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. 1996 Mar;16(3):943–951. doi: 10.1128/mcb.16.3.943

Overexpression of human insulin receptor substrate 1 induces cellular transformation with activation of mitogen-activated protein kinases.

T Ito 1, Y Sasaki 1, J R Wands 1
PMCID: PMC231076  PMID: 8622697

Abstract

The receptor insulin substrate 1 protein (IRS-1) is a specific substrate for insulin receptor tyrosine kinase. Expression and tyrosyl phosphorylation of IRS-1 play an important role during normal hepatocyte growth, and the gene is overexpressed in hepatocellular carcinoma tissue. We determined if IRS-1 overexpression directly contributes to cellular transformation. The human IRS-1 gene was subcloned into a mammalian expression vector driven by the cytomegalovirus early promoter. NIH 3T3 cells transiently transfected with this vector subsequently developed transformed foci. Several stably transfected cell lines were established, and they grew efficiently under low-serum conditions and formed colonies when plated in soft agar. Cell lines overexpressing IRS-1 displayed increased tyrosyl phosphorylation of IRS-1 and association with Grb2 but not with the p85 subunit of phosphatidylinositol 3' kinase. Since Grb2 is a component of the son-of-sevenless-Ras pathway and upstream in the mitogen-activated protein kinase (MAPK) cascade, enzymatic activities of the major components of this cascade, such as MAPK kinase and MAPK were evaluated and found to be substantially increased in three independent cell lines with IRS-1 protein overexpression. Such cells, when injected into nude mice, were highly tumorigenic, and there may be a correlation between the degree of MAPK activation and tumor growth rate. This report describes the generation of a transformed phenotype by overexpression of a molecule without a catalytic domain far upstream in the signal transduction cascade and suggests that prolonged activation of MAPKs by this mechanism may be one of the molecular events related to hepatocellular transformation.

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Selected References

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  1. Avruch J., Zhang X. F., Kyriakis J. M. Raf meets Ras: completing the framework of a signal transduction pathway. Trends Biochem Sci. 1994 Jul;19(7):279–283. doi: 10.1016/0968-0004(94)90005-1. [DOI] [PubMed] [Google Scholar]
  2. Backer J. M., Myers M. G., Jr, Shoelson S. E., Chin D. J., Sun X. J., Miralpeix M., Hu P., Margolis B., Skolnik E. Y., Schlessinger J. Phosphatidylinositol 3'-kinase is activated by association with IRS-1 during insulin stimulation. EMBO J. 1992 Sep;11(9):3469–3479. doi: 10.1002/j.1460-2075.1992.tb05426.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Baltensperger K., Kozma L. M., Cherniack A. D., Klarlund J. K., Chawla A., Banerjee U., Czech M. P. Binding of the Ras activator son of sevenless to insulin receptor substrate-1 signaling complexes. Science. 1993 Jun 25;260(5116):1950–1952. doi: 10.1126/science.8391166. [DOI] [PubMed] [Google Scholar]
  4. Boguski M. S., McCormick F. Proteins regulating Ras and its relatives. Nature. 1993 Dec 16;366(6456):643–654. doi: 10.1038/366643a0. [DOI] [PubMed] [Google Scholar]
  5. Bucher M. L., Swaffield M. N. Regulation of hepatic regeneration in rats by synergistic action of insulin and glucagon. Proc Natl Acad Sci U S A. 1975 Mar;72(3):1157–1160. doi: 10.1073/pnas.72.3.1157. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Bucher N. L., Patel U., Cohen S. Hormonal factors concerned with liver regeneration. Ciba Found Symp. 1977;(55):95–107. doi: 10.1002/9780470720363.ch5. [DOI] [PubMed] [Google Scholar]
  7. Cantley L. C., Auger K. R., Carpenter C., Duckworth B., Graziani A., Kapeller R., Soltoff S. Oncogenes and signal transduction. Cell. 1991 Jan 25;64(2):281–302. doi: 10.1016/0092-8674(91)90639-g. [DOI] [PubMed] [Google Scholar]
  8. Cowley S., Paterson H., Kemp P., Marshall C. J. Activation of MAP kinase kinase is necessary and sufficient for PC12 differentiation and for transformation of NIH 3T3 cells. Cell. 1994 Jun 17;77(6):841–852. doi: 10.1016/0092-8674(94)90133-3. [DOI] [PubMed] [Google Scholar]
  9. Crews C. M., Erikson R. L. Extracellular signals and reversible protein phosphorylation: what to Mek of it all. Cell. 1993 Jul 30;74(2):215–217. doi: 10.1016/0092-8674(93)90411-i. [DOI] [PubMed] [Google Scholar]
  10. Davis R. J. The mitogen-activated protein kinase signal transduction pathway. J Biol Chem. 1993 Jul 15;268(20):14553–14556. [PubMed] [Google Scholar]
  11. Fantl W. J., Muslin A. J., Kikuchi A., Martin J. A., MacNicol A. M., Gross R. W., Williams L. T. Activation of Raf-1 by 14-3-3 proteins. Nature. 1994 Oct 13;371(6498):612–614. doi: 10.1038/371612a0. [DOI] [PubMed] [Google Scholar]
  12. Fei Z. L., D'Ambrosio C., Li S., Surmacz E., Baserga R. Association of insulin receptor substrate 1 with simian virus 40 large T antigen. Mol Cell Biol. 1995 Aug;15(8):4232–4239. doi: 10.1128/mcb.15.8.4232. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Freed E., Symons M., Macdonald S. G., McCormick F., Ruggieri R. Binding of 14-3-3 proteins to the protein kinase Raf and effects on its activation. Science. 1994 Sep 16;265(5179):1713–1716. doi: 10.1126/science.8085158. [DOI] [PubMed] [Google Scholar]
  14. Gotoh Y., Nishida E., Matsuda S., Shiina N., Kosako H., Shiokawa K., Akiyama T., Ohta K., Sakai H. In vitro effects on microtubule dynamics of purified Xenopus M phase-activated MAP kinase. Nature. 1991 Jan 17;349(6306):251–254. doi: 10.1038/349251a0. [DOI] [PubMed] [Google Scholar]
  15. Harlan J. E., Hajduk P. J., Yoon H. S., Fesik S. W. Pleckstrin homology domains bind to phosphatidylinositol-4,5-bisphosphate. Nature. 1994 Sep 8;371(6493):168–170. doi: 10.1038/371168a0. [DOI] [PubMed] [Google Scholar]
  16. Hu Q., Milfay D., Williams L. T. Binding of NCK to SOS and activation of ras-dependent gene expression. Mol Cell Biol. 1995 Mar;15(3):1169–1174. doi: 10.1128/mcb.15.3.1169. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Irie K., Gotoh Y., Yashar B. M., Errede B., Nishida E., Matsumoto K. Stimulatory effects of yeast and mammalian 14-3-3 proteins on the Raf protein kinase. Science. 1994 Sep 16;265(5179):1716–1719. doi: 10.1126/science.8085159. [DOI] [PubMed] [Google Scholar]
  18. Jhun B. H., Meinkoth J. L., Leitner J. W., Draznin B., Olefsky J. M. Insulin and insulin-like growth factor-I signal transduction requires p21ras. J Biol Chem. 1994 Feb 25;269(8):5699–5704. [PubMed] [Google Scholar]
  19. Kuhné M. R., Pawson T., Lienhard G. E., Feng G. S. The insulin receptor substrate 1 associates with the SH2-containing phosphotyrosine phosphatase Syp. J Biol Chem. 1993 Jun 5;268(16):11479–11481. [PubMed] [Google Scholar]
  20. Kyriakis J. M., App H., Zhang X. F., Banerjee P., Brautigan D. L., Rapp U. R., Avruch J. Raf-1 activates MAP kinase-kinase. Nature. 1992 Jul 30;358(6385):417–421. doi: 10.1038/358417a0. [DOI] [PubMed] [Google Scholar]
  21. Leevers S. J., Marshall C. J. Activation of extracellular signal-regulated kinase, ERK2, by p21ras oncoprotein. EMBO J. 1992 Feb;11(2):569–574. doi: 10.1002/j.1460-2075.1992.tb05088.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Li W., Nishimura R., Kashishian A., Batzer A. G., Kim W. J., Cooper J. A., Schlessinger J. A new function for a phosphotyrosine phosphatase: linking GRB2-Sos to a receptor tyrosine kinase. Mol Cell Biol. 1994 Jan;14(1):509–517. doi: 10.1128/mcb.14.1.509. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Mansour S. J., Matten W. T., Hermann A. S., Candia J. M., Rong S., Fukasawa K., Vande Woude G. F., Ahn N. G. Transformation of mammalian cells by constitutively active MAP kinase kinase. Science. 1994 Aug 12;265(5174):966–970. doi: 10.1126/science.8052857. [DOI] [PubMed] [Google Scholar]
  24. Maroney A. C., Qureshi S. A., Foster D. A., Brugge J. S. Cloning and characterization of a thermolabile v-src gene for use in reversible transformation of mammalian cells. Oncogene. 1992 Jun;7(6):1207–1214. [PubMed] [Google Scholar]
  25. Myers M. G., Jr, Backer J. M., Sun X. J., Shoelson S., Hu P., Schlessinger J., Yoakim M., Schaffhausen B., White M. F. IRS-1 activates phosphatidylinositol 3'-kinase by associating with src homology 2 domains of p85. Proc Natl Acad Sci U S A. 1992 Nov 1;89(21):10350–10354. doi: 10.1073/pnas.89.21.10350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Myers M. G., Jr, Sun X. J., White M. F. The IRS-1 signaling system. Trends Biochem Sci. 1994 Jul;19(7):289–293. doi: 10.1016/0968-0004(94)90007-8. [DOI] [PubMed] [Google Scholar]
  27. Nishida E., Gotoh Y. The MAP kinase cascade is essential for diverse signal transduction pathways. Trends Biochem Sci. 1993 Apr;18(4):128–131. doi: 10.1016/0968-0004(93)90019-j. [DOI] [PubMed] [Google Scholar]
  28. Nishiyama M., Wands J. R. Cloning and increased expression of an insulin receptor substrate-1-like gene in human hepatocellular carcinoma. Biochem Biophys Res Commun. 1992 Feb 28;183(1):280–285. doi: 10.1016/0006-291x(92)91640-c. [DOI] [PubMed] [Google Scholar]
  29. Rose D. W., Saltiel A. R., Majumdar M., Decker S. J., Olefsky J. M. Insulin receptor substrate 1 is required for insulin-mediated mitogenic signal transduction. Proc Natl Acad Sci U S A. 1994 Jan 18;91(2):797–801. doi: 10.1073/pnas.91.2.797. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Sasaki Y., Wands J. R. Ethanol impairs insulin receptor substrate-1 mediated signal transduction during rat liver regeneration. Biochem Biophys Res Commun. 1994 Feb 28;199(1):403–409. doi: 10.1006/bbrc.1994.1243. [DOI] [PubMed] [Google Scholar]
  31. Sasaki Y., Zhang X. F., Nishiyama M., Avruch J., Wands J. R. Expression and phosphorylation of insulin receptor substrate 1 during rat liver regeneration. J Biol Chem. 1993 Feb 25;268(6):3805–3808. [PubMed] [Google Scholar]
  32. Skolnik E. Y., Batzer A., Li N., Lee C. H., Lowenstein E., Mohammadi M., Margolis B., Schlessinger J. The function of GRB2 in linking the insulin receptor to Ras signaling pathways. Science. 1993 Jun 25;260(5116):1953–1955. doi: 10.1126/science.8316835. [DOI] [PubMed] [Google Scholar]
  33. Starzl T. E., Francavilla A., Halgrimson C. G., Francavilla F. R., Porter K. A., Brown T. H., Putnam C. W. The origin, hormonal nature, and action of hepatotrophic substances in portal venous blood. Surg Gynecol Obstet. 1973 Aug;137(2):179–199. [PMC free article] [PubMed] [Google Scholar]
  34. Sun X. J., Crimmins D. L., Myers M. G., Jr, Miralpeix M., White M. F. Pleiotropic insulin signals are engaged by multisite phosphorylation of IRS-1. Mol Cell Biol. 1993 Dec;13(12):7418–7428. doi: 10.1128/mcb.13.12.7418. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Sun X. J., Miralpeix M., Myers M. G., Jr, Glasheen E. M., Backer J. M., Kahn C. R., White M. F. Expression and function of IRS-1 in insulin signal transmission. J Biol Chem. 1992 Nov 5;267(31):22662–22672. [PubMed] [Google Scholar]
  36. Tobe K., Kadowaki T., Tamemoto H., Ueki K., Hara K., Koshio O., Momomura K., Gotoh Y., Nishida E., Akanuma Y. Insulin and 12-O-tetradecanoylphorbol-13-acetate activation of two immunologically distinct myelin basic protein/microtubule-associated protein 2 (MBP/MAP2) kinases via de novo phosphorylation of threonine and tyrosine residues. J Biol Chem. 1991 Dec 25;266(36):24793–24803. [PubMed] [Google Scholar]
  37. Touhara K., Inglese J., Pitcher J. A., Shaw G., Lefkowitz R. J. Binding of G protein beta gamma-subunits to pleckstrin homology domains. J Biol Chem. 1994 Apr 8;269(14):10217–10220. [PubMed] [Google Scholar]
  38. Troppmair J., Bruder J. T., Munoz H., Lloyd P. A., Kyriakis J., Banerjee P., Avruch J., Rapp U. R. Mitogen-activated protein kinase/extracellular signal-regulated protein kinase activation by oncogenes, serum, and 12-O-tetradecanoylphorbol-13-acetate requires Raf and is necessary for transformation. J Biol Chem. 1994 Mar 4;269(9):7030–7035. [PubMed] [Google Scholar]
  39. Waters S. B., Yamauchi K., Pessin J. E. Functional expression of insulin receptor substrate-1 is required for insulin-stimulated mitogenic signaling. J Biol Chem. 1993 Oct 25;268(30):22231–22234. [PubMed] [Google Scholar]
  40. White M. F., Kahn C. R. The insulin signaling system. J Biol Chem. 1994 Jan 7;269(1):1–4. [PubMed] [Google Scholar]
  41. Yonezawa K., Ando A., Kaburagi Y., Yamamoto-Honda R., Kitamura T., Hara K., Nakafuku M., Okabayashi Y., Kadowaki T., Kaziro Y. Signal transduction pathways from insulin receptors to Ras. Analysis by mutant insulin receptors. J Biol Chem. 1994 Feb 11;269(6):4634–4640. [PubMed] [Google Scholar]
  42. Zhang X. F., Settleman J., Kyriakis J. M., Takeuchi-Suzuki E., Elledge S. J., Marshall M. S., Bruder J. T., Rapp U. R., Avruch J. Normal and oncogenic p21ras proteins bind to the amino-terminal regulatory domain of c-Raf-1. Nature. 1993 Jul 22;364(6435):308–313. doi: 10.1038/364308a0. [DOI] [PubMed] [Google Scholar]

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