Skip to main content
NCBI home page
The Journal of Clinical Investigation logoLink to The Journal of Clinical Investigation
. 1996 Nov 1;98(9):2100–2108. doi: 10.1172/JCI119016

A carboxy-terminal truncated insulin receptor substrate-1 dominant negative protein reverses the human hepatocellular carcinoma malignant phenotype.

S Tanaka 1, J R Wands 1
PMCID: PMC507655  PMID: 8903330

Abstract

Insulin receptor substrate-1 (IRS-1), a substrate of various receptor tyrosine kinases transmits mitogenic signals initiated by extracellular ligands. This protein is involved in normal hepatocyte growth and has been found to be overexpressed in human hepatocellular carcinoma. Expression of a carboxy-terminal truncated IRS-1 molecule containing the pleckstrin homology and phosphotyrosine-binding domains associates with the insulin receptor and prevents tyrosyl phosphorylation of endogenous IRS-1 and Shc proteins. Thus, subsequent activation of downstream signaling molecules induced by insulin and IGF-1 such as phosphatidylinositol-3 kinase and mitogen activated protein kinase is inhibited. The morphologic features of transformed human hepatocellular carcinoma cells change to a differentiated hepatocyte appearance and characteristics of the malignant phenotype as manifested by anchorage independent cell growth and tumor formation in nude mice are lost. These studies demonstrate that signal transduction pathways mediated through or by IRS-1 are important in hepatocyte and human hepatocellular carcinoma cell growth.

Full Text

The Full Text of this article is available as a PDF (798.2 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Aaronson S. A. Growth factors and cancer. Science. 1991 Nov 22;254(5035):1146–1153. doi: 10.1126/science.1659742. [DOI] [PubMed] [Google Scholar]
  2. Araki E., Lipes M. A., Patti M. E., Brüning J. C., Haag B., 3rd, Johnson R. S., Kahn C. R. Alternative pathway of insulin signalling in mice with targeted disruption of the IRS-1 gene. Nature. 1994 Nov 10;372(6502):186–190. doi: 10.1038/372186a0. [DOI] [PubMed] [Google Scholar]
  3. Argetsinger L. S., Hsu G. W., Myers M. G., Jr, Billestrup N., White M. F., Carter-Su C. Growth hormone, interferon-gamma, and leukemia inhibitory factor promoted tyrosyl phosphorylation of insulin receptor substrate-1. J Biol Chem. 1995 Jun 16;270(24):14685–14692. doi: 10.1074/jbc.270.24.14685. [DOI] [PubMed] [Google Scholar]
  4. 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]
  5. 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]
  6. Barrett J. C., Ts'o P. O. Evidence for the progressive nature of neoplastic transformation in vitro. Proc Natl Acad Sci U S A. 1978 Aug;75(8):3761–3765. doi: 10.1073/pnas.75.8.3761. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Bissell D. M., Arenson D. M., Maher J. J., Roll F. J. Support of cultured hepatocytes by a laminin-rich gel. Evidence for a functionally significant subendothelial matrix in normal rat liver. J Clin Invest. 1987 Mar;79(3):801–812. doi: 10.1172/JCI112887. [DOI] [PMC free article] [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. D'Ambrosio C., Keller S. R., Morrione A., Lienhard G. E., Baserga R., Surmacz E. Transforming potential of the insulin receptor substrate 1. Cell Growth Differ. 1995 May;6(5):557–562. [PubMed] [Google Scholar]
  10. DiPersio C. M., Jackson D. A., Zaret K. S. The extracellular matrix coordinately modulates liver transcription factors and hepatocyte morphology. Mol Cell Biol. 1991 Sep;11(9):4405–4414. doi: 10.1128/mcb.11.9.4405. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Furusaka A., Nishiyama M., Ohkawa K., Yamori T., Tsuruo T., Yonezawa K., Kasuga M., Hayashi S., Tanaka T. Expression of insulin receptor substrate-1 in hepatocytes: an investigation using monoclonal antibodies. Cancer Lett. 1994 Aug 29;84(1):85–92. doi: 10.1016/0304-3835(94)90361-1. [DOI] [PubMed] [Google Scholar]
  12. Guo D., Donner D. B. Tumor necrosis factor promotes phosphorylation and binding of insulin receptor substrate 1 to phosphatidylinositol 3-kinase in 3T3-L1 adipocytes. J Biol Chem. 1996 Jan 12;271(2):615–618. doi: 10.1074/jbc.271.2.615. [DOI] [PubMed] [Google Scholar]
  13. Gustafson T. A., He W., Craparo A., Schaub C. D., O'Neill T. J. Phosphotyrosine-dependent interaction of SHC and insulin receptor substrate 1 with the NPEY motif of the insulin receptor via a novel non-SH2 domain. Mol Cell Biol. 1995 May;15(5):2500–2508. doi: 10.1128/mcb.15.5.2500. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. 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]
  15. He L., Isselbacher K. J., Wands J. R., Goodman H. M., Shih C., Quaroni A. Establishment and characterization of a new human hepatocellular carcinoma cell line. In Vitro. 1984 Jun;20(6):493–504. doi: 10.1007/BF02619623. [DOI] [PubMed] [Google Scholar]
  16. Ito T., Sasaki Y., Wands J. R. Overexpression of human insulin receptor substrate 1 induces cellular transformation with activation of mitogen-activated protein kinases. Mol Cell Biol. 1996 Mar;16(3):943–951. doi: 10.1128/mcb.16.3.943. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Kaleko M., Rutter W. J., Miller A. D. Overexpression of the human insulinlike growth factor I receptor promotes ligand-dependent neoplastic transformation. Mol Cell Biol. 1990 Feb;10(2):464–473. doi: 10.1128/mcb.10.2.464. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Kavanaugh W. M., Williams L. T. An alternative to SH2 domains for binding tyrosine-phosphorylated proteins. Science. 1994 Dec 16;266(5192):1862–1865. doi: 10.1126/science.7527937. [DOI] [PubMed] [Google Scholar]
  19. Koch K. S., Leffert H. L. Increased sodium ion influx is necessary to initiate rat hepatocyte proliferation. Cell. 1979 Sep;18(1):153–163. doi: 10.1016/0092-8674(79)90364-7. [DOI] [PubMed] [Google Scholar]
  20. Koch K. S., Shapiro P., Skelly H., Leffert H. L. Rat hepatocyte proliferation is stimulated by insulin-like peptides in defined medium. Biochem Biophys Res Commun. 1982 Dec 15;109(3):1054–1060. doi: 10.1016/0006-291x(82)92046-0. [DOI] [PubMed] [Google Scholar]
  21. 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]
  22. Kuhné M. R., Zhao Z., Rowles J., Lavan B. E., Shen S. H., Fischer E. H., Lienhard G. E. Dephosphorylation of insulin receptor substrate 1 by the tyrosine phosphatase PTP2C. J Biol Chem. 1994 Jun 3;269(22):15833–15837. [PubMed] [Google Scholar]
  23. Macaulay V. M. Insulin-like growth factors and cancer. Br J Cancer. 1992 Mar;65(3):311–320. doi: 10.1038/bjc.1992.65. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. 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]
  25. Musacchio A., Gibson T., Rice P., Thompson J., Saraste M. The PH domain: a common piece in the structural patchwork of signalling proteins. Trends Biochem Sci. 1993 Sep;18(9):343–348. doi: 10.1016/0968-0004(93)90071-t. [DOI] [PubMed] [Google Scholar]
  26. 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]
  27. Myers M. G., Jr, Grammer T. C., Brooks J., Glasheen E. M., Wang L. M., Sun X. J., Blenis J., Pierce J. H., White M. F. The pleckstrin homology domain in insulin receptor substrate-1 sensitizes insulin signaling. J Biol Chem. 1995 May 19;270(20):11715–11718. doi: 10.1074/jbc.270.20.11715. [DOI] [PubMed] [Google Scholar]
  28. 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]
  29. Nakabayashi H., Taketa K., Miyano K., Yamane T., Sato J. Growth of human hepatoma cells lines with differentiated functions in chemically defined medium. Cancer Res. 1982 Sep;42(9):3858–3863. [PubMed] [Google Scholar]
  30. 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]
  31. Pawson T. Protein modules and signalling networks. Nature. 1995 Feb 16;373(6515):573–580. doi: 10.1038/373573a0. [DOI] [PubMed] [Google Scholar]
  32. Pelicci G., Lanfrancone L., Grignani F., McGlade J., Cavallo F., Forni G., Nicoletti I., Grignani F., Pawson T., Pelicci P. G. A novel transforming protein (SHC) with an SH2 domain is implicated in mitogenic signal transduction. Cell. 1992 Jul 10;70(1):93–104. doi: 10.1016/0092-8674(92)90536-l. [DOI] [PubMed] [Google Scholar]
  33. Platanias L. C., Uddin S., Yetter A., Sun X. J., White M. F. The type I interferon receptor mediates tyrosine phosphorylation of insulin receptor substrate 2. J Biol Chem. 1996 Jan 5;271(1):278–282. doi: 10.1074/jbc.271.1.278. [DOI] [PubMed] [Google Scholar]
  34. Rotin D., Honegger A. M., Margolis B. L., Ullrich A., Schlessinger J. Presence of SH2 domains of phospholipase C gamma 1 enhances substrate phosphorylation by increasing the affinity toward the epidermal growth factor receptor. J Biol Chem. 1992 May 15;267(14):9678–9683. [PubMed] [Google Scholar]
  35. Sabe H., Hata A., Okada M., Nakagawa H., Hanafusa H. Analysis of the binding of the Src homology 2 domain of Csk to tyrosine-phosphorylated proteins in the suppression and mitotic activation of c-Src. Proc Natl Acad Sci U S A. 1994 Apr 26;91(9):3984–3988. doi: 10.1073/pnas.91.9.3984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. 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]
  37. 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]
  38. Sasaoka T., Rose D. W., Jhun B. H., Saltiel A. R., Draznin B., Olefsky J. M. Evidence for a functional role of Shc proteins in mitogenic signaling induced by insulin, insulin-like growth factor-1, and epidermal growth factor. J Biol Chem. 1994 May 6;269(18):13689–13694. [PubMed] [Google Scholar]
  39. Sugimoto S., Wandless T. J., Shoelson S. E., Neel B. G., Walsh C. T. Activation of the SH2-containing protein tyrosine phosphatase, SH-PTP2, by phosphotyrosine-containing peptides derived from insulin receptor substrate-1. J Biol Chem. 1994 May 6;269(18):13614–13622. [PubMed] [Google Scholar]
  40. 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]
  41. Sun X. J., Wang L. M., Zhang Y., Yenush L., Myers M. G., Jr, Glasheen E., Lane W. S., Pierce J. H., White M. F. Role of IRS-2 in insulin and cytokine signalling. Nature. 1995 Sep 14;377(6545):173–177. doi: 10.1038/377173a0. [DOI] [PubMed] [Google Scholar]
  42. Tamemoto H., Kadowaki T., Tobe K., Yagi T., Sakura H., Hayakawa T., Terauchi Y., Ueki K., Kaburagi Y., Satoh S. Insulin resistance and growth retardation in mice lacking insulin receptor substrate-1. Nature. 1994 Nov 10;372(6502):182–186. doi: 10.1038/372182a0. [DOI] [PubMed] [Google Scholar]
  43. Tanaka S., Ito T., Wands J. R. Neoplastic transformation induced by insulin receptor substrate-1 overexpression requires an interaction with both Grb2 and Syp signaling molecules. J Biol Chem. 1996 Jun 14;271(24):14610–14616. doi: 10.1074/jbc.271.24.14610. [DOI] [PubMed] [Google Scholar]
  44. 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]
  45. Vuori K., Ruoslahti E. Association of insulin receptor substrate-1 with integrins. Science. 1994 Dec 2;266(5190):1576–1578. doi: 10.1126/science.7527156. [DOI] [PubMed] [Google Scholar]
  46. Welham M. J., Learmonth L., Bone H., Schrader J. W. Interleukin-13 signal transduction in lymphohemopoietic cells. Similarities and differences in signal transduction with interleukin-4 and insulin. J Biol Chem. 1995 May 19;270(20):12286–12296. doi: 10.1074/jbc.270.20.12286. [DOI] [PubMed] [Google Scholar]
  47. 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]

Articles from Journal of Clinical Investigation are provided here courtesy of American Society for Clinical Investigation

RESOURCES