INSULIN REGULATION OF RAS-DEPENDENT SIGNALING PATHWAYS

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DESCRIPTION (Adapted from the applicant's abstract): The investigator has observed that several agents which activate the various MAP kinase pathways (including insulin, platelet-derived growth factor (PDGF), T-cell receptor activation, serum, phorbol esters, v-ras and v-raf) result in a rapid serine/threonine phosphorylation of the ras GTP/GDP exchange factor SOS. Subsequent to this phosphorylation, there is a dissociation of SOS from Grb2 that appears to correlate with the inactivation phase of ras/GTP loading. This phenomenon, however, is not universal, as stimulation of the epidermal growth factor (EGF) and nerve growth factor receptors cause a phosphorylation of SOS, yet fails to induce the dissociation of the Grb2-SOS complex. The principal investigator proposes to determine the mechanism(s) for these apparent divergent signaling events and to directly assess the physiological consequences of these processes in terms of ras activation and downstream biological responsiveness. This will be accomplished by determining the specific sites of serine/threonine phosphorylation of SOS from insulin and EGF-stimulated cells and by expressing various deletion and point mutations of SOS. The principal investigator will use the MAP kinase phosphatase (MKP1) and various negative-dominant and constituitively active kinases in the ERK/JNK/p38 kinase pathways to determine the in vivo pathways responsible for basal and hormonal stimulated changes in SOS phosphorylation, association with Grb2 and effect on ras/GTP loading. The principal investigator will investigate the potential presence of other associated effector proteins involved in the uncoupling of the Grb2-SOS complex by expression of epitope tagged mutant SOS and Grb2 proteins that are defective in specific interactions motifs. In addition, in vivo metabolic labeling experiments will be used to identify potential associated effector proteins. Finally, the principal investigator will assess the effect of membrane targeting of Shc, Grb2 and SOS on the site-specific phosphorylation of SOS and Shc, and their specific interactions with each other as well as with the EGF and insulin receptor tyrosine kinases.