(from the application): The polo family of protein kinases is now known to have a critical role in proliferation. In higher eukaryotes, flies, and yeast polo kinase homologues are essential for cell cycle progression through M phase. In mammals, distinct polo kinases -for example, Snk-are also expressed early in the cell cycle. The mammalian enzymes, particularly Plk and Snk are the focus of the studies proposed in this application. IN addition to the catalytic kinase domain, polo kinases have a conserved sequence in the C-terminus, denoted the polo box. Polo box mutations disrupt the cellular localization observed with wild-type Plk and compromise Plk mitotic functions. Polo box interacting proteins will be identified and their potential to influence centrosome biogenesis and function, as well as septin localization and organization, will be determined. A general search for Snk and Plk substrates will be conducted by screening expression libraries and by engineering these enzymes to attain the capacity to utilize ATP analogues that will not be used efficiently by other kinases. In order to more precisely characterize Snk functions, embryonic stem cells will be generated with a targeted gene disruption. These cells will be used to obtain mice with germline transmission in order to determine the potential role of Snk in embryonic development. These studies have direct relevance to cancer, as the ordered segregation of chromosomes to two daughter cells requires unimpaired Plk function. Until recently, aneuploidy, the condition in cancer cells in which they display an abnormal number of chromosomes, was considered a consequence, rather than a cause, of the malignancy. However, recent studies suggest aneuploidy can cause cancer. Centrosomes play a critical role in the maintainance of the integrity of the human genome. Aberrant centrosome structure and aneuploidy are tightly correlated in advanced tumors. The studies proposed here address the molecular events controlled by Plk involving centrosome duplication and initiation of cleavage furrow formation and the potential of Snk to influence these and other events in specific cell types.
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