repressor). far, approximately 650 human miRNAs have been iden-
Many of the Myc targets code for components that act tified. The task of establishing microRNA properties and
to regulate the cell cycle. function of individual miRs is ongoing and already there
are indications that each miR can modulate the activity
Myc targets of up to 100 mRNAs to influence a large number of key
Many of the targets activated or repressed by Myc play biological processes:
a critical role in promoting cell cycle progression and cell
growth during the early G1 phase (Module 9: Figure prolif- Maintenance of embryonic stem (ES) cell pluripotency.
eration signalling network). Myc functions in a transcrip- MicroRNA modulation of cell-cycle regulatory mech-
tional pathway that results in the expression of various anisms
components of the proliferative signalling pathways, such p53 functions and microRNAs
as activators of the cell cycle [cyclin D1 , cyclin D2 and MicroRNA regulation of differentiation
cyclin-dependent kinase 4 (CDK4)] that operate during Differentiation of cardiac cells
G1 to drive the cell into S phase (Module 4: Figure Myc as Differentiation of smooth muscle cells
a gene activator). Myc also increases the expression of the Cell proliferation
inhibitor of DNA binding (Id) proteins (Ids) that help pro- Apoptosis
mote proliferation by switching off the cyclin-dependent Stress responses.
kinase (CDK) inhibitor p16INK4a (Module 8: Figure prolif- MicroRNA dysregulation and cancer
eration-differentiation switch). In addition, it can repress
genes that normally inhibit cell cycle progression, such as
the CDK inhibitors p15 and p21 (Module 4: Figure Myc MicroRNA biogenesis
as a gene repressor). The first step in microRNA biogenesis is the transcription
Another important target for Myc is to activate the of the miR gene by RNA polymerase II (Pol II) to form
expression of the alternative reading frame (ARF) tu- the primary miRNA (pri-miRNA) transcripts, which has
mour repressor, which inhibits the mouse double minute-2 a characteristic stem-loop structure and a 5 capped poly-
(MDM2) E3 ligase that degrades p53 (Module 9: Figure adenylated (poly A) tail (Module 4: Figure microRNA
proliferation signalling network). In this way, Myc activ- biogenesis). This pri-miRNA is recognized by Drosha,
ates the p53 surveillance mechanism, which can thus result which is a double-stranded RNA-specific nuclease that
in apoptosis. This indicates that Myc can activate both pro- acts together with DiGeorge syndrome critical region 8
liferation and apoptosis. This apparent paradox can prob- (DGCR8, also known as Pasha) to cleave pri-miRNA
ably be explained by a concentration effect of Myc. Under to form pre-miRNA. This pre-miRNA is then exported
normal growth stimulation, the level of Myc may not in- from the nucleus by Exportin 5, which is a RAN-GTP-
crease enough to activate ARF to trigger apoptosis, but this dependent nuclear transport receptor. Once it enters the
pathway may come into play when Myc levels are abnor- cytoplasm, the pre-miRNA is recognized by the trans-
mally increased as might occur during Myc dysregulation activator RNA-binding protein (TRBP) and the ribonuc-
and cancer development. lease Dicer, which cleaves the precursor to form the mature
With regard to cell growth, Myc plays a central role microRNA (miR).
by controlling the biogenesis of ribosomes. Myc has been The miR acts by binding to Argonaute (Ago14) pro-
shown to increase the transcription of all three of the RNA teins to form a RNA-silencing complex (RISC) that re-
polymerases (RNA Pol I, RNA Pol II and RNA Pol III). cognizes and inhibits target messenger RNAs (mRNAs).
Associated with this activation of cell growth, Myc can A short seed region between bases 2 and 7 at the 5 end of
also stimulate cell metabolism by increasing the expression the miRNA is responsible for recognizing and binding to
of lactate dehydrogenase (LDH), which may be particu- the 3 untranslated region (UTR) of their target mRNAs to
larly important during cancer development. Myc can also inhibit protein synthesis through a number of mechanisms
increase the expression of serine hydroxymethyl trans- such as translational repression, mRNA deadenylation and
ferase (SHMT), which results in an increase in the meta- mRNA degradation. The translational repression of pro-
bolic pathway that generates one-carbon units. tein synthesis can occur through inhibition of either EIF4E
to prevent initiation or the ribosomes to block elonga-
tion. Expression can also be inhibited by deadenylation
MicroRNA (miRNA) of the poly-A tail by activation of the CCR4-NOT. The
The microRNAs (miRs) are a large class of highly con- RISC complex can also bring about direct degradation of
served non-coding small RNAs (approximately 2026 mRNA.
nucleotides) that function as post-transcriptional regulat-
ors. They are key regulators of gene silencing by binding to MicroRNA properties and function
the 3 untranslated region (UTR) of specific target mRNAs The standard process of microRNA biogenesis generates
to influence both their stability and translation. Uncover- approximately 650 microRNAs (miRs) with widely differ-
ing the processes responsible for microRNA biogenesis ent properties and functions. The properties and functions
have revealed a highly regulated sequence of events with of some of the well established miRs are described below:

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Module 4: Figure Myc as a gene activator
SIN3/HDAC HDAC1
SAP30
complex HDAC2 SIN3
SAP18
SDS3
RbAp46/48 Skp2
SCF
Fbw7
SCF
P
HDA
C1
SAP30
T58
S62
HDAC SIN3 Myc
2 GSK3 Myc
/48 SAP18
SDS3
46
Mad
X
Ap
Deacetylation or Myc P Usp28

Max
Rb
Mnt
E-Box ERK1/2 Myc Degradation
ARF
Mad Cyclin D1
or Cell
Mnt
Cyclin D2
HAT CDK4 cycle
Myc
Max Myc target Ids Cell
genes proliferation
E-Box
RNA Pol I
RNA Pol II
Ac Ac Ac Ac RNA Pol III Cell
LDHA growth
Acetylation
SHMT
Myc activation of target genes that function in cell proliferation.

There are a large number of Myc target genes that are activated by an increase in the level of Myc. Many of these Myc targets have an E-box element
containing the 5-CACGTG-3 sequence. In quiescent G0 or differentiated cells, the many Myc target genes are repressed by Max/Mad or Max/Mnt
dimers, together with the SIN3/HDAC transcriptional co-repressor complex. The HDACs deacetylate chromatin, making it inaccessible to transcription.
Myc activates transcription by replacing the Mad/Mnt, which removes the repressor complex. The Myc/Max dimer bind to the E-box and provides a
complex to bring in various coactivators. The histone acetyltransferases (HATs), such as CBP/p300, CGN5 and TIP60, acetylate histones to open up
the chromatin to make it accessible so that the Myc target genes can be transcribed. These target genes contribute to cell growth and activation of
the cell cycle.
Let-7 Insulin-like growth factor II mRNA binding protein 1

The family of let-7 miRNAs has twelve members (let-7-a1, (IMP1)
let-7-a2, let-7-a3, let-7-b, let-7-c, let-7-d, let-7-f1, let-7-f2, The insulin-like growth factor II mRNA-binding protein 1
let-7-g, let-7-I and miR-98). They are located on eight (IMP) family has three members, IMP13, that function in
different chromosomal locations and are related to each post-transcriptional regulation including processes such as
other by having identical seed sequences and may thus RNA trafficking, stabilization and translation. They have
act on similar targets. The level of let-7 is very low in been identified as a target of the miRNA let-7 that helps to
embryonic stem (ES) cells and progenitor cells, but is an orchestrate developmental processes by controlling the ex-
important contributor to the microRNA regulation of dif- pression of various embryonic genes. IMP1, which is also
ferentiation (Module 8: Figure ES cell miRNAs). The level known as the coding region determinant-binding protein
of let-7 in ES cells is kept low through the RNA-binding (CRD-BP), plays a role in cell proliferation and survival
proteins LIN28 and LIN28B through a double-negative- by stabilizing various target mRNAs (e.g. IGF-II, c-myc,
feedback loop. Down-regulation of let-7 by LIN28 and tau, FMR1, semaphorin and TrCP1) by shielding them
LIN28B has an important role maintaining the pluripo- against degradation.
tency of embryonic stem cells (ES). IMP1 is overexpressed in various human neoplasias
The increase in let-7 that occurs during differentiation
is reversed during the development of cancer, where a pro- Mlin41
gressive decline in the level of let-7 coincides with an in- Like C. elegans lin-41, mouse lin41 (Mlin41) is regulated
crease in the expression of the genes that control prolif- by let-7 and miR-125 miRNAs. In a reciprocal manner,
eration. There appears to be a direct correlation between Mlin41 co-operates with the pluripotency factor Lin-28 in
these events because many of the let-7 target proteins are suppressing let-7 activity, revealing a dual control mech-
proliferative signals such as Ras and cMyc and cell-cycle anism regulating let-7 in stem cells. The ability on Mlin41
components such as CDC25A and CDK6. They also reg- to silence let-7 may depend on its interaction with Dicer
ulate various embryonic genes such as high mobility group and the Argonaute proteins.
box A2 (HMGA2), insulin-like growth factor II mRNA The human homologue of lin41 (Hlin41), which is also
binding protein 1 (IMP-1) and Mlin-41. known as tripartite motif-containing 71 (TRIM71), is a

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Module 4: Figure Myc as a gene repressor
Max Myc
A.
UV p300
DNMT3a
DNMT3a
P P Max Myc
P P
P p300 P
p21 Miz1 X p21
P53
Miz1
P53
INR INR
SRE SRE
B.
TGF Max Myc
_
Max Myc
Smad1/4
P
Miz1 p15
P
Miz1 X p15
INR INR
SRE SRE
Myc represses genes activated by Miz1.

Activation of some of the cyclin-dependent kinase (CDK) inhibitors, such as p15 and p21, are repressed by Myc. A. The p21 gene, which is activated
through UV irradiation and p53, is also activated at the initiator (INR) site by Miz1, which binds p300 that acts to acetylate chromatin to open up the
locus for transcription. Myc/Max dimers inhibit this transcription by binding to Miz1 to displace the p300, and it also brings in the DNA methyltransferase
DNMT3a. B. The p15 gene is also activated by Miz1. This gene is also controlled by transforming growth factor (TGF-), which has two actions. It
uses activated Smad1/4 to bind to the serum response element (SRE) site, and it also inhibits Myc, which is a potent repressor of Miz1. An increase
in Myc levels can silence this gene.
potential human developmental and disease gene because 1 helps to promote differentiation (see step 2 in Module 8:
Mlin41 is required for neural tube closure and survival. Figure skeletal muscle myogenesis).
miR-1 and miR-133 miR-15 and miR-16

Closely related forms of miR-1 and miR-133 occur as One of the functions of these two miRNAs is to inhibit
clusters on the same chromosomal locus and are tran- the activin receptor type IIA (ACVRIIA) (Module 2: Table
scribed together to control muscle differentiation. For ex- Smad signalling toolkit), which is stimulated during early
ample, the miR-1-1/miR-133a-2 gene cluster is transcribed development by Nodal that acts through the Smad sig-
together during the differentiation of cardiac cells (Module nalling pathway (Module 2: Figure Smad signalling). Ex-
8: Figure cardiac development). The miR-133a continues pression of miR-15 and miR-16 is inhibited by the ca-
to function in differentiated cardiac muscle cells where it nonical Wnt/-catenin signalling pathway, which thus in-
restrains the expression of Ca2 + signalling components creases the Nodal-ACVRIIA activation gradient that con-
such as the type 2 inositol 1,4,5-trisphosphate receptor tributes to dorsoventral specification.
(InsP3 R2), calcineurin (CaN) and the nuclear factor of ac- Another role for miR-15 and miR-16 is to regulate ap-
tivated T cells 3 (NFAT3) (Module 12: Figure miRNA optosis by repressing the expression of Bcl-2. These two
and cardiac hypertrophy). However, in response to sig- miRs are often deleted or down-regulated in many cases of
nals that trigger hypertrophy, the Ca2 + released by the chronic lymphocytic leukaemia (CLL). The level of miR-
InsP3 R2 reduces the expression of miR-133a and results in 16-1 is enhanced by p53 (Module 4: Figure microRNAs
a co-ordinated increase in the expression of the InsP3 R2s, and p53 function).
CaN and NFAT3 to set up a positive-feedback loop that
strongly promotes the onset of hypertrophy. miR-21
During the differentiation of skeletal muscle, MyoD en- miR-21 has a general role to enhance signalling through
hances the expression of these miRNAs that then have dif- protein tyrosine kinase-linked receptors (PTKRs) by in-
ferent roles in promoting the proliferation-differentiation hibiting both PTEN and sprouty (SPRY). Up-regulation
switch in that miR-133 inhibits proliferation whereas miR- of miR-21, which will enhance signalling through these

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Module 4: Figure microRNA biogenesis
Degradation Ago1-4
Transcription
factors (e.g. SRF)
AAAA
mRNA
miR
Pol II transcription Ago1-4

A
pri-miRNA mRNA Deadenylation
CCR4-NOT
polyA
Drosha DGCR8 Ago1-4

mRNA
AAAA
EIF4E Ribosome
Dr 8 Translational repression
os
ha CR RNA-induced
Ago1-4
DG silencing
complex (RISC)
Dicer
TRBP Ago1-4
Exportin 5
Dicer
pre-miRNA
TRBP miR
pre-miRNA
Biogenesis and function of microRNA.

RNA polymerase II (Pol II) transcribes miRNA genes to form primary miRNA (pri-miRNA) transcripts, which has a characteristic stem-cell structure. The
latter is recognized by the ribonuclease Drosha that acts together with DiGeorge syndrome critical region 8 (DGCR8, also known as Pasha) to cleave
pri-miRNA to form pre-miRNA, which is then exported from the nucleus by Exportin 5. Once it enters the cytoplasm, the pre-miRNA is recognized by
TAR RNA-binding protein (TRBP) and the ribonuclease called Dicer, which cleaves the precursor to form the mature microRNA (miR). The miR acts
by binding to Argonaute (Ago14) proteins to form a RNA-silencing complex (RISC) that recognizes and inhibits target messenger RNAs (mRNAs)
through a number of mechanisms, such as translational repression, mRNA deadenylation and mRNA degradation.
receptors, has been found in various tumours and may also miR-125b
contribute to heart disease by increasing the proliferation A component of the relationship between p53 function
of cardiac fibroblasts leading to fibrosis. and microRNA is the role of miR-125b to suppress the
activity of p53 (Module 4: Figure microRNAs and p53
miR-23b function). During genotoxic stress associated with DNA
The miR-23b cluster acts to enhance formation of Smad3, damage, miR-125b is repressed and this then allows p53 to
Smad4 and Smad5, which operate in the TGF--sensitive arrest the cell cycle by inhibiting G1 (Module 9: Figure G1
Smad signalling pathway (Module 2: Figure Smad sig- checkpoint signalling).
nalling). This control mechanism may operate during liver
stem cell differentiation.
miR-126
miR-26a There is a major role for miR-126 in controlling vascular
This miRNA acts specifically to inhibit PTEN to enhance development by modulating VEGF-dependent angiogen-
signalling through the PtdIns 3-kinase signalling pathway esis. Endothelial cells have large amounts of miR-126,
and this was shown to have pathological consequences by which acts to maintain VEGF signalling by inhibiting the
enhancing the emergence of tumours that are derived from expression of the Class I PtdIns 3-kinase regulatory p85
glial cells in brain. subunit (also known as PIK3R2) that inhibits PtdIns 3-k-
inase signalling and sprouty-related, EVH1 domain-con-
miR-29b taining protein 1 (SPRED1) that inhibits the MAP kinase
The anti-apoptotic protein Mcl-1 is regulated by miR-29b. signalling pathway. In this way, miR-126 will inhibit two of
A decrease in the expression of mir-29b would result in the main signalling mechanisms that regulate angiogensis
an up-regulation of Mcl-1 which could have implications (Module 9: Figure VEGF-induced proliferation).
for cancer in much the same way as miR-15 and miR16
regulate apoptosis in various cancers.
miR-137
miR-34 miR-137 functions in neurogenesis and neuronal matura-
The expression of Sirtuin 1 (SIRT1) is regulated by miR-34. tion. MIR137 is a schizophrenia-associated gene.

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miR-143 and miR-145 the expression of E-cadherin and are of central importance
The miR-143 and miR-145 genes, which are arranged for the epithelial to mesenchymal transition (EMT).
as a cluster, are transcribed together as occurs during One of the functions of ZEB1 is to regulate the expres-
the differentiation of smooth muscle (Module 8: Figure sion of IL-2
smooth muscle cell differentiation). In the case of smooth In the mouse, miR-200c represses gene activity by acting
muscle cells, miR-145 and miR-143 regulate the genes re- on TCF and on evi, which is required for the secretion of
sponsible for initiating and maintaining the differentiated Wnt.
state. What is remarkable about these two microRNAs Mutations in the ZEB1 gene have been associated with
is that they can single-handedly direct the proliferation posterior polymorphous corneal dystrophy-3 (PPCD)
differentiation switch, which is such a feature of the and late-onset Fuchs endothelial corneal dystrophy.
smooth muscle cell phenotype. When arteries are injured,
there is a decline in the levels of miR-145 and miR-143 miR-302
and this may result in the differentiated contractile cells The miR-302 cluster has eight related miRNAs that are
switching back into a proliferative state and could account regulated by the stem cell transcription factors Oct4 and
for the development of atherosclerotic blood vessels with Sox2 as part of the ES cell cycle miRNA regulatory mech-
thickened walls. anisms (Module 8: Figure ES cell miRNAs). Expression
These two microRNAs also have a role in embryonic of miR-302 can convert human skin cancer cells back into
stem (ES) cells where they are of critical importance in reg- pluripotent ES cells.
ulating the microRNA regulation of differentiation (Mod-
ule 8: Figure ES cell miRNAs). One primary target of miR-324-5p
miR-145 is the proto-oncogene c-Myc, whose enhanced The miR-324-5p suppresses the GLI1 transcription factor
expression is associated with aggressive tumors. that operates in the Hedgehog signalling pathway (Module
The fact that miR-145 mRNA is markedly reduced in 2: Figure Hedgehog signalling pathway).One of the func-
many cancer cells could explain Myc dysregulation and tions of GLI1 is to control the proliferation of cerebellar
cancer development. granule progenitor cells.
Mutations in miR-324-5p result in the development of
miR-152 medulloblastomas.
One of the functions of miR-152 is to repress the activity
of the type 2 sarco/endo-plasmic reticulum Ca2+ -ATPase miR-372 and miR-373
2 (SERCA2), which functions in Ca2 + homoeostasis as These two microRNAs act to reduce the inhibitory ef-
part of the Ca2+ OFF reactions (Module 2: Figure Ca2+ fect of the large tumour suppressor (Lats), which is a
signalling dynamics). serine/threonine protein kinase that phosphorylates the
Yes-associated protein (YAP) and transcriptional coactiv-
ator with PDZ-binding motif (TAZ), also known as Ta-
miR-192
fazzin, that are transcription factors which operate in the
The transcription of miR-192, which acts to inhibit expres-
Hippo signalling pathway (Module 2: Figure hippo sig-
sion of the zinc-finger E-box binding homeobox 2 (ZEB2),
nalling pathway).
is enhanced by transforming growth factor (TGF-)
that acts through the Smad signalling pathway. One of the
actions of ZEB2 is to repress transcription of miR-216a
and miR-217 that inhibit the activity of PTEN resulting
Signalsome stability
During the final phase of development, each cell type be-
in an increase in the activity of the PtdIns 3-kinase sig-
gins a process of differentiation during which a specific
nalling pathway. This action of miR-192 illustrates how
set of genes are expressed that define the phenotype of the
the miRNAs can function in the cross-talk that occurs
different cells found in the body. A key component of this
between signalling pathways. Such a mechanism might ac-
process of differential gene transcription is signalsome ex-
count for the hypertrophy and survival of mesangial cells
pression (Module 8: Figure signalsome expression), during
during diabetic nephrology.
which the cell puts in place a cell-type-specific signalling
system. One of the features of the differentiated state is
miR-199 its relative stability, which includes stability of the cell-
The expression of Sirtuin 1 (SIRT1) is regulated by miR- type-specific signalsomes. This stability depends on the
199. turnover of signalsome components being tightly regu-
lated, which depends on a balance between the degrada-
miR-200 tion of signalsome components and their replacement. In
There is a miR-200 family that has five members (miR- the light of continuous signalsome degradation, stability is
200a, miR-200b, miR-200c, miR-141 and miR-429). Some maintained by ongoing transcription processes. However,
of the main targets of the miR-200 family are zinc finger E- signalsomes are not fixed in stone, but can be remodelled
box binding homeobox 1 (ZEB1), which is also known as during both normal and pathological conditions. In the
transcription factor 8 (TCF8), and zinc finger E-box bind- latter case, phenotypic remodelling of the signalsome is a
ing homeobox 2 (ZEB2), also known as Smad-interacting major cause of disease and may contribute to the process
protein 1 (SIP1). These two transcription factors regulate of ageing.

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How do cells regulate the transcription of signalsome 6. The gene DYRK1A encodes the dual-specificity
components in order to maintain the stability of their sig- tyrosine-phosphorylation regulated kinase 1A
nalling systems?It seems that they may operate a quality (DYRK1A), which is a serine/threonine protein
assessment system whereby the properties of the output kinase located in the nucleus and is responsible for
signals are constantly monitored and any deviations are fed phosphorylating NFAT to promote its export from the
back to the transcriptional system so that adjustments can nucleus. Like DSCR1 described above, DYRK1A is
be made to various signalling components. Such autoreg- also located on human chromosome 21, where trisomy
ulatory mechanisms are beginning to emerge for the Ca2 + occurs in patients with Downs syndrome.
signalling system. 7. NFAT induces the transcription of the
What is remarkable about the stability of the Ca2 + sig- sarco/endo-plasmic reticulum Ca2+ -ATPase (SERCA)
nalsome is that Ca2 + itself plays a key role in regulating the pump that transports cytosolic Ca2 + back into the
phenotypic expression of its signalling pathway (Module endoplasmic reticulum (ER)/sarcoplasmic reticulum
4: Figure signalsome transcription). The Ca2 + signalsome (SR) store (Module 2: Figure Ca2+ signalling toolkit).
is a self-regulatory system with an inherent compensatory 8. Expression of the large-conductance (BK) channels,
mechanism that enables the signalsome to adapt to im- which are activated by Ca2 + (Module 3: Figure K+
posed changes. Each cell-specific signalsome is set up to channel domains), are controlled by NFAT3.
deliver a characteristic Ca2 + transient, and any alteration 9. NFAT reduces the expression of the KCa 1 subunit
in this output signal tends to induce subtle alterations in that functions to control the Ca2 + sensitivity of the
the signalsome so that normal delivery is restored. In gen- large-conductance (BK) channels (Module 3: Figure K+
eral, a decline in the level of Ca2 + signalling results in an channel domains).
up-regulation of the signalsome, and vice versa.
Similar regulatory mechanisms may function to control
There are a number of examples of such a homoeostatic
other signalling systems. For example, the section on mi-
mechanism based on Ca2 + -induced transcription of Ca2 +
togen-activated protein kinase (MAPK) signalling prop-
signalling components:
erties reveals that the extracellular-signal-regulated kinase
(ERK) pathway (Module 2: Figure ERK signalling) and the
c-Jun N-terminal kinase (JNK) pathway (Module 2: Fig- Overexpressing calsequestrin (CSQ) in mouse cardiac
ure JNK signalling) can induce the transcription of their myocytes strongly reduces the amplitude of Ca2 +
own signalling components. In the case of the Ca2 + sig- spikes that activate contraction and results in congest-
nalling system, there are numerous examples of Ca2 + - ive heart failure (CHF). This increase in CSQ greatly
induced transcription of Ca2 + signalling components op- enhances the amount of stored Ca2 + , and this would
erating to compensate for alterations in the signalling path- result in an increase in Ca2 + signal amplitude if it were
way (Module 4: Figure signalsome transcription). This not for a marked down-regulation of components of the
Ca2 + -dependent regulation of Ca2 + signalling pathways release mechanism [ryanodine receptor type 2 (RYR2),
is particularly evident in the relationship between Ca2+ triadin and junctin].
signalling and cardiac hypertrophy. A reduction in the Ca2 + content of the ER/SR results
Support for such a mechanism is evident from the fact in the activation of the activating transcription factor
that the genes that encode components of Ca2 + signalling 6 (ATF6), which then acts to increase the expression of
pathways are themselves regulated by Ca2 + -dependent the SERCA2 pump as a compensatory mechanism to re-
transcription factors such as the calcineurin/nuclear factor store the level of luminal Ca2 + . Disruption of one copy
of activated T cells (NFAT) system (Module 4: Figure of the SERCA2 gene results in altered Ca2 + homoeo-
NFAT control of Ca2+ signalling toolkit): stasis, as shown by a decrease in the amplitude of the
Ca2 + transient in isolated cardiomyocytes (Module 4:
1. Activated NFAT binds to a site on the promoter of Figure Ca2+ signals in SERCA+/ cardiac cells). This de-
endothelin, which is a potent activator of receptors that cline in spike amplitude was due to a 50% decline in the
generate Ca2 + signals. Ca2 + content of the SR. However, the rate of recovery
2. The transient receptor potential (TRP) family member was normal, and this seems to result from compensat-
TRPC3 is up-regulated by NFAT. ory alterations in both phospholamban (PLN) and the
3. Activated NFAT induces the transcription of the inos- Na+ /Ca2+ exchanger (NCX). The amount of PLN was
itol 1,4,5-trisphosphate receptor type 1 (InsP3 R1) re- reduced and there was an increase in its phosphorylated
sponsible for releasing internal Ca2 + . state; both changes would reduce its inhibitory activ-
4. NFAT is capable of inducing its own transcription in ity on SERCA2. In addition, the Na + /Ca2 + exchanger
that it can increase the expression of NFAT2. was up-regulated.
5. Activated NFAT increases the expression of Downs When triadin 1 is overexpressed in mice, there is
syndrome critical region 1 (DSCR1) gene, which en- a compensatory decline in the expression of both
codes a protein that inhibits the activity of calcineurin RYR2 and junctin (Module 12: Figure Ca2+ in triad-
(CaN), thereby setting up a negative-feedback loop. in-1-overexpressing mice).
This gene is located within the critical region of human Overexpression of the L-type channel in cardiac cells
chromosome 21 where trisomy occurs in patients with results in an up-regulation of the NCX accompanied by
Downs syndrome. a modest hypertrophy.

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Module 4: Figure signalsome transcription
Calcium signalling
toolkit
Receptors Cardiac specific

calcium
signalsome
Transducers Et-1 Calcium
PLC1
transient
Channels
L-type
RESPONSE
SERCA2a
Pumps
PV
CAM, TnC
Buffers
+
OR
Sensors -
Ca 2+-induced transcription of
Ca 2+ signalling components
Ca2 + regulates the phenotypic expression of the Ca2 + signalsome.

During differentiation, each cell type assembles a specific Ca2 + signalsome, which delivers a Ca2 + transient exactly suited to control its particular
cellular responses. In addition, this Ca2 + transient may also contribute to a feedback loop whereby a process of Ca2 + -induced transcription of Ca2 +
signalling components functions to stabilize the signalsome.
Module 4: Figure NFAT control of Ca2 + signalling toolkit
Endothelin 2+ +
Ca K
TRPC3 and TRPC6 BK K Ca 1
InsP3 R1 P
2+ NFAT P
ER Ca P
+
CaN NFAT
SERCA DSCR1 shuttle
NFAT DYRK1A
Endothelin
1
TRPC3 and TRPC6
2 InsP 3 R1
3 NFAT2 2+
4 Ca Signalling
DSCR1 toolkit
5
DYRK1A components
6
SERCA
7
BK
8 K Ca 1
9
Regulated expression of the Ca2 + signalling toolkit by nuclear factor of activated T cells (NFAT).
The calcineurin (CaN)/nuclear factor of activated T cells (NFAT) transcriptional cascade plays a direct role in a process of Ca2 + -induced transcription
of components of the Ca2 + signalling toolkit. (See the text for details of the different components.)

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Module 4: Figure Ca2 + signals in SERCA + / cardiac cells tion signalling pathway in kidney cells, the primary cilia
respond to fluid flow by a large increase in intracellu-
lar Ca2 + . This process of mechanotransduction in kidney
cells depends upon the activation of the polycystin-2 chan-
nels, which are highly concentrated on these primary cilia
where they function as mechanosensors.
The formation and function of primary cilia requires
a tight integration of the microtubule cytoskeleton with
the processes of membrane and protein trafficking. The
Rab signalling mechanism (Module 2: Figure Rab sig-
nalling) controls the transport of key ciliary components
that are carried into the cilium by various molecular mo-
tors. Rab8a, Rab17, and Rab23 appear to have a role in
many of these trafficking events. One of the functions
of Rab8a is to interact with cenexin/ODF2, which is a
microtubule-binding protein that is essential for cilium
biogenesis.
The developmental and multiple cellular function of the
primary cilium is very dependent on inositol polyphos-
phate 5E-phosphatase (INPP5E), which removes the 5-
phosphate from PtdIns4,5P2 and PtdIns3,4,5P3 (Step 12 in
Module 2: Figure phosphoinositide metabolism). Muta-
tions in the INNP5E gene have been linked to MORM
syndrome. Other mutations that affect primary cilia have
been linked to a number of diseases, such as Bardet-Biedl
syndrome, neural tube defects, polycystic kidney disease
and retinal degeneration.
Contractions and Ca2 + transients in wild-type (WT) and sarco/endo-
plasmic reticulum Ca2 + -ATPase (SERCA) + / heterozygous (HET)
ventricular myocytes.
The myocytes from the heterozygous mice had smaller Ca2 + transients Actin remodelling
(A) and contractions as measured by the percentage of cell shortening
(B). Reproduced from Ji, Y., Lalli, M.J., Babu, G.J., Xu, Y., Kirckpatrick, Reorganization of the cytoskeleton, which depends in part
D.L., Liu, L.H., Chiamvimonvat, N., Walsh, R.A., Shull, G.E. and Peri- on a process of actin remodelling, is controlled by a num-
asamy, M. (2000) Disruption of a single copy of the SERCA2 gene results ber of signalling systems. An important aspect of actin
in altered Ca2 + homeostasis and cardiomyocyte function. J. Biol. Chem.
275:3807338080, with permission from the American Society for Bio- remodelling is the process of actin tread milling, which de-
chemistry and Molecular Biology; see Ji et al. 2000. pends on the polymerization of ATP-G-actin into the end
of existing actin filaments at the barbed end (plus end) and
the removal of ADP-actin at the pointed-end (minus-end).
Such remodelling of the actin cytoskeleton has multiple
functions such as the regulation of cell shape, adhesion, cy-
Ciliary beating tokinesis, gastrulation, cell migration and morphogenesis.
There are two types of cilia, as determined by the organ- The ERM protein family have a special role in attaching
ization of the axoneme, which can be arranged into either actin filaments to the plasma membrane. There also is an
a 9 + 2 pattern or a 9 + 0 pattern, as found in the interesting relationship operating between actin dynamics
primary cilium. The former are found on ciliated epithelial and gene transcription. A number of signalling mechan-
cells, where they beat rhythmically. This form of ciliary isms contribute to regulation of the many processes that
beating can be regulated, and this has been studied in some control this polymerization and depolymerization of actin
detail in airway epithelial cells. (Module 4: Figure actin remodelling).
The monomeric G proteins, such as the Rho family
Primary cilium members Rho, Rac and Cdc42, are of particular import-
The 9 + 0 cilia, also known as primary cilia, are usually ance for such actin remodelling. For example, the Rac
immotile except when located within the nodal region of signalling mechanism is activated by a number of stim-
the developing embryo, where they have a twirling mo- uli that act through guanine nucleotide exchange factors
tion that sets up the fluid flow responsible for determining (GEFs) to convert inactive Rac-GDP into active Rac-
left-right asymmetry (Module 8: Figure nodal flow hypo- GTP, which then has a number of functions (Module 2:
thesis). Figure Rac signalling). One of these functions is to activ-
The immotile primary cilia (9 + 0), at least one of ate the WiskottAldrich syndrome protein (WASP) ver-
which is present in most cells (Module 4: Figure primary prolin homologous (WAVE), which orchestrates the act-
cilium), have important sensory functions involved in de- in-related protein 2/3 complex (Arp2/3 complex) (Mod-
velopment, liquid flow in the kidney, mechanosensation, ule 4: Figure actin remodelling proteins). Cdc42 is another
sight, and smell. In the case of the mechanotransduc- monomeric G protein that functions in actin remodelling

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Module 4: Figure primary cilium
Example of a single primary cilium on a cultured cell.

This human retinal pigmented epithelial cell was labelled with an antibody against acetylated tubulin that has picked out the single primary cilium
shown in green. Reproduced from Curr. Opin. Cell Biol., Vol. 15, Pazour, G.J. and Witman, B.G., The vertebrate primary cilium is a sensory organelle,
pp. 105110. Copyright (2003), with permission from Elsevier; see Pazour and Witman 2003.
Module 4: Figure actin remodelling
PTKR GPCR
PtdIns4,5P2 2+
Ca
+
Rac Gelsolin
GTP
Uncapping
IRS p53 Capping

Arp2/3
WAVE
Polymerization
Cofilin
Capping
Severing
Uncapping
Profilin-actin T4-actin
Depolymerization
Profilin
MONOMERIC Thymosin-4 (T4)

ACTIN POOL
Actin remodelling mechanisms.

Actin remodelling is regulated by different signalling pathways. Protein tyrosine kinase-linked receptors (PTKRs) and G protein-coupled receptors
(GPCRs) act through the Rac signalling pathway (Module 2: Figure Rac signalling) and initiate polymerization by recruiting the Arp2/3 complex. An
increase in Ca2 + activates Gelsolin that inhibits polymerization by capping the growing end and also functions to sever existing filaments into short
capped fragments that have two fates. The pointed-end can be depolymerized with the help of cofilin that then donates actin monomers to the pool
of actin bound to profilin and thymosin-4 (T4). Some of these short fragments can also be uncapped through a mechanism that depends on the
formation of PtdIns4,5P2 that inactivates gelsolin resulting in uncapping such that polymerization can continue. This Figure is based on the information
shown in Figure 10 in Shalini et al. (2013).

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(Module 2: Figure Cdc42 signalling). In this case, a key teins function has come from the study of gelsolin that
component of the action of the active Cdc42/GTP com- has a characteristic structure consisting of six (G1G6)
plex is WiskottAldrich syndrome protein (WASP) that closely similar gelsolin domains (97118 amino acids) that
controls the actin assembly (Module 4: Figure actin re- are fairly equally spaced along the length of the molecule.
modelling proteins). Many of the other members of the gelsolin superfam-
The cytoplasm contains a monomeric actin pool in ily have a similar distribution of domains whereas others
which individual actin molecules are bound to various have either fewer domains or domains that are fused to
actin-binding proteins. The profilinactin pool is used for other protein domains. These molecular variations result
the polymerization reaction. The actin monomers bound in the family members having slightly different properties
to thymosin-4 (T4) act as a reservoir that can transfer to control a variety of process in different cell types.
actin into the profilinactin pool as required (Module 4:
Figure actin remodelling). These profilinactin complexes Adseverin
then feed actin monomers into the growing barbed end Adseverin, which is also known as scinderin (SCIN),
during the processive actin polymerization reaction. is a member of the gelsolin/villin superfamily of actin-
This polymerization process is modulated by gelsolin modulating proteins. It is closely related to gelsolin and
through two important reactions. In response to an elev- is expressed in various endocrine cells and in the skin.
ation in Ca2 + , gelsolin undergoes a conformation change In the case of chromaffin cells located within the adrenal
that exposes its actin-binding region that can then either medulla, it functions to disintegrate the layer of cortical
cap or sever actin. By capping the growing barber ends actin filaments to enable the secretory vesicles to approach
it prevents further polymerization. Gelsolin can also the membrane where they fuse to release adrenalin (Mod-
bind to regions down the length of the filaments to ule 7: Figure chromaffin cell secretion). Adseverin has also
produce a pool of shorter capped segments that can influ- been implicated in the differentiation of chondrocytes.
ence subsequent actin formation through two pathways.
Firstly, these shorter segments can be uncapped through Gelsolin
the PtdIns4,5P2 signalling cassette. The PtdIns4,5P2 dis- Gelsolin belongs to the gelsolin/villin superfamily of actin-
rupts the gelsolinactin interaction to provide uncapped modulating proteins. It is a Ca2 + -sensitive protein that
barbed ends that can nucleate the formation of new fil- modulates actin by promoting the nucleation of new actin
aments. Since this uncapping occurs near the membrane, filaments, capping of the barbed ends of growing filaments
it will enable actin filaments to be formed rapidly near and the severing of existing filaments (Module 4: Figure
the surface to change the shape of the membrane and to actin remodelling).
promote cell movement. Alternatively, the pointed end The gelsolin protein contains six (G1G6) closely sim-
of these short segments can be depolymerized through a ilar gelsolin domains (97118 amino acids) that are fairly
mechanism facilitated by cofilin to replenish the pool of equally spaced along the length of the molecule. The Ca2 + -
actin monomers. binding sites of gelsolin are distributed down the length of
Remodelling of the cortical actin cytoskeleton plays an the molecule: the G1 and G4 domains contain the Type-1
important role in a number of cellular responses: Ca2 + -binding site whereas a Type 2 site is located on each
of the six gelsolin domains. The calcium-binding affinities
Control of cytokinesis at the time of cell division (Mod- (K d ) for these different sites vary between 0.2 and 600 M,
ule 9: Figure cytokinesis). which indicates that gelsolin may be able to respond to a
Formation of pseudopodia during phagocytosis. wide range of Ca2 + concentrations.
Construction of the focal adhesion complex (Module 6: The activity of gelsolin is regulated by either the Ca2 +
Figure integrin signalling ). signalling pathway or the PtdIns4,5P2 signalling cassette.
Formation of the osteoclast podosomes (Module 7: Fig- In its inactive state, gelsolin is folded up in such a way
ure osteoclast podosome). as to shield the major actin-binding regions located on
Actin assembly, pseudopod formation and uropod con- domains G1, G2 and G4. Elevation in Ca2 + induces a
traction during neutrophil chemotaxis, which is a classic major conformational change in gelsolin that exposes these
example of cell migration (Module 11: Figure neutrophil actin-binding regions thus enabling it to carry out its actin
chemotactic signalling). remodelling functions of either capping or severing actin
Growth of the spine during the action of Ca2+ in syn- filaments (Module 4: Figure actin remodelling). Capping
aptic plasticity (Module 10: Figure Ca2+ -induced syn- of the growing filaments terminates polymerization. The
aptic plasticity). severing activity of gelsolin results in the formation of
Regulation of hormone release by pituicytes in the pos- large numbers of shorts capped actin filaments that have
terior pituitary (Module 10: Figure magnocellular neur- two fates. They can either be depolymerized through the
ons). activity of cofilin that then feeds actin monomers into the
monomeric actin store where they are bound to either
Gelsolin/villin superfamily profilin or thymosin-4 (T4) or they can be uncapped to
The gelsolin/villin superfamily of actin-modulating pro- provide short segments that can be polymerized to form
teins contains eight members: adseverin, CapG, gelsolin, new filaments.
flightless I, advillin, villin, villin-like protein, and supervil- The uncapping of actin filaments is driven by forma-
lin. Much of our current information on how these pro- tion of PtdIns4,5P2 , which binds to three binding sites

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located in the linker region between G1 and G2, a region These multiple functions of supervillin depend on its
on G2 that overlaps the actin-binding site and the third ability to bind to many different proteins:
is found in the linker region between G5 and G6. When
PtdIns4,5P2 binds to these sites it disrupts the interaction The ability of supervillin isoforms 1 and 4 to promote
beween gelsolin and actin. cell survival depends on its ability to decrease levels
Familial amyloidosis is caused by a mutation in the type- of the tumour suppressor protein p53. Supervillin acts
2 calcium-binding site of domain G2. on the ubiquitin ligase that participates in p53 ubiquit-
ination and degradation (Module 4: Figure p53 func-
tion). In particular, it reduces the activity of the ubiquit-
Flightless I in-specific protease Usp7, which is also known as herpes
Flightless I is a member of the gelsolin/villin superfam- virus-associated ubiquitin-specific protease (HAUSP),
ily of actin-modulating proteins. Its name reflects the fact which is a p53-binding protein that functions to de-
that flightless I was first identified in Drosophila linked ubiquitinate p53. The ability of supervillin to interact
to a mutation causing defects in flight. Typical of many with USP7/HAUSP results in its own deubiquitination
members of the gelsolin/villin superfamily, the mammalian and stability.
flightless I protein has six gelsolin domains (G1G6), but Supervillin can regulate the reactivity of blood platelets.
is unusual in that it has an extensive N-terminal leucine- For example, it inhibits the rapid activation and spread-
rich repeat (LRR) domain, which normally functions in ing of platelets that occurs during thrombus formation
proteinprotein interactions. This LRR domain enables (Module 11: Figure thrombus formation). Supervillin
flightless I to bind to other proteins such as LRR Flightless may act to link together the GP1bGPIXGPV com-
I-interacting proteins 1 and 2 (LLRFIP1 and LRRFIP2) plex on the platelet surface to the actin cytoskeleton (See
and the TAR RNA-interacting protein (TRIP), which is step 1 in Module 11: Figure platelet activation).
a double-stranded RNA-binding protein. Flightless I and Supervillin has a myosin II regulatory N-terminus that
LLRFIP1 seem to be a component of the blood platelet seems to have a role in regulating the turnover of podo-
cytoskeleton. somes, which are specialized cell adhesion complexes
Flightless I is expressed in a large number of cell types used by cells to form attachments with the extracellular
and has been implicated in many different functions such matrix (ECM) or to interact with other cells. Osteoclast
as development, control of gene transcription (coactiva- podosomes are an example of such a podosome (Module
tion of nuclear hormone receptors and regulation of - 7: Figure osteoclast podosome) that contain many of the
catenin-dependent transcription), inflammation, cell mi- same components that are found in the focal adhesion
gration and wound healing. In the case of wound healing, complex (Module 6: Figure integrin signalling). How-
flightless I seems to impair cellular proliferation and the re- ever, podosomes differ from the latter in that they are
epithelialization necessary to repair large wounds. Some of much more labile in that they have a lifetime of 212 min.
these effects may be caused by an increase in inflammation This rapid turnover may depend, in part, on the ability
and this may be a significant factor in the reduced healing of supervillin to modulate the dynamics of actomyosin-
of foot ulcers in diabetic patients. Flightless I may con- dependent contractility. Supervillin can interact with
tribute to this enhanced inflammation through its ability cytoskeletal proteins such as actin, non-muscle my-
to sequester the adaptor protein MyD88 that is a com- osin II (NMII) and myosin light chain kinase (MLCK)
ponent of the Toll receptor signalling pathway (Module 2: (Module 7: Figure osteoclast podosome). Dissolution
Figure Toll receptor signalling). of the podosomes may be triggered when supervillin
activates myosin II.
Supervillin
Supervillin is a member of the gelsolin/villin superfamily Villin
of actin-modulating proteins. Supervillin has five of the Villin is a member of the gelsolin/villin superfamily of
gelsolin domains (G2G6) and also contains the types 1 actin-modulating proteins. It is closely related to gel-
and 2 Ca2 + -binding sites. There is a C-terminal headpiece solin. It is expressed in various epithelial tissues such as
resembling that of villin, but it may not bind actin. There gastrointestinal tract, gall bladder and kidney. It seems to
are a number of isoforms: Isoform 1 (a canonical non- be located primarily within the microvilli where it contrib-
muscle 200 kDa isoform), isoform 2 also known as archvil- utes to the reorganization of the actin bundles. Like some
lin (striated muscle), isoform 3 (smooth muscle archvillin) of the other members of the superfamily it contains the six
and isoform 4 (non-muscle isoform). Supervillin has an (G1G6) closely similar gelsolin domains, which contain
important property of being able to link actin filaments the Ca2 + -binding domains.
to membranes. It can also increase myosin II contractil- A characteristic feature of villin is a C-terminal head-
ity and can induce rapid integrin recycling by reducing piece that has an F-actin-binding domain that is respons-
integrin-mediated cell adhesion. It is widely distributed ible for the cross-linking and bundling of actin especially
and has been implicated in multiple processes such as cy- at low concentrations of Ca2 + . At higher concentrations
tokinesis, myogenesis, cell-substrate adhesion and spread- of Ca2 + , villin both caps and severs actin much as gelsolin
ing, regulation of the tumour suppressor protein p53 levels does during actin remodelling (Module 4: Figure actin re-
to control cell survival and formation of podosomes and modelling). Villin also has three phosphatidylinositol 4,5-
invadosomes. bisphosphate (PtdIns4,5P2 ) binding sites: one is located

C
at the head piece and the other two are in the core. The interactive binding (CRIB) domain, reflecting the fact that
binding of PtdIns4,5P2 inhibits actin capping and severing it is this site that binds to Cdc42 or Rac. The GBD domain
to enhance actin bundling cross-linking. Tyrosine phos- is followed by a proline-rich region (Pro), two verprolin
phorylation of villin can enhance the Ca2 + sensitivity of (V) homology regions, a cofilin-like (C) and an acidic (A)
villin and this is likely to play a significant role in regulating region.
its ability to modulate actin dynamics. WASP seems to be particularly important for carrying
out the actin remodelling function of Cdc42 (Module 2:
Villin-like protein Figure Cdc42 signalling). Following cell stimulation, the
Villin-like protein is a member of the gelsolin/villin su- GTP-bound form of Cdc42 binds to WASP and appears
perfamily of actin-modulating proteins. It has a domain to open up the molecule such that the C-terminal region
structure that resembles that of advillin and villin. It is becomes free to associate with the actin-related protein 2/3
strongly expressed in epithelia (gall-bladder and intest- complex (Arp2/3 complex), which binds to the C/A region
ine). There are indications that it may also play a role in and begins to polymerize actin (Module 4: Figure actin re-
spermatogenesis. modelling protein). The actin monomers are brought in as
a complex with profilin, which binds to the verprolin ho-
Advillin mology region. The profilin/actin complex dissociates, and
Advillin is a member of the gelsolin/villin superfamily of the actin monomer is added to the growing tail, whereas
actin-modulating proteins. It is closely related to adseverin the profilin is released to the cytoplasm.
and villin and is expressed in various tissues such as uterus, The PtdIns4,5P2 regulation of actin remodelling can be
testis, taste buds, brain, gastrointestinal tract and skeletal accommodated in this mechanism because this lipid binds
muscle. Like villin, advillin has a C-terminal headpiece that to the basic (B) region and may act together with Cdc42
is responsible for actin binding. to activate WASP.
It seems to play an important role in the outgrowth of WASP plays an important role in T cell cytoskeletal re-
neurons from the dorsal root ganglia and trigeminal ganglia organization during formation of the immunological syn-
and may also function in neuronal regeneration. apse (Module 9: Figure immunological synapse structure).
Mutations in WASP cause Wiskott-Aldrich syndrome.
WiskottAldrich syndrome protein (WASP)
The WiskottAldrich syndrome protein (WASP) family
of proteins plays a critical role in cytoskeletal remodel- WiskottAldrich syndrome protein (WASP)
ling by functioning as intermediaries between upstream verprolin homologous (WAVE)
signalling events and the downstream regulators of actin. There are three WiskottAldrich syndrome protein
One of the first components to be identified was the pro- (WASP) verprolin homologous (WAVE) isoforms that
tein WASP. A closely related neural WASP (N-WASP) closely resemble each other with regard to their domain
is now known to be ubiquitous. In addition, there are structure. The N-terminal region begins with a WAVE ho-
three WiskottAldrich syndrome protein (WASP) verpro- mology domain (WHD), followed by a basic (B) region,
lin homologous (WAVE) isoforms that have similar func- a proline-rich region (Pro), a verprolin (V) homology re-
tions. IRSp53, an insulin receptor substrate (IRS), func- gion, a cofilin-like (C) and an acidic (A) region (Module
tions as an intermediary between Rac and WAVE during 4: Figure actin remodelling protein). The main difference
the process of membrane ruffling (Module 2: Figure Rac from WiskottAldrich syndrome protein (WASP) is that
signalling). The adaptor protein Abelson-interactor (Abi), WAVE lacks the GTPase-binding domain (GBD). The ac-
which functions in the Abl signalling pathway (Module 1: tivator Rac is connected to WAVE through a number of
Figure Abl signalling), plays an important role in linking adaptors. The insulin receptor substrate (IRS) protein has
Rac to WAVE. a Src homology 3 (SH3) domain that binds to the proline-
The WASP family plays a critical role in orchestrat- rich (Pro) region and a Rac-binding domain (RCB) that
ing the processes of actin remodelling. The N-termini of links it to Rac. In addition, Abelson-interactor (Abi) is
both WAVE and WASP have a verprolin (V) homology an adaptor that links WAVE to the actin-related protein
region, a cofilin-like (C) and an acidic (A) region, which 2/3 complex (Arp2/3 complex) (Module 1: Figure Abl sig-
play key roles in promoting actin polymerization. The V nalling).
region binds the profilinactin complex to release free actin WAVE seems to be particularly important for carrying
monomers that are then used by the Arp2/3 complex at- out the actin remodelling function of Rac (Module 2: Fig-
tached to the C/A region to polymerize actin. The WAVE ure Rac signalling). Following cell stimulation, the GTP-
complex favours actin branching, as occurs during mem- bound form of Rac binds to IRSp53, which functions as
brane ruffling, whereas the WASP complex forms long an adaptor protein, to link Rac to WAVE (Module 4: Fig-
actin filaments that produce filopodia. ure actin remodelling). This binding of the Rac/IRSp53
WASP and N-WASP are fairly similar proteins with re- complex appears to open up the molecule such that the C-
spect to their domain structure (Module 4: Figure actin re- terminal region becomes free to associate with the Arp2/3
modelling protein). In the case of N-WASP, the N-terminal complex, which binds to the C/A region to begin actin
region begins with a WASP homology 1 (WH1) domain, polymerization. The actin monomers are brought in as a
followed by a basic (B) region and a GTPase-binding do- complex with profilin, which binds to the verprolin (V) ho-
main (GBD). The latter is also called the Cdc42- and Rac- mology region. The profilin/actin complex dissociates, and

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Module 4: Figure actin remodelling proteins
Filopodium
PtdIns4,5P2 IRSp53 PtdIns4,5P2 Cdc42

Rac
GTP
RC 3
SH GTP
B
WHD B Wh1 B GBD
Abi1/2 WASP Pro
Pro VV
WAVE V C
C Actin A Arp2/3
A Arp2/3 branching
Actin-Profilin Ena/VASP
Actin-Profilin
Actin
Profilin Profilin assembly
WiskottAldrich syndrome protein (WASP) and WASP verprolin homologous (WAVE) proteins orchestrate remodelling of the actin cytoskeleton.
The WiskottAldrich syndrome protein (WASP) verprolin homologous (WAVE) protein is activated by both the phospholipid PtdIns4,5P2 and by the
G protein Rac, which is linked to WAVE through the insulin receptor substrate IRSp53. The cofilin (C) homology and the acidic (A) regions bind
actin-related protein 2/3 complex (Arp2/3 complex), which is responsible for actin polymerization that favours a branching pattern as is seen during
membrane ruffling. The related WASP protein is regulated by PtdIns4,5P2 and the G protein Cdc42, which binds to the GTPase-binding domain (GBD).
The N-terminal C/A domain binds Arp2/3 to initiate actin polymerization, as does the WAVE protein. However, the WASP configuration seems to favour
the formation of long filaments to form filopodia.
the actin monomer is added to the growing tail, whereas Formation of the actin cytoskeleton during integrin sig-
the profilin is released to the cytoplasm. nalling (Module 6: Figure integrin signalling).
The PtdIns4,5P2 regulation of actin remodelling can be Assembly of actin filaments in the postsynaptic dens-
accommodated in this mechanism because this lipid binds ity (PSD) of neurons (Module 10: Figure postsynaptic
to the basic (B) region and may act together with Rac density). Remodelling of this complex plays a role in
to activate WAVE. This WAVE-dependent remodelling of synaptic plasticity (See steps 6 and 7 in Module 10: Fig-
the actin cytoskeleton plays a critical role in regulating ure Ca2+ -induced synaptic plasticity).
the store-operated entry of Ca2 + into T cells (Module 3: Regulation of the cytoskeleton during ephrin (Eph) re-
Figure STIM-induced Ca2+ entry). If the level of WAVE2 ceptor signalling (Module 1: Figure Eph receptor sig-
is reduced in Jurkat T cells, there is a marked reduction in nalling).
the amplitude of Ca2 + (Module 3: Figure WAVE2 effects Formation of actin during endothelial cell contraction
on Ca2+ entry). (Module 7: Figure endothelial cell contraction).
Assembly of actin in the osteoclast podosome (Module
Actin-related protein 2/3 complex (Arp2/3 7: Figure osteoclast podosome).
complex) Assembly of actin in the pseudopod during neutrophil
The actin-related protein 2/3 complex (Arp2/3 complex) chemotaxis (Module 11: Figure neutrophil chemotactic
is made up of a collection of seven proteins that initiate signalling).
actin polymerization to form Y-branched actin filaments. Assembly of actin during membrane invagination and
In effect, it attaches to a pre-existing filament to form a scission during the process of endocytosis (Module 4:
nucleation site from which a new actin filament begins Figure scission of endocytic vesicles).
to polymerize (Module 4: Figure actin remodelling). Two
of the proteins are actin-related proteins (Arp2 and Arp3),
whereas the others are called the actin-related protein com-
plex (Arpc-1Arpc-5). Cortactin
The Arp2/3 complex plays a central role in the regula- Cortactin functions as an activator of the actin-related pro-
tion of a number of cellular processes that depend upon tein 2/3 complex (Arp2/3 complex). It plays an important
actin remodelling: role in assembling the plume of actin that is attached to

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Module 4: Figure Ena/VASP family
Profilin G-actin F-actin

PKA phosphorylation
site
P
Mena EVH1 LERER PRO FAB EVH2 CCD
P
EVL EVH1 PRO FAB EVH2 CCD
P
VASP EVH1 PRO FAB EVH2 CCD
Structural organization of the Ena/vasodilator-stimulated phosphoprotein (VASP) family.

The three members of the Ena/vasodilator-stimulated phosphoprotein (VASP) family function in remodelling the actin cytoskeleton. They are particularly
important in linking various cell signalling pathways to the processes responsible for actin assembly. EVH, Ena/VASP homology; PRO, proline-rich
domain; CCD, coiled-coil region.
the neck of the vesicular bulb during membrane invagina- One of the functions of VASP is to promote actin re-
tion and scission (Module 4: Figure scission of endocytic modelling during clot formation in blood platelets. Phos-
vesicles). It also is one of the postsynaptic density (PSD) phorylation of VASP by protein kinase A (PKA) is one
signalling elements that play an important role in neur- of the cyclic AMP-dependent inhibitory mechanisms for
onal actin remodelling (Module 10: Figure postsynaptic blocking clot formation (Step 12 in Module 11: Figure
density). platelet activation). Ena/VASP binds to FAT1, which is
the mammalian orthologue of the Drosophila atypical cad-
herin Fat (Ft) that functions in planar cell polarity (PCP)
Ena/vasodilator-stimulated phosphoprotein (Module 8: Figure planar cell polarity signalling).
(VASP) family
The Ena/VASP family contains three closely related fam-
ily members: Mena (mammalian Enabled), EVL (Ena- ERM protein family
VASP-like) and VASP (vasodilator-stimulated phosphop- The ERM protein family consists of ezrin, radixin and
rotein) (Module 4: Figure Ena/VASP family). Ena/VASP moesin that act as molecular cross-linkers between actin
functions in the dynamics of actin assembly during both filaments and proteins in the cell membrane. They are char-
cellcell interactions and during the protrusion of la- acterized by their N-terminal FERM domain, which en-
mellipodia and filopodia during cell migration. There is ables these ERM proteins to interact with the proteins in
an N-terminal Ena/VASP homology 1 (EVH1) domain, the membrane. The C-terminal region, which is highly
a central proline-rich (PRO) domain and a C-terminal charged, binds to actin.
EVH2 domain, which binds to both G- (globular) and
F- (filamentous) actin. The central PRO domain binds to Actin dynamics and gene transcription
profilin. All members of the family have a conserved pro- There is a close relationship between actin dynamics,
tein kinase A (PKA) site. which occurs during actin remodelling, and gene transcrip-
Ena/VASP proteins have been shown to interact with tion. It is the balance between actin assembly and disas-
many of the proteins associated with actin assembly such sembly that acts to regulate gene transcription that depends
as WiskottAldrich syndrome protein (WASP) and pro- on the transcriptional coactivator myocardin-related tran-
filin. One way in which it might alter actin dynamics is to scription factor (MRTF), which is an actin-binding pro-
bind to the barbed ends of actin, where it antagonizes the tein (Module 4: Figure actin dynamics and gene transcrip-
activity of capping proteins while promoting addition of tion). When bound to actin, MRTF is located in the cyto-
actin monomers (Module 4: Figure actin remodelling). plasm, but when actin is removed by its assembly into actin

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Module 4: Figure actin dynamics and gene transcription
Stimulus Stimulus
PTKR
Rho
GDP
+
+
Rho
+
ROCK GTP
mDia
LIM-K1
_ Cofilin
F-actin
Actin Actin
disassembly assembly STARS
G-actin MRTF
MRTF STARS
MRTF MRTF
Actin Actin-related protein 3

Caldesmon Cofilin 1
MRTF Dystrophin Gelsolin
SRF Myosin heavy Trpomyosin 1
chains 9 & 11 Villin 1
CArG
Vinculin
Actin dynamics and gene transcription.

The relationship between actin dynamics and gene transcription depends on the transcriptional coactivator myocardin-related transcription factor
(MRTF), which is an actin-binding protein. When bound to actin, MRTF is located in the cytoplasm, but when actin is removed by assembly into
actin filaments, the free MRTF is released and enters the nucleus where it acts as a coactivator of the serum response factor (SRF) that regulates
expression of a large number of proteins that regulate the function of actin. The striated muscle activator of Rho signalling (STARS) contributes to this
gene activation by binding to free actin thus contributing to the liberation of MRTF.
filaments, the free MRTF is released and enters the nuc- factors to nucleate actin polymerization. Most of them
leus where it acts as a coactivator of the serum response have an N-terminal GTPase-binding domain (GBD) fol-
factor (SRF) which regulates expression of a large number lowed by a diaphanous inhibitory domain (DID), a coiled-
of proteins that regulate the function of actin. The striated coil domain (CC), a variable number of three formin-
muscle activator of Rho signalling (STARS) contributes to homology domains (FH1, FH2 and FH3) and finally a
this gene activation by binding to free actin thus contrib- diaphanous autoregulatory domain (DAD). Under resting
uting to the liberation of MRTF. conditions, the C-terminal DAD interacts with the DID
to induce a conformational change that effectively blocks
Striated muscle activator of Rho signalling (STARS) the FH domains. Activation by the Ras family of small
The striated muscle activator of Rho signalling (STARS), G proteins (e.g. Rho, Rac and Cdc42), which bind to the
which is also known as Myocyte Stress 1 (MS1) or actin- GBD region, disrupts the DIDDAD interaction thereby
binding Rho-activating protein (ABRA), is expressed opening up the molecule such that the FH2 domain can
mainly in cardiac and skeletal muscle. One of its func- initiate filament assembly. The formins remain associated
tions is to bind to actin, which facilitates the release with the fast-growing barbed end, enabling rapid insertion
of myocardin-related transcription factors (MRTFs) that of actin subunits while protecting the end from capping
contribute to the activation of serum response factor (SRF) proteins. Elongation proceeds as profilinactin complexes
(Module 4: Figure actin dynamics and gene transcription). are recruited by the adjacent FH1 domain.
The formin family is divided into the formins and
Diaphanous-related formins (DRFs).
Formins The formins consist of Formin-1 (FMN1), Formin-2
The formins, which consist of 15 members, are a widely ex- (FMN2), delphin, FHDC1 (INF1) and INF2.
pressed family of proteins that contribute to actin remod- The Diaphanous-related formins (DRFs) consist of
elling. This large formin family consists of large multido- dishevelled-associated activator of morphogenesis 1
main proteins that associate with a variety of other cellular (DAAM1), Formin-related gene in leukocytes 1 (FRL1)

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and mammalian diaphanous-like formin proteins (mDia1, Neuronal Ca2 + sensor proteins (NCSs)
mDia2 and mDia3). Burgoyne, R.D., OCallaghan, D.W., Hasdemir, B., Haynes, L.P. and
Tepikin, A.V. (2004) Neuronal Ca2 + -sensor proteins: multitalented
regulators of neuronal function. Trends Neurosci. 27:203209.
Formin-1 (FMN1)
Formin-1 (FMN1) belongs to the formin family of actin Annexins
remodelling proteins. FMN1 plays a role in the formation Babiychuk, E.B. and Draeger, A. (2000) Annexins in cell membrane
of both dendrites and synapses. Another important role dynamics: Ca2 + -regulated association of lipid microdomains. J. Cell
Biol. 150:11131123.
for FMN1 is in cell adhesion where it regulates the actin Gerke, V., Creutz, C.E. and Moss, S.E. (2005) Annexins: linking Ca2 +
filaments that bind to members of the classical cadherins signalling to membrane dynamics. Nat. Rev. Mol. Cell Biol. 6:449461.
(Module 6: Figure classical cadherin signalling). Song, G., Harding, S.E., Duchen, M.R., Tunwell, R., OGara, P., Hawkins,
T.E., Hawkins, T.E., Das, D., Young, B. and Moss, S.E. (2002) DT40
cells lacking the Ca2 + -binding protein annexin 5 are resistant to
Dishevelled-associated activator of morphogenesis 1 Ca2 + -dependent apoptosis. Proc. Natl. Acad. Sci. U.S.A.
(DAAM1) 99:80548059.
Dishevelled-associated activator of morphogenesis 1 Moss, S.E. (2002) Altered mechanical properties and intracellular
calcium signaling in cardiomyocytes from annexin 6 null-mutant mice.
(DAAM1) belongs to the formin family of actin remod- FASEB J. 16:622624.
elling proteins. DAAM1 has an unusual activation mech- Rescher, U. and Gerke, V. (2004) Annexins- unique membrane binding
anism in that it is linked to some of the Wnt signalling proteins with diverse functions. J. Cell Sci. 117:26312639.
pathways (Module 2: Figure Wnt signalling pathways). In

particular, it seems to play a role in the Wnt/planar cell S100 proteins
polarity (PCP) pathway (Module 2: Figure Wnt signalling Barraclough, R. (1998) Calcium-binding protein S100 A4 in health and
disease. Biochim. Biophys. Acta 1338:190199.
pathways) as illustrated in the Frizzled (Fz)/Flamingo Broom, A., Pochet, R., Authelet, M., Pradier, L., Borghgraef, P., Van
(Fmi) polarity signalling pathway (Module 8: Figure planar Leuven, F., Heizmann, C.W. and Brion, J.-P. (2004) Astrocytic
cell polarity signalling). The ability of DAAM1 to pro- calcium/zinc binding protein S100A6 over expression in Alzheimers
disease and in PS1/APP transgenic mice models. Biochim. Biophys.
mote actin formation seems to play a central role in tissue Acta 1742:161168.
morphogenesis. For example, it controls cardiac morpho- Donato, R. (2001) S100: a multigene family of calcium-modulated
genesis by assembling the actin filaments of the contractile proteins of the EF-hand type with intracellular and extracellular
functions. Int. J. Biochem. Cell. Biol. 33:637668.
machinery. Heizmann, C.W., Schafer, B.W. and Fritz, G. (2003) The family of S100
cell signalling proteins. Handbook of Cell Signaling, Vol. 2 (Bradshaw,
Mammalian diaphanous-like formin 1 (mDia1) R.A. and Dennis, E.A., eds), pp. 8793, Academic Press,
There are three mammalian diaphanous-like formin pro- San Diego.
teins (mDia1, mDia2 and mDia3) that belong to the
Diaphanous-related formin (DRFs) family. They contain Ca2 + /calmodulin-dependent protein kinases
three formin homology domains that are used to bind to (CaMKs)
various effectors. mDia1 is activated by theRho signalling Corcoran, E.F. and Maens, A.R. (2001) Defining
Ca2 + /calmodulin-dependent protein kinase cascades in
mechanism and functions to control actin polymerization transcriptional regulation. J. Biol. Chem. 276:29752978.
by binding to profilin (Module 2: Figure Rho signalling). Fujisawa, H. (2001) Regulation of the activities of multifunctional
mDia1 has also been implicated in the control of polycystin Ca2 + /calmodulin-dependent protein kinases. J. Biochem. (Tokyo)
129:193199.
2. Hook, S.S. and Means, A.R. (2001) Ca2 + /CaM-dependent kinases: from
activation to function. Annu. Rev. Pharmacol. Toxicol. 41:471505.
Hudmon, A. and Schulman, H. (2002) Structurefunction of the
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004 Sensors and Effectors

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004 Sensors and Effectors

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Cell Signalling Biology Michael J.

Berridge r Module 4 r Sensors and Effectors 4 r1

Sensors and Effectors

Sensors Ca2 + sensors

Module 4: Figure EF-hand motif

Structural organization of the EF-hand.

Module 4: Figure CaM structure

The EF-hand Ca2 + -binding domain.

Module 4: Figure annexin structure

Annexin structure and membrane association.

Module 4: Figure annexin molecular structures

Molecular structure of different annexins.

in Module 4: Figure annexin structure and panel c in Mod- Annexin A7

Module 4: Figure S100 phylogenetic tree

Phylogenetic tree and clustering of many S100 genes on human chromosome 1.

Gene transcription Ca2 + /calmodulin-dependent protein kinase I (CaMKI)

Module 4: Figure structure of CaMKs

A C CaMKK Autoinhibitory domain

A C CaMKK Ca 2+/CaM-binding domain

A C CaMK I ATP-binding site (A)

Module 4: Figure activation of CaMKs

Ca2 + activation of Ca2 + /calmodulin-dependent protein kinases I and IV (CaMKI/CaMKIV).

Module 4: Figure structure of CaMKII

Three-dimensional structure of Ca2 + /calmodulin-dependent protein kinase II (CaMKII).

Module 4: Figure CaMKII activation

Ca2 + activation of Ca2 + /calmodulin-dependent protein kinase II (CaMKII).

Module 4: Figure calcineurin

Calcineurin (CaN) is a Ca2 + /calmodulin-sensitive serine/threonine protein phosphatase.

of CaN is particularly evident in the inhibition of nuc- Cyclosporin A (CsA)

Module 4: Figure membrane and protein trafficking

Membrane and protein trafficking pathways.

Module 4: Figure COPII-coated vesicles

Cargo transport from the ER to the Golgi using COPII-coated vesicles.

Module 4: Figure COPI-coated vesicle

p23/p24 Cargo SNARE

1. Transmembrane golgins such as Giantin, Golgin-84

Module 4: Figure Ca2 + -dependent exocytosis

Two types of Ca2 + -dependent exocytosis.

Module 4: Figure vesicle cycle

The exocytotic vesicle cycle.

Module 4: Figure Ca2 + -induced membrane fusion

SNAP-25 Synaptotagmin VII Synaptotagmin I/II

Exocytotic machinery: a hypothetical mechanism for Ca2 + -induced membrane fusion.

Exocytotic machinery allel arrays (Module 4: Figure Ca2+ -induced membrane

Synaptobrevin [also known as vesicle-associated mem- Ca2 + -dependent exocytosis

Module 4: Figure endocytosis

TFR Cargo sorting

PtdIns4P P PIPKI PtdIns4,5P2 P P P P

Scission Dynamin ring

Dynamin P CaN Actin plume

Control of clathrin-mediated endocytosis.

Module 4: Figure cargo sorting signals

Cargo recognition by sorting proteins.

Amphiphysins its cofactor auxilin. The auxilin is recruited by a lipid sig-

Module 4: Figure scission of endocytic vesicles

Endocytic vesicle scission and removal of the clathrin coat.

micro-organism to initiate phagocytosis to form a pha- Endosome vesicle fusion to early

Module 4: Figure PtdIns3P formation in phagosomes

Module 4: Figure phagosome maturation

Module 4: Figure endosome vesicle fusion