Purification of a 15-kDa cdk4- and cdk5-binding protein.
Yeasts p13suc1/p18CKS and their human homologues, p9CKShs1/p9CKShs2, strongly interact with p34cdc2 and p34cdk2. While attempting to purify the starfish oocyte p13suc1 homologue, we discovered a 15-kDa protein cross-reactive with anti-p9CKShs2/anti-p13suc1 antibodies. p15cdk-BP-Sepharose binds an anti-PSTAIRE cross-reactive protein of 33 kDa when loaded with starfish oocyte extracts. The p15cdk-BP-bound "PSTAIRE signal" is part of a 250-kDa complex distinct from p34cdc2/cyclin B. p15cdk-BP-Sepharose beads retain a kinase phosphorylating HMG I/Y, P1, and myelin basic protein (among 24 substrates tested). Major cdc2 kinase substrates are not phosphorylated by the p15cdk-BP-bound kinase. Phosphopeptide maps of P1 phosphorylated by the p15cdk-BP-bound kinase, p34cdc2/cyclin B, p 33cdk5/p25, and casein kinase 2 showed that these kinases phosphorylate P1 on different sites. Phosphopeptide maps of P1 phosphorylated by the p15cdk-BP-bound starfish kinase and p15cdk-BP-bound human p34cdk4/cyclin D are largely coincident. To investigate the nature of the p15cdk-BP-bound kinase, extracts of mammalian tissues and cells were loaded on p9CKShs1- and p15cdk-BP-Sepharose and the bound proteins were analyzed using specific anti-cdk antibodies. cdc2 and cdk2 bind to p9CKShs1-Sepharose, but not to p15cdk-BP. cdk4 and cdk5 bind to p15cdk-BP-Sepharose, but not to p9CKShs1-Sepharose. We conclude that p15cdk-BP specifically binds the cdk4/cyclin D and cdk5 kinases and, along with p13suc1 and p9CKShs, may be part of a larger family of cdk-binding proteins.
Azzi
L
L
CNRS, Station Biologique, Roscoff, France.
Meijer
L
L
Ostvold
A C
AC
Lew
J
J
Wang
J H
JH
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D002850:Chromatography, Gel; D003429:Cross Reactions; D051358:Cyclin-Dependent Kinase 4; D051360:Cyclin-Dependent Kinase 5; D018844:Cyclin-Dependent Kinases; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D010766:Phosphorylation; D011485:Protein Binding; D011494:Protein Kinases / Q000378:metabolism*; D011506:Proteins / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism; D011518:Proto-Oncogene Proteins; D013215:Starfish
D011506:Proteins; D011518:Proto-Oncogene Proteins; D011494:Protein Kinases; D051360:Cyclin-Dependent Kinase 5; C495900:CDK4 protein, human; C495905:CDK5 protein, human; D051358:Cyclin-Dependent Kinase 4; D018844:Cyclin-Dependent Kinases