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Biochem. J. (2005) 387, 639647 (Printed in Great Britain)

The yeast cyclin-dependent kinase inhibitor Sic1 and mammalian p27Kip1

are functional homologues with a structurally conserved inhibitory domain
Matteo BARBERIS*, Luca DE GIOIA*, Maria RUZZENE, Stefania SARNO, Paola COCCETTI*, Piercarlo FANTUCCI*,
Marco VANONI* and Lilia ALBERGHINA*1
*Dipartimento di Biotecnologie e Bioscienze, Universit`a degli Studi di Milano-Bicocca, Piazza della Scienza 2, 20126 Milano, Italy, and Dipartimento di Chimica Biologica,
Universit`a di Padova, Viale G. Colombo 3, 35121 Padova, Italy

In Saccharomyces cerevisiae, Sic1, an inhibitor of Cdk (cyclindependent kinase), blocks the activity of S-Cdk1 (Cdk1/Clb5,6)
kinase that is required for DNA replication. Deletion of Sic1
causes premature DNA replication from fewer origins, extension
of the S phase and inefficient separation of sister chromatids during anaphase. Despite the well-documented relevance of Sic1
inhibition of S-Cdk1 for cell cycle control and genome instability,
the molecular mechanism by which Sic1 inhibits S-Cdk1 activity
remains obscure. In this paper, we show that Sic1 is functionally
and structurally related to the mammalian Cki (Cdk inhibitor)
p27Kip1 of the Kip/Cip family. A molecular model of the inhibitory
domain of Sic1 bound to the Cdk2cyclin A complex suggested
that the yeast inhibitor might productively interface with the mammalian Cdk2cyclin A complex. Consistent with this, Sic1 is able
to bind to, and strongly inhibit the kinase activity of, the Cdk2
cyclin A complex. In addition, comparison of the different inhi-

INTRODUCTION

Cdks (cyclin-dependent kinases) play an essential regulatory role

in cell cycle progression: it is in fact the sequential activation of
Cdks by specific, unstable, regulatory subunits, named cyclins,
that first triggers the onset of DNA replication and later initiates
mitosis [1,2]. The evolutionary conservation of Cdks and cyclins
from yeast to mammalian cells is well established [1,2]. In budding yeast a single Cdk (Cdc28; now renamed Cdk1) is involved
in the control of the cell cycle. In the cell cycle there are cyclins
associated with G1 (Cln1, Cln2 and Cln3 in budding yeast; cyclin D in mammals), S phase (Clb5,6 in yeast; cyclins E and A in
mammals) and mitosis (Clb1,2 in yeast; cyclin B in mammals).
Both cyclins and Cdks have a wide degree of redundancy, and
it is currently believed that their specificity in driving the cell
cycle is dependent more on their timing of expression and subcellular localization than on substrate specificity embedded in their
molecular structure (reviewed in [2]).
Cdk activity is tightly regulated by various molecular mechanisms [3] that include regulatory phosphorylation, differential
expression and/or localization, and interaction with regulatory
proteins, such as Ckis (Cdk inhibitors), which inhibit Cdk activity
by binding to Cdkcyclin complexes.
In budding yeast, the Cki Sic1 blocks the activity of Clb-containing complexes: both Cdk1/Clb5,6 required for DNA replication, and Cdk1/Clb1,2 required in mitosis [4]. The S-Cdk1
(Cdk1/Clb5,6) kinase activity required for the initiation of DNA
replication is present only when Sic1 is removed [4]. Sic1 is

bitory patterns obtained using histone H1 or GST (glutathione

S-transferase)pRb (retinoblastoma protein) fusion protein as
substrate (the latter of which recognizes both the docking site and
the catalytic site of Cdk2cyclin A) offers interesting suggestions
for the inhibitory mechanism of Sic1. Finally, overexpression of
the KIP1 gene in vivo in Saccharomyces cerevisiae, like overexpression of the related SIC1 gene, rescues the cell cycle-related
phenotype of a sic1 strain. Taken together, these findings strongly indicate that budding yeast Sic1 and mammalian p27Kip1 are
functional homologues with a structurally conserved inhibitory
domain.
Key words: cell division cycle, cyclin-dependent kinase inhibitor
(Cki), molecular modelling, p27Kip1 , Sic1, surface plasmon
resonance.

degraded by ubiquitin-dependent proteolysis that is regulated by

multi-site phosphorylation performed by Cdk1/Cln1,2; this targets Sic1 to be specifically recognized by the F-box protein Cdc4,
which recruits Sic1 for ubiquitination by the Cdc34SCF (Skp1/
Cullin/F-box) complex [5]. Deletion of Sic1 causes premature
DNA replication from fewer origins, extension of the duration
of S phase and inefficient separation of sister chromatids during
anaphase. The ensuing double-strand breaks generate genomic
instability and gross chromosomal rearrangements. Delaying
S-Cdk1 activation rescues defects in both S and M phases [6].
Despite the well-documented relevance of Sic1-mediated inhibition of S-Cdk1 for cell cycle control and genomic instability,
the molecular mechanism by which Sic1 inhibits S-Cdk1 activity
remains obscure. Sic1 has been proposed to be a functional homologue of the mammalian Cki p21Cip1 [7], which in turn is characterized by significant sequence similarity with the Cki p27Kip1
(42 % identity). Progression through S phase in mammalian cells
is promoted by a Cdk2cyclin A complex, whose activity is
inhibited by p27Kip1 . The X-ray structure of the inhibitor domain
of p27Kip1 bound to the Cdk2cyclin A complex has been reported
[8]. The three-dimensional structure reveals that the N-terminus
of p27Kip1 is extended over the surface of the Cdk2cyclin A
complex, forming hydrophobic contacts with regions on both
cyclin and kinase. The inhibitory mechanism of p27Kip1 is multifaceted: it occupies a secondary substrate recruitment site on
cyclin A; it binds to the N-terminal lobe of Cdk2, flattening it
out and disrupting the active site; and it inserts itself into the
ATP binding pocket, blocking ATP binding to Cdk2 [8]. The fact

Abbreviations used: Cki, cyclin-dependent kinase inhibitor; Cdk, cyclin-dependent kinase; S-Cdk1, Cdk1/Clb5,6 (Cdk activity required to start DNA
replication); GST, glutathione S-transferase; pRb, retinoblastoma protein; SPR, surface plasmon resonance.
1
To whom correspondence should be addressed (email [email protected]).


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M. Barberis and others

that the inhibitory domain of Sic1 [9] does not have significant
sequence identity with either p27Kip1 or p21Cip1 (as shown in this
paper) has hampered a comparative analysis of the relationship
between the structure and function of Cdk and Cki in budding
yeast and in mammalian cells, despite remarkable similarity and
functional interchangeability of their cyclins and Cdk [1,2].
Here we show that the inhibitory domain of Sic1 is functionally
and structurally related to the inhibitory domain of mammalian
p27Kip1 of the Kip/Cip family. Molecular modelling of the inhibitory domain of Sic1 suggested that the protein interface of
the yeast inhibitor should be able to interact productively with the
mammalian Cdk2cyclin A complex. Consistently, Sic1 was
shown to bind to, and strongly inhibit the kinase activity of, the
Cdk2cyclin A complex. In addition, comparison of the different
inhibitory patterns obtained using as substrates histone H1 or
GST (glutathione S-transferase)pRb (retinoblastoma protein),
the latter of which recognizes both the docking site and the catalytic site of Cdk2cyclin A, offers interesting suggestions for the
inhibitory mechanism of Sic1. The physiological relevance of
these experimental results was confirmed by overexpressing the
KIP1 gene in vivo in Saccharomyces cerevisiae. The yeast and
mammalian Ckis in fact showed equivalent ability to rescue the
cell cycle-related phenotype of a sic1 strain. Taken together,
these findings strongly indicate that Sic1 from budding yeast and
mammalian p27Kip1 are functional homologues with a structurally
conserved inhibitory domain.

EXPERIMENTAL
Molecular modelling of Sic1 protein

Initial alignments of the Sic1 sequence (SwissProt accession number P38634) were carried out using CLUSTALW [10], and secondary structures were predicted using the PHD [11], PSIPRED
[12] and JPRED [13] methods. A partial model of the Sic1 inhibitory domain (residues 215284) was obtained by using the
Swiss-Pdb Viewer 3.7 program [14] performing in silico mutagenesis of the Cdkcyclin-interacting portion of p27Kip1 , abridged
from the structure of the p27Kip1 Cdk2cyclin A complex (PDB ID
1JSU). The Sic1 model was docked on the Cdk2cyclin A complex, and the model of the ternary complex was subsequently
refined by 5000 steps of conjugate gradients energy minimization using the GROMOS force field [15].
Recombinant DNA techniques

Escherichia coli DH5 strain was used as host cell in cloning

experiments, and standard recombinant DNA manipulations were
performed [16]. E. coli BL21 (DE3)[pLysE] strain was used to
generate recombinant Sic1His6 protein.
Plasmid pIVEX2.4a containing the SIC1 gene was generated by
cloning the entire open reading frame of the SIC1 gene obtained
as a BamHIBamHI fragment from plasmid pQE-30 Xa (Qiagen)
into the BamHI restriction site of pIVEX2.4a MCS. Plasmid
pEMBLyex4 containing the SIC1 gene under the control of the
GAL1 promoter (pyex-SIC1) was generated by cloning the entire
open reading frame of the SIC1 gene with one haemagglutinin
epitope, obtained as an EcoRIXbaI fragment from plasmid 2801
(kindly provided by E. Schwob, Institute of Molecular Genetics,
CNRS UMR 5535 and Universite Montpellier II, Montpellier,
France), in plasmid pEMBLyex4 MCS. Plasmid pEMBLyex4
containing the KIP1 gene under the control of the GAL1 promoter
(pyex-KIP1) was generated by a fusion of the SalIBamHI GAL1
promoter fragment to the KIP1 open reading frame, cloned as an


c 2005 Biochemical Society

EcoRIBamHI fragment derived from a pcDNA3-derived plasmid

carrying the whole KIP1 open reading frame (Invitrogen).
The yeast strain used in this study was 4245 (MATa, ade2-1,
leu2-3, ura3, trp1-1, his3-11, GAL, psi + , bar1::LEU2, sic1::
HIS3, can1-100), kindly supplied by E. Schwob. The strain was
subsequently transformed with empty plasmid pEMBLyex4 or
with the pyex-SIC1 and pyex-KIP1 derivatives. Yeast cells were
transformed by a modification of the lithium acetate procedure
[17].
Yeast growth conditions

Yeast cells transformed with pEMBLyex4, pyex-SIC1 or pyexKIP1 plasmids were grown at 30 C in a synthetic medium containing 6.7 g/l YNB (yeast nitrogen base) without amino acids
(Difco), 100 mg/l adenine, 50 mg/l leucine and tryptophan, and
2 % (w/v) galactose as carbon source to induce expression of
plasmid-borne genes.
The cell density of liquid cultures was determined with a
Coulter counter using mildly sonicated and diluted samples. The
fraction of budded cells was scored microscopically from samples
of at least 300 cells after mild sonication.
Flow cytofluorimetric analysis

A total of 2 107 cells in mid-exponential growth [cell density

approx. (28) 106 cells/ml] were harvested by filtration, mildly
sonicated, fixed in 70 % (v/v) ethanol and subsequently processed
for FACS analysis. Cells were washed three times with PBS,
resuspended in 1 ml of PBS with 1 mg/ml RNAse and incubated
overnight at 37 C. After incubation, cells were washed once with
PBS, resuspended in DNA staining solution (0.46 mM propidium
iodide/50 mM Tris/15 mM MgCl2 , pH 7.7) and incubated in ice
in the dark for 30 min. All centrifugations were performed at
18 000 g for 5 min at 4 C. Cell suspensions were transferred
into FACS Falcon tubes and sonicated prior to FACS analysis.
To measure cell size, forward scatter was recorded using the
same cells. Analyses were performed with a FACScan (Becton
Dickinson), and plots were generated with WinMDI 2.8.
Production and purification of recombinant proteins
and protein complex

E. coli strain BL21 (DE3)[pLysE] was transformed with plasmid

pIVEX2.4a-SIC1, cultured in LuriaBertani broth with 100 g/
ml ampicillin and 34 g/ml chloramphenicol at 37 C (A600 = 0.5),
and induced for 4 h with 50 M isopropyl -D-thiogalactoside
at 25 C. Sic1His6 protein was purified on Ni2+ /nitrilotriacetate
beads as described in the QIA Expressionist Handbook (Qiagen)
and eluted with 100 mM imidazole. Protein concentration was
measured by the Bradford method using a Bio-Rad protein assay
kit. The purified protein was stored at 20 C in 50 mM Tris/HCl,
pH 7.5, and 100 mM NaCl.
Baculovirus cells were transformed with plasmids pVL1392GST-Cdk2 and pVL1392-GST-cyclin A separately to express
Cdk2 or cyclin A proteins, or by co-expression of the two plasmids for expression of the Cdk2cyclin A complex. GSTCdk2
and GSTcyclin A proteins and the GSTCdk2cyclin A kinase
complex were purified on glutathioneSepharose 4 Fast Flow
(Amersham Biosciences) and subsequently cleaved with PreScissionTM Protease (Amersham Biosciences) to remove the GST
tags. PreScissionTM and cleaved GST tags were removed by rechromatography on glutathioneSepharose 4 Fast Flow. Purified
proteins were kindly provided by Dr Simon Plyte (PharmaciaPfizer, Nerviano, Milan, Italy).

Yeast and mammalian Cdk inhibitors share a conserved inhibitory domain

Figure 1

641

Sequence alignment and secondary structure predictions for p27Kip1 , p21Cip1 and Sic1 inhibitory domains

(A) Schematic view of the full-length Sic1 protein, indicating the sites of phosphorylation by Cdk1 (black lines) and the Cdk1-inhibitory domain (grey). (B) Secondary structure predictions computed
by PHD, PSIPRED and JPRED programs for the inhibitory domains of p27Kip1 (residues 3860), p21Cip1 (residues 2749) and Sic1 (residues 215284) are shown (H, -helix; c, coil; E, -sheet).
Residues involved in binding to either Cdk2 or cyclin A are underlined. Residues predicted to be in an -helix within the Cdk2cyclin A-interacting region are shown as white-on-black characters.
Secondary structure deduced from X-ray crystallography is shown for p27Kip1 . Corresponding expected secondary structures for p21Cip1 and Sic1 in the Cdk2cyclin A complex are also shown.

BIAcore analysis

A BIAcore X system was used to analyse molecular interactions

by means of SPR (surface plasmon resonance) [18]. Sic1 protein
was covalently coupled to a Sensor Chip CM5 (carboxymethylated dextran surface), using amine-coupling chemistry [19]. A
surface density of 3500 resonance units was generated for Sic1
protein by using 100 l of a 50 g/ml protein solution in acetate
buffer, pH 3.5. Solutions of each interacting protein (Cdk2, cyclin
A or their complex) were injected over the surface at 25 C in a
volume of 35 l at a flow rate of 10 l/min in HBS running buffer
[10 mM Hepes, pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.005 %
(v/v) surfactant P20]. After injection, protein solutions were replaced by HBS at a continuous flow rate of 10 l/min. Surface
regeneration was accomplished by injecting successively 1 M
NaCl, 100 mM NaOH and 10 mM HCl (1 min contact time). All
injected solutions were run simultaneously over a control flow cell
containing a blank surface (with no immobilized protein). The
response observed in the control flow cell was subtracted for each
sensorgram (time course of the SPR signal) and the baseline was
normalized to 0 resonance units. Apparent kinetic (kon and koff )
and thermodynamic (K D = koff /kon ) parameters of the interactions
studied were obtained by simultaneous fitting of binding curves
obtained with different concentrations of each interacting protein

or protein complex using the BIAevaluation 3.1 SPR kinetic software (BIAcore).
In vitro Cdk2 kinase assays

In vitro assays of the phosphorylation activity of the Cdk2

cyclin A kinase complex were run in a final volume of 30 l
of 50 mM Tris/HCl, pH 7.5, 12 mM MgCl2 , 1 mM dithiothreitol,
40 nM Cdk2cyclin A kinase complex and an appropriate concentration of substrate: histone H1 (Roche) from 1 to 30 M or GST
pRb (residues 769921) [Rb (769); sc-4112; Santa Cruz Biotechnology, Inc.] from 0.5 to 8 M. When required, an appropriate
Sic1 concentration (from 1.5 to 5 M) was added. The reaction
was started by adding 500 M [ -32 P] ATP (specific radioactivity
2000 c.p.m./pmol) and stopped after a 10 min incubation at 30 C
by cooling samples on ice and adding 4 SDS sample buffer
[0.25 M Tris/HCl, pH 6.8, 40 % (v/v) glycerol, 9.2 % (w/v) SDS,
4 % (v/v) -mercaptoethanol, 0.01 % (w/v) Bromophenol Blue].
Proteins were separated by SDS/PAGE on 12 % (w/v) polyacrylamide gels. Phosphorylated bands were identified by autoradiography and quantified using Scion Image software (Scion Corp.)
on a 256 grey level Tiff-scanned image of the autoradiographic
film.


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Figure 2

M. Barberis and others

Molecular visualization of surface interactions of p27Kip1 and Sic1 inhibitory domains with the Cdk2cyclin A complex

-Helices of p27Kip1 (residues 3860) (A) and Sic1 (residues 226248) (B) are shown in red; Cdk2 and cyclin A are visualized in green and blue respectively. (C) Analysis of the interface contacts
of p27Kip1 and Sic1 long amphipathic -helixes with the Cdk2cyclin A complex were conducted within the 5 A range. The indicated residues of the Sic1 -helix are involved in the same contacts
as the corresponding residues of the p27Kip1 -helix (see Figure 1B) with regard to the Cdk2cyclin A complex.

Kinase activities of Cdk2cyclin A towards histone H1 and

GSTpRb, in the presence or absence of Sic1, were analysed with
GraphPad Prism software. Kinetic parameters were calculated by
fitting the data to the MichaelisMenten equation. For histone H1
kinase assays only, the K i value was calculated by plotting the
apparent K m values obtained against the cognate Sic1 concentration.
Protein extraction and immunoblotting

A total of 2 108 exponentially growing cells were harvested

by filtration and lysed using ice-cold HB1 buffer [25 mM Mops,
pH 7.2, 15 mM MgCl2 , 15 mM EDTA, 0.5 % (v/v) Triton X-100]
plus protease inhibitor mix (Complete EDTA-free Protease Inhibitor Cocktail Tablets; Roche). An equal volume of acid-washed
glass beads (Sigma) was added, and cells were broken by ten
vortex/ice cycles of 1 min each. Extracts were clarified by centrifugation. Protein concentration was measured by the Bradford
method using a Bio-Rad protein assay kit. Typically, samples of
70100 g of protein were used for direct Western blotting. Protein extracts were separated using a 12 % (w/v) polyacrylamide
gel, and Western blots to detect Sic1 or p27Kip1 proteins were
performed with purified anti-Sic1 polyclonal antibody kindly
provided by Dr Oriano Marin (Dipartimento di Chimica Biologica, Universit`a di Padova, Padova, Italy) and anti-p27Kip1
monoclonal antibody (Transduction Laboratories) respectively
(1:1000 dilution). An enhanced chemiluminescence system


c 2005 Biochemical Society

(ECL Western Blotting Detection Reagents; Amersham Biosciences) was used for antibody detection after immunoblotting.
RESULTS
The inhibitory domain of yeast Sic1 is structurally related
to that of a mammalian Cki

Sic1 has no obvious sequence homologues in the databanks, as

indicated by the very low scores obtained using BLAST [20],
PSI-BLAST [21] and FASTA [22] (results not shown). Due to its
very low sequence similarity to p27Kip1 , conventional homology
modelling could not be applied to Sic1. Analysis of the inhibitory domain of Sic1 by fold recognition methods such as
GenTHREADER [23,24] and 3D-PSSM [25,26] did not identify
any suitable compatible fold (results not shown). Since it has
been suggested that Sic1 may be a functional homologue of Ckis
of the Kip/Cip family [7], we reasoned that local properties at the
interaction surface with the Cdkcyclin complex might be conserved. Secondary structure predictions for the sequence corresponding to the inhibitory domain of Sic1 (residues 215284 [9]),
as well as for the corresponding domains of p27Kip1 (residues
2593 [8]) and p21Cip1 (residues 1482 [8]), were computed as
described in the Experimental section, and compared with the
secondary structure deduced from the X-ray structure of p27Kip1
(Figure 1B). This analysis revealed that the secondary structures
of these protein domains should be very similar. Notably, a long

Yeast and mammalian Cdk inhibitors share a conserved inhibitory domain

Figure 3

643

Analysis of the binding of Sic1 to Cdk2, cyclin A and the Cdk2cyclin A complex by BIAcore

Sic1 was immobilized on the sensor chip and the binding curves were recorded by flowing the analytes Cdk2 (A), cyclin A (B) and the Cdk2cyclin A complex (C) on the chip surface. (D)
Values for apparent association rate (k on ), dissociation rate (k off ) and affinity constant (K D ) were obtained by fitting of data in (A)(C), as detailed in the Experimental section. (E) Analysis of
stoichiometry of binding between Sic1 and the analytes, obtained by plotting ln [d(RU)/d(time)] against time. Trend line(s) approximating each curve are also shown. The original graph is shown to the
right.

-helix predicted in Sic1 (residues 226248) shares a similar

amphiphilic profile with the corresponding -helix of p27Kip1 and
p21Cip1 .
On the basis of secondary structure predictions, the inhibitory
domain of Sic1 was modelled using the corresponding domain of
p27Kip1 as a template, and the -helix of p27Kip1 (residues 3860;
Figure 2A) was mutated in silico to generate the predicted -helix
of Sic1 (residues 226248). The Sic1 model was then docked on to
the Cdk2cyclin A binary complex and optimized by energy minimization, as described in the Experimental section (Figure 2B).

Analysis of the ternary complex reinforced the reliability of this

structural prediction. In fact, the interface between the inhibitory
domains of Sic1, Cdk2 and cyclin A is characterized by proper
steric and electronic contacts that should allow the formation of
a stable ternary complex. Five residues within the amphipathic
-helix of p27Kip1 establish hydrophobic contacts with either cyclin A or Cdk2 [8]; corresponding amino acids in the predicted Sic1
-helix make contact with a significant sub-group of the interacting residues in the computed model (Figure 2C). For instance, the
hydrophobic properties of residue Leu41 of p27Kip1 positioned for


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M. Barberis and others

kinetic (kon and koff ) and thermodynamic (K D = koff /kon ) parameters

of the studied interactions were determined by fluxing several
concentrations of each chosen interactor. Typical sensorgrams
obtained using Cdk2, cyclin A or the Cdk2cyclin A complex are
reported in Figures 3(A)3(C) respectively. Apparent kinetic and
thermodynamic parameters obtained after fitting sensorgrams are
summarized in Figure 3(D). These data indicate that the affinity of
Sic1 for Cdk2 is very low, while binding of Sic1 to cyclin A and,
much more so, to the Cdkcyclin complex is more favourable.
These findings suggest that Sic1 could realize its inhibitory function by interacting first with the cyclin and then extending on the
surface of the kinase complex to reach the binding site on
the kinase. A two-step mechanism of binding of Sic1 to the Cdk2
cyclin A complex is supported by the secondary plot of
ln [d(RU)/d(time)] against time (where RU = response) derived by
sensorgrams shown for clarity on the side of the secondary plots
(Figure 3E). The interactions of Sic1 with cyclin A and Cdk2 individually show a curve with a unique slope (indicative of a single
binding site), whereas the interaction with the Cdk2cyclin A
complex is better described by a curve with two slopes, indicative
of Sic1 making contact with two sites on the complex.
Inhibition by Sic1 of Cdk2cyclin A kinase activity

Figure 4

Inhibition of Cdk2cyclin A kinase activity by Sic1

Cdk2cyclin A kinase activity was measured with various histone H1 (A) or GSTpRb
(B) concentrations at several fixed Sic1 concentrations: 0 M (), 1.5 M (), 3.5 M ()
and 5 M (). The ATP concentration used in the assay was 500 M, and the Cdk2cyclin A
concentration was 40 nM.

specific interaction with the cyclin subunit of the Cdk2cyclin A

complex [8] are conserved in Sic1 by residue Ile229 , supporting
the notion that the three Ckis share a common structural fold in
the Cdkcyclin-interacting region. Interestingly, residue Arg233 of
Sic1, although not itself hydrophobic, appears able to be threaded
within the cyclin A structure in the correct orientation, making
use of the alkyl moiety to effect specific hydrophobic interactions.
In summary, these findings indicate that, despite their low sequence similarity, the inhibitory domains of yeast Sic1 and mammalian p27Kip1 may be structurally related.
The yeast Sic1 protein binds to the mammalian
Cdk2cyclin A complex

The results reported above predict that the inhibitory domain

of Sic1 has the ability to interact with the mammalian Cdk2
cyclin A complex. To test this hypothesis directly, we tested the
interactions of Sic1 with mammalian Cdk2 and cyclin A (alone
or in complex) by SPR using a BIAcore X. In all experiments
described, Sic1 was immobilized on the sensor chip and apparent


c 2005 Biochemical Society

To investigate the mechanism of inhibition of Cdk2cyclin A

activity by Sic1, a kinetic analysis using the purified proteins was
conducted using either histone H1 or GSTpRb as substrate, as
described in the Experimental section. Substrate recruitment to
Cdk2 has been shown to rely upon interactions with a docking site
located on cyclin A for substrates such as E2F1, p21Cip1 , p27Kip1 ,
p107 and pRb [27], while histone H1, which is also phosphorylated by Cdk2cyclin A, binds only to the catalytic site on Cdk2
and not to the docking site [28]. It was therefore of interest to
compare the effects of adding Sic1 to two assays of Cdk2cyclin
A activity, one utilizing pRb as substrate and the other using histone H1.
Since it has been shown that pRb contains in the C-terminal region (beginning at residue 870) the motif able to bind the docking
site, a commercially available GSTpRb fusion protein (spanning
residues 769921 of pRb), which contains both the cyclin A-binding motif and Cdk/cyclin phospho-acceptor sites [27], was used
in the assay.
The kinase activity of Cdk2cyclin A towards histone H1 and
GSTpRb was determined by in vitro kinase assays in the presence
of [ -32 P]ATP. The initial velocities of histone H1 and GSTpRb
phosphorylation, obtained in the presence of a fixed ATP concentration (500 M) at different Sic1 concentrations, were calculated
by densitometry and plotted as a function of substrate concentration. Experimental data for the uninhibited and inhibited reactions are reported in Figures 4(A) (histone H1 as substrate) and
4(B) (GSTpRb as substrate), together with the corresponding
MichaelisMenten curves. The histone H1 kinase assay indicated
that, under our experimental conditions, Sic1 is acting as a competitive inhibitor. The apparent K m values of the Cdk2cyclin A
complex were used to calculate the K i in a secondary plot of apparent K m against Sic1 concentration. The obtained K i value for Sic1
(500 nM) was found to be of the same order of magnitude as the
K D value obtained by BIAcore analysis (Figure 3D). In contrast,
the GSTpRb kinase assay showed that both kinetic parameters
(V max and K m ) changed in the presence of Sic1, with a very marked
decrease in the V max value (Figure 4B).
In summary, our analysis shows that Sic1 presents different
inhibitory patterns with regard to the Cdk2cyclin A complex depending on whether histone H1 or GSTpRb protein is used as
substrate.

Yeast and mammalian Cdk inhibitors share a conserved inhibitory domain

Figure 5

645

Overexpression of KIP1 rescues the cell cycle-related phenotype in a sic1 strain

sic1 strains transformed with the URA3 -based plasmid pEMBLyex4 or the same plasmid expressing the SIC1 and KIP1 genes under the control of a galactose-inducible promoter were grown in
a synthetic medium containing 2 % (w/v) galactose, collected in mid-exponential phase (approx. 4 106 cells/ml) and scored for expression of Sic1 (A) or p27Kip1 (B) protein by immunoblots
with antibodies as described in the Experimental section, for the fraction of unbudded cells (C) by direct microscopic examination, for the fraction of cells in G1 by FACS analysis (C and D),
and for cell size by forward scatter (E). Data in (C) are means +
S.D. of at least three independent experiments. Representative DNA and size distributions and immunoblots are shown in the
other panels.

Overexpression of KIP1 in yeast rescues the cell cycle-related

phenotype of a sic1 strain

To ascertain the physiological significance of promiscuous interactions among yeast and mammalian Ckis, Cdk and cyclins, the
ability of the yeast and mammalian Ckis to rescue the cell cyclerelated phenotype of yeast cells deleted for the SIC1 gene was
examined. Cells carrying disruption or deletion of SIC1 are viable
[29], but display phenotypes indicative of aberrant cell cycle
progression. In fact, cultures of sic1 cells have a low fraction
of G1 unbudded cells and display an altered cell size ([30] and
Figure 5). The effects of Sic1 and p27Kip1 overexpression, driven
by a GAL1 promoter (Figures 5A and 5B), on cell cycle-related
parameters were thus investigated in cells growing exponentially
in galactose-supplemented media.
Overexpression of either Sic1 or p27Kip1 significantly increased
the fraction of unbudded cells (Figure 5C) and cells with a G1 DNA
content (Figures 5C and 5D), as one may expect for a Cki that
expresses its function at the G1 /S transition. Analysis of forward
scatter (a measure of cell size) showed that overexpression of
either Sic1 or p27Kip1 resulted in a shift in the size distribution.
Figure 5(E) shows that sic1 cells expressing either the yeast or
the mammalian Cki had size distributions shifted towards larger
cells compared with pEMBLyex4-transformed sic1 cells.
DISCUSSION

In budding yeast, Sic1 blocks the activity of the S-Cdk1 kinase required for the initiation of DNA replication, the onset of which
requires removal of Sic1 [4]. A relevant role for Sic1 in the control
of the G1 -to-S transition is indicated by the fact that deletion of

Sic1 causes premature DNA replication from fewer origins (sparse

DNA firing), extension of S phase duration and inefficient separation of sister chromatids during anaphase. Genomic instability
is generated by the ensuing double-strand breaks and gross chromosomal rearrangements [6].
The novelty of our results lies in the indication that Sic1 is structurally and functionally related to mammalian p27Kip1 , a member
of the Kip/Cip family of Ckis, sharing a conserved inhibitory
domain. Moreover, we have collected evidence showing that Sic1,
like p27Kip1 , interacts with both the docking site and the catalytic
site of the Cdk2cyclin A complex.
The lack of sufficient sequence identity between Sic1 and
p27Kip1 , and the failure of fold recognition methods to identify any
suitable compatible fold, hampered the possibility of using conventional modelling techniques. In order to ascertain the degree
of evolutionary conservation of the inhibitory domain of yeast
and mammalian Ckis, we used in silico mutagenesis guided by
the results of secondary structure prediction experiments to
build the putative Cdkcyclin-interacting helix of Sic1 and later
to dock it to the mammalian Cdk2cyclin A complex. Although
our docking experiment does not have a resolution high enough
to unambiguously assign inter-residue contacts, inspection of the
computed ternary complex (in a form after energy minimization
by molecular mechanics) suggested that the inhibitory domain of
Sic1 has a surface that is able to interact with both subunits of the
mammalian Cdk2cyclin A complex.
A sequential mechanism for the inhibition of Cdk2cyclin complexes has been proposed for Kip/Cip inhibitors on a structural
basis [8,31], and more recently confirmed by studies of binding of
p27Kip1 to Cdk2cyclin A by SPR and NMR [32]. The latter study


c 2005 Biochemical Society

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M. Barberis and others

showed that the intrinsic flexibility of the mammalian inhibitor in

solution affects the kinetics of its interaction with Cdk2, cyclin
A and the Cdk2cyclin A complex. In particular, the different affinities observed for binding of p27Kip1 to Cdk2 and cyclin A
strongly suggest that p27Kip1 recognizes and binds to the cyclin
A subunit of Cdk2cyclin A complex as the first step in a sequential mechanism, and then folds into an elongated structure that
extends to the kinase subunit [32].
Our computational data suggest that Sic1 may bind to the mammalian Cdk2cyclin A complex with a similar mechanism and
thus share a similar mode of inhibition with Kip/Cip proteins.
Consistently, the interaction of Sic1 with mammalian Cdk2 and
cyclin A (alone or in complex), tested by BIAcore, indicated that
the affinity of Sic1 for Cdk2 is quite low, while binding of Sic1 to
cyclin A and (to a much greater extent) to the Cdk2cyclin A complex is strong. In addition, secondary plots derived by BIAcore
sensorgrams indicate that Sic1 makes contact with two sites on
the complex.
A two-site mechanism for binding of Sic1 to the Cdkcyclin
complex is further supported by the results of experiments testing
the inhibition of pRb phosphorylation by Sic1. In fact, while
histone H1 (which lacks a cyclin A binding motif) at high concentrations overcomes the inhibitory activity of Sic1, pRb at high
concentrations is not able to reverse the inhibition by Sic1, the
major effect of which is thus a consistent decrease in the apparent
V max of the Cdk2cyclin A complex with regard to the pRb substrate (Figure 4B). Proteins that have a cyclin A binding motif,
such as pRb, have to interact first with the docking site on cyclin
A in order to be phosphorylated; in fact, both site-directed mutagenesis of the pRb cyclin binding motif and competition with a
short peptide spanning the Cdk2cyclin A binding motif present
in the E2F1 substrate prevents its phosphorylation by Cdk2 [27].
Thus binding of pRb to the catalytic site of the kinase may displace
the phosphorylatable Sic1 domain from the Cdk2 catalytic site,
without resulting in significant pRb phosphorylation, so explaining the observed decrease in apparent V max .
Cross and Jacobson [33] showed previously that Clb5 and
p27Kip1 interact in vivo in yeast in two-hybrid experiments,
and proposed that a conserved region of cyclins may provide interactions with their targets. Our experiments go one step further by
showing that the p27Kip1 protein can functionally substitute in vivo
for Sic1, since overexpression of the KIP1 gene in Saccharomyces
cerevisiae was able to rescue the cell cycle-related phenotype of a
sic1 strain, in a manner indistinguishable from overexpression
of the homologous yeast Cki gene. It should also be remembered
that Sic1 has been shown previously to be a functional homologue
of the Schizosaccharomyces pombe Cdk inhibitor Rum1, despite
the very limited sequence identity between the Ckis from the two
yeasts [34].
Taken together, the results presented in this paper support
the notion that Sic1, although not showing significant sequence
identity with p27Kip1 , shares a conserved inhibitory domain, interacts with the Cdkcyclin complex by a similar two-site mechanism and can be functionally replaced in vivo by p27Kip1 .
We particularly thank Professor Lorenzo A. Pinna for suggestions and critical reading
of the manuscript. We are grateful to Dr Oriano Marin for purified anti-Sic1 polyclonal
antibody from rabbit, and to Dr Simon Plyte (Pharmacia-Pfizer) for the kind supply of
Cdk2cyclin A kinase complex purified from baculovirus. This work was supported by
research grants AIRC, FIRB 2001 and PSO CNR-MIUR to L. A., and COFIN 2002 to M. V.

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Received 2 August 2004/1 December 2004; accepted 17 December 2004

Published as BJ Immediate Publication 14 January 2005, DOI 10.1042/BJ20041299

c 2005 Biochemical Society