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.
Kaldis
P
P
Yale University School of Medicine, Department of Molecular Biophysics and Biochemistry, New Haven, Connecticut 06520-8114, USA. kaldis@ncifcrf.gov
Cheng
A
A
Solomon
M J
MJ
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