Effect of nutritional state on the formation of a complex involving insulin receptor IRS-1, the 52 kDa Src homology/collagen protein (Shc) isoform and phosphatidylinositol 3'-kinase activity.

The Src homology and collagen protein (Shc) is tyrosine phosphorylated in response to insulin; however, evidence for its interaction with insulin receptor (IR) in normal tissues is missing. Interactions between IR, Shc and regulatory subunits of the phosphatidylinositol 3'-kinase (PI 3'-kinase) were characterized in the present study in liver and muscles of chickens submitted to various nutritional states. A chicken liver Shc cDNA fragment encoding a 198 amino acid long fragment, including the phosphotyrosine binding domain was sequenced. It shows 89% homology with the corresponding human homologue. The amounts of the three Shc isoforms (66, 52 and 46 kDa) and Shc messenger were not altered by the nutritional state. Shc tyrosine phosphorylation was decreased by fasting in both liver and muscle. Importantly, Shc was immunoprecipitated by IR antibody (mostly the 52 kDa isoform) or by alphaIRS-1(mostly the 46 kDa isoform). IR-Shc association was decreased by fasting and restored by refeeding. In liver, alphaShc immunoprecipitated the three forms of regulatory subunits of PI 3'-kinase and a PI 3'-kinase activity which was decreased by fasting. In muscle, alphaShc immunoprecipitated only the p85 isoform; the associated PI 3'-kinase activity was not altered by the nutritional state. Conversely, in both tissues anti-p85 antibody precipitated only the 52 kDa Shc isoform. In liver, antibodies to insulin receptor substrate-1 (alphaIRS-1), Shc or IR immunoprecipitated the three regulatory subunits of PI 3'-kinase and an equal PI 3'-kinase activity, without any residual activity left in the supernatants, suggesting the presence of a large complex involving IR, IRS-1, Shc (mainly the 52 kDa isoform) and PI 3'-kinase activity. The presence of another complex containing IRS-1 and the 46 kDa Shc isoform, but no PI 3'-kinase activity, is suggested.

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