Inappropriate regulation of a variety of signal transduction processes can result in a multitude of diseases due to improper differentiation and development or malignant transformation. PI3K plays a critical role in cell proliferation. Until recently, however, little was known regarding how PI3K signaled. P70S6K1 was the first signaling protein kinase shown to act downstream of PI3K. Since then, S6K1 activation has been shown to require several lipid-dependent kinases, binding to activated rho family G proteins (Cdc42/rac1), and phosphorylation at multiple sites by distinct inputs. Underscoring its importance, oncogene products such as Dbl, TIAM-1, and Akt specifically activate S6K1, and the T cell immunosuppressants rapamycin and dexamethasone specifically antagonize its activation. The two isoforms of S6K1, alpha-1 and alpha-2, are believed to regulate gene expression and protein translation, respectively. However, little is known of their downstream targets. The research proposed in this application addresses issues regarding the regulation of S6K1 activation, downstream signaling and its role in cell proliferation. The first objective is to define the mechanisms of S6K1 activation by determining how phosphorylation of the autoinhibitory domain and two critical sites in the linker region, S371 and T389, are regulated, and the consequences of these phosphorylations to S6K1 activation. In addition, how these events are coordinated with and cooperate with the phosphorylation of T229 in the activation loop by PDK1 will be examined. The second objective is to identify, using molecular and biochemical approaches, S6K1 associated proteins and assess their roles in regulation of S6K1 or signaling by S6K1. These proteins are expected to be either upstream regulators, downstream targets, or scaffolding proteins that assemble the S6K1 signaling complex. The third objective is to characterize the biological function of S6K1 in a cell system that will allow elucidation of the linkage between S6K1 and the cell cycle machinery. The fourth objective is to characterize transgenic mice expressing S6K1 in a transgenic T-cell model, to assess the role of the protein kinase in murine T-cell proliferation, differentiation, and ultimately function. Since several hematopoietic oncogenes and T cell immunosuppressants modulate S6K1 activity this will allow the examination of the functions of S6K.
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