The identification of the molecular connections between obesity, high fat diet and sedentary lifestyle and the promotion of insulin resistance, type 2 diabetes (T2D) and the metabolic syndrome is an area of intense investigation. Numerous studies from many labs indicate that potential pathogenic mediators converge at the level of Ser/Thr phosphorylation of the insulin receptor (IR) and/or its substrates (IRSs), including activation of insulin signaling itself (negative feedback), inflammatory cytokines and other mediators of inflammation (cross-talk), free fatty acids and cellular and oxidative stress. A large number of Ser/Thr kinases have been shown to lead to IR and/or IRS phosphorylation and by doing so potentially participate in mediating insulin resistance. However, most of these studies have utilized over-expression in cultured cells, and since most Ser/Thr kinases function in cascades, it has not been possible to determine primary vs. secondary effects. Specific mechanisms for inhibition are unknown if virtually every case. We are employing a proteomics approach to identify (1) discrete kinases that disrupt IP/IRS interactions and (2) sites of phosphorylation and mechanisms responsible for disruption of insulin signaling. Our approach utilizes a disruptive yeast tri-hybrid (Y3H) method specifically designed and developed for this purpose and mass spectrometry to identify discrete sites, the Y3H method and x-ray crystallography to determine mechanism, and analyses of rodent and patient tissue samples to assess potential relevance under physiological and pathological conditions.
Specific aims i nclude the use of (1) Y3H to screen a broad panel of Ser/Thr kinases to identify those that disrupt IR/IRS interactions, (2) mass spectrometry to identify specific sites of phosphorylation on IR and IRSs isolated from Y3H, (3) Y3H to identify and confirm specific sites of Ser/Thr phosphorylation and mechanisms of disruption of IR/IRS interactions, (4) newly developed phospho-specific antibodies to validate sites of phosphorylation in cells, animals and patient samples to determine physiological and pathophysiological conditions under which phosphorylation occurs, and (5) x-ray crystallography to visualize specific pathophysiological mechanisms for disruption of IR/IRS interactions. These studies should provide a growing framework for understanding major mechanisms of insulin resistance in obesity, T2D and the metabolic syndrome.
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