The effects of changing the site of activating phosphorylation in CDK2 from threonine to serine.
Cyclin-dependent kinases (CDKs) that control cell cycle progression are regulated in many ways, including activating phosphorylation of a conserved threonine residue. This essential phosphorylation is carried out by the CDK-activating kinase (CAK). Here we examine the effects of replacing this threonine residue in human CDK2 by serine. We found that cyclin A bound equally well to wild-type CDK2 (CDK2(Thr-160)) or to the mutant CDK2 (CDK2(Ser-160)). In the absence of activating phosphorylation, CDK2(Ser-160)-cyclin A complexes were more active than wild-type CDK2(Thr-160)-cyclin A complexes. In contrast, following activating phosphorylation, CDK2(Ser-160)-cyclin A complexes were less active than phosphorylated CDK2(Thr-160)-cyclin A complexes, reflecting a much smaller effect of activating phosphorylation on CDK2(Ser-160). The kinetic parameters for phosphorylating histone H1 were similar for mutant and wild-type CDK2, ruling out a general defect in catalytic activity. Interestingly, the CDK2(Ser-160) mutant was selectively defective in phosphorylating a peptide derived from the C-terminal domain of RNA polymerase II. CDK2(Ser-160) was efficiently phosphorylated by CAKs, both human p40(MO15)(CDK7)-cyclin H and budding yeast Cak1p. In fact, the k(cat) values for phosphorylation of CDK2(Ser-160) were significantly higher than for phosphorylation of CDK2(Thr-160), indicating that CDK2(Ser-160) is actually phosphorylated more efficiently than wild-type CDK2. In contrast, dephosphorylation proceeded more slowly with CDK2(Ser-160) than with wild-type CDK2, either in HeLa cell extract or by purified PP2Cbeta. Combined with the more efficient phosphorylation of CDK2(Ser-160) by CAK, we suggest that one reason for the conservation of threonine as the site of activating phosphorylation may be to favor unphosphorylated CDKs following the degradation of cyclins.
Yale University School of Medicine, Department of Molecular Biophysics and Biochemistry, New Haven, Connecticut 06520-8114, USA. kaldis@ncifcrf.gov
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D019943:Amino Acid Substitution; D042846:CDC2-CDC28 Kinases; D051357:Cyclin-Dependent Kinase 2; D018844:Cyclin-Dependent Kinases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012694:Serine; D013379:Substrate Specificity; D013912:Threonine; D000095043:Cyclin-Dependent Kinase-Activating Kinase
D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D013912:Threonine; D012694:Serine; D017346:Protein Serine-Threonine Kinases; D042846:CDC2-CDC28 Kinases; C495894:CDK2 protein, human; D051357:Cyclin-Dependent Kinase 2; D018844:Cyclin-Dependent Kinases; D000095043:Cyclin-Dependent Kinase-Activating Kinase; C000722416:CDK7 protein, human