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J. Biol. Chem., Vol. 275, Issue 42, 32578-32584, October 20, 2000

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The Effects of Changing the Site of Activating Phosphorylation in CDK2 from Threonine to Serine^*

Philipp Kaldis, Aiyang Cheng, and Mark J. Solomon

From the Yale University School of Medicine, Department of Molecular Biophysics and Biochemistry, New Haven, Connecticut 06520-8114

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 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.

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