BIOCHEMISTRY OF CELL CYCLE REGULATION

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DESCRIPTION: Progression through the cell cycle is regulated by the sequential activation and inactivation of a number of cyclin-dependent protein kinase (cdks). Many cdks, such as p34cdc2, and cdc4 require an activating phosphorylation on a site equivalent to Thr-161 in human p34cdc2. This phosphorylation is carried out by CAK, the Cdk-Activating Kinase. Solomon and others purified vertebrate CAK and found that it contained at least two proteins, a catalytic subunit termed p40MO15 (or cdk7), and a regulatory subunit, cyclin H. Both proteins are also subunits of the general transcriptio factor TFIIH. He recently purified CAK from the budding yeast, and found that it was composed of a single subunit (Cak1p) with unusual protein kinase motifs and only distant similarity to previously identified CAKs. Since the transcription function of p40MO15 is performed in yeast by Kin28p, the identification of Cak1p as the physiological yeast CAK suggested that there might be an additional vertebrate CAK, one more like Cak1p than p40MO15. These studies are aimed at furthering our understanding of CAK, both in yeast and in vertebrates. The Specific Aims of this project are: 1) To determine the range of Cak1p's substrates, how it is regulated, and its three-dimensional structure. 2) To characterize the end-product inhibition of Cak1p and to determine its physiological role. 3) To determine whether p40MO15 is a physiological CAK in vertebrates, to screen other fungi for evidence of a Cak1p-like CAK, to search for an additional vertebrate CAK, similar to yeast Cak1p, and to assess its functional importance. 4) To identify and study the phosphatase that counters Cak1p by dephosphorylating Thr-169 of Cdc28p.