Assessment of the in situ tyrosine kinase activity of mutant insulin receptors lacking tyrosine autophosphorylation sites 1162 and 1163.
In the present studies mutant insulin receptors with regulatory tyrosine residues 1162 and 1163 changed to phenylalanines were tested for tyrosine kinase activity. In agreement with prior studies, this mutant receptor was found to exhibit almost no insulin-stimulated exogenous kinase activity when assayed in vitro. In contrast, this mutant receptor was found in situ to have a significant, albeit reduced, ability to mediate the tyrosine phosphorylation of various endogenous proteins, as assessed by Western blotting with antiphosphotyrosine antibodies. In addition, extracts of insulin-treated cells overexpressing this mutant receptor exhibited increased amounts of tyrosine phosphorylated phosphatidylinositol 3-kinase compared to control cells. Finally, this mutant receptor, like the wild-type receptor, was found to mediate an increase in the activity of a membrane-associated phosphatidylinositol 4,5-biphosphate kinase. These results indicate that 1) in vitro assessments of the tyrosine kinase activity of mutant insulin receptors may not accurately reflect their in vivo activities; and 2) the ability of the mutant receptor lacking tyrosine autophosphorylation sites 1162 and 1163 to mediate insulin-stimulated tyrosine phosphorylation of various endogenous substrates may account for the reported ability of this receptor to mediate various biological responses.
Yonezawa
K
K
Department of Pharmacology, Stanford University School of Medicine, California 94305.
Roth
R A
RA
D000818:Animals; D001665:Binding Sites; D015153:Blotting, Western; D002460:Cell Line; D006224:Cricetinae; D006801:Humans; D007328:Insulin / Q000494:pharmacology; D007700:Kinetics; D009154:Mutation; D019869:Phosphatidylinositol 3-Kinases; D010766:Phosphorylation; D010770:Phosphotransferases / Q000378:metabolism; D017853:Phosphotransferases (Alcohol Group Acceptor); D019000:Phosphotyrosine; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D011972:Receptor, Insulin / Q000235:genetics* / Q000378:metabolism; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000378:metabolism
D007328:Insulin; D019000:Phosphotyrosine; D014443:Tyrosine; D010770:Phosphotransferases; D019869:Phosphatidylinositol 3-Kinases; D017853:Phosphotransferases (Alcohol Group Acceptor); C068961:phosphatidylinositol 4,5-biphosphate kinase; C020077:1-phosphatidylinositol-4-phosphate 5-kinase; D011505:Protein-Tyrosine Kinases; D011972:Receptor, Insulin