Mechanism of Cdk2/Cyclin E inhibition by p27 and p27 phosphorylation.

The biochemical interactions between the Cdk2/Cyclin E kinase and its inhibitor p27, were investigated using purified, recombinant p27 and CAK-phosphorylated Cdk2/Cyclin E. From kcat/Km determinations using either histone H1 or pRb as substrates, we found that Cdk2/Cyclin E has 60-fold higher specificity for pRb than for histone H1. The IC50 ...
value of p27 increased with increasing Cdk2/Cyclin E concentrations while it remained constant at various ATP and histone H1 concentrations, suggesting that p27 acts as a tight binding inhibitor of Cdk2/Cyclin E. We also found that p27 could be phosphorylated by Cdk2/Cyclin E only at high enzyme concentrations, and that p27 forms a stable interaction with Cdk2/Cyclin E regardless of its phosphorylation state. Our results further indicate that the Cdk2/Cyclin E/p27 ternary complex is kinetically inactive as an enzyme; instead it serves as a substrate for Cdk2/Cyclin E. These results suggest that if phosphorylation of p27 by Cdk2/Cyclin E is involved in its ubiquitin-dependent degradation, as previously suggested, then the target for such event is the phosphorylated p27 bound to Cdk2/Cyclin E and not free p27.
Mesh Terms:
Adenosine Triphosphate, Amino Acid Sequence, Animals, Binding Sites, CDC2-CDC28 Kinases, Cell Cycle Proteins, Cyclin E, Cyclin-Dependent Kinase 2, Cyclin-Dependent Kinase Inhibitor p27, Cyclin-Dependent Kinases, Dose-Response Relationship, Drug, Enzyme Inhibitors, Humans, Kinetics, Macromolecular Substances, Mice, Microtubule-Associated Proteins, Molecular Sequence Data, Protein Binding, Protein-Serine-Threonine Kinases, Recombinant Proteins, Substrate Specificity, Tumor Suppressor Proteins
Biochemistry
Date: Jul. 06, 1999
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