Biochemistry of Cell Cycle Regulation

Yale University, New Haven CT

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Abstract

DESCRIPTION (provided by applicant): Progression through the cell cycle is regulated by the sequential activation and inactivation of cyclin-dependent protein kinase (cdks). All of the cell cycle cdks, such as human Cdc2, Cdk2, and Cdk4, and yeast Cdc2Sp, require an activating phosphorylation on a site equivalent to Thr-160 in human Cdk2. This phosphorylation is carried out by CAK, the Cdk-Activating Kinase. In most species, CAK consists of a catalytic subunit, Cdk7, a regulatory subunit, cyclin H, and an assembly factor, MAT 1. All three of these proteins are also subunits of the general transcription factor TFIIH. In yeast, however, CAK consists of a single polypeptide, Cakip. The phosphorylation that is carried out by CAK is removed by Type 2C phosphatases (PP2Cs). These studies are aimed at furthering our understanding of activating phosphorylations of cdks, both in yeast and in vertebrates. The Specific Aims of this project are: 1) To use "analog-sensitive" (asi) and "analog-specific" (as2) forms of Cakip and CDK7 to study CAK functions and substrates in yeast and humans. 2)To characterize the regulation of PP2C activity by N-terminal myristoylation and to identify additional PP2C substrates in yeast. 3)To test the hypothesis that the differences in the CDK activation pathways of budding yeast and of most other species result from the closed vs. open mitoses in these organisms, respectively. 4)To investigate why CDKs have been designed to require activating phosphorylations and to determine the consequences of not dephosphorylating a mammalian CDK following cyclin degradation.