title: 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.
abstract: 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 beta-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 (approximately 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 (approximately 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.
authors.affiliation: Third Department of Internal Medicine, Faculty of Medicine, University of Tokyo, Japan.
authors.forename: A
authors.initials: A
authors.lastname: Ando
authors.forename: K
authors.initials: K
authors.lastname: Momomura
authors.forename: K
authors.initials: K
authors.lastname: Tobe
authors.forename: R
authors.initials: R
authors.lastname: Yamamoto-Honda
authors.forename: H
authors.initials: H
authors.lastname: Sakura
authors.forename: Y
authors.initials: Y
authors.lastname: Tamori
authors.forename: Y
authors.initials: Y
authors.lastname: Kaburagi
authors.forename: O
authors.initials: O
authors.lastname: Koshio
authors.forename: Y
authors.initials: Y
authors.lastname: Akanuma
authors.forename: Y
authors.initials: Y
authors.lastname: Yazaki
mesh_terms: D000818:Animals; D000936:Antigen-Antibody Complex; D001483:Base Sequence; D016466:CHO Cells; D006224:Cricetinae; D004247:DNA; D003847:Deoxyglucose / Q000378:metabolism; D004705:Endocytosis; D004789:Enzyme Activation; D006133:Growth Substances / Q000378:metabolism; D015151:Immunoblotting; D007328:Insulin / Q000378:metabolism*; D037102:Lectins; D008969:Molecular Sequence Data; D009154:Mutation; D010649:Phenylalanine / Q000378:metabolism*; D010766:Phosphorylation; D011233:Precipitin Tests; D011494:Protein Kinases / Q000378:metabolism; D011972:Receptor, Insulin / Q000235:genetics / Q000378:metabolism*; D012995:Solubility; D013936:Thymidine / Q000378:metabolism; D014162:Transfection; D014443:Tyrosine / Q000378:metabolism*
chemical_list: D000936:Antigen-Antibody Complex; D006133:Growth Substances; D007328:Insulin; D037102:Lectins; D014443:Tyrosine; D010649:Phenylalanine; D004247:DNA; D003847:Deoxyglucose; D011494:Protein Kinases; D011972:Receptor, Insulin; D013936:Thymidine