During the past funding period, we have several important advances in our understanding of the proximal insulin receptor signaling events mediating downstream insulin biological responsiveness. In this proposal we plan to focus onto two related insulin signaling events that utilize ras as the central target for regulation. In addition, the mechanisms controlling ras function have not only broad implications for our understanding of insulin action at the level of gene transcription and macromolecular synthesis but also directly relate to other tyrosine and non-tyrosine kinase signaling events as well as in the control of differentiation and cell development. First, we have observed that insulin stimulation results in a MEK-dependent feedback phosphorylation of SOS, the guanylnucleotide exchange factor for ras. This feedback phosphorylation correlates with a disassociation of the Grb2-SOS complex and appears to account for the desensitization of ras activation. Since other tyrosine kinase and non-tyrosine kinase pathways are also linked to the ERK pathway, we will investigate their interaction at the level of heterologous desensitization of ras activation. Furthermore, we have also obtained evidence that the uncoupling of the Grb2-SOS complex requires MEK-activation but is independent of ERK activity. Since SOS does not contain any consensus MEK phosphorylation sites and does not phosphorylate SOS in vitro, we will identify the MEK- dependent ERK-like kinase responsible for SOS phosphorylation. Second, we and others have also established that-the tyrosine-specific phosphatase, SHPTP2 is an important positive mediator of insulin action and functions upstream of ras in mediating the raf/MEK/ERK pathway. We have recently identified a 115 kDa protein as the major insulin-stimulated tyrosine phosphorylated docking protein for SHPTP2. Thus, we plan to further characterize and identify the molecular nature of this protein and determine its role as an upstream effector for ras function.
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