The goal of this project is to describe in molecular detail one limb of the insulin signal transduction system. Nearly all of the classical metabolic responses to insulin require activation of the phosphatidylinositol 3' OH kinase (PI-3 Kinase). Thus to understand insulin signalling, the identity of the effectors of the PI-3 Kinase relevant to insulin must be identified and characterized. This project focuses on an outflow from PI-3 kinase to the protein (Ser/Thr) kinase known as p70 S6 kinase. This enzyme appears to be critical to the insulin control of protein synthesis, in particular to the process of ribosomal biogenesis, a crucial component of the anabolic response to insulin. We showed previously that p70 is regulated by insulin through a complex multisite phosphorylation directed at several different domains involving Thr252, Ser394, Thr412 and others, and catalyzed by an array of PI-3 Kinase-regulated protein kinases situated upstream. The activity of the p7O S6 kinase is also regulated by the availability of amino acids through a novel signal transduction pathway, largely distinct from that controlled by insulin, but which converges with the pathway controlled by the mTOR kinase, the target of the immuno- suppressant drug, rapamycin. This novel amino and mTOR responsive pathway may utilize protein phosphatases to control cell function. We recently discovered one of the enzymes responsible for the insulin activation of p70 S6 kinase. This enzyme, called PDK1 is also one of the activators of the cAkt/PKB kinase. PDK1 must work in conjunction with other, as yet unidentified Ptd Ins (3,4,5)P3 activated kinases, in order to activate effectively both p70 and PKB. The goals of this project for the next period are to: 1) Isolate and characterize the other PI-3 kinase-regulated protein kinase necessary for activation of p70, the p70 Thr412 kinase; 2 ) Characterize the protein kinase active on p70 Ser394 in vivo, and the nature and significance of mTO- catalyzed phosphorylation of p70; 3) Characterize the protein phosphatases that act on p70 and their regulation by insulin, amino acids an mTOR; 4) Characterize the regulation, cellular itinerary and cellular targets of PDK1, an important multifunctional PI-3 kinase effector. These studies will clarify several of the major new signalling outflows of the insulin receptor, and will provide new insights into the regulation of protein synthesis. These results will have implications for the design of new pharmacologic interventions in diabetes mellitus and will have wide implications for the nutritional regulation of body protein turnover.
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