One hallmark of cancer is the loss of regulatory mechanisms that control cell proliferation. Among the controls that are attenuated or lost is the capacity to arrest cell cycle progression in response to ionizing radiation and consequent DNA damage or other insult to the cell. Such checkpoints include arrest at the G1/S boundary, during the S phase, during the G2/M transition, and at the point of mitotic spindle assembly. The Polo kinases are a family of kinases that retain an evolutionarily conserved amino acid sequence, designated the polo box, at the carboxy end of the protein. They participate in the transit of cells from late G2 into mitosis, through mitosis and exit from mitosis. The polo kinase examined in this application is Plk3 (former by designated Prk), one of three known mammalian polo kinases, whose function remains ill defined. We have shown that this protein is expressed relatively constantly throughout the cell cycle but find that its kinase activity fluctuates. Based on co-immunoprecipitation studies, it associates with the amphase promoting complex (APC), but the proteins with which it interacts are not known. It is also very rapidly phosphorylated in response to ionizing radiation and DNA damage, suggestive of a role in cell cycle arrest or DNA repair, and it becomes phosphorylated following exposure of cells to nocodazole, suggestive of a role in the spindle assembly checkpoint. Curiously, unlike its close relative Plk1, which transforms NIH 3T3 cells following transfection, Plk3 causes mammalian cells to undergo apoptosis following transfection and arrests cells during cytokinesis. Whereas overexpressed Plk3 associates with a post-mitotic midbody, endogenous Plk3 does not, suggesting that interpretation of results derived from Plk3 overexpression may not accurately reflect the Plk3 true function(s). The immediate goals of this application are to characterize Plk3 and to elucidate its function(s) in cellular regulation and in response radiation damage and other insult. Specifically we propose to establish phosphorylation patterns of Plk3 following irradiation and nocodazole treatment, identify substrates for its kinase activity and other proteins with which it interacts, and isolate mutant Plk3 that circumvent apoptosis and permit survival following transfection. Using this information and mouse embryo fibroblasts with mutant or null Plk3 alleles, we propose to elucidate the pathway(s) in which Plk3 participates in the regulation of cell proliferation and in response to radiation and consequent DNA damage.
