Stoichiometry of cyclin A-cyclin-dependent kinase 2 inhibition by p21(Cip1/Waf1)

Progression through the eukaryotic cell cycle is regulated by phosphorylati on, which is catalyzed by cyclin-dependent kinases. Cyclin-dependent kinase s are regulated through several mechanisms, including negative regulation b y p21 (variously called CAP20, Cip1, Sdi1, and WAF1). It has been proposed that multiple p21 molecules are required to inhibit cyclin-dependent kinase s, such that p21 acts as a sensitive buffer of cyclin-dependent kinase acti vity or as an assembly factor for the complexes formed by the cyclins and c yclin-dependent kinases. Using purified, full-length proteins of known conc entration (determined by absorbance) and cyclin A-Cdk2 of known activity (c alibrated with staurosporine), we find that a 1:1 molar ratio of p21 to cyc lin A-Cdk2 is able to inhibit Cdk2 activity both in the binary cyclin A-Cdk 2 complex and in the presence of proliferating cell nuclear antigen (PCNA). Our results indicate that the mechanism of p21 inhibition of cyclin A-Cdk2 does not involve multiple molecules of bound p21.