Cak1 Is Required for Kin28 Phosphorylation and Activation In Vivo

ABSTRACT

Complete activation of most cyclin-dependent protein kinases (CDKs) requires phosphorylation by the CDK-activating kinase (CAK). In the budding yeast, Saccharomyces cerevisiae, the major CAK is a 44-kDa protein kinase known as Cak1. Cak1 is required for the phosphorylation and activation of Cdc28, a major CDK involved in cell cycle control. We addressed the possibility that Cak1 is also required for the activation of other yeast CDKs, such as Kin28, Pho85, and Srb10. We generated three new temperature-sensitive cak1mutant strains, which arrested at the restrictive temperature with nonuniform budding morphology. All three cak1 mutants displayed significant synthetic interactions with loss-of-function mutations in CDC28 and KIN28. Loss of Cak1 function reduced the phosphorylation and activity of both Cdc28 and Kin28 but did not affect the activity of Pho85 or Srb10. In the presence of the Kin28 regulatory subunits Ccl1 and Tfb3, Kin28 was phosphorylated and activated when coexpressed with Cak1 in insect cells. We conclude that Cak1 is required for the activating phosphorylation of Kin28 as well as that of Cdc28.

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Cak1 Is Required for Kin28 Phosphorylation and Activation In Vivo

ACKNOWLEDGMENTS

We thank Fred Cross for his generous gift of the Cak1-independentcdc28 mutants and for valuable discussions. We also thank Julia Charles, Rick Young, Erin Peckol, Kayvan Roayaie, and Jean Gabriel Valay for reagents and technical advice.

This work was supported by funding from the National Institute of General Medical Sciences (to D.O.M.), a postgraduate scholarship from the Natural Sciences and Engineering Research Council of Canada (to A.F.), and a Damon Runyon-Walter Winchell Postdoctoral Fellowship (to J.L.N.).