The past few years have seen outstanding progress in the understanding of the signal transduction mechanisms that underlie the cellular responses to insulin and other ligands that act through receptor tyrosine kinases; nevertheless, much remains to be elucidated. It is now clear that activation of the insulin receptor tyrosine kinase initiates an array of intracellular signalling pathways, primarily through the phosphorylation of specific Tyr residues on the IR and its substrates. IRS-1 and Shc, and perhaps other polypeptides; each TyrP site can then associate with one or more signalling proteins through their SH2 domains. Engagement of these signalling proteins (e.g. Grb/Sos, PI-3 kinase, etc.) riggers independent, parallel signalling pathways that are composed of small GTPases and cascades of protein Ser/Thr kinases. Each pathway serves to transmit, amplify and further diversify the initial receptor- generated signal. The pathway best understood currently involves Ras.GTP activation of the protein kinase cascade involving C-Raf-1, Mek, MAP kinase, Rsk (S6 kinase). The studies proposed herein focus on an equally important but less well-understood group of receptor outflows largely distinct from the Ras cascade, that directs activation of the p70 S6 kinase. In contrast to its closest homolog, the Rsk S6 kinase, which requires only a single upstream activator (i.e. MAP kinase) in situ and in vitro, work in the PI's lab during the prior award period has provided strong evidence that p70 S6 kinase requires (at least) three independent upstream inputs for insulin activation: 1) multiple phosphorylation of a p70 autoinhibitory domain by an array of proline-directed kinases (including Erks and Cdks); 2) phosphorylation of p70 by casein kinase-2; and 3) a Wortmannin-inhibitable signal presumably initiated by the insulin-activated phosphatidyl inositol 3-kinase. We propose to determine the molecular basis by which each of these inputs participates in the activation of p70 S6 kinase, and more importantly, to use the mutant and recombinant p70 S6 kinase cDNAs and polypeptides we have engineered in a variety of biochemical and cloning strategies, to unravel the signal transduction pathways that contribute to the normal insulin regulation of p70 S6 kinase. Specifically, we will elucidate the biochemical steps that couple the PI-3 kinase to p70 kinase, as well as the regulatory mechanisms by which receptor tyrosine kinases engage casein kinase-2 and G1 cyclin-dependent kinases. These studies will clarify several of the major unelucidated signalling outflows from the insulin receptor, and provide new insights into the hormonal regulation of cell metabolism and growth. These results will have implications for the design of new pharmacologic interventions in diabetes mellitus, and in understanding the disordered growth control that characterizes cancer.
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