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A bipartite substrate recognition motif for cyclin-dependent kinases
D Y Takeda1, J A Wohlschlegel, A Dutta
1Department of Pathology, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts 02115, USA.
The Journal of Biological Chemistry
|November 9, 2000
Summary
The Cy motif significantly enhances substrate binding to cyclin-dependent kinases (Cdks) without altering catalytic rates. This motif, alongside SPXK, forms a bipartite recognition sequence crucial for Cdk phosphorylation.
Area of Science:
- Biochemistry
- Molecular Biology
- Enzymology
Background:
- Cyclin-binding motifs like Cy and RXL are found in proteins interacting with cyclin-Cdk complexes.
- Cyclin-dependent kinases (Cdks) regulate cell cycle progression through phosphorylation.
Purpose of the Study:
- To kinetically analyze the role of the Cy motif in substrate phosphorylation by cyclin-dependent kinases.
- To investigate the combined function of Cy and SPXK motifs in substrate recognition by Cdks.
Main Methods:
- Kinetic analysis of cyclin A-Cdk2 and cyclin E-Cdk2 enzymes.
- Systematic variation of the linker length between the Cy motif and the phosphoacceptor site.
- Assessment of peptide PS100 as a competitive inhibitor.
Main Results:
- The Cy motif decreased the K(m(peptide)) 75-120-fold for both cyclin A-Cdk2 and cyclin E-Cdk2, while k(cat) remained unchanged.
- Evidence suggests a bipartite substrate recognition sequence involving the Cy motif and the (S/T)PX(K/R) site.
- Peptide PS100 competitively inhibited cyclin-Cdk complexes, with higher affinity for cyclin E-Cdk2.
Conclusions:
- A Cy motif, positioned near the SPXK sequence, is essential for efficient Cdk-mediated phosphorylation.
- The Cy motif and SPXK site likely bind simultaneously to the cyclin-Cdk complex.
- Cy motif mimetics represent potential specific inhibitors of Cdk substrate recognition.