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.
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December 1991
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Research Article|
December 15 1991
Phosphorylation in vitro of the 85 kDa subunit of phosphatidylinositol 3-kinase and its possible activation by insulin receptor tyrosine kinase
H Hayashi;
H Hayashi
*Department of Enzyme Genetics, Institute for Enzyme Research, The University of Tokushima, 3-18-15 Kuramoto-cho, Tokushima 770
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N Miyake;
N Miyake
*Department of Enzyme Genetics, Institute for Enzyme Research, The University of Tokushima, 3-18-15 Kuramoto-cho, Tokushima 770
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F Kanai;
F Kanai
*Department of Enzyme Genetics, Institute for Enzyme Research, The University of Tokushima, 3-18-15 Kuramoto-cho, Tokushima 770
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F Shibasaki;
F Shibasaki
†Department of Biosignal Research, Tokyo Metropolitan Institute of Gerontology, Sakae-cho, Itabashi-ku, Tokyo 173, Japan
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T Takenawa;
T Takenawa
†Department of Biosignal Research, Tokyo Metropolitan Institute of Gerontology, Sakae-cho, Itabashi-ku, Tokyo 173, Japan
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Y Ebina
Y Ebina
*Department of Enzyme Genetics, Institute for Enzyme Research, The University of Tokushima, 3-18-15 Kuramoto-cho, Tokushima 770
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Publisher: Portland Press Ltd
Online ISSN: 1470-8728
Print ISSN: 0264-6021
© 1991 The Biochemical Society, London
1991
Biochem J (1991) 280 (3): 769–775.
Citation
H Hayashi, N Miyake, F Kanai, F Shibasaki, T Takenawa, Y Ebina; Phosphorylation in vitro of the 85 kDa subunit of phosphatidylinositol 3-kinase and its possible activation by insulin receptor tyrosine kinase. Biochem J 15 December 1991; 280 (3): 769–775. doi: https://doi.org/10.1042/bj2800769
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