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,

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... DK2 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 PP2C␤. 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. Cyclin-dependent kinases (CDKs), 1 a subfamily of protein kinases, promote cell cycle progression. In mammals, nine different CDKs (CDK1 to CDK9; CDK1 is better known as CDC2) have been identified (for a review, see Ref. 1). The activity of