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 re gulated in many ways, including activating phosphorylation of a conserved t hreonine residue. This essential phosphorylation is carried out by the CDK- activating kinase (CAK). Here we examine the effects of replacing this thre onine 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 )). I, the absence of activating phosphorylation, CDK2(Ser-160)-cyclin A co mplexes were more active than wild-type CDK2(Thr-160)-cyclin A complexes. I n contrast, following activating phosphorylation, CDK2(Ser-160)-cyclin A co mplexes were less active than phosphorylated CDK2(Thr-160)-cyclin A complex es, reflecting a much smaller effect of activating phosphorylation on CDK2( Ser-160). The kinetic parameters for phosphorylating histone H1 were simila r for mutant and wild-type CDK2, ruling out a general defect in catalytic a ctivity. Interestingly, the CDK2(Ser-160) mutant was selectively defective in phosphorylating a peptide derived from the C-terminal domain of RNA poly merase II. CDK2(Ser-160) was efficiently phosphorylated by CAKs, both human p40(MO15)(CDK7)-cyclin H and budding yeast Caklp. In fact, the k(cat) valu es 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, dephosph orylation proceeded more slowly with CDK2(Ser-160) than with wild-type CDK2 , either in HeLa cell extract or by purified PP2C beta. Combined with the m ore efficient phosphorylation of CDK2(Ser-160) by CAK, we suggest that one reason for the conservation of threonine as the site of activating phosphor ylation may be to favor unphosphorylated CDKs following the degradation of cyclins.