From the CNRS UMR 8525, Institut de Biologie de
Lille/Pasteur Institute of Lille, 59019 Lille Cedex, France, the
§ Unité de Microbiologie, Faculté Universitaire
des Sciences Agronomiques de Gembloux, 5030 Gembloux, Belgium,
and
Laboratorium voor Genetica, Department
of Plant Genetics, Flanders Interuniversity Institute for Biotechnology
(VIB), Universiteit Gent, B-9000 Gent, Belgium
Received for publication, July 19, 2000, and in revised form, September 25, 2000
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ABSTRACT |
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The WW domain of the human PIN1 and
p13SUC1, a subunit of the cyclin-dependent kinase
complex, were previously shown to be involved in the regulation of the
cyclin-dependent kinase complex activity at the entry into
mitosis, by an unresolved molecular mechanism. We report here
experimental evidence for the direct interaction of p13SUC1
with a model CDC25 peptide, dependent on the phosphorylation state of
its threonine. Chemical shift perturbation of backbone 1HN, 15N, and
13C Conserved Ser/Thr kinase complexes drive progression through the
different cell cycle phases in eukaryotic cells. The complex consists
of a regulatory subunit, the cyclin, and a catalytic subunit, the
cyclin-dependent protein kinase
(CDK).1 This latter
specifically recognizes the serine/threonine-proline motif, and a
structural basis for the proline preference has been recently described
(1). A complex interplay of phosphorylation and dephosphorylation by
and of the complex regulates tightly the cell cycle; at the
G2/M transition, for example, the activation of the complex
requires dephosphorylation of the CDK Thr-14 and Tyr-15 residues by the
phosphatase CDC25. CDC25 is highly phosphorylated at mitosis, in part
by the CDK complex that carries out the up-regulation of CDC25
activity. The hyperphosphorylated CDC25 activates in turn the CDK
complex, creating a positive feedback loop.
The kinase complex contains, in addition to the cyclin and the CDK, a
small essential regulatory protein, called CKS
(cyclin-dependent kinase subunit), whose function is not
precisely known. Depletion and overexpression of CKS caused a
G2 delay or abolished entry into mitosis, and in the latter
case, accumulation of inactive kinase molecules phosphorylated on
Tyr-15 (2, 3). Moreover, in vitro CKS enhances the
phosphorylation of the CDC25, even though the kinase activity of the
CDK complex is not directly modified by the CKS binding, suggesting a
role for CKS in substrate recognition (4). A similar stimulation of
phosphorylation of a CDK substrate by CKS was demonstrated in the case
of the CDC27 substrate in the proteasome (5). Furthermore, the CKS from
Schizosaccharomyces pombe, commonly called the
p13SUC1 protein, can bind the proteasome in a
phosphorylation-dependent way (6). This evidence leads to a
commonly accepted picture where the CKS subunit of the CDK complex
targets the activated complex (7) to specific phosphoproteins, such as
the CDC25 phosphatase. The presence of a conserved anion-binding site
at the surface of CKS molecule, occupied in the crystal structure of
human p9CKSHs2 by a sulfate molecule (8, 9), gives some
structural basis to this hypothesis, although no structural data on CKS
with a bona fide substrate have been reported.
PIN1 is another essential regulator at mitotic entry that binds
mitosis-specific phosphoproteins such as CDC25 (10-14). The human PIN1
protein contains two domains, a 100-residue prolyl cis/trans-isomerase C-terminal catalytic domain
and a small N-terminal WW domain (15). The WW domain of the human PIN1
is responsible for the binding to the Thr(P)/Ser(P)-proline motifs
(16). The catalytic domain shows specificity for the Thr(P)/Ser(P)-Pro
bond. Both site-specific catalytic activity and binding are essential for PIN1 biological activity (16, 17). A functional interplay between
the PIN1 protein and the CDK complex was recently demonstrated, as PIN1
completely abolishes the stimulation of the CDK-mediated phosphorylation of CDC25 by CKS (4). Intriguingly, only the WW domain
seems to be involved in this antagonistic action (4). Similar to the
CKS protein, PIN1 does not affect the CDK kinase activity directly, at
least in vitro, suggesting that competition between CKS and
the WW domain for the phosphorylated substrate might be at the origin
of the antagonistic action of PIN1.
NMR spectroscopy is most suitable to investigate potential molecular
interactions and can lead to accurate values of interaction constants
but also to mapping of the residues implicated in the interaction (both
on the protein target and the peptidic ligand), kinetic parameters
related to interaction and/or catalysis, and eventually a structural
model of the interaction complex. We report here the results of our NMR
study to determine whether the CKS protein indeed binds phosphorylated
substrates, such as a model CDC25 peptide containing a Thr(P). We found
a phosphorylation-dependent binding of p13SUC1 to
the peptide substrate, and we mapped the interaction site by a chemical
shift perturbation method. The importance of the proline residue at
position +1 following the Thr(P), as in the threonine-proline motif
corresponding to the minimal CDK recognition site, was demonstrated.
The affinity of p13SUC1 for a phosphopeptide containing an
alanine/proline substitution at position +1 following the Thr(P) was
indeed significantly decreased. Finally, we checked whether the
antagonism between the WW domain of PIN1 and CKS for the
phosphorylation of CDC25 could be due to direct interaction between
both proteins or, alternatively, to their competition for binding the
phosphorylated substrate. No direct interaction was detected,
confirming that the competition for the same substrate is the
underlying molecular mechanism of the observed antagonism.
Protein Expression, Peptide Synthesis, and
Purifications--
p13SUC1 (Swissprot accession number
P08463) and p13PA90 were expressed in Escherichia coli
BL21(DE3) using the T7 promoter-based vector pRK172 (18).
p13SUC1 mutagenesis will be described elsewhere.
15N,13C-Labeled proteins were prepared by
growing cells in M9 minimal medium (19) with
15NH4Cl (1 g l NMR Spectroscopy--
All the NMR experiments were performed in
a buffer of 50 mM deuterated Tris-HCl, pH 6.3 (Cambridge
Isotope Laboratories), 100 mM NaCl, and 1 mM
DTT. The spectra were recorded at 20 °C on a Bruker 600-MHz DMX
spectrometer (Bruker, Karlsruhe, Germany). Sequential backbone
resonance assignment of p13PA90 was achieved using the following three
pairs of triple resonance (three-dimensional) experiments:
HNCA/HN(CO)CA, HNCO/HN(CA)CO, and 15N-edited HSQC
NOESY/15N-edited HSQC TOCSY as will be described
elsewhere.2 Increasing
amounts of unlabeled synthetic peptide of sequence EQPLpTPVTDL
(phospho-CDC25 peptide) or EQPLpTAVTDL (Ala/Pro-substituted phospho-CDC25 peptide) were added to a
[15N,13C]p13PA90 sample. Final concentrations
were successively 0.83/0.22, 0.82/0.40, 0.78/0.80, 0.70/1.7, 0.66/2.2,
and 0.66/3.5 mM for the p13PA90/phospho-CDC25 peptide
sample and 0.66/0.33, 0.66/0.66, 0.66/1.32, 0.66/2.0, and 0.66/3.3
mM for the p13PA90/Ala/Pro-substituted phospho-CDC25
peptide sample. 1H-15N heteronuclear single
quantum coherence (HSQC) spectra were recorded at each titration point.
The backbone 1HN, 15N, and
13C Mapping of the Phospho-CDC25-binding Site on
p13SUC1--
In a first phase of this work, we performed the
sequence assignment of the wild-type p13SUC1 molecule by triple
resonance spectroscopy on a 15N,13C doubly
labeled sample.2 However, due to conformational
heterogeneity, 10 amide functions proved to be broadened beyond
detection in the 1H-15N HSQC spectra. Mutation
of the
To analyze the interaction between p13PA90 and a phosphorylated
substrate, we titrated a synthetic phosphorylated peptide of CDC25 into
a 15N,13C-labeled p13PA90 protein sample, and
we observed chemical shift changes in the corresponding HSQC spectra
(Fig. 1, A and B,
and Fig. 2 A and
B). Peptide addition affected the p13PA90 amide
1HN and/or 15N chemical shift
values in either of the following ways. Gradual, be it large, chemical
shift changes were observed upon binding for some residues as follows:
His-26, Arg-30, Tyr-31, His-40, Thr-77, Leu-80, Gly-81, Phe-97, Arg-99,
and Glu-100 showed composite chemical shift perturbations above 0.5 ppm
(Fig. 1B and Fig. 2). Other signals of peptidic groups
disappeared from the spectra during the titration (Gln-78, Ser-79,
Trp-82, lateral chain of Trp-82) until an excess of ligand was added
(Fig. 1A). The Gln-78 amide group did not emerge until our
maximum molar ratio of 1:5. Although for one interacting system, the
thermodynamic (Kd) and kinetic
(kon and koff) parameters
are constant, the NMR behavior of the resonances can be quite different
according to the change in chemical shift that a nucleus undergoes upon
interaction. For nuclei that are not involved directly in the
interaction, we expect a relatively small chemical shift perturbation,
leading to the case of rapid exchange (kexch >
The interactions are mainly localized in the The CKS Protein Is Binding a Phosphothreonine within a
Threonine-Proline CDK Epitope--
As the motif threonine-proline is
the minimal recognition site of the CDK, we wanted to test if the CKS
had a similar specificity for the proline at position +1. The NMR
titration experiment was repeated with a modified phospho-CDC25 peptide
that contained an alanine instead of a proline at the position +1
following the Thr(P). The residues that showed major changes upon
binding of the original phospho-CDC25 peptide (Gln-78, Ser-79, and
Trp-82) were also affected by addition of the Pro/Ala-substituted
phospho-CDC25 peptide, but at an equivalent molar ratio of peptide to
p13PA90 protein, the chemical shift perturbations observed upon
addition of Pro/Ala-substituted phospho-CDC25 peptide were far inferior to the equivalent results obtained with the phospho-CDC25 peptide (Fig.
1 and Fig. 2B). The dissociation constant
Kd was estimated by a titration experiment, based on
the chemical shift change observed for 5 residues (Thr-77, Gln-78,
Gly-81, Trp-82, and Lys-98) to be 5.4 ± 1 mM, a
6-fold increase compared with the dissociation constant for the
phospho-CDC25 peptide (Fig. 2B). The proline residue at
position +1 is thus clearly involved in the recognition of the
substrate by the p13SUC1 protein, as could be expected for an
adaptor protein of the CDK complex to its substrate.
Competition of WW and p13SUC1 for Binding to a
Phosphorylated CDC25 Peptide--
We next asked whether
p13SUC1 and PIN1 interact through the WW and/or the catalytic
domain of the latter, affecting potentially the binding of
p13SUC1 to the phospho-CDC25 peptide. The resonances of p13PA90
in the 1H-15N HSQC were not significantly
affected by addition of an equimolar amount of WW domain alone, and no
catalytic activity of the prolyl cis/trans-isomerase domain
PIN1At from A. thaliana on the Glu-91
The WW domain of PIN1 was previously shown to bind to the phospho-CDC25
peptide (16). This interaction was indeed verified by NMR spectroscopy,
and the molecular contacts between the CDC25 peptide and the WW domain
agreed with the recent structural data (30) on the complex between PIN1
and a peptide representing a heptad repeat of the RNA polymerase II
C-terminal domain of the large
subunits.3 More importantly, we could follow the molecular
competition between PIN1 and CKS by adding the WW domain to a 0.8 mM [15N,13C]p13PA90/0.8
mM phospho-CDC25 sample. After addition of non-labeled WW,
resulting in final concentrations of 0.5 mM p13PA90/0.5
mM WW/0.5 mM phospho-CDC25 peptide, we observed
a significant decrease in the chemical shift perturbation of the
p13PA90 resonances (Fig. 4). The p13PA90
resonances observed in the presence of WW were shifted back at a
position roughly equivalent to the ones observed in the sample 0.8 mM p13PA90/0.25 mM phospho-CDC25. This showed that an important fraction of the phospho-CDC25 peptide is unavailable to p13PA90 binding, due to competitive binding of the WW domain.
Addition of the prolyl cis/trans-isomerase
catalytic domain PIN1At of A. thaliana to a
p13PA90/phospho-CDC25 sample to final concentrations of 0.35 mM p13PA90/0.35 mM PIN1At/1 mM
phospho-CDC25 did not result in significant modifications of the HSQC
spectra compared with the equivalent spectra of a 1:3
p13PA90/phospho-CDC25 sample without PIN1At, although the phospho-CDC25
peptide was a good substrate for the PIN1At enzyme (data not shown
(20)). We therefore conclude that the catalytic domain of PIN1At alone does not affect directly the binding of the p13PA90 to the
phospho-CDC25 peptide.
We have shown by NMR chemical shift perturbation mapping that
p13SUC1 is able to bind a phosphopeptide corresponding to a
fragment of CDC25 centered on Thr-48. As revealed by large chemical
shift perturbations, the residues involved in the binding correspond primarily to the conserved sulfate-binding site (Arg-30, Gln-78, Trp-82, and Arg-99) reported in an earlier structural study of the
p9CKSHs2 homologue (8, 9). The largest perturbations were
observed in the loop connecting the Based on our results, we propose that the stimulation of the CDC25
phosphorylation by the CDK complex in the presence of CKS (4) is
mediated by binding of the CKS to the CDC25 substrate. The estimated
dissociation constant of the order of 1 mM is rather low
but could be higher when the full protein rather than a peptide is the
substrate. A second factor contributing potentially to an increased
affinity might be the stabilization of the highly flexible It was recently shown that PIN1 completely abolishes the stimulation of
the CDK-mediated phosphorylation of CDC25 by CKS, although neither
p13SUC1 nor PIN1 affect directly the CDK activity, at least
in vitro (4). We first tested if PIN1 had any interaction
with the CKS protein. The conserved EP motif found in the Not only the catalytic domain but also the WW domain of human PIN1 does
not interact directly with p13SUC1. However, it does bind to
the same Thr(P)-Pro motif as CKS, classifying both of them as
proline-directed phosphothreonine-binding modules. This differentiates
them from another sequence-specific phosphopeptide-binding protein, the
14-3-3 protein (26, 27), that is also involved in CDC25 binding and
regulation of the entry into mitosis (28, 29) but binds an
Arg-Ser-X-Ser(P)-X-Pro motif, where X
can be any amino acid (27). The distinction can be further extended to
the structural level, with WW and CKS both being mainly The structure of the protein PIN1 in complex with a
phosphoserine-containing peptide shows that the proline is recognized by aromatic residues Tyr-23 and Trp-34 on the WW domain and that the
Ser(P) is in direct contact with residues Ser-16 and Arg-17 located in
the loop I connecting the strand Our data are consistent with a previous biochemical study that strongly
suggests that PIN1 could slow down the entry into mitosis by
competition with the CKS protein for binding to substrates of the CDK
complex (4). In vivo, PIN1 was shown to interact with CDC25
(13, 14), and the overexpression of CKS leading to accumulation of
inactive kinase molecules phosphorylated on Tyr-15 indicates a direct
interaction between CKS and CDC25 (2, 3). Moreover, recent studies have
shown that the PIN1 protein regulates negatively the entry into mitosis
(10, 13, 14) and is necessary for the replication checkpoint control
(17). Therefore, although the previously described in vitro
competition for the same substrate (4) and our direct interaction
mapping do not prove of itself that PIN1 and CKS compete in
vivo, the combined evidence points toward a balance between both
proteins with the level of phosphorylation of mitotic phosphoproteins
ensuring a precise control of the timing of entry into mitosis. The
role of the prolyl cis/trans-isomerase catalytic
domain remains an unclarified point, as we were not able to detect any
effect of this domain on binding of phosphorylated substrate by the
CKS. The entire PIN1 protein is necessary to perform its essential function in vivo (34) and to ensure the replication
checkpoint at mitosis (16, 17), and this point will be the subject of further research.
resonances during NMR titration experiments allows
accurate identification of the binding site, primarily localized around the anion-binding site, occupied in the crystal structure of the homologous p9CKSHs2 by a sulfate molecule. The epitope
recognized by p13SUC1 includes the proline at position +1 of
the phosphothreonine, as was shown by the decrease in affinity for a
mutated CDC25 phosphopeptide, containing an alanine/proline
substitution. No direct interaction between the PIN1 WW domain or its
catalytic proline cis/trans-isomerase domain
and p13SUC1 was detected, but our study showed that in
vitro the WW domain of the human PIN1 antagonizes the binding of
the p13SUC1 to the CDC25 phosphopeptide, by binding to the same
phosphoepitope. We thus propose that the full
cyclin-dependent kinase complex stimulates the
phosphorylation of CDC25 through binding of its p13SUC1 module
to the phosphoepitope of the substrate and that the reported WW
antagonism of p13SUC1-stimulated CDC25 phosphorylation is
caused by competitive binding of both protein modules to the same phosphoepitope.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1) and
13C glucose (2 g l
1) (Cambridge
Isotope Laboratories, Cambridge, MA) as the sole nitrogen and carbon
sources, respectively. P13PA90 was purified by anion exchange with a
Q-HyperD column (Biosepra, Marlborough, MA) equilibrated in 50 mM Tris-HCl, pH 8.0, followed by gel filtration with a
Superdex 200 column (Amersham Pharmacia Biotech) in Tris-HCl, 50 mM, pH 8.0, and finally by reverse phase chromatography
with a Poros 50R1 column (Perspective Biosystems, Framingham, MA)
equilibrated in 0.1% trifluoroacetic acid and developed with an
acetonitrile gradient. Synthesis of phosphopeptides is as described
(20). The 10-amino acid phospho-CDC25 peptide used in this study is derived from the conserved Thr(P)-Pro site at Thr-48 of CDC25 of
Xenopus laevis (Swissprot accession number P30309);
the exact sequence is QPLpTPVTDL. The human WW domain from PIN1
(Swissprot accession number Q13526) was obtained by chemical
synthesis and purified by reverse phase with a C18 Hyperprep column.
resonances of the ligand-bound p13PA90 protein were
unambiguously assigned by three-dimensional HNCA experiments. The
binding constant was calculated by fitting the formula,
ppm = 0.5
ppmmax (1 + X + Kd/[p13PA90)
((1 + X + Kd/[p13PA90])2
4X)1/2 to the observed resonances, with
ppm
as the observed 1HN and 15N
combined chemical shift (see Fig. 2 legend),
ppmmax as
the maximum shift at saturation with the peptide, Kd
as the dissociation constant, and X as the molar ratio of
phosphorylated peptide on protein concentration [p13PA90]. Control
experiments were performed by adding 1 mM EQPLTPVTDL
(unphosphorylated control peptide) to a 0.27 mM
[15N,13C]p13PA90 sample or 2.2 mM
EQPLpTPVTDL to a 0.85 mM
[15N,13C]p13SUC1 sample. For the
phosphate titration, an increasing amount of sodium phosphate, pH 6.3, was added to a 2 mM p13PA90 sample. In the competition
experiment against the WW domain, the sample contained 0.48 mM p13PA90, 0.48 mM WW, and 0.48 mM
phospho-CDC25 peptide. To check the effect of the catalytic domain of
PIN, the samples contained 0.48 mM p13PA90 and 0.48 mM PIN1At from Arabidopsis thaliana (20) and
0.35 mM p13PA90/0.35 mM PIN1At/1 mM
phospho-CDC25.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-hinge Pro-90 to Ala, previously described to enhance the
stability of the protein (21), allowed us to almost complete the
assignment, with only Arg-39 that could not be attributed. All
resonances observed in both the wild-type p13SUC1 and the
mutant p13PA90 protein are virtually identical, confirming that both
proteins share a common three-dimensional structure. Chemical shift
analysis and observation of medium range nuclear Overhauser effect
signals indicated that the secondary structure in solution for both
proteins is similar to the crystal structure of the monomeric globular
form of p13SUC1 (22).2
), with the observation of an averaged resonance line (Fig. 1,
B-D). For resonances that undergo larger chemical shift
perturbations, intermediate exchange will contribute significantly to
the apparent line broadening. Upon saturation with ligand, all binding
sites become constitutively occupied, leading to reappearance of the
lines (Fig. 1A). Based on the gradual chemical shift
perturbation observed for 5 residues during the peptide titration
(residues Glu-37, Val-41, Gly-81, Glu-83, and Lys-98), we estimated the
affinity constant to be on the order of 900 ± 260 µM (Fig. 2B). The combined
1HN-15N perturbations were
confirmed by the chemical shift perturbation of the 13C
(data not shown), which also gave information on the proline residues.
None of the 7 prolines found in p13PA90 showed perturbation of the
13C
chemical shift upon phospho-CDC25 titration.
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Fig. 1.
Interaction of p13PA90 with the
phospho-CDC25 peptide (A and B) or
Ala/Pro-substituted phospho-CDC25 peptide (C and
D). Overlaid sections of 15N-HSQC
spectra acquired on p13PA90, before (in black contours) and
after addition of 0.25 (orange); 0.5 (red), 0.75 (pink), 1 (violet); 2 (dark blue); 3 (light blue) and 5 (green) molar equivalent of
phospho-CDC25 peptide (A and B) or
Ala/Pro-substituted phospho-CDC25 peptide (C and
D). Resonances are labeled. B-D, a gradual
chemical shift perturbation of residue Gly-81 (B and
D) or residue Trp-82 (C) can be observed, due to
fast exchange between bound and unbound form of p13PA90 on the NMR time
scale. A, an intermediate exchange leads to disappearance of
residue Trp-82 signal.
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Fig. 2.
Interaction of p13PA90 with the phospho-CDC25
peptide (A and B) or
Ala/Pro-substituted phospho-CDC25 peptide (B).
A, a plot of the combined chemical shift changes in backbone
amide 1HN and 15N of p13PA90 upon
binding of phospho-CDC25 peptide (3 molar equivalents)
versus the residue number of the protein. The combined
1HN and 15N shift changes were
calculated as (ppm) = ((
1HN)2 + 0.2·(
15N)2)1/2. The Gln-78
amide group, indicated by an asterisk, is only detected at a
molar ratio of 1:5. LC82 corresponds to the lateral chain of residue
Trp-82. Secondary structure elements are indicated in boxes
at the bottom (22). B, combined
1HN and 15N chemical shift
perturbation of residue Gly-81 (in ppm) observed upon addition of
increasing amount of phospho-CDC25 peptide (diamond) or
Ala/Pro-substituted phospho-CDC25 peptide (triangles) to
p13PA90. Solid line corresponds to the fitted function (see
"Experimental Procedure"). The molar ratio of peptide on protein
concentration is shown on the abscissa.
-sheet of p13PA90.
Large chemical shift modifications were observed for the conserved
surface residues forming the sulfate-binding site (8, 9), which are
residues Arg-30, Arg-39, Gln-78, Trp-82, and Arg-99 in p13SUC1.
These residues are most likely in direct contact with the
phosphorylated CDC25 peptide. The largest perturbations were observed
in the loop connecting helix-
3 and the
3-strand of the
-sheet
(Fig. 2A and Fig. 3). The
other smaller resonance perturbations corresponded to neighboring
residues, with similar small chemical shift perturbation of residues
located in the
3-helix (Glu-68, Glu-69, Glu-70, Arg-72, and Gly-73).
As a control, we also recorded an HSQC spectra of the p13SUC1
wild-type protein in the presence of an excess of phosphorylated CDC25
peptide, and we found identical perturbations as those of the mutant
protein for all the resonances that could be observed (data not shown).
The observed interaction is specific for the phosphorylated peptide, as
no modification of the p13PA90 resonances was observed upon addition of
the non-phosphorylated control peptide (data not shown). However, the
addition of inorganic phosphate did cause chemical shift perturbation
of the same residues implicated in the phospho-peptide binding. The
perturbations were, however, less important, and a binding constant of
40 mM was estimated. This indicated that in addition to the
phosphate group on the threonine, other contacts were involved in the
binding of the phosphopeptide by the p13PA90.
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Fig. 3.
Map of the chemical shift perturbation data
onto a three-dimensional Connolly solid surface model (left
panel) and a ribbon model representation (right
panel) of p13SUC1. The orientation of both
models is identical. Residues are colored according to a scale from
blue to white/white to red (0 < (ppm) < 2.75) based on observed
1HN and 15N combined chemical shift
perturbation on phospho-CDC25 peptide addition (3 molar equivalents).
The figure was generated with the program InsightII (Molecular
Simulations, Inc.). Crystallographic structure of
p13SUC1 is from Ref. 22.
Pro-92 motif of
p13PA90 was observed. This precludes a direct interaction between PIN1
and CKS to be at the basis of the functional antagonism between both
molecules (4).
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Fig. 4.
Competition of the human WW domain and
p13PA90 for binding a phospho-CDC25 peptide. Colors are as in Fig.
1, and resonances are labeled. The resonance represented in
yellow and indicated by an arrow was extracted
from a 15N-HSQC spectra of the sample 1:1:1
p13PA90/phospho-CDC25 peptide/WW.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
3-helix and the
3-strand
(Thr-77, Gln-78, Ser-79, Leu-80, Gly-81, and Trp-82). The other smaller observed perturbations correspond to neighboring residues in the
-sheet and to residues from helix-
3. The interaction was shown to
be dependent on the phosphorylation of the Thr and to involve the Pro
at position +1 following the Thr(P). Although the exact motif
recognized by the CKS could be even more specific, the Ser/Thr kinase
that generates this motif was previously shown to be proline-directed (1).
-sheet of
CKS by the binding to the CDK kinase. The affinity of the CKS for the
CDK complex is high (18) (the Kd is estimated to be
about 100 nM (23)), and most probably the CKS will only
interact with its substrate when it is itself bound to the CDK kinase.
Finally, if the CKS has to target the CDK complex to its substrate, a
high turnover might be necessary, with preference of weak over strong
binding constants.
-hinge of
the CKS could indeed be a potential substrate of the catalytic prolyl cis/trans-isomerase domain of PIN1 (11, 12). This
-hinge plays an important role in the binding of CKS to the CDK (24, 25) and in the stability and folding of the CKS protein
(21).2 We did not observe by NMR chemical shift
perturbation analysis any significant binding to or modification of
p13PA90 by the PIN1At catalytic domain of A. thaliana. The
PIN1At enzyme had also no effect on the binding of the phospho-CDC25
peptide by p13PA90, although we observed catalysis of the
cis/trans-isomerization of the Thr(P)-Pro bound
contained in the phospho-CDC25 peptide (20).
-sheet proteins that use their
-sheet to construct the binding site (16),
whereas the anion-binding site on the 14-3-3 protein is located on a
surface composed of
-helices (27).
1 and
2 (30). The recognition is
highly selective but of low affinity, consistent with our results.
Recognition of Pro-rich ligand peptides by a different type of WW
domain was shown to be similar to that found in SH3 complexes, although
both protein modules have different structures (31). That this might
result in a molecular competition is indicated by the polyproline
region of formin binding interchangeably to the WW or SH3 domain
of a formin-binding protein (32, 33). The WW domains of
formin might therefore regulate the function of the SH3 domains
by modulating their interaction with ligand peptides through direct
competition for the same Pro-rich sequence (32, 33). The presented
evidence of direct competition between the WW domain of PIN1 and CKS
for Thr(P)-Pro-containing peptides could in a very similar way regulate
the substrate binding of CKS and hence the CDK activity.
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ACKNOWLEDGEMENTS |
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We thank A. Kasprowiak and J.-S. Fruchart for peptide synthesis, Dr. E. Buisine for help with the image generation, and G. Schuurman-Wolters from the Department of Biochemistry of the University of Groningen, The Netherlands, for protein purification. The 600 MHz NMR facility used in this study was funded by the European community (FEDER), the Région Nord-Pas de Calais, the Center National de la Recherche Scientifique, and the Institut Pasteur de Lille.
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FOOTNOTES |
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* This work was supported by Tournesol Grant 98.110.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ Chargé de Recherches from the Fonds National de la Recherche Scientifique (Belgium).
To whom correspondence may be addressed. Tel.: 33 3 20871229;
Fax: 33 3 20871233; E-mail: isabelle.landrieu@pasteur-lille.fr.
** Present address: Biophysical Chemistry Dept., University of Groningen, 9747 Groningen, The Netherlands.
§§ To whom correspondence may be addressed. Tel.: 33 3 20871229; Fax: 33 3 20871233; E-mail: guy.lippens@pasteur-lille.fr.
Published, JBC Papers in Press, September 29, 2000, DOI 10.1074/jbc.M006420200
2 B. Odaert, I. Landrieu, G. Schuurman-Wolters, G. Lippens, and R. Scheek, manuscript in preparation.
3 R. Wintjens, G. Lippens, and I. Landrieu, submitted for publication.
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ABBREVIATIONS |
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The abbreviations used are: CDK, cyclin-dependent kinase; CKS, cyclin-dependent kinase subunit; HSQC, heteronuclear single quantum coherence; NOE, nuclear Overhauser effect; Ser(P)/Thr(P), phosphoserine/phosphothreonine.
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