From the Department of Pathology, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts 02115
Received for publication, June 29, 2000, and in revised form, October 26, 2000
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ABSTRACT |
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Cy or RXL motifs have been previously
shown to be cyclin binding motifs found in a wide range of
cyclin-Cdk interacting proteins. We report the first kinetic
analysis of the contribution of a Cy motif on a substrate to
phosphorylation by cyclin-dependent kinases. For both
cyclin A-Cdk2 and cyclin E-Cdk2 enzymes, the presence of a Cy motif
decreased the Km(peptide) 75-120-fold while the kcat remained unchanged. The large
effect of the Cy motif on the
Km(peptide) suggests that the Cy motif
and (S/T)PX(K/R) together constitute a bipartite substrate
recognition sequence for cyclin-dependent kinases.
Systematic changes in the length of the linker between the Cy motif and
the phosphoacceptor serine suggest that both sites are engaged
simultaneously to the cyclin and the Cdk, respectively, and eliminate a
"bind and release" mechanism to increase the local concentration of
the substrate. PS100, a peptide containing a Cy motif, acts as a
competitive inhibitor of cyclin-Cdk complexes with a 15-fold lower
Ki for cyclin E-Cdk2 than for cyclin A-Cdk2. These
results provide kinetic proof that a Cy motif located a minimal
distance from the SPXK is essential for optimal
phosphorylation by Cdks and suggest that small chemicals that mimic the
Cy motif would be specific inhibitors of substrate recognition by
cyclin-dependent kinases.
Timely progression through the cell cycle depends upon the well
orchestrated activation and deactivation of
cyclin-dependent kinases. Each of these kinases is active
for only a short period of the cell cycle during which time it
phosphorylates a number of substrates required for entry into the next
phase of the cell cycle. Cyclin
A-Cdk21 and cyclin E-Cdk2
both play a major role in the G1/S transition of the cell
cycle by the phosphorylation of various substrates including pRb, E2F,
and CDC6 (1, 2). Despite their critical role in this process, little is
known about how these substrates are targeted to specific cyclin-Cdk complexes.
Because the (S/T)PX(K/R) consensus phosphorylation site is
broadly applicable to all substrates of all Cdks, it would be incapable of conferring the substrate specificity seen within a cellular context. An alternate mechanism by which this specificity could be
achieved is through the presence of a docking site on the substrate that recruits the appropriate cyclin-Cdk to the protein. The resulting high localized concentration of the cyclin-Cdk then facilitates the
phosphorylation of potential Ser-Thr phosphorylation sites that have
been brought into close proximity. In previous work, we and others have
identified a sequence motif present in a number of cellular proteins
that interact with cyclin-Cdk complexes and could potentially perform
this function (3-5). These cyclin binding motifs (Cy or
RXL) have been found in substrates such as E2F and CDC6,
activators like Cdc25a and inhibitors of the p21-27 family, and are
absolutely required for the association of cyclin-Cdk complexes with
these proteins (3, 4, 6-8). The importance of this motif in the
interaction of these proteins with cyclin-Cdks is further highlighted
by the crystal structure of the Cdk inhibitor p27 complexed with cyclin
A-Cdk2 (9). In this structure, the N-terminal half of the inhibitor p27
was shown to associate with cyclin A-Cdk2 through two distinct regions
of the protein, a C-terminal region buried in the ATP-binding cleft of
the Cdk2 active site and a N-terminal Cy motif bound to a shallow
hydrophobic groove on the surface of the cyclin. Although there is no
structural evidence to confirm it, it seems likely that substrates
containing a Cy motif would bind in a fashion similar to the inhibitor.
The Cy motif of the substrate would bind to the same groove on the cyclin and allow potential phosphorylation sites on the protein to
associate with the nearby Cdk2 subunit and become phosphorylated.
One substrate that we propose acts in this fashion is the human
replication factor, CDC6. This factor is involved in the formation of a
prereplication complex and is required for the initiation of DNA
replication (10, 11). At the onset of S-phase mammalian CDC6 is
phosphorylated by cyclin A-Cdk2, which inactivates it by exporting it
from the nucleus into the cytoplasm (7, 12, 13). Phosphopeptide
analysis has shown that this phosphorylation by cyclin A-Cdk2 occurs on
Ser-54, Ser-74, and Ser-106 (13). This requires the presence of a
nearby Cy motif at residues 94-98 as evidenced by the fact that its
mutation abolishes phosphorylation at these
sites.2
In this report, we describe the first kinetic analysis of a Cy
motif-containing substrate to determine the contribution of the Cy
motif to the catalytic efficiency of cyclin-Cdk complexes. Using a
series of peptides derived from CDC6 that contain a consensus SPXK phosphorylation site and either a wild-type or mutated
Cy motif, we show that an intact Cy motif plays a critical role in targeting the peptide to cyclin-Cdk complexes. We have also examined the effect of changing the length of the linker between the Cy motif
and the Cdk phosphorylation site to show that both sites must be
simultaneously bound to the cyclin-Cdk to maximize phosphorylation of
the substrate.
Expression and Purification of Cyclin-Cdk
Complexes--
Baculoviruses expressing GST-cyclin E, GST-cyclin A,
and Cdk2 were gifts from Helen Piwnica-Worms. Sf9 cells were
coinfected with the appropriate cyclin-Cdk pair and affinity-purified
as described previously (15) with the following changes. After affinity
binding to glutathione-agarose beads, the complexes were cleaved from
GST using Novagen's Thrombin Cleavage Capture Kit.
Kinase Assays--
Phosphorylation reactions were performed in a
total volume of 15 µl containing 50 mM Hepes (pH 7.4), 10 mM MgCl2, 0.5 mM dithiothreitol, 0.02% Triton X-100, 1 µCi of [ Peptide Synthesis and Purification--
The peptides containing
a polyglycine linker of either two or six residues, PS100 and DTM101,
were commercially synthesized by Research Genetics, Inc. All other
peptides were synthesized using the Trp-LE expression system.
Oligonucleotide cassettes based on CDC6 were subcloned into the vector
pMM (a gift from Stephen Blacklow), which expresses the peptide as a
fusion protein with a Trp-LE peptide leader sequence. The
peptides were then purified to homogeneity as described in Blacklow and
Kim (15). Peptide purity was assessed by high performance liquid
chromatography and identity was confirmed by matrix-assisted laser
desorption/ionization time-of-flight mass spectroscopy. The
sequences of PS100 (a peptide derived from the Cdk inhibitor p21) and
DTM101 (a p21-derived peptide with a scrambled Cy motif) are
ACRRLFGPVDSE and ACRFGRLPVDSE, respectively. The sequences of the
CDC6-derived peptides are shown in Fig. 1.
Purification of Enzymes and Substrates--
To determine the
contribution of the Cy motif to a cyclin-Cdk substrate, we constructed
a series of recombinant peptide substrates derived from the replication
factor, HsCDC6 (11). These peptides all contain a cyclin-Cdk
phosphorylation site at the N terminus and either a wild-type Cy motif
(CDC6(wt)), a mutated Cy motif (CDC6(mut)), or a null Cy motif
(CDC6(null)) (Fig. 1). We postulated that
these peptides would be ideal substrates for this study considering that 1) the N-terminal SPXK is known to be phosphorylated by
cyclin-Cdk complexes in vitro and 2) the phosphorylation of
this site in vivo is dependent upon an intact Cy motif. The
two sites are in close proximity in the amino acid sequence of HsCDC6
(~20 residues) allowing a peptide to easily span this region. After
expression of these peptides in Escherichia coli, they were
purified to homogeneity before their use in the kinetic studies (data
not shown). Cyclin A-Cdk2 and cyclin E-Cdk2 were also purified to
homogeneity as determined by SDS-polyacrylamide gel electrophoresis and
Coomassie Blue staining (Fig. 2). The
identities of the proteins were confirmed by Western blotting with the
appropriate antibodies (data not shown). Phosphorylation of cyclin
A-Cdk2 and cyclin E-Cdk2 with bacterially expressed CIV1 resulted in a
2-fold increase in velocity suggesting that the purified cyclin-Cdk
complexes were not completely phosphorylated on Thr-160.
Determination of Kinetic Parameters--
Using purified enzyme and
the peptide substrates, we developed a highly reproducible kinase
assay. Phosphorylation of the peptide substrates by both cyclin A-Cdk2
and cyclin E-Cdk2 followed hyperbolic kinetics and increased linearly
as a function of both enzyme concentration and time when substrate
concentrations were not limiting (data not shown). All further
experiments were carried out using conditions within this linear range
to ensure that the results could be interpreted using
Michaelis-Menten-based equations.
Initial velocities were determined for both cyclin A-Cdk2 and cyclin
E-Cdk2 complexes using our CDC6-based peptides as substrates. These
velocities were plotted against ATP concentrations on a double-reciprocal plot using various fixed concentrations of peptide substrate. A representative plot in which cyclin E-Cdk2 was used to
phosphorylate the CDC6(wt) peptide is shown in Fig.
3A. In all of these plots, the
intersecting pattern of initial velocities is consistent with a
sequential kinetic mechanism in which both substrates (ATP and peptide)
must be bound before any products are released. From these data,
however, we are unable to show whether substrate addition is an ordered
or random process. kcat and
Km for a given substrate-enzyme pair were determined by secondary plots of the slopes and intercepts of the initial velocity
lines versus reciprocal substrate concentration (Fig. 3,
B and C). A summary of the data for all of
the enzymes and substrates can be found in Table
I.
The wild-type substrate was efficiently bound by both cyclin A-Cdk2 and
cyclin E-Cdk2 as demonstrated by Km values of 1.7 and 7.9 µM, respectively. Upon mutation of the Cy motif in the N terminus from RRLVF to RAARA, these values increased 75-fold
to 145 µM for cyclin A-Cdk2 and 120-fold to 970 µM for cyclin E-Cdk2. These dramatic increases in
Km demonstrate the importance of the Cy motif in
targeting substrates to these enzyme complexes. The
Km values for CDC6(mut) were 27 µM and
165 µM for cyclin A-Cdk2 and cyclin E-Cdk2, respectively, a 15- and 20-fold increase compared with the wild-type peptide. Thus,
this mutation produces a partially functional Cy motif rather than a
completely nonfunctional motif.
In contrast to the Km values for the peptide
substrates, the kcat and the
Km(ATP) values for the enzymes remained
very similar with less than a 4-fold change between substrates. This
would suggest that although the Cy motif plays a critical role in
increasing the affinity of cyclin-Cdk complexes for a particular
substrate, it does not significantly increase the efficiency of
phosphoryl transfer from ATP to the peptide.
Competition with Cy Motif-containing Peptides--
To further
demonstrate that the Cy motif acts as a docking site for the
interaction of substrate with enzyme, we tested the ability of a Cy
motif-containing peptide, PS100, to inhibit the phosphorylation of our
peptide substrates. If the Cy motif truly directs substrates in this
manner, then the PS100 peptide is expected to inhibit the
phosphorylation of Cy motif-containing substrates such as our CDC6(wt)
and CDC6(mut) peptides but unable to inhibit CDC6(null), which lacks a
Cy motif. The data are shown in Fig. 4,
A and B. The concentration of the substrate
peptides had to be adjusted to obtain equivalent phosphorylation by
cyclin-Cdk complexes with more of CDC6(null) being used relative to
CDC6(wt) or CDC6(mut). Despite this, a comparison of the ratio of the
inhibitor to substrate for any given peptide substrate shows that PS100 selectively inhibits the phosphorylation of only Cy motif-containing substrates, CDC6(wt) and CDC6(mut), but not that of CDC6(null). DTM101,
a peptide containing a scrambled Cy motif, does not inhibit the
phosphorylation of any of the substrates (data not shown) consistent
with our previous results that a negative control inhibitory peptide
containing a mutation in the Cy motif does not inhibit the
phosphorylation of Rb (4).
Considering the unusual shape for the inhibition curve of cyclin A-Cdk2
with the CDC6(wt) peptide and PS100, we carried out a systematic
inhibition study to determine the mode of inhibition of PS100 for both
cyclin A-Cdk2 and cyclin E-Cdk2 using the CDC6(wt) peptide as the
substrate. Lineweaver-Burk plots for these inhibition experiments are
shown in Fig. 5, A and
B, for cyclin E-Cdk2 and cyclin A-Cdk2, respectively. Visual
inspection of these plots shows that PS100 competitively inhibits the
phosphorylation of the CDC6(wt) peptide by both cyclin E-Cdk2 and
cyclin A-Cdk2. From these data, we were also able to determine the
inhibition constant (Ki) for PS100 which was
7.5 ± 0.5 µM for cyclin E-Cdk2 and 117.5 ± 11.6 µM for cyclin A-Cdk2.
Effects of Linker Length on Substrate
Phosphorylation--
Previous studies on the mechanism of action of Cy
motifs have been unable to determine whether both the Cy motif and the
Cdk phosphorylation site must be simultaneously engaged with the
cyclin-Cdk complex or whether the Cy motif binds first to the cyclin to
increase the local concentration of the substrate around the enzyme and is then released to allow the Cdk phosphorylation site to bind the
kinase active site (16). To distinguish between these two possibilities, we reasoned that simultaneous engagement of both binding
sites would require the Cy motif and the phosphorylation site to be
separated by an amino acid linker of sufficient length to span the
40-Å distance from the binding site on the surface of the cyclin to
the catalytic site on the Cdk. The bind and release mechanism on the
other hand would be independent of the length of the amino acid linker.
To test this hypothesis, we systematically replaced the wild-type amino
acid linker (16 residues) connecting the Cdk phosphorylation site and
Cy motif of our CDC6 peptide with flexible, predominantly polyglycine
linkers of 2, 6, 12, or 18 residues. Assuming the flexible linkers
would extend on the average 4 Å/residue, the distance separating the
two sites on these substrate peptides would be 8, 24, 48, and 72 Å,
respectively. These substrates were made in the context of both the
CDC6(wt) and CDC6(null) peptides and then tested for their
ability to be phosphorylated by cyclin A-Cdk2 and cyclin E-Cdk2.
By comparing the phosphorylation of the wild-type
versus the null peptides, we were able to specifically
determine the contribution of the Cy motif for a given linker length
and thus eliminate any artifacts that may arise from differential
binding of the shorter peptides to p81 phosphocellulose. As shown in
Fig. 6, A and B, we
found that only substrates containing both an intact Cy motif and
either the 12- or the 18-residue linker were effectively
phosphorylated. Substrates that either lacked a Cy motif or contained a
linker that was unable to span the distance from the Cdk binding site to the cyclin binding site were phosphorylated extremely poorly. This
length dependence of the linker strongly suggests that both the Cy
motif and the Cdk phosphorylation site must be simultaneously bound to
cyclin-Cdk complex to promote its efficient phosphorylation and
eliminates the bind and release model of substrate phosphorylation.
We have used a series of peptide substrates derived from HsCDC6 to
determine the contribution of a Cy motif to the phosphorylation of a
substrate by cyclin-Cdk complexes. This detailed kinetic analysis of
the phosphorylation of these substrates reveals its importance in
substrate recognition by cyclin-Cdks and provides additional insight
into its mechanism of action.
The CDC6 wild-type peptide was efficiently phosphorylated in
vitro by both cyclin E-Cdk2 and cyclin A-Cdk2 complexes. The measured Km for the peptide was less than 10 µM for both enzymes suggesting the existence of a high
affinity interaction between the enzyme and our substrate. This is in
contrast to previously characterized substrates whose
Km values were no lower than 200 µM,
100-fold greater than our peptide (17). Because these previously
characterized substrates contained only the consensus (S/T)PX(K/R) phosphorylation site, this reduction in
Km for our peptides can likely be attributed to the
presence of a Cy motif. Indeed, the presence of this Cy motif makes the
wild-type CDC6 peptide the most efficient peptide substrate of
cyclin-Cdk complexes characterized to date.
The extremely efficient phosphorylation of our CDC6(wt) peptide is
surprising considering a study by Solomon et al. (17), which
defined the sequence requirements of the consensus Cdk phosphorylation site. They showed that a SPPK phosphorylation site, like that present
in CDC6, is phosphorylated at <5% of the level of the SPRK
phosphorylation site of their wild-type peptide. This decrease in
phosphorylation can be attributed to the enzyme's inability to
tolerate a proline at the third position of the sequence. Their result
is consistent with our data for the CDC6(null) peptide, which is poorly
phosphorylated by cyclin-Cdk complexes. Hence, we conclude that the
addition of a Cy motif is sufficient to convert a peptide whose
phosphorylation site would normally make it a poor substrate into a
very efficient substrate, emphasizing the contribution of the Cy motif
to the enzyme-substrate interaction. Therefore, substrate recognition
by cyclin-Cdks occurs through a bipartite recognition sequence on the
substrate consisting of both the Cdk phosphorylation site
((S/T)PX(K/R)) and the cyclin binding Cy motif.
We had earlier reported that the Cy motif of p21 inhibited the
phosphorylation of pRb but not histone H1 (4). Now we show that a Cy
motif-containing peptide (PS100) is able to selectively inhibit only Cy
motif-containing substrates. This is consistent with PS100 competing
with substrate for the binding site on the cyclin and confirms our
model in which the Cy motif targets substrates to the enzyme via a
docking site on the cyclin. If the physiological targets of cyclin-Cdks
necessarily use the Cy motif-cyclin interaction, peptides or
chemicals that mimic the Cy motif are likely to be specific inhibitors
of Cdks and will differ from existing inhibitors that target the ATP
binding site. Indeed, preliminary studies show that such peptides lead
to the selective killing of only transformed cells in which the E2F
pathway has been deregulated (18).
Not much is known about how the specificity of cyclin-Cdk complexes is
determined. Our results suggest one mechanism by which this specificity
could be achieved. The Km for CDC6(wt) was 1.7 µM for cyclin A-Cdk2 and 7.9 µM for cyclin
E-Cdk2, suggesting that both enzymes have a high affinity for the Cy
motif present in this particular peptide. In contrast, CDC6(mut) had a
Km of 27 µM for cyclin A-Cdk2 but 163 µM for cyclin E-Cdk2. Therefore, cyclin A-Cdk2 but not
cyclin E-Cdk2 could effectively phosphorylate the mutant substrate.
Thus, although the wild-type Cy motif interacted strongly with both
enzymes, mutations could be made in the Cy motif that confer
specificity to cyclin A-Cdk2 over cyclin E-Cdk2. We also observed that
the inhibitory PS100 peptide containing the RRLFG Cy motif was a far
better inhibitor of cyclin E-Cdk2 (Ki = 7.5 µM) than cyclin A-Cdk2 (Ki = 117.5 µM). Based on these results, it seems likely that
different Cy motifs will preferentially associate with a specific
cyclin-Cdk complex and thereby target that substrate for
phosphorylation by only that enzyme.
By studying the effects of linker length on substrate phosphorylation,
we have shown that both the Cy motif and the Cdk phosphorylation site
must be simultaneously bound to the cyclin-Cdk complex. Previous work
suggests that the purpose of the Cy motif was to increase the local
concentration of the substrate around the enzyme (19). Our results
suggest that in addition to this role, the Cy motif may also be
responsible for orientating specific Cdk phosphorylation sites with
respect to the active site of Cdk2 to further facilitate their
phosphorylation, a mechanism that requires the concurrent binding of
the Cy motif and Cdk phosphorylation site to the enzyme as seen with
the CDC6-derived substrates. For example, binding of the Cy motif of a
substrate to the cyclin might conformationally restrain the substrate
such that only particular Cdk phosphorylation sites are accessible to
the Cdk. In this way, the Cy motif would not only increase the overall
affinity of the cyclin-Cdk for the substrate, it would also specify
which phosphorylation sites would be targeted by the kinase.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-32P]ATP, and various
concentrations of ATP and peptide dissolved in deionized water. Five
different peptide and ATP concentrations were used that ranged from
0.5 × Km to 5 × Km for each substrate. Reactions were initiated by the addition of 1 µl
of enzyme diluted in reaction buffer and incubated at 30 °C for 20 min. Reactions were terminated with 1 µl of 0.5 M EDTA and 10 µl of the reaction mixture spotted onto a 2- × 2-cm square of
Whatman phosphocellulose P-81 filter paper. Papers were washed in 0.5%
H3PO4 three times for 5 min, once in 50% EtOH,
0.5% H3PO4 for 5 min, and dried under a heat
lamp. Incorporation of [
-32P]ATP into the
phosphoacceptor peptide was then quantified by liquid scintillation
counting of the paper squares. Under these conditions <10% of peptide
was phosphorylated upon termination of the reaction, and velocities
were linear with respect to both time and enzyme concentration.
Assuming steady state kinetics, initial velocity data and ATP
concentrations were fitted to the Michaelis-Menten equation using the
Kaleidagraph program, and kcat and
Km were determined. All experiments were done at
least twice in duplicate. Protein quantitation was determined by
Bio-Rad protein assay.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
View larger version (18K):
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Fig. 1.
Schematic of substrate peptides derived from
HsCDC6. Peptides were constructed that spanned the consensus Cdk
phosphorylation site from residues 74-77 and the Cy motif from
residues 94-98. The N-terminal Cdk phosphorylation site and the
C-terminal Cy motif are highlighted in bold.
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Fig. 2.
SDS-polyacrylamide gel electrophoresis of
cyclin E-Cdk2 and cyclin A-Cdk2. Purified cyclin E-Cdk2
(lane 1) and cyclin A-Cdk2 (lane 2) were loaded
on a 12% gel, and the proteins were stained with Coomassie Blue.
View larger version (13K):
[in a new window]
Fig. 3.
Representative initial velocity patterns and
secondary plots. A, initial velocity pattern was
determined for cyclin E-Cdk2 using ATP as the varied substrate and the
following fixed concentrations of CDC6 wild-type peptide: 2.5 µM ( ), 5 µM (
), 10 µM
(
), 20 µM (X), and 40 µM (
).
B, secondary plot of primary slopes versus
reciprocal peptide concentration. C, secondary plot of
primary intercepts versus reciprocal peptide
concentration.
Kinetic parameters for cyclin/cdk complexes and CDC6-derived peptides
1.
View larger version (12K):
[in a new window]
Fig. 4.
Cy motif-containing peptide
selectively inhibits the phosphorylation of only Cy
motif-containing substrates by cyclin E-Cdk2 (A) and
cyclin A-Cdk2 (B). The Cy motif-containing
peptide PS100 is able to inhibit the phosphorylation of 5 µM CDC6(wt) ( ) and 50 µM CDC6(mut) (
)
but not of 1 mM CDC6(null) (
) substrate.
View larger version (12K):
[in a new window]
Fig. 5.
PS100 competitively inhibits the
phosphorylation of the CDC6(wt) substrate by cyclin E-Cdk2
(A) and cyclin A-Cdk2 (B).
Initial velocities were determined in the presence of different fixed
concentrations of the PS100 peptide: 0 µM ( ), 6.25 µM (
), 12.5 µM (
), and 25 µM (
) for cyclin E-Cdk2 and 0 µM (
),
100 µM (
), 200 µM (
), and 500 µM (
) for cyclin A-Cdk2.
View larger version (14K):
[in a new window]
Fig. 6.
Phosphorylation of peptide substrates by
cyclin E-Cdk2 (A) and cyclin A-Cdk2
(B) is dependent on the length of the linker
connecting the N-terminal Cdk phosphorylation site and the C-terminal
Cy motif. For each given linker length, the velocities were
determined for both the wild-type Cy motif (gray bar) and
the null Cy motif (black bar).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
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We thank Stephen C. Blacklow and Brian Dwyer for their advice and helpful discussions.
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FOOTNOTES |
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* This work was supported by funds from the United States Army Medical Research and Materiel Command (DAMD 17-97-1-7314) and a predoctoral fellowship (to J. A. W.) from United States Dept. of Defense (National Defense Science and Engineering Graduate Fellowship Program).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.
Both authors contributed equally to this work.
§ To whom correspondence should be addressed. Tel.: 617-278-0468; Fax: 617-732-7449; E-mail: adutta@rics.bwh.harvard.edu.
Published, JBC Papers in Press, November 6, 2000, DOI 10.1074/jbc.M005719200
2 L. Delmolina and A. Dutta, unpublished resulst.
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ABBREVIATIONS |
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The abbreviations used are: Cdk, cyclin-dependent kinases; GST, glutathione S-transferase.
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REFERENCES |
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