Insulin stimulation of phosphatidylinositol 3-kinase activity maps to insulin receptor regions required for endogenous substrate phosphorylation

We have studied the phosphatidylinositol 3-kinase (Ptdlns 3-kinase) in insulin-stimulated Chinese hamster ovary (CHO) cells expressing normal (CHO/IR) and mutant human insulin receptors. Insulin stimulation of CHO/IR cells results in an increase in PtdIns 3-kinase activity associated with anti-phosphotyrosine (αPY) immunoprecipitates, which has been previously shown to correlate with the in vivo production of PtdIns(3,4)P₂, and PtdIns(3,4,5)P₃ (Ruderman, N., Kapeller, R., White, M. F., and Cantley, L. C. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 1411-1415). Stimulation was maximal within l min and showed a dose response identical to that of insulin receptor autophosphorylation. The PtdIns 3-kinase also associated with the insulin receptor in an insulin-stimulated manner, as approximately 50% of the total αPY-precipitable activity could be specifically immunoprecipitated with anti-insulin receptor antibody. Mutant insulin receptors displayed variable ability to stimulate the Ptdlns 3-kinase, but in all cases the presence of Ptdlns 3-kinase in αPY immunoprecipitates correlated closely with the tyrosyl phosphorylation of the endogenous substrate pp185. In CHO cells expressing a kinase-deficient mutant (IR_A1018), there was no observable insulin stimulation of PtdIns 3-kinase activity in αPY immunoprecipitates and no tyrosyl phosphorylation of pp185. Substitution of Tyr¹¹⁴⁶ in the insulin receptor regulatory region with phenylalanine partially impaired receptor autophosphorylation, pp185 phosphorylation, and insulin-stimulated increases in αPY-precipitable Ptdlns 3-kinase activity. In contrast, a deletion mutant lacking 12 amino acids from the juxtamembrane region (IR_Δ960) displayed normal in vivo autophosphorylation but failed to stimulate the Ptdlns 3-kinase or phosphorylate pp185. Finally, a mutant receptor from which the C-terminal 43 amino acids had been deleted (IR_ΔCT) exhibited normal insulin-stimulated autophosphorylation, pp185 phosphorylation, and stimulation of the PtdIns 3-kinase activity in αPY immunoprecipitates. These data suggest that the Ptdlns 3-kinase is itself a substrate of the insulin receptor kinase or associates preferentially with a substrate. A comparison of the biological activities of the mutant receptors with their activation of the Ptdlns 3-kinase furthermore suggests that the Ptdlns 3-kinase may be linked to insulin's ability to regulate DNA synthesis and cell growth.