Cyclin dependent kinases (Cdks) are the key regulators of cell cycle transitions in eukaryotic cells. Precise coordination of each cell cycle step with the others is essential for cells to correctly transmit an intact genome to each daughter cell. Failure to do so can lead to polyploidy, genomic instability and cancer. Recently work has shown that the Cdc 14-family of protein phosphatases may be important regulators of Cdk activity and play a crucial role in coordinating late mitotic events to maintain genomic stability. We have found that the fission yeast S. pombe Cdc 14 ortholog Clp 1p functions to block further rounds of cell cycle progression if cytokinesis is delayed. Clp 1p carries out this function by regulating Cdk activity and a conserved signaling pathway termed the SIN. Deletion of clp1 makes cells unable to restrain cell cycle progression if cytokinesis is delayed resulting in polyploid cells. The long-term goal of this project is to understand how Clp1p regulates Cdk activity, and how Clp1p is regulated to ensure that mitosis is properly coordinated with cytokinesis.
My specific aims are: (1) To determine how release of Clp1p from the nucleus in early mitosis is regulated and how in late mitosis the SIN functions to keep Clp1p out of the nucleus, as well as the significance of this regulation for Clp1p function. (2) To characterize the role of Clp1p phosphorylation in regulating Clp1p activity and/or localization by (A) determining the effect of phosphorylation of on Clp1p phosphatase activity, (B) mapping and mutagenesis of Clp1p phosphorylation sites followed by characterization of the phenotype of Clp1p phosphorylation site mutants. (3) To identify (A) Clp1p interacting protein(s) at different cell cycle stages, as well as (B) Clp1p regulated processes using biochemical and genetic analysis. (4) To test the model that Clp1p regulates Cdk activity by antagonizing a self-activating mechanism by which Cdk1p promotes its own activity by phosphorylating and activating its activator Cdc25p and inhibiting its inhibitor Wee 1p.
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