Skip to main content
NCBI home page
The EMBO Journal logoLink to The EMBO Journal
. 1989 Jan;8(1):159–166. doi: 10.1002/j.1460-2075.1989.tb03360.x

A chimeric EGF-R-neu proto-oncogene allows EGF to regulate neu tyrosine kinase and cell transformation.

H Lehväslaiho 1, L Lehtola 1, L Sistonen 1, K Alitalo 1
PMCID: PMC400785  PMID: 2565807

Abstract

The neu oncogene, characterized by Weinberg and colleagues, is a transforming gene found in ethylnitrosourea-induced rat neuro/glioblastomas; its human proto-oncogene homologue has been termed erbB2 or HER2 because of its close homology with the epidermal growth factor receptor (EGF-R) gene (c-erbB1). Expression of the rat neu oncogene is sufficient for transformation of mouse NIH 3T3 fibroblasts in culture and for the development of mammary carcinomas in transgenic mice, but the neu proto-oncogene has not been associated with cell transformation. We constructed a vector for expression of a chimeric cDNA and hybrid protein consisting of the EGF-R extracellular, transmembrane and protein kinase C-substrate domains linked to the intracellular tyrosine kinase and carboxyl terminal domain of the rat neu cDNA. Upon transfection with the construct, NIH 3T3 cells gave rise to EGF-R antigen-positive cell clones with varying amounts of specific EGF binding. Immunofluorescence and immunoprecipitation using neu- and EGF-receptor specific antibodies demonstrated a correctly oriented and positioned chimeric EGF-R-neu protein of the expected apparent mol. wt on the surface of these cells. EGF or TGF alpha induced tyrosine phosphorylation of the chimeric receptor protein, stimulated DNA synthesis of EGF-R-neu expressing cells and led to a transformed cell morphology and growth in soft agar. In contrast, the neu proto-oncogene did not show kinase activity or transforming properties when expressed at similar levels in NIH 3T3 cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Full text

PDF
159

Images in this article

161Image
on p.161
162Image
on p.162
162Image
on p.162
163Image
on p.163

Selected References

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

  1. Bargmann C. I., Hung M. C., Weinberg R. A. Multiple independent activations of the neu oncogene by a point mutation altering the transmembrane domain of p185. Cell. 1986 Jun 6;45(5):649–657. doi: 10.1016/0092-8674(86)90779-8. [DOI] [PubMed] [Google Scholar]
  2. Bargmann C. I., Hung M. C., Weinberg R. A. The neu oncogene encodes an epidermal growth factor receptor-related protein. Nature. 1986 Jan 16;319(6050):226–230. doi: 10.1038/319226a0. [DOI] [PubMed] [Google Scholar]
  3. Bargmann C. I., Weinberg R. A. Increased tyrosine kinase activity associated with the protein encoded by the activated neu oncogene. Proc Natl Acad Sci U S A. 1988 Aug;85(15):5394–5398. doi: 10.1073/pnas.85.15.5394. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bargmann C. I., Weinberg R. A. Oncogenic activation of the neu-encoded receptor protein by point mutation and deletion. EMBO J. 1988 Jul;7(7):2043–2052. doi: 10.1002/j.1460-2075.1988.tb03044.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bernard O., Fazekas de St Groth B., Ullrich A., Green W., Schlessinger J. High-affinity interleukin 2 binding by an oncogenic hybrid interleukin 2-epidermal growth factor receptor molecule. Proc Natl Acad Sci U S A. 1987 Apr;84(8):2125–2129. doi: 10.1073/pnas.84.8.2125. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Coussens L., Yang-Feng T. L., Liao Y. C., Chen E., Gray A., McGrath J., Seeburg P. H., Libermann T. A., Schlessinger J., Francke U. Tyrosine kinase receptor with extensive homology to EGF receptor shares chromosomal location with neu oncogene. Science. 1985 Dec 6;230(4730):1132–1139. doi: 10.1126/science.2999974. [DOI] [PubMed] [Google Scholar]
  7. Di Fiore P. P., Pierce J. H., Fleming T. P., Hazan R., Ullrich A., King C. R., Schlessinger J., Aaronson S. A. Overexpression of the human EGF receptor confers an EGF-dependent transformed phenotype to NIH 3T3 cells. Cell. 1987 Dec 24;51(6):1063–1070. doi: 10.1016/0092-8674(87)90592-7. [DOI] [PubMed] [Google Scholar]
  8. Di Fiore P. P., Pierce J. H., Kraus M. H., Segatto O., King C. R., Aaronson S. A. erbB-2 is a potent oncogene when overexpressed in NIH/3T3 cells. Science. 1987 Jul 10;237(4811):178–182. doi: 10.1126/science.2885917. [DOI] [PubMed] [Google Scholar]
  9. Giard D. J., Aaronson S. A., Todaro G. J., Arnstein P., Kersey J. H., Dosik H., Parks W. P. In vitro cultivation of human tumors: establishment of cell lines derived from a series of solid tumors. J Natl Cancer Inst. 1973 Nov;51(5):1417–1423. doi: 10.1093/jnci/51.5.1417. [DOI] [PubMed] [Google Scholar]
  10. Gluzman Y. SV40-transformed simian cells support the replication of early SV40 mutants. Cell. 1981 Jan;23(1):175–182. doi: 10.1016/0092-8674(81)90282-8. [DOI] [PubMed] [Google Scholar]
  11. Graham F. L., van der Eb A. J. A new technique for the assay of infectivity of human adenovirus 5 DNA. Virology. 1973 Apr;52(2):456–467. doi: 10.1016/0042-6822(73)90341-3. [DOI] [PubMed] [Google Scholar]
  12. Gullick W. J., Berger M. S., Bennett P. L., Rothbard J. B., Waterfield M. D. Expression of the c-erbB-2 protein in normal and transformed cells. Int J Cancer. 1987 Aug 15;40(2):246–254. doi: 10.1002/ijc.2910400221. [DOI] [PubMed] [Google Scholar]
  13. Haigler H., Ash J. F., Singer S. J., Cohen S. Visualization by fluorescence of the binding and internalization of epidermal growth factor in human carcinoma cells A-431. Proc Natl Acad Sci U S A. 1978 Jul;75(7):3317–3321. doi: 10.1073/pnas.75.7.3317. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Hanks S. K., Quinn A. M., Hunter T. The protein kinase family: conserved features and deduced phylogeny of the catalytic domains. Science. 1988 Jul 1;241(4861):42–52. doi: 10.1126/science.3291115. [DOI] [PubMed] [Google Scholar]
  15. Hudziak R. M., Schlessinger J., Ullrich A. Increased expression of the putative growth factor receptor p185HER2 causes transformation and tumorigenesis of NIH 3T3 cells. Proc Natl Acad Sci U S A. 1987 Oct;84(20):7159–7163. doi: 10.1073/pnas.84.20.7159. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Hung M. C., Schechter A. L., Chevray P. Y., Stern D. F., Weinberg R. A. Molecular cloning of the neu gene: absence of gross structural alteration in oncogenic alleles. Proc Natl Acad Sci U S A. 1986 Jan;83(2):261–264. doi: 10.1073/pnas.83.2.261. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Hunter T., Cooper J. A. Protein-tyrosine kinases. Annu Rev Biochem. 1985;54:897–930. doi: 10.1146/annurev.bi.54.070185.004341. [DOI] [PubMed] [Google Scholar]
  18. Hunter T., Ling N., Cooper J. A. Protein kinase C phosphorylation of the EGF receptor at a threonine residue close to the cytoplasmic face of the plasma membrane. Nature. 1984 Oct 4;311(5985):480–483. doi: 10.1038/311480a0. [DOI] [PubMed] [Google Scholar]
  19. Jainchill J. L., Aaronson S. A., Todaro G. J. Murine sarcoma and leukemia viruses: assay using clonal lines of contact-inhibited mouse cells. J Virol. 1969 Nov;4(5):549–553. doi: 10.1128/jvi.4.5.549-553.1969. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. King C. R., Borrello I., Bellot F., Comoglio P., Schlessinger J. Egf binding to its receptor triggers a rapid tyrosine phosphorylation of the erbB-2 protein in the mammary tumor cell line SK-BR-3. EMBO J. 1988 Jun;7(6):1647–1651. doi: 10.1002/j.1460-2075.1988.tb02991.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. King C. R., Kraus M. H., Aaronson S. A. Amplification of a novel v-erbB-related gene in a human mammary carcinoma. Science. 1985 Sep 6;229(4717):974–976. doi: 10.1126/science.2992089. [DOI] [PubMed] [Google Scholar]
  22. Kraus M. H., Popescu N. C., Amsbaugh S. C., King C. R. Overexpression of the EGF receptor-related proto-oncogene erbB-2 in human mammary tumor cell lines by different molecular mechanisms. EMBO J. 1987 Mar;6(3):605–610. doi: 10.1002/j.1460-2075.1987.tb04797.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  24. Lasfargues E. Y., Coutinho W. G., Redfield E. S. Isolation of two human tumor epithelial cell lines from solid breast carcinomas. J Natl Cancer Inst. 1978 Oct;61(4):967–978. [PubMed] [Google Scholar]
  25. Muller W. J., Sinn E., Pattengale P. K., Wallace R., Leder P. Single-step induction of mammary adenocarcinoma in transgenic mice bearing the activated c-neu oncogene. Cell. 1988 Jul 1;54(1):105–115. doi: 10.1016/0092-8674(88)90184-5. [DOI] [PubMed] [Google Scholar]
  26. Nestor J. J., Jr, Newman S. R., DeLustro B., Todaro G. J., Schreiber A. B. A synthetic fragment of rat transforming growth factor alpha with receptor binding and antigenic properties. Biochem Biophys Res Commun. 1985 May 31;129(1):226–232. doi: 10.1016/0006-291x(85)91426-3. [DOI] [PubMed] [Google Scholar]
  27. Padhy L. C., Shih C., Cowing D., Finkelstein R., Weinberg R. A. Identification of a phosphoprotein specifically induced by the transforming DNA of rat neuroblastomas. Cell. 1982 Apr;28(4):865–871. doi: 10.1016/0092-8674(82)90065-4. [DOI] [PubMed] [Google Scholar]
  28. Riedel H., Dull T. J., Schlessinger J., Ullrich A. A chimaeric receptor allows insulin to stimulate tyrosine kinase activity of epidermal growth factor receptor. Nature. 1986 Nov 6;324(6092):68–70. doi: 10.1038/324068a0. [DOI] [PubMed] [Google Scholar]
  29. Riedel H., Massoglia S., Schlessinger J., Ullrich A. Ligand activation of overexpressed epidermal growth factor receptors transforms NIH 3T3 mouse fibroblasts. Proc Natl Acad Sci U S A. 1988 Mar;85(5):1477–1481. doi: 10.1073/pnas.85.5.1477. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Russell W. C., Newman C., Williamson D. H. A simple cytochemical technique for demonstration of DNA in cells infected with mycoplasmas and viruses. Nature. 1975 Feb 6;253(5491):461–462. doi: 10.1038/253461a0. [DOI] [PubMed] [Google Scholar]
  31. Schechter A. L., Stern D. F., Vaidyanathan L., Decker S. J., Drebin J. A., Greene M. I., Weinberg R. A. The neu oncogene: an erb-B-related gene encoding a 185,000-Mr tumour antigen. Nature. 1984 Dec 6;312(5994):513–516. doi: 10.1038/312513a0. [DOI] [PubMed] [Google Scholar]
  32. Seki J., Owada M. K., Sakato N., Fujio H. Direct identification of phosphotyrosine-containing proteins in some retrovirus-transformed cells by use of anti-phosphotyrosine antibody. Cancer Res. 1986 Feb;46(2):907–916. [PubMed] [Google Scholar]
  33. Semba K., Kamata N., Toyoshima K., Yamamoto T. A v-erbB-related protooncogene, c-erbB-2, is distinct from the c-erbB-1/epidermal growth factor-receptor gene and is amplified in a human salivary gland adenocarcinoma. Proc Natl Acad Sci U S A. 1985 Oct;82(19):6497–6501. doi: 10.1073/pnas.82.19.6497. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Shih C., Padhy L. C., Murray M., Weinberg R. A. Transforming genes of carcinomas and neuroblastomas introduced into mouse fibroblasts. Nature. 1981 Mar 19;290(5803):261–264. doi: 10.1038/290261a0. [DOI] [PubMed] [Google Scholar]
  35. Sistonen L., Keski-Oja J., Ulmanen I., Hölttä E., Wikgren B. J., Alitalo K. Dose effects of transfected c-Ha-rasVal 12 oncogene in transformed cell clones. Exp Cell Res. 1987 Feb;168(2):518–530. doi: 10.1016/0014-4827(87)90024-3. [DOI] [PubMed] [Google Scholar]
  36. Slamon D. J., Clark G. M., Wong S. G., Levin W. J., Ullrich A., McGuire W. L. Human breast cancer: correlation of relapse and survival with amplification of the HER-2/neu oncogene. Science. 1987 Jan 9;235(4785):177–182. doi: 10.1126/science.3798106. [DOI] [PubMed] [Google Scholar]
  37. Stanley K. K., Luzio J. P. Construction of a new family of high efficiency bacterial expression vectors: identification of cDNA clones coding for human liver proteins. EMBO J. 1984 Jun;3(6):1429–1434. doi: 10.1002/j.1460-2075.1984.tb01988.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Stern D. F., Heffernan P. A., Weinberg R. A. p185, a product of the neu proto-oncogene, is a receptorlike protein associated with tyrosine kinase activity. Mol Cell Biol. 1986 May;6(5):1729–1740. doi: 10.1128/mcb.6.5.1729. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Stern D. F., Kamps M. P., Cao H. Oncogenic activation of p185neu stimulates tyrosine phosphorylation in vivo. Mol Cell Biol. 1988 Sep;8(9):3969–3973. doi: 10.1128/mcb.8.9.3969. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Stern D. F., Kamps M. P. EGF-stimulated tyrosine phosphorylation of p185neu: a potential model for receptor interactions. EMBO J. 1988 Apr;7(4):995–1001. doi: 10.1002/j.1460-2075.1988.tb02906.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Ullrich A., Coussens L., Hayflick J. S., Dull T. J., Gray A., Tam A. W., Lee J., Yarden Y., Libermann T. A., Schlessinger J. Human epidermal growth factor receptor cDNA sequence and aberrant expression of the amplified gene in A431 epidermoid carcinoma cells. 1984 May 31-Jun 6Nature. 309(5967):418–425. doi: 10.1038/309418a0. [DOI] [PubMed] [Google Scholar]
  42. Velu T. J., Beguinot L., Vass W. C., Willingham M. C., Merlino G. T., Pastan I., Lowy D. R. Epidermal-growth-factor-dependent transformation by a human EGF receptor proto-oncogene. Science. 1987 Dec 4;238(4832):1408–1410. doi: 10.1126/science.3500513. [DOI] [PubMed] [Google Scholar]
  43. Venter D. J., Tuzi N. L., Kumar S., Gullick W. J. Overexpression of the c-erbB-2 oncoprotein in human breast carcinomas: immunohistological assessment correlates with gene amplification. Lancet. 1987 Jul 11;2(8550):69–72. doi: 10.1016/s0140-6736(87)92736-x. [DOI] [PubMed] [Google Scholar]
  44. Yamamoto T., Ikawa S., Akiyama T., Semba K., Nomura N., Miyajima N., Saito T., Toyoshima K. Similarity of protein encoded by the human c-erb-B-2 gene to epidermal growth factor receptor. Nature. 1986 Jan 16;319(6050):230–234. doi: 10.1038/319230a0. [DOI] [PubMed] [Google Scholar]
  45. Yarden Y., Ullrich A. Growth factor receptor tyrosine kinases. Annu Rev Biochem. 1988;57:443–478. doi: 10.1146/annurev.bi.57.070188.002303. [DOI] [PubMed] [Google Scholar]
  46. Yokota J., Yamamoto T., Miyajima N., Toyoshima K., Nomura N., Sakamoto H., Yoshida T., Terada M., Sugimura T. Genetic alterations of the c-erbB-2 oncogene occur frequently in tubular adenocarcinoma of the stomach and are often accompanied by amplification of the v-erbA homologue. Oncogene. 1988 Mar;2(3):283–287. [PubMed] [Google Scholar]
  47. Yokota J., Yamamoto T., Toyoshima K., Terada M., Sugimura T., Battifora H., Cline M. J. Amplification of c-erbB-2 oncogene in human adenocarcinomas in vivo. Lancet. 1986 Apr 5;1(8484):765–767. doi: 10.1016/s0140-6736(86)91782-4. [DOI] [PubMed] [Google Scholar]
  48. van de Vijver M., van de Bersselaar R., Devilee P., Cornelisse C., Peterse J., Nusse R. Amplification of the neu (c-erbB-2) oncogene in human mammmary tumors is relatively frequent and is often accompanied by amplification of the linked c-erbA oncogene. Mol Cell Biol. 1987 May;7(5):2019–2023. doi: 10.1128/mcb.7.5.2019. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from The EMBO Journal are provided here courtesy of Nature Publishing Group

RESOURCES