Enhanced insulin-induced mitogenesis and mitogen-activated protein kinase activities in mutant insulin receptors with substitution of two COOH-terminal tyrosine autophosphorylation sites by phenylalanine

We have studied the function of a mutant human insulin receptor in which two COOH-terminal autophosphorylation sites (Tyr-1316 and -1322) were replaced by phenylalanine (F/Y COOH-terminal 2 tyrosines (CT₂)). In addition, we have also constructed a mutant receptor in which Lys-1018 in the ATP-binding site was changed to arginine (R/K 1018). Both the wild type insulin receptor (HIR) and the mutant receptors were expressed in Chinese hamster ovary (CHO) cells by stable transfection. Autophosphorylation of solubilized and partially purified F/Y CT₂ was decreased by approximately 30% compared with the HIR. Tyrosine kinase activities of F/Y CT₂ and HIR toward exogenous substrates were almost equal. When CHO cells transfected with F/Y CT₂ (CHO-F/Y CT₂) were stimulated with insulin, autophosphorylation of the β-subunit of the insulin receptor and the phosphorylation of an endogenous substrate (pp185) in the intact cell were normal compared with cells expressing HIR (CHO-HIR). CHO-F/Y CT₂ exhibited the same insulin sensitivity as CHO-HIR with respect to 2-deoxyglucose uptake. However, the dose-response curve of insulin-stimulated thymidine incorporation in CHO-F/Y CT₂ was shifted to the left (∼5-7-fold) compared with that in CHO-HIR. There was no significant difference in insulin-like growth factor 1-stimulated thymidine incorporation between CHO-F/Y CT₂ and CHO-HIR. Furthermore, the dose-response curve of insulin-stimulated kinase activity toward myelin basic protein in CHO-F/Y CT₂ was also shifted to the left (∼5-fold) compared with that in CHO-HIR. Kinase assays in myelin basic protein-containing gels revealed that both species of MAP kinases (M_r 44,000, 42,000) were more sensitive to activation by insulin in CHO-F/Y CT₂ than in CHO-HIR. This observation was confirmed in immune complex kinase assays toward microtubule-associated protein 2 (MAP2) using specific antibodies against mitogen-activated protein (MAP) kinase. R/K 1018 mutant insulin receptors showed an absence of insulin-stimulated kinase activity and CHO cells transfected with R/K 1018 (CHO-R/K 1018) failed to enhance 2-deoxyglucose uptake or thymidine incorporation in response to insulin. In addition, R/K 1018 kinase-defective insulin receptors were unable to mediate insulin-stimulated MAP kinase activation. These data suggest that: 1) tyrosine kinase activity of the insulin receptor is required for activation of insulin-stimulated MAP kinases and 2) phosphorylation of COOH-terminal tyrosine residues may play an inhibitory role in mitogenic signaling through regulation of MAP kinases.