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J. Biol. Chem., Vol. 276, Issue 3, 1993-1997, January 19, 2001
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From the Department of Pathology, Brigham and Women's Hospital,
Harvard Medical School, Boston, Massachusetts 02115
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.
A Bipartite Substrate Recognition Motif for
Cyclin-dependent Kinases*
,, and
*
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.
Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.
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