BIOCHEMICAL-CHARACTERIZATION OF THE HUMAN CYCLIN-DEPENDENT PROTEIN-KINASE ACTIVATING KINASE - IDENTIFICATION OF P35 AS A NOVEL REGULATORY SUBUNIT

The activation of cyclin-dependent protein kinases (Cdks) is dependent upon site specific phosphorylation and dephosphorylation reactions, a s well as positive and negative regulatory subunits. The human Cdk-act ivating protein kinase (Cak1) is itself a Cdc2-related cyclin-dependen t protein kinase that associates with cyclin Il. The present study uti lized specific anti-Cak1 antibodies and immunoaffinity chromatography to identify additional Cak1-associated proteins and potential target s ubstrates. Immunoprecipitation of metabolically labeled human osteosar coma cells revealed a number of Cak1-associated proteins, including p9 5, p37 (cyclin H), and a 35-kDa protein that was further characterized herein. Microsequence analysis obtained after limited proteolysis rev ealed peptide fragments that are similar, but not identical to, human and yeast cyclins, thus identifying p35 as a cyclin-like regulatory su bunit. The greatest sequence similarity of human p35 is with Mcs2, a y east cyclin that is essential for cell cycle progression. Immunoaffini ty chromatography performed under nondenaturing conditions afforded th e isolation of enzymatically active Cak1 from cell lysates, enabling s tudies of kinase autophosphorylation and comparative substrate utiliza tion. Immunoaffinity-purified Cak1 phosphorylated monomeric Cdc2 and C dk2, but not Cdk4; the phosphorylation of both Cdc2 and Cdk2 were incr eased in the presence of recombinant cyclin A. These studies indicate that the Cak1 catalytic subunit, like Cdc2 and Cdk2, associates with m ultiple regulatory partners and suggests that subunit composition may be an important determinant of this multifunctional enzyme.