Mechanism of Cdk2/cyclin E inhibition by p27 and p27 phosphorylation

The biochemical interactions between the Cdk2/Cyclin E kinase and its inhib itor p27, were investigated using purified, recombinant p27 and CAK-phospho rylated Cdk2/Cyclin E. From k(cat)/K-m determinations using either histone H1 or pRb as substrates, we found that Cdk2/Cyclin E has 60-fold higher spe cificity for pRb than for histone H1. The IC50 value of p27 increased with increasing Cdk2/Cyclin E concentrations while it remained constant at vario us ATP and histone H1 concentrations, suggesting that p27 acts as a tight b inding inhibitor of Cdk2/Cyclin E. We also found that p27 could be phosphor ylated by Cdk2/Cyclin E only at high enzyme concentrations, and that p27 fo rms a stable interaction with Cdk2/Cyclin E regardless of its phosphorylati on state. Our results further indicate that the Cdk2/Cyclin E/p27 ternary c omplex is kinetically inactive as an enzyme; instead it serves as a substra te for Cdk2/Cyclin E. These results suggest that if phosphorylation of p27 by Cdk2/Cyclin E is involved in its ubiquitin-dependent degradation, as pre viously suggested, then the target for such event is the phosphorylated p27 bound to Cdk2/Cyclin E and not free p27.