just parking........
Biochemistry 1999 Jul 6;38(27):8713-22
Mechanism of Cdk2/Cyclin E inhibition by p27 and p27 phosphorylation.
Xu X, Nakano T, Wick S, Dubay M, Brizuela L
Mitotix Inc., One Kendall Square, Cambridge, Massachusetts 02139, USA.
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. |
