Characterization of a novel cdk1‐related kinase

Abstract

The p13 suc1 /p9 CKShs proteins bind tightly to the cyclin-dependent kinases cdk1 and cdk2. The distantly related protein, p15 cdk-BP , binds cdk4/6, cdk5 and cdk8. We now show that immobilized p15 cdk-BP binds both an HMG-I kinase and a 35-kDa protein that cross-reacts with anti-PSTAIRE antibodies (PSTAIRE is a totally conserved motif located in subdomain III of cdk). This`cdkX' and the HMG-I kinase also bind to an immobilized inhibitor of cdks (HD). Several properties clearly distinguish cdkX, and its associated HMG-I kinase, from known anti-PSTAIRE cross-reactive cdks: (a) cdkX migrates, in SDS/PAGE, in a position intermediate between prophase phosphorylated cdk1 and metaphase dephosphorylated cdk1; (b) in contrast with cdk1, cdkX and associated HMG-I kinase activity do not decrease following successive depletions on p9 CKShs1 ±sepharose; (c) cdkX and associated HMG-I kinase activity, but not cdk1, decrease following depletions on immobilized inhibitor; (d) cdkX is expressed during the early development of sea urchin embryos; in contrast with cdk1/cyclin B kinase, the p15 cdk-BP -bound HMG-I kinase is active throughout the cell cycle; compared with cdk1 it is active later in development; (e) p15 cdk-BP -bound HMG-I kinase is essentially insensitive to powerful inhibitors of cdk such as purvalanol, roscovitine, olomoucine, p21 cip1 and p16 INK4A ; HD is only moderately inhibitory. Altogether these results suggest the existence of a new cdk1-related kinase, possibly involved in the regulation of early development. The presence of this kinase in all organisms investigated so far, from plants to mammals, calls for its definitive identification.Keywords: cdc2; cdk-BP; CKS; CKShs1; cyclin-dependent kinases; HMG-I; cell cycle; PSTAIRE; suc1.The cyclin-dependent kinases (cdk), regulate the cell division cycle (for reviews, see [1,2]). Cdc2 was the first cdk to be described, as a gene essential for G 1 /S and G 2 /M transitions in Schizosaccharomyces pombe [3]. In addition to cdc2 and CDC28, its homologue in Saccharomyces cerevisiae [4], the yeast genomes contain several related kinases: Mop1 (Sc. pombe), PHO85, KIN28, CTK1, SVG1 and SRB10 in S. cerevisiae (for a review see [5]). A human cdc2 homologue, cdk1, was identified by complementation [6], followed by homologues in all species from yeast and plants to mammals [2,7]. They all share a totally conserved motif, known as the PSTAIRE motif, located in subdomain III of the kinase. Antibodies directed against the PSTAIRE motif are widely used in the immunodetection of cdk1 and cdk2 [8]; this domain is involved in the binding of cyclins and has been used to design oligonucleotides by which to identify and name new cdk1-related kinases [9]. So far, nine mammalian cdks have been described: cdk1, 2, 3, 4, 6 and 7 regulate the cell cycle, cdk5 is involved in neural tissue and cdk7, 8 and 9 regulate transcription (for a review see [2,10] whereas PCTAIRE-2 is expressed in postmitotic neurones [18]. The PITSLRE kinase family appears to play an important role in apoptosis [19,...

Evidence for a pre‐restriction point Cdk3 activity

“…Since a monoclonal anti-cdk3 was not available, we used monoclonal anti-PSTAIRE to verify that the anti-cdk3 immunoprecipitates contained a 36-kDa cdk protein. The PSTAIRE sequence is conserved among cdks and distinguishes them from other protein kinases [Morgan, 1997;Tournier et al, 1997;Detivaud et al, 1999]. Anti-PSTAIRE antibody revealed four prominent bands in immunoblots of whole cell extracts (Fig.…”

Cellular effects of purvalanol A: A specific inhibitor of cyclin‐dependent kinase activities

“…Besides, cdk sensitive to this inhibitor may not be all known, and new cdkrelated enzymes are still being discovered. 31 Further development of cdk-specific inhibitors active in living cells will help to resolve this question and identify more precisely the functions of cdk in the different cellular processes.…”