Cellular response to an external stimulus requires the appropriate cascade of signals from the cell surface to the nucleus. The integrated actions of both protein kinases and protein phosphatases govern the phosphorylation state of key intracellular proteins which are crucial to normal cellular signaling. In contrast to the protein serine/threonine kinases (PKs), which have been extensively studied in this context, much less attention has been given to the protein serine/threonine phosphatases (PPs). It is clear, however, that this group of proteins, especially nuclear PPs, play an integral role in the control of cell growth and differentiation. The catalytic subunits of two such phosphatases, PP2A and PPX, are highly homologous and presumably complexed with similar or related regulatory subunits. The subcellular distribution of the PP2A and PPX holoenzymes is, however, different since PP2A is predominantly cytosolic, whereas PPX is predominantly nuclear. The overall goals of this proposal are to determine the localization, regulation, and function of nuclear protein serine/threonine phosphatase 2A and X holoenzymes. A multidisciplinary approach employing biochemical, immunological, and molecular biology approaches will be used to accomplish these goals. A combination of independent experimental strategies, including tissue immunoblotting, immunofluorescence, subcellular fractionation and immunoblotting will be used to define the tissue distribution and subcellular localization of PP2A and PPX. Nuclear PP2A and PPX holoenzymes will be purified in order to establish the oligomeric composition of these enzymes. The mechanism(s) for regulation of PP2A and PPX will be elucidated by analyzing changes in the phosphorylation state of phosphatase subunits, changes in subcellular localization of phosphatase subunits, and/or changes in oligomeric structure of phosphatase holoenzymes. In parallel, three different, yet complementary, techniques are proposed for assaying phosphatase activity and function in intact cells. Finally, a combination of mutagenesis and molecular genetic strategies will be used to deduce the structural basis for regulation and nuclear localization of these enzymes. It is anticipated that the proposed studies, with their blend of predictably productive as well as high-risk efforts, will reveal the structure and regulation of nuclear phosphatases, their structural and regulatory relationship with cytosolic phosphatases, and their importance in nuclear events, including hormone-regulated transcription. Naturally, these studies will enhance our knowledge of the fundamental process of cell cycle control and nuclear transcription as well as the regulation of these processes by phosphorylation/dephosphorylation events. However, insights gained might also offer some molecular enlightenment of the defective nuclear events controlling perturbed development or oncogenesis. Finally, identification of the precise protein-protein interactions responsible for localization and regulation of phosphatase activity may suggest novel therapeutic strategies to foster or interfere with these processes in various pathological states.
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