Cyclin A-Cdk2 inhibition by p21, p27, and p57

Abstract

Progression through the eukaryotic cell cycle is regulated by phosphorylation, which is catalyzed by cyclin-dependent kinases. Cyclin-dependent kinases are regulated through several mechanisms, including negative regulation by the p21 (variously called CAP20, Cip1, Sdi1, and WAF1) family of inhibitors. It has been proposed that multiple p21 molecules are required to inhibit cyclin-dependent kinases, such that p21 acts as a sensitive buffer of cyclin-dependent kinase activity or as an assembly factor for the complexes formed by the cyclins and cyclin-dependent kinases. Using purified, full-length proteins of known concentration (determined by absorbance) and cyclin A-Cdk2 of known activity (calibrated with staurosporine), we find that a 1:1 molar ratio of p21 to cyclin A-Cdk2 is able to inhibit Cdk2 activity both in the binary cyclin A-Cdk2 com plex 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. p27 contributes to cell-cycle regulation by inhibiting the enzymatic activity of cyclin-dependent kinases. In contrast to the ordered conformation observed in the crystal structure of the p27-cyclin A-Cdk2 complex, we find that the human p27 Cdk-inhibitor is intrinsically disordered in isolation under conditions where the domain inhibits cyclin A-Cdk2. Using proline- and alanine-mapping mutatgenesis, we show the p27 domain contains marginally stable helical structure that is localized to the region that folds as an α-helix, but not the 310 helix, upon binding cyclin A-Cdk2. Reducing or increasing the helical propensity in the isolated p27 domain with proline and alanine mutagenesis did not affect formation of the p27-inhibited cyclin A-Cdk2 complex in either energetic or functional terms. We conclude that non-local (quaternary) contacts between p27 and cyclin A-Cdk2 are more important determinants of p27-cyclin A-Cdk2 assembly than pre-existing local (helical) interactions in the intrinsically disordered p27. Biophysical characterization and functional analysis of human p57 and the isolated inhibition domain of p57 completes a general characterization of this family of proteins. The inhibition domain of p57 is based on the homologous residues of p27 that were used in the crystal structure of p27-cyclin A-Cdk2. Circular dichroism spectra of both full-length p57 and the excised inhibition domain exhibit similar features expected of an unfolded protein. Sedimentation equilibrium and gel filtration indicate this protein is monomeric and in an extended conformation. Cdk-inhibition by both full-length p57 and the inhibition domain indicates that these proteins are active in a 1:1 stoichiometry with slightly greater Kd as compared to p27ID. We conclude p57 is unordered in isolation and behaves in a similar manner to that of p21 and p27.