The study of cellular proteins that are specifically bound by the tumor antigens of papovaviruses is proving increasingly important to our understanding of human cancer, yielding important insights into signal transduction pathways that play a role in cancer. Protein phosphatase 2A (PP2A) and 14-3-3 proteins are highly conserved proteins ubiquitous in eukaryotes. They are known to be involved in the control of cell proliferation and are cellular targets of the papovavirus oncogene, polyomavirus middle tumor antigen (MT), with which they form stable complexes. The long range goal of this proposal is to understand both the role(s) and regulation of PP2A and 14-3-3 proteins in MT functions, especially MT-mediated transformation. The approach will be threefold: 1) First, to directly investigate the role of PP2A activity in PP2A/MT/pp60c-src complex formation and function, MT and pp60c-src associated with catalytically inactive PP2A point mutants will be analyzed biochemically and functionally to determine the type, location, and functional importance of novel modifications. 2) Second, because understanding MT regulation of PP2A requires further delineation of normal PP2A regulation, three novel PP2A-associated proteins that are likely to be involved in PP2A function and are also likely to shed light on MT effects on PP2A will be identified and/or cloned and initially characterized. Each of these proteins will be purified by immunoaffinity purification, microsequenced, cloned if novel, and characterized biochemically and functionally in regard to their involvement in PP2A and MT functions. 3) Third, to determine if 14-3-3 proteins contribute to oncogenic transformation by MT, perhaps by affecting association of other proteins with MT, MT mutants defective in binding 14-3-3 proteins will be isolated and analyzed for complex formation with cellular proteins and in transformation assays.
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