The proposed studies will the fundamental molecular processes underlying the control of eukaryotic cell division. These experiments will provide the basic foundation for an understanding of the defects in cell cycle control that occurs in cancer cells. The events of the cell division are controlled by the cyclin-dependent kinases (Cdks), a highly conserved family of protein kinases whose activation requires association with cyclin regulatory subunit. The complexes in late mitosis is accomplished primary by ubiquitin-dependent proteolysis of mitotic cyclins. The rate-limiting regulatory step in this process is catalyzed by a multi-subunit ubiquitin ligase known as the anaphase-promoting complex (APC). The proposed experiments will explore the mechanisms controlling activation of the APC in late mitosis, primary using biochemical approaches in the budding yeast Saccharomyces cerevisiae. The APC was purified from S. cerevisiae and found to be activated in vitro by the regulatory protein Hct1/Cdh1. Preliminary evidence supports the hypothesis that phosphorylation of Hct1 at multiple sites regulates its ability to activate the APC. We will analyze the phosphorylation of Hct1 in vivo, assess its role in Hct function, and identify the regulatory enzymes that control Hct phosphorylation. Preliminary results have also led to the hypothesis that cyclin destruction by the APC is controlled in late mitosis by a complex network of regulatory proteins, including the Polo-related protein kinase Cdc5. We will explore the mechanisms controlling the activation of Cdc5 in mitosis. Finally, the proposed work will address the hypothesis that the subunits of the APC itself are controlled by modifications that change its responsiveness to the Hct1 regulator. In addition, the long term goal in these studies is to complete our understanding of APC regulation by reconstituting the APC from individual recombinant subunits.
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