PURIFICATION OF A 15-KDA CDK4-BINDING AND CDK5-BINDING PROTEIN

Yeasts p13(suc1)/p18(CKS) and their human homologues, p9(CKShs1)/p9(CK Shs2), strongly interact with p34(cdc2) and p34(cdk2). While attemptin g to purify the starfish oocyte p13(suc1) homologue, we discovered a 1 5-kDa protein crossreactive with anti-p9(CRShs2)/anti-p13(suc1) antibo dies. p15(cdk-BP)-Sepharose binds an anti-PSTAIRE cross-reactive prote in of 33 kDa when loaded with starfish oocyte extracts. The p15(cdk-BP )-bound ''PSTAIRE signal'' is part of a 250-kDa complex distinct from p34(cdc2)/cyclin B. p15(cdk-BP)-Sepharose beads retain a kinase phosph orylating HMG I/Y, P1, and myelin basic protein (among 24 substrates t ested). Major cdc2 kinase substrates are not phosphorylated by the p15 (cdk-BP)-bound kinase. Phosphopeptide maps of P1 phosphorylated by the p15(cdk-BP)-bound kinase, p34(cdc2)/cyclin B, p33(cdk5)/p25, and case in kinase 2 showed that these kinases phosphorylate P1 on different si tes. Phosphopeptide maps of P1 phosphorylated by the p15(cdk-BP)-bound starfish kinase and p15(cdk-BP)-bound human p34(cdh4)/cyclin D are la rgely coincident. To investigate the nature of the p15(cdk-BP)-bound k inase, extracts of mammalian tissues and cells were loaded on p9(CKShs 1)- and p15(cdk-BP)-Sepharose and the bound proteins were analyzed usi ng specific anti-cdk antibodies. cdc2 and cdk2 bind to p9(CKShs1)-Seph arose, but not to p15(cdk-BP). cdk4 and cdk5 bind to p15(cdk-BP)-Sepha rose, but not to p9(CKShs1)-Sepharose. We conclude that p15(cdk-BP) sp ecifically binds the cdk4/cyclin D and cdk5 kinases and, along with p1 3(suc1) and p9(CKShs), may be part of a larger family of cdk-binding p roteins.