Insulin stimulates association of insulin receptor substrate-1 with the protein abundant Src homology/growth factor receptor-bound protein 2

K. Tobe, K. Matuoka, H. Tamemoto, K. Ueki, Y. Kaburagi, S. Asai, T. Noguchi, M. Matsuda, S. Tanaka, S. Hattori, Y. Fukui, Y. Akanuma, Y. Yazaki, T. Takenawa, T. Kadowaki

Research output: Contribution to journalArticle

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Abstract

Insulin activates the ras proto-oncogene product p21(ras) (Ras) by stimulating conversion of the inactive GDP-bound form of Ras to the active GTP-bound form. The protein ASH (for abundant Src homology) (Matuoka, K., Shibata, M., Yamakawa, A., and Takenawa, T. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 9015-9019) is composed of one Src homology (SH)2 and two SH3 domains and highly homologous to the Caenorhabditis elegans protein sem-5 that couples a tyrosine kinase to a Ras protein. We have studied an interaction of ASH with insulin-stimulated tyrosine-phosphorylated proteins in Chinese hamster ovary cells overexpressing human insulin receptors (CHO- HIR cells). In an anti-ASH (αASH) immunoprecipitates, we detected a 170-kDa phosphoprotein that was recognized by an anti-phosphotyrosine antibody and an anti-insulin receptor substrate 1 antibody (αIRS-1) from the insulin- stimulated [32P]orthophosphate-labeled CHO-HIR cells. We failed to detect the tyrosine phosphorylation of the protein ASH. These data suggested that insulin stimulates IRS-1·ASH complex formation in intact cells. Incubation of an ASH fusion protein with the lysates of insulin-stimulated CHO-HIR cells revealed that the fusion protein of ASH was able to bind the tyrosine- phosphorylated 170-kDa protein that was recognized by αIRS-1. We also demonstrated that fusion protein of ASH was able to bind the fusion protein of tyrosine-phosphorylated IRS-1 fragments, suggesting that ASH is able to bind tyrosine-phosphorylated IRS-1 directly. These data suggest that IRS- 1·ASH complex formation may play a role in coupling the insulin receptor kinase to a Ras signaling pathway. Furthermore, we observed an insulin- stimulated phosphatidylinositol (PI) 3-kinase activity in αASH immunoprecipitates, suggesting the formation of an ASH·IRS-1·PI 3-kinase complex. This complex formation was detected as early as 10 s after insulin stimulation in intact CHO-HIR cells. This is the first report that supports the notion that IRS-1 binds several signal transducing molecules containing SH2 domains, thus serves as an SH2 docking protein that transduces insulin's signal multidirectionally.

Original languageEnglish (US)
Pages (from-to)11167-11171
Number of pages5
JournalJournal of Biological Chemistry
Volume268
Issue number15
StatePublished - Jan 1 1993

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GRB2 Adaptor Protein
Insulin Receptor Substrate Proteins
CHO Cells
Tyrosine
Association reactions
Insulin
Fusion reactions
Proto-Oncogene Proteins p21(ras)
src Homology Domains
Proteins
Antibodies
Phosphotransferases
Caenorhabditis elegans Proteins
Phosphatidylinositol 3-Kinase
Insulin Antibodies
ras Proteins
Immunoglobulin Fragments
Phosphotyrosine
Phosphorylation
Cell Fusion

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Tobe, K., Matuoka, K., Tamemoto, H., Ueki, K., Kaburagi, Y., Asai, S., ... Kadowaki, T. (1993). Insulin stimulates association of insulin receptor substrate-1 with the protein abundant Src homology/growth factor receptor-bound protein 2. Journal of Biological Chemistry, 268(15), 11167-11171.

Insulin stimulates association of insulin receptor substrate-1 with the protein abundant Src homology/growth factor receptor-bound protein 2. / Tobe, K.; Matuoka, K.; Tamemoto, H.; Ueki, K.; Kaburagi, Y.; Asai, S.; Noguchi, T.; Matsuda, M.; Tanaka, S.; Hattori, S.; Fukui, Y.; Akanuma, Y.; Yazaki, Y.; Takenawa, T.; Kadowaki, T.

In: Journal of Biological Chemistry, Vol. 268, No. 15, 01.01.1993, p. 11167-11171.

Research output: Contribution to journalArticle

Tobe, K, Matuoka, K, Tamemoto, H, Ueki, K, Kaburagi, Y, Asai, S, Noguchi, T, Matsuda, M, Tanaka, S, Hattori, S, Fukui, Y, Akanuma, Y, Yazaki, Y, Takenawa, T & Kadowaki, T 1993, 'Insulin stimulates association of insulin receptor substrate-1 with the protein abundant Src homology/growth factor receptor-bound protein 2', Journal of Biological Chemistry, vol. 268, no. 15, pp. 11167-11171.
Tobe, K. ; Matuoka, K. ; Tamemoto, H. ; Ueki, K. ; Kaburagi, Y. ; Asai, S. ; Noguchi, T. ; Matsuda, M. ; Tanaka, S. ; Hattori, S. ; Fukui, Y. ; Akanuma, Y. ; Yazaki, Y. ; Takenawa, T. ; Kadowaki, T. / Insulin stimulates association of insulin receptor substrate-1 with the protein abundant Src homology/growth factor receptor-bound protein 2. In: Journal of Biological Chemistry. 1993 ; Vol. 268, No. 15. pp. 11167-11171.
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AU - Tobe, K.

AU - Matuoka, K.

AU - Tamemoto, H.

AU - Ueki, K.

AU - Kaburagi, Y.

AU - Asai, S.

AU - Noguchi, T.

AU - Matsuda, M.

AU - Tanaka, S.

AU - Hattori, S.

AU - Fukui, Y.

AU - Akanuma, Y.

AU - Yazaki, Y.

AU - Takenawa, T.

AU - Kadowaki, T.

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N2 - Insulin activates the ras proto-oncogene product p21(ras) (Ras) by stimulating conversion of the inactive GDP-bound form of Ras to the active GTP-bound form. The protein ASH (for abundant Src homology) (Matuoka, K., Shibata, M., Yamakawa, A., and Takenawa, T. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 9015-9019) is composed of one Src homology (SH)2 and two SH3 domains and highly homologous to the Caenorhabditis elegans protein sem-5 that couples a tyrosine kinase to a Ras protein. We have studied an interaction of ASH with insulin-stimulated tyrosine-phosphorylated proteins in Chinese hamster ovary cells overexpressing human insulin receptors (CHO- HIR cells). In an anti-ASH (αASH) immunoprecipitates, we detected a 170-kDa phosphoprotein that was recognized by an anti-phosphotyrosine antibody and an anti-insulin receptor substrate 1 antibody (αIRS-1) from the insulin- stimulated [32P]orthophosphate-labeled CHO-HIR cells. We failed to detect the tyrosine phosphorylation of the protein ASH. These data suggested that insulin stimulates IRS-1·ASH complex formation in intact cells. Incubation of an ASH fusion protein with the lysates of insulin-stimulated CHO-HIR cells revealed that the fusion protein of ASH was able to bind the tyrosine- phosphorylated 170-kDa protein that was recognized by αIRS-1. We also demonstrated that fusion protein of ASH was able to bind the fusion protein of tyrosine-phosphorylated IRS-1 fragments, suggesting that ASH is able to bind tyrosine-phosphorylated IRS-1 directly. These data suggest that IRS- 1·ASH complex formation may play a role in coupling the insulin receptor kinase to a Ras signaling pathway. Furthermore, we observed an insulin- stimulated phosphatidylinositol (PI) 3-kinase activity in αASH immunoprecipitates, suggesting the formation of an ASH·IRS-1·PI 3-kinase complex. This complex formation was detected as early as 10 s after insulin stimulation in intact CHO-HIR cells. This is the first report that supports the notion that IRS-1 binds several signal transducing molecules containing SH2 domains, thus serves as an SH2 docking protein that transduces insulin's signal multidirectionally.

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