Cyclin-Stimulated Binding of Cks Proteins to Cyclin-Dependent Kinases

ABSTRACT

Although Cks proteins were the first identified binding partners of cyclin-dependent protein kinases (cdks), their cell cycle functions have remained unclear. To help elucidate the function of Cks proteins, we examined whether their binding to p34^cdc2(the mitotic cdk) varies during the cell cycle in Xenopusegg extracts. We observed that binding of human CksHs2 to p34^cdc2 was stimulated by cyclin B. This stimulation was dependent on the activating phosphorylation of p34^cdc2 on Thr-161, which follows cyclin binding and is mediated by the cdk-activating kinase. Neither the inhibitory phosphorylations of p34^cdc2 nor the catalytic activity of p34^cdc2 was required for this stimulation. Stimulated binding of CksHs2 to another cdk, p33^cdk2, required both cyclin A and activating phosphorylation. Our findings support recent models that suggest that Cks proteins target active forms of p34^cdc2 to substrates.

ACKNOWLEDGMENTS

We thank Philipp Kaldis for providing purified recombinant Cak1p and HA-p33^cdk2, Zach Pitluk for providing affinity-purified polyclonal anti-GST antibodies, Jennifer Holmes for providing purified GST protein, Steve Reed for providing CksHs1 and CksHs2 expression clones, Helen Piwnica-Worms for providing the GST-cyclin A expression clone, and Debra Patra and Bill Dunphy for providing the Xe-p9-containing vector from which Xe-p9 was cloned. We also thank Janet Burton, Deborah Enke, Philipp Kaldis, Jonathan Kimmelman, Jeffrey Peterson, and Karen Ross for critical reading of the manuscript and the entire Solomon lab for experimental advice.

This work was supported by MSTP grant GM07205 from NIH, grant GM47830 from NIH, and the Searle Scholars Program/The Chicago Community Trust. M.J.S. is a Leukemia Society of America Scholar.