IN-VITRO ASSOCIATION OF THE PHOSPHATIDYLINOSITOL 3-KINASE REGULATORY SUBUNIT (P85) WITH THE HUMAN INSULIN-RECEPTOR

The insulin receptor, as a consequence of ligand binding, undergoes au tophosphorylation of critical tyrosyl residues within the cytoplasmic portion of its beta-subunit. The 85 kDa regulatory subunit of phosphat idylinositol (PI) 3-kinase (p85), an SH2 domain protein, has been impl icated as a regulatory molecule in the insulin signal transduction pat hway. For the present study, glutathione S-transferase (GST) fusion pr oteins of p85 SH2 domains were used to determine if such motifs associ ate directly with the autophosphorylated human insulin receptor, The p 85 N + C (amino plus carboxyl) SH2 domains were demonstrated to associ ate with the autophosphorylated beta-subunit, while neither the GTPase activator protein (GAP)N SH2 domain nor the phospholipase C-gamma 1 ( PLC gamma 1) N + C SH2 domains exhibited measurable affinity for the a ctivated receptor. The p85 N SH2 domain demonstrated weak association with the insulin receptor, while the p85 C SH2 domain alone formed no detectable complexes with the insulin receptor. The association of p85 N + C SH2 domains with the autophosphorylated receptor was competed e fficiently by a 15-residue tyrosine-phosphorylated peptide correspondi ng to the carboxyl-terminal region of the insulin receptor, but not by phosphopeptides of similar length derived from the juxtamembrane or r egulatory regions. The insulin receptor C domain phosphopeptide inhibi ted the p85 N + C SH2 domain-insulin receptor complex with an IC0.5 of 2.3 +/- 0.35 mu M, whereas a 10-residue phosphopeptide derived from t he insulin receptor substrate 1 (IRS-1) competed with an IC0.5 of 0.54 +/- 0.10 mu M. These results demonstrate that, in vitro, there is an association between the p85 regulatory protein and the carboxyl-termin al region of the activated insulin receptor that requires the presence of both the N and C SH2 domains. Furthermore, formation of the p85/in sulin receptor complex may lead to signaling pathways independent of I RS-1. (C) Munksgaard 1995.