Phosphorylation in vitro of the 85 kDa subunit of phosphatidylinositol 3-kinase and its possible activation by insulin receptor tyrosine kinase.
Insulin causes a dramatic and rapid increase in phosphatidylinositol 3-kinase activity in the anti-phosphotyrosine immunoprecipitates of cells overexpressing the human insulin receptor. This enzyme may therefore be a mediator of insulin signal transduction [Endemann, Yonezawa & Roth (1990) J. Biol. Chem. 265, 396-400; Ruderman, Kapeller, White & Cantley (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 1411-1415]. At least two questions remain to be elucidated. Firstly, does the insulin receptor tyrosine kinase phosphorylate phosphatidylinositol 3-kinase directly, or does it phosphorylate a protein associated with the 3-kinase? Second, if the enzyme is a direct substrate for the insulin receptor tyrosine kinase, does tyrosine phosphorylation of phosphatidylinositol 3-kinase by the kinase alter the specific enzyme activity, or does the amount of the tyrosine-phosphorylated form of the phosphatidylinositol 3-kinase increase, with no change in the specific activity? We report here evidence that the 85 kDa subunit of highly purified phosphatidylinositol 3-kinase is phosphorylated on the tyrosine residue by the activated normal insulin receptor in vitro, but not by a mutant insulin receptor which lacks tyrosine kinase activity. We found that an increase in enzyme activity was detected in response to insulin not only in the anti-phosphotyrosine immunoprecipitates of the cytosol, but also in the cytosolic fraction before immunoprecipitation. In addition, we partially separated the tyrosine-phosphorylated form from the unphosphorylated form of the enzyme, by using a f.p.l.c. Mono Q column. The insulin-stimulated phosphatidylinositol 3-kinase activity was mainly detected in the fraction containing almost all of the tyrosine-phosphorylated form. This result suggests that tyrosine phosphorylation of phosphatidylinositol 3-kinase by the insulin receptor kinase may increase the specific activity of the former enzyme in vivo.
Hayashi
H
H
Department of Enzyme Genetics, University of Tokushima, Japan.
Miyake
N
N
Kanai
F
F
Shibasaki
F
F
Takenawa
T
T
Ebina
Y
Y
D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003600:Cytosol / Q000201:enzymology; D004789:Enzyme Activation; D006801:Humans; D066298:In Vitro Techniques; D007328:Insulin / Q000494:pharmacology; D046911:Macromolecular Substances; D019869:Phosphatidylinositol 3-Kinases; D010766:Phosphorylation; D010770:Phosphotransferases / Q000187:drug effects / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D019000:Phosphotyrosine; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D011972:Receptor, Insulin / Q000378:metabolism*; D014443:Tyrosine / Q000031:analogs & derivatives / Q000276:immunology / Q000378:metabolism*
D007328:Insulin; D046911:Macromolecular Substances; D019000:Phosphotyrosine; D014443:Tyrosine; D010770:Phosphotransferases; D019869:Phosphatidylinositol 3-Kinases; D011505:Protein-Tyrosine Kinases; D011972:Receptor, Insulin