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 in sulin 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 m uscles of chickens submitted to various nutritional states. A chicken liver Shc cDNA fragment encoding a 198 amino acid long fragment, inclu ding the phosphotyrosine binding domain was sequenced. It shows 89% ho mology with the corresponding human homologue. The amounts of the thre e Shc isoforms (66, 52 and 46 kDa) and Shc messenger were not altered by the nutritional state. Shc tyrosine phosphorylation was decreased b y fasting in both liver and muscle. Importantly, Shc was immunoprecipi tated by IR antibody (mostly the 52 kDa isoform) or by alpha IRS-1 (mo stly the 46 kDa isoform). IR-Shc association was decreased by fasting and restored by refeeding. In liver, alpha Shc immunoprecipitated the three forms of regulatory subunits of PI 3'-kinase and a PI 3'-kinase activity which was decreased by fasting. In muscle, aShc immunoprecipi tated only the p85 isoform; the associated PI 3'-kinase activity was n ot altered by the nutritional state. Conversely, in both tissues anti- p85 antibody precipitated only the 52 kDa Shc isoform. In liver, antib odies to insulin receptor substrate-1 (alpha IRS-1), Shc or IR immunop recipitated the three regulatory subunits of PI 3'-kinase and an equal PI 3'-kinase activity, without any residual activity left in the supe rnatants, suggesting the presence of a large complex involving IR, IRS -I, Shc (mainly the 52 kDa isoform) and PI 3'-kinase activity. The pre sence of another complex containing IRS-1 and the 46 kDa Shc isoform, but no PI 3'-kinase activity, is suggested.