Principles of Biochemistry

Third Edition
International Student Version
Donald Voet • Judith G. Voet • Charlotte W. Pratt

Chapter 13
Biochemical Signaling

Copyright © 2008 by John Wiley & Sons, Inc.

 HORMONES

• Maintenir Homeostase
• Répondre à des stimuli externes
• Réguler des cycles (maturation,
cycle mentruel, differentiation, etc)

Figure 13-1
Figure 13-2
   
 Receptors
= Proteins that bind signals
and initiate a signaling cascade

Cell membrane receptors

-integral membrane
proteins that bind an
extracellular signal and
start a signal cascade

Intracellular receptors
-nuclear hormone receptors
 Nuclear hormone receptors

Examples include
-steroid hormone receptor and
-thyroid hormone receptor
-Retinoic acid receptor
-Vitamine D receptor

NHRs are transcription factors that respond to

specific ligands

Ligands alter the ability to bind to specific DNA

regulatory elements
 Receptor Properties
A hormone binds to a Receptor
- soluble (cytoplasmic) for lipophilic hormones:
* arachidonic acid derivatives (leukotrienes, retinoic acid, prostaglandines)
* vitamine D3 and steroids
* thyroid hormones (T3 & T4)
- membrane bound (at the plasma membrane): for peptides and neurotransmitters

Often the same Hormone can bind different Receptors

=> different signaling pathways => different responses
=> receptor subtypes (role of agonists/antagonists)

ex: Adrenaline binds to a (α1, α2, α3) or (β1, β2) receptors

 Liaison Ligand-Recepteur

(R)(L) ((R)T – (R*L))(L)

KL = __________ = _____________________
(R*L) (R*L)

Diagramme hyperbolique Diagramme de Scatchard

B (Bmax – B)
__ = _____________

F KL

Box 13-2a
 General Principles of Signal Transduction
1. Communication usually
involves
(i) a signaling molecule,
(ii) a receptor,
(iii) intracellular signal
transducers and
(iv) targets
 General Principles of Signal Transduction
2. Each cell responds to a complex profile of
signaling molecules (crosstalk)
General Principles of Signal Transduction
3. Different cells respond differently to a
particular signaling molecule
Protein kinases
 Receptor tyrosine kinases
Most common type of receptor for many common
protein hormones including EGF, PDGF, FGF, HGF,
IGF-1, VEGF, NGF.
Domain organization in a variety of receptor
tyrosine kinase (RTK) subfamilies.
 Recepteurs à activité Tyrosine Kinase
Recepteur d’Insuline Recepteur de hGH
(hormone de croissance)

Figure 13-4
 Recepteurs tyrosine kinases
Le Recepteur possede une activité intrinsèque
tyrosine kinase

Lorsque le ligand se lie, le recepteur se dimerise

et développe une activité tyrosine kinase

Il s’auto-phosphoryle (autophosphorylation),
provoquant:
1. une activité kinase plus forte
2. une plus forte affinité pour d’autres proteines
Une fois liées ces protéines seront phosphorylées
Schematic diagrams of RTKs.
 1. Recepteurs à activité Tyrosine Kinase

Analogue d’ATP
Structure du Domaine Tyrosine Kinase (Recepteur d’Insuline)

Jaune=déphosphorylé
Vert=phophorylé

PTK Domain
undergoes major conformation change &
autophosphorylation (1 to 3 Tyr residues)
 Structure des Domaines SH2 & SH3 (de Grb2)

Figure 13-9
 Structure des Domaines SH2 & SH3 (Recepteur d’Insuline)

SH2 SH3
Relaying the signal:
Binding Modules, Adaptors, GEF, GAP
SH2 P
• SH2 domains mediate signal Transduction T
• PTB domains bind pY-containing peptides B
• SH3 domains bind Pro-rich peptides SH3
 Structure du domaine SH3 de Abl dans le complexe le decapeptide
Pro-rich (APTMPPPLPP).
Page 693

SH3 domain:
Molecular velcro: mediate interactions between kinases & regulatory proteins
present in great variety of proteins GF
•  receptor Tyrosine Kinases P SH2
•  non-Receptor Tyrosine Kinases, GRB2
SH3 P SOS Ras
•  adaptor proteins (ex. Grb2) P
•  structural proteins (myosin, spectrin) P
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 Relaying the signal:
Binding Modules, Adaptors, GEF, GAP

• SH2 domains mediate signal Transduction

• PTB domains bind pY-containing peptides
• SH3 domains bind Pro-rich peptides

Other Binding Modules

WW domain (2 Trp residues)
Plekstrin homology domain (PH domain)
PDZ domain
Relay:
Grb2, Shc & IRS:
• adaptor proteins
• recruit Sos to the vicinity of Ras
Ras is activated by RTK via Grb2-SOS complex
Activation of Ras
 Ras GTPase Cycle
Ras-GTP
GTP H2 O

GTPase Activating
Guanine Nucleotide Proteins
Exchange Factors
(GAPs)
(GEFs)

Pi
GDP Ras-GDP

-GAPs discovered biochemically

-GEFs discovered genetically- first in yeast and then drosophila
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 Ras Superfamily
Ras Rho Rab Arf Ran
H-Ras RhoA Rab1-N Arf1-6 Ran
N-Ras RhoB
K-Ras RhoC

TC21 RhoG
RhoE
Rap1
Rap2 CDC42

R-Ras Rac1
Rac2
RalA
RalB

Growth/ Cytoskeleton
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 Functions of Ras Proteins

1) Promote Cell Proliferation

-fibroblasts, epithelial cells, lymphocytes
-mediate actions of growth factors

2) Promote Cell Differentiation

-neuronal progenitor cells (PC12)
-mediate action of neurotrophins

3) Contribute to Differentiated Cell Functions

-CNS neurons
-mediate effects of calcium signaling

Voet Biochemistry 3e
© 2004 John Wiley &
Sons, Inc.
 Complexe Ras-GDP-GAP43-AlF3

Figure 13-10
 The Ras-activated MAP kinase cascade

Raf

Mek

Erk

Figure 13-7
 MAP KINASE
•  The mitogen-activated protein kinase (MAPK) pathways
are typically comprised of a three-member protein
kinase cascade.
•  Specificity of MAPK responses is achieved by
activation of different three-kinase modules.
•  There are at least three sets of mammalian MAPK
modules.
–  the extracellular-signal-regulated kinases (ERKs),
–  the Jun N-terminal kinases (JNKs)
–  the p38 kinases.
•  As a group, the MAPKs are major players in mediating a
variety of signals for cell proliferation and differentiation.
 Cascade MAP-kinase

Figure 13-11
Scaffolding proteins help organize MAPKs
Scaffold proteins that modulate mammalian MAP
kinase cascades. (a) JIP-1 (b) MEKK1.
(JNK-interacting protein).
 Structure de Src (= non-Receptor Tyrosine kinase)
•  Many NRTK are activated by tyrosine kinase-associated receptors of the Scr family
•  Examples of TK-associated receptors: Src, Fyn, Lck

•  Autoinhibitory mechanims of Src

Structure de Src (=non Receptor Tyrosine kinase)
Modèle d’activation
 Abl

Abl = Protein Tyrosine Kinase - role in cancer

inhibitor of abl (gleevec) may inhibit cell proliferation

Structure of the Abl PTK domain in complex with a

truncated derivative of gleevec (anticancer drug).
 PROTEIN TYROSINE PHOSPHATASES
Protein Tyr Phosphatases
• SHP-2
Protein Ser/Thr Phosphatases
• PP1
• PP2A
• PP2B (=calcineurin / target of
immunosuppressant drugs)
• PP2C

Protein tyrosine phosphatase SHP-2.

PP2A
• Structurally variable
• Functionally diverse
• Catalytic subunit
• Scaffold subunit (A)(PR65)
• Four different regulatory subunits (B, B’, B’’, B’’’), bind to A & C
subunits

A subunit of PP2A.
 PP2A

Scaffold subunit: HEAT repeats

 Calcineurin (=PP2B protein phosphatase)
(a) human FKBP12·FK506–CaN. (b) Human CaN with CaNA yellow, its autoinhibitory
segment red, and CaNB cyan.

Cyclosporin & FK506: highly effective immunosuppressants

CALCINEURIN: target of immunosuppressive drugs

ex. immunophilins (cyclophilins, FKBP12, etc)
•  subunits : Catalytic A (CaNA) + regulatory B (CaNB)
•  binds NFAT --> translocated into the nucleus --> gene expression
 JAK-STAT Pathway
JAK: = Janus kinases - contiennent 2 domaines Tyrosine Kinase
STAT: Signal Transducers and Activators of Transduction
= Transcription Factors, activated by JAK

1.  - Ligand binding dimerizes the receptor (β1 and β2 subunits)

2.  - Receptor dimers bind JAK and induce phosphorylation of JAK
3.  - Phosphorylated JAK phosphorylate the Receptor Subunits
4.  - Phophorylated Receptor can phosphorylate STAT
5.  - Phophorylated STAT dimerizes
6.  - Dimeric, phosphorylated STAT moves to the nucleus and act as
Transcription Factors
7.  - ---> gene expression
The JAK-STAT pathway for the intracellular
relaying of cytokine signals.
JAK-STAT
JAK-STAT Pathway
PI3K
 Tensin=Structure of PTEN (Phosphatase and Tensin homolog).
cytoskeletal actin binding protein

PTEN = inositol-polyphosphate phosphatase

•  Tumor suppressor: loss of function results in cancer
•  downregulates Akt - controls the levels of Ptd-Inositol-3,4-Phosphate
•  dephosphorylates P-Ser & P-Thr
Heterotrimeric G-Proteins
 GPCR = G-Protein Coupled Receptor

Très grande famille de récepteurs

Recepteurs monomèriques
Figure 13-17 7 domaines transmembranaires (7TM)
 G-protein linked receptors
Ligand: Diverse ligands, such as epinephrine

Receptor: Integral membrane protein with 7TM

(7 transmembrane domains)

G-protein: trimeric protein (α, β, γ) attached to

the cell membrane by lipid anchors

Effectors: Target proteins that show altered

activity when they interact with activated G-
protein subunits (α, or βγ)
G-protein coupled Receptor
Figure 13-18: Rhodopsine
G-protein linked receptors and G-proteins

Receptor

G-protein
 hormone A G-protein that is part of a pathway that stimulates
signal Adenylate Cyclase is called Gs & its α subunit Gsα.

outside

GPCR plasma
membrane

α γ γ + α cytosol
AC
GDP β β GTP

GTP GDP ATP cAMP + PP i

The α subunit of a G-protein (Gα) binds GTP, & can hydrolyze it to GDP + Pi.
α & γ subunits have covalently attached lipid anchors that bind a G-protein
to the plasma membrane cytosolic surface.
Adenylate Cyclase (AC) is a transmembrane protein, with cytosolic domains
forming the catalytic site.
Gs & Gi Pathways
G-protein dissociation
GTP hydrolysis ends signaling and induces
trimerization
Figure 13-19 Protéine-G trimérique -
sous unités alpha/beta/gamma
Variety of G-proteins

•  Gs are stimulatory
•  Gi/0 are inhibitory
•  Gq act on PLC
•  G12/13 act on ion channels

–  22 α subunits
–  5 β subunits
–  12 γ subunits
VARIETY OF G-PROTEINS: Gα, -β, -γ
 ADP-ribosylation

par cholera toxine de Gsα

par pertussis toxine de Giα
Box 13-4b
Figure 13-23 Système Adenylate Cyclase
 Heterotrimeric G-Proteins
The cAMP cascade

Page 428
 Figure 13-20 Structure de Adenylate Cyclase -
M1/M2: =6TM / C1a/C2a:= domaines catalytiques pseudosymétriques
Box 13-4a
PKA activation by cAMP
PKA activates gene expression
CASCADE de PHOSPHORYLATION
EXEMPLE: Métabolisme du Glycogène
 R2C2 + 4cAMP  2C + R2(cAMP)4
Inactif actif
Figure 13-21 PKA : domaine catalytique domaine régulatoire
 CREB = 
cAMP responsive
Elements Binding
Protein
 Inactivation of PKA pathway

The G-protein -PKA pathway is inactivated by:

– Receptor desensitization (phophorylation by PKA)
– GTP hydrolysis in G-protein (GTPase of a-subunit)
– cAMP hydrolysis by phosphodiesterase
– PKA inhibition
– Phosphatase action on PKA targets
– Activation of an antagonistic pathway (Gi)
Turn off of the signal:
1. Gα hydrolyzes GTP to GDP + Pi. (GTPase).
The presence of GDP on Gα causes it to rebind
to the inhibitory βγ complex.
Adenylate Cyclase is no longer activated.
2. Phosphodiesterase catalyzes hydrolysis of
cAMP  AMP.
Turn off of the signal (cont.):
3. Hormone Receptor desensitization occurs.
This process varies with the hormone.
  Some receptors are phosphorylated via G-
protein-coupled receptor kinases.
  The phosphorylated receptor may then bind to
a protein arrestin that blocks receptor-G-
protein activation & promotes removal of the
receptor from the membrane by clathrin-
mediated endocytosis.
4. Protein Phosphatase catalyzes removal by
hydrolysis of phosphates that were attached to
proteins via Protein Kinase A.
 NNNNNH2OOHOHHHH2CHOPOO-1'3
Phosphodiesterase enzymes catalyze: cAMP NH2
cAMP + H2O  AMP
N
The phosphodiesterase that cleaves N
cAMP is activated by phosphorylation
catalyzed by Protein Kinase A. N N

Thus cAMP stimulates its own O

degradation, leading to rapid turnoff C
of a cAMP signal. O
P O
O O
Inhibiteur de Phosphodiesterase
cAMP  AMP
PDE

Inhibiteur spécifique de PDE5

Figure 13-24 Système IP3-DAG
 Phospholipases
PLC generates DAG and
phosphoinositides, such as IP3
(inositol 1, 4, 5- triphosphate)
Figure 13-25
 Domain organization of the four classes of
phosphoinositide-specific PLCs.
Page 709
PKC Inhibiteur de PKC
 Role du Calcium:
Calcium-calmoduline Protéine Kinase
Calmoduline Domaine EF-hand
Calmoduline Domaine EF-hand
 secretion
metabolism contraction/motility

apoptosis
Ca 2+ fertilization

proliferation differentiation

growth
 Ca2+ activates PLA2
that triggers Arachidonic Acid Metabolism

Ca2+
R
PLC G PLA2
AA
InsP3

Ca

Ca
Linoleic Acid

(20:4 Δ5, Δ8, Δ11, Δ14)

Prostagandin biosynthesis pathways are a
common drug target.
Interactions between G-proteins and RTKs
 KINASES: RESUME
A/Ser-Thr kinases
– PKA: cAMP dépendantes RRXSX
– PKCα,β,γ XRXXSSRS
– PK-Ca-CAM-I NYLRRLSDSNF
– PK-Ca-CAM-II XRXXSX
– PK-Ca-CAM-III RAGETRFTDTRK
– PKG: cGMP dépendantes RXXSRX
B/ Tyr Kinases
-PKY lient –SH2/activent –SH3
INSULIN SIGNAL TRANSDUCTION
Anthrax Spores

Anthrax Lethal Factor