The reversible phosphorylation of proteins on serine (ser) and threonine (thr) residues is an important mechanism for regulation of diverse pathways controlling cellular physiology. The protein ser/thr phosphatase 2A (PP2A) family of enzymes plays an integral role in many of these processes. The heterotrimeric PP2A holoenzyme consists of a core dimer of A and C (catalytic) subunits associated with one of many divergent variable subunits in three gene families (B, B', and B""""""""). PP2A activity and function are controlled by a complex hierarchy of regulatory mechanisms; however, the combination of mechanisms occurring in a specific cell type, or subcellular region, remains to be elucidated. The current proposal focuses on three important aspects of regulation of PP2A: 1) the functional significance of carboxyl methylation and phosphorylation of the catalytic subunit of PP2A; 2) the impact of B subunits on substrate specificity and/or localization of PP2A; 3) characterization of cellular protein kinases that interact with PP2A, that also may serve as PP2A substrates. Complementary biochemical, immunological, and mutagenesis approaches will be used to determine the role that post-translational modifications and variable B subunits play in regulating PP2A activity, association with interacting proteins, and cellular localization. PP2A/protein kinase complexes will be isolated and phosphatase/kinase activities will be examined using known inhibitors and activators of these enzymes. Finally, the regulation (i.e. by cellular stimuli) and potential function of these complexes will be examined both in vitro and in intact cells by protein purification and co-localization strategies. Together, these studies will provide a better molecular understanding of multi-level regulation of PP2A and the influence of PP2A/interacting proteins in cellular signaling pathways. Additional studies aimed at identifying protein-protein interaction domains of PP2A subunits and/or interacting proteins will provide insights, and cellular reagents, for future exploration of the functional relevance of these interactions on cell growth, differentiation, and apoptosis. Moreover, the identification of the precise protein-protein interactions responsible for localization and regulation of PP2A complexes may suggest novel therapeutic strategies to foster or interfere with these processes in various pathological states, including perturbed development and oncogenesis.
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