1992 Fiscal Year Final Research Report Summary
Molecular mechanisms of insulin signal transduction and its disorder
| Project/Area Number |
03454161
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| Research Category |
Grant-in-Aid for General Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Research Field |
Pathological medical chemistry
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| Research Institution | The University of Tokushima |
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Principal Investigator |
EBINA Yousuke The University of Tokushima, Institute for Enzyme Research, Professor, 酵素科学研究センター, 教授 (00112227)
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| Co-Investigator(Kenkyū-buntansha) |
HAYASHI Hideki The University of Tokushima, Institute for Enzyme Research, Research Associate, 酵素科学研究センター, 助手 (10218589)
MURAKAMI Takashi The University of Tokushima, Institute for Enzyme Research, Research Associate, 酵素科学研究センター, 助手 (40210009)
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| Project Period (FY) |
1991 – 1992
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| Keywords | Insulin Receptor / Insulin signal transduction / PI 3-kinase |
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| Research Abstract |
After adding insulin to cells overexpressing the insulin receptor, the activity of phosphatidylinositol (PI) 3-kinase in the anti-phosphotyrosine immunoprecipitates was rapidly and greatly increased. This enzyme may therefore be a substrate for the insulin receptor tyrosine kinase and may be one of the mediators of insulin signal transduction. However, it is unclear whether or not activated tyrosine kinase of the insulin receptor directly phosphorylates PI 3-kinase at tyrosine residue(s) and whether insulin stimulates the specific activity of PI 3-kinase. We reported previously that the 85-kDa subunit of purified PI 3-kinase was phosphorylated at tyrosine residue(s) by the insulin receptor in vitro.
To examine the tyrosine phosphorylation of PI 3-kinase and change of its activity by insulin treatment in vivo, we used a specific antibody to the 85-kDa subunit of PI 3-kinase. The activity of PI 3-kinase in immunoprecipitates with the antibody against the p85 subunit of PI 3-kinase was inc
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reased about 3-fold by insulin treatment of cells overexpressing insulin receptors. Insulin treatment also stimulated the tyrosine, serine, and threonine phosphorylations of the alpha-type 85-kDa subunit of PI 3-kinase in vivo. Phosphatase treatment of the immunoprecipitates abolished the increase in PI 3-kinase activity. The phosphorylation(s) of the kinase itself, tyrosine phosphorylation(s) of associated protein(s), or the complex formation of the phosphorylated PI 3-kinase with associated proteins may increase the activity of PI 3-kinase.
In the next work, we identified the major tyrosine phosphorylation sites of the alpha-type p85 by the insulin receptor. [^<32>P]Phosphopeptides obtained from lysylendopeptidase digestion of phosphorylated alpha-type p85 in intact cells after insulin treatment were analyzed using reverse-phase high performance liquid chromatography and thin layer electrophoresis. The alpha-type p85 of PI 3-kinase was phosphorylated at tyrosines 368, 580, and 607 by the insulin receptor in vivo. Less
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