The insulin resistance associated with type II diabetes is thought to have arisen from selective defects in protein kinase mediated signal transduction pathways utilized by the hormone. An understanding of the components that normally govern the regulation of these pathways is required if new therapies are to be developed to treat this disease. A focus in our laboratory is to under-stand the hormonal regulation of protein phosphatase 1 (PP-1) by insulin. Early research showed that PP-1 has significant regulatory roles in most of the major metabolic pathways controlled by the hormone, including glycolysis, glycogen synthesis, fatty acid synthesis, cholesterol bio- synthesis, protein synthesis and lipolysis. More recent studies with the phenotypes of genetic mutants in which PP-1 was deleted has shown the enzyme is also essential in the regulation of processes as diverse as the transport of ions and nutrients into cells, gene transcription and cell cycle. The sequencing of the human genome has revealed that about 2 percent of the expressed genome is devoted to protein kinases. However, the human genome encodes about 20 times fewer Ser/Thr protein phosphatases than Ser/Thr protein kinases. The question then arises as to how a relatively small protein like the catalytic subunit of PP-1 (PP-1 C) can coordinately regulate so many dephosphorylation events in vivo? The key to this paradox is the finding that the functions of PP-1 are closely linked to subcellular localization with regulatory targeting subunits that confer substrate specificity to a common catalytic subunit. About 20 of these have been identified, but given the involvement of PP-1 in so many cellular events others must exist. The physiological targets of PP-1 targeting subunits has only been defined in 1 or 2 cases. Identifying the physiological substrates of most protein phosphatases in cells has remained an intractable problem. The completion of human genome, and the near completion of other genomes such as mouse, in combination with advanced microsequencing technologies affords new opportunities for probing the functions of phosphatases in vivo. Using advanced microsequencing technologies it is now possible to identify any protein in a complex mixture with incredible sensitivity and rapidity. In this proposal we will utilize a combination of affinity capture technology, cell permeable PP-1 holoenzyme disrupting peptides and functional proteomics to determine the molecular mechanisms by which insulin and adrenaline coordinately regulate dephosphorylation events in skeletal muscle cells. Four important goals will be achieved; first, increased understanding of the molecular mechanisms of action of insulin; second, a re- established role for PP-1 in insulin and adrenaline action; third, an understanding of the mechanisms by which targeting subunits alter PP-1 activity in vivo; fourth, a demonstration of the utility of the proteomics in delineating signal transduction pathways in vivo.
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