Skip to main content
PMC home page
Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1990 Aug;10(8):4035–4044. doi: 10.1128/mcb.10.8.4035

Evidence for epidermal growth factor (EGF)-induced intermolecular autophosphorylation of the EGF receptors in living cells.

A M Honegger 1, A Schmidt 1, A Ullrich 1, J Schlessinger 1
PMCID: PMC360914  PMID: 2164634

Abstract

In response to epidermal growth factor (EGF) stimulation, the intrinsic protein tyrosine kinase of EGF receptor is activated, leading to tyrosine phosphorylation of several cellular substrate proteins, including the EGF receptor molecule itself. To test the mechanism of EGF receptor autophosphorylation in living cells, we established transfected cell lines coexpressing a kinase-negative point mutant of EGF receptor (K721A) with an active EGF receptor mutant lacking 63 amino acids from its carboxy terminus. The addition of EGF to these cells caused tyrosine phosphorylation of the kinase-negative mutant by the active receptor molecule, demonstrating EGF receptor cross-phosphorylation in living cells. After internalization the kinase-negative mutant and CD63 have separate trafficking pathways. This limits their association and the extent of cross-phosphorylation of K721A by CD63. The coexpression of the kinase-negative mutant together with active EGF receptors in the same cells suppressed the mitogenic response toward EGF as compared with that in cells that express active receptors alone. The presence of the kinase-negative mutant functions as a negative dominant mutation suppressing the response of active EGF receptors, probably by interfering with EGF-induced signal transduction. It appears, therefore, that crucial events of signal transduction occur before K721A and active EGF receptors are separated by their different endocytic itineraries.

Full text

PDF
4035

Images in this article

4037Image
on p.4037
4038Image
on p.4038
4040Image
on p.4040

Selected References

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

  1. Cartwright C. A., Eckhart W., Simon S., Kaplan P. L. Cell transformation by pp60c-src mutated in the carboxy-terminal regulatory domain. Cell. 1987 Apr 10;49(1):83–91. doi: 10.1016/0092-8674(87)90758-6. [DOI] [PubMed] [Google Scholar]
  2. Chen W. S., Lazar C. S., Poenie M., Tsien R. Y., Gill G. N., Rosenfeld M. G. Requirement for intrinsic protein tyrosine kinase in the immediate and late actions of the EGF receptor. 1987 Aug 27-Sep 2Nature. 328(6133):820–823. doi: 10.1038/328820a0. [DOI] [PubMed] [Google Scholar]
  3. Chou C. K., Dull T. J., Russell D. S., Gherzi R., Lebwohl D., Ullrich A., Rosen O. M. Human insulin receptors mutated at the ATP-binding site lack protein tyrosine kinase activity and fail to mediate postreceptor effects of insulin. J Biol Chem. 1987 Feb 5;262(4):1842–1847. [PubMed] [Google Scholar]
  4. Cochet C., Kashles O., Chambaz E. M., Borrello I., King C. R., Schlessinger J. Demonstration of epidermal growth factor-induced receptor dimerization in living cells using a chemical covalent cross-linking agent. J Biol Chem. 1988 Mar 5;263(7):3290–3295. [PubMed] [Google Scholar]
  5. Ellis L., Clauser E., Morgan D. O., Edery M., Roth R. A., Rutter W. J. Replacement of insulin receptor tyrosine residues 1162 and 1163 compromises insulin-stimulated kinase activity and uptake of 2-deoxyglucose. Cell. 1986 Jun 6;45(5):721–732. doi: 10.1016/0092-8674(86)90786-5. [DOI] [PubMed] [Google Scholar]
  6. 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]
  7. Honegger A. M., Dull T. J., Felder S., Van Obberghen E., Bellot F., Szapary D., Schmidt A., Ullrich A., Schlessinger J. Point mutation at the ATP binding site of EGF receptor abolishes protein-tyrosine kinase activity and alters cellular routing. Cell. 1987 Oct 23;51(2):199–209. doi: 10.1016/0092-8674(87)90147-4. [DOI] [PubMed] [Google Scholar]
  8. Honegger A. M., Kris R. M., Ullrich A., Schlessinger J. Evidence that autophosphorylation of solubilized receptors for epidermal growth factor is mediated by intermolecular cross-phosphorylation. Proc Natl Acad Sci U S A. 1989 Feb;86(3):925–929. doi: 10.1073/pnas.86.3.925. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Honegger A. M., Schmidt A., Ullrich A., Schlessinger J. Separate endocytic pathways of kinase-defective and -active EGF receptor mutants expressed in same cells. J Cell Biol. 1990 May;110(5):1541–1548. doi: 10.1083/jcb.110.5.1541. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Honegger A. M., Szapary D., Schmidt A., Lyall R., Van Obberghen E., Dull T. J., Ullrich A., Schlessinger J. A mutant epidermal growth factor receptor with defective protein tyrosine kinase is unable to stimulate proto-oncogene expression and DNA synthesis. Mol Cell Biol. 1987 Dec;7(12):4568–4571. doi: 10.1128/mcb.7.12.4568. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Honegger A., Dull T. J., Bellot F., Van Obberghen E., Szapary D., Schmidt A., Ullrich A., Schlessinger J. Biological activities of EGF-receptor mutants with individually altered autophosphorylation sites. EMBO J. 1988 Oct;7(10):3045–3052. doi: 10.1002/j.1460-2075.1988.tb03169.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Honegger A., Dull T. J., Szapary D., Komoriya A., Kris R., Ullrich A., Schlessinger J. Kinetic parameters of the protein tyrosine kinase activity of EGF-receptor mutants with individually altered autophosphorylation sites. EMBO J. 1988 Oct;7(10):3053–3060. doi: 10.1002/j.1460-2075.1988.tb03170.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Hunter T., Cooper J. A. Epidermal growth factor induces rapid tyrosine phosphorylation of proteins in A431 human tumor cells. Cell. 1981 Jun;24(3):741–752. doi: 10.1016/0092-8674(81)90100-8. [DOI] [PubMed] [Google Scholar]
  14. 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]
  15. Kmiecik T. E., Shalloway D. Activation and suppression of pp60c-src transforming ability by mutation of its primary sites of tyrosine phosphorylation. Cell. 1987 Apr 10;49(1):65–73. doi: 10.1016/0092-8674(87)90756-2. [DOI] [PubMed] [Google Scholar]
  16. Koland J. G., Cerione R. A. Growth factor control of epidermal growth factor receptor kinase activity via an intramolecular mechanism. J Biol Chem. 1988 Feb 15;263(5):2230–2237. [PubMed] [Google Scholar]
  17. Kris R. M., Lax I., Gullick W., Waterfield M. D., Ullrich A., Fridkin M., Schlessinger J. Antibodies against a synthetic peptide as a probe for the kinase activity of the avian EGF receptor and v-erbB protein. Cell. 1985 Mar;40(3):619–625. doi: 10.1016/0092-8674(85)90210-7. [DOI] [PubMed] [Google Scholar]
  18. Lax I., Bellot F., Honegger A. M., Schmidt A., Ullrich A., Givol D., Schlessinger J. Domain deletion in the extracellular portion of the EGF-receptor reduces ligand binding and impairs cell surface expression. Cell Regul. 1990 Jan;1(2):173–188. doi: 10.1091/mbc.1.2.173. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Lax I., Kris R., Sasson I., Ullrich A., Hayman M. J., Beug H., Schlessinger J. Activation of c-erbB in avian leukosis virus-induced erythroblastosis leads to the expression of a truncated EGF receptor kinase. EMBO J. 1985 Dec 1;4(12):3179–3182. doi: 10.1002/j.1460-2075.1985.tb04062.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Livneh E., Prywes R., Kashles O., Reiss N., Sasson I., Mory Y., Ullrich A., Schlessinger J. Reconstitution of human epidermal growth factor receptors and its deletion mutants in cultured hamster cells. J Biol Chem. 1986 Sep 25;261(27):12490–12497. [PubMed] [Google Scholar]
  21. Livneh E., Reiss N., Berent E., Ullrich A., Schlessinger J. An insertional mutant of epidermal growth factor receptor allows dissection of diverse receptor functions. EMBO J. 1987 Sep;6(9):2669–2676. doi: 10.1002/j.1460-2075.1987.tb02558.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Margolis B. L., Lax I., Kris R., Dombalagian M., Honegger A. M., Howk R., Givol D., Ullrich A., Schlessinger J. All autophosphorylation sites of epidermal growth factor (EGF) receptor and HER2/neu are located in their carboxyl-terminal tails. Identification of a novel site in EGF receptor. J Biol Chem. 1989 Jun 25;264(18):10667–10671. [PubMed] [Google Scholar]
  23. Margolis B., Rhee S. G., Felder S., Mervic M., Lyall R., Levitzki A., Ullrich A., Zilberstein A., Schlessinger J. EGF induces tyrosine phosphorylation of phospholipase C-II: a potential mechanism for EGF receptor signaling. Cell. 1989 Jun 30;57(7):1101–1107. doi: 10.1016/0092-8674(89)90047-0. [DOI] [PubMed] [Google Scholar]
  24. McClain D. A., Maegawa H., Lee J., Dull T. J., Ulrich A., Olefsky J. M. A mutant insulin receptor with defective tyrosine kinase displays no biologic activity and does not undergo endocytosis. J Biol Chem. 1987 Oct 25;262(30):14663–14671. [PubMed] [Google Scholar]
  25. Meckling-Hansen K., Nelson R., Branton P., Pawson T. Enzymatic activation of Fujinami sarcoma virus gag-fps transforming proteins by autophosphorylation at tyrosine. EMBO J. 1987 Mar;6(3):659–666. doi: 10.1002/j.1460-2075.1987.tb04805.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Meisenhelder J., Suh P. G., Rhee S. G., Hunter T. Phospholipase C-gamma is a substrate for the PDGF and EGF receptor protein-tyrosine kinases in vivo and in vitro. Cell. 1989 Jun 30;57(7):1109–1122. doi: 10.1016/0092-8674(89)90048-2. [DOI] [PubMed] [Google Scholar]
  27. Moolenaar W. H., Bierman A. J., Tilly B. C., Verlaan I., Defize L. H., Honegger A. M., Ullrich A., Schlessinger J. A point mutation at the ATP-binding site of the EGF-receptor abolishes signal transduction. EMBO J. 1988 Mar;7(3):707–710. doi: 10.1002/j.1460-2075.1988.tb02866.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Northwood I. C., Davis R. J. Activation of the epidermal growth factor receptor tyrosine protein kinase in the absence of receptor oligomerization. J Biol Chem. 1988 Jun 5;263(16):7450–7453. [PubMed] [Google Scholar]
  29. Piwnica-Worms H., Saunders K. B., Roberts T. M., Smith A. E., Cheng S. H. Tyrosine phosphorylation regulates the biochemical and biological properties of pp60c-src. Cell. 1987 Apr 10;49(1):75–82. doi: 10.1016/0092-8674(87)90757-4. [DOI] [PubMed] [Google Scholar]
  30. Rosen O. M., Herrera R., Olowe Y., Petruzzelli L. M., Cobb M. H. Phosphorylation activates the insulin receptor tyrosine protein kinase. Proc Natl Acad Sci U S A. 1983 Jun;80(11):3237–3240. doi: 10.1073/pnas.80.11.3237. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Russell D. S., Gherzi R., Johnson E. L., Chou C. K., Rosen O. M. The protein-tyrosine kinase activity of the insulin receptor is necessary for insulin-mediated receptor down-regulation. J Biol Chem. 1987 Aug 25;262(24):11833–11840. [PubMed] [Google Scholar]
  32. Schlessinger J. Allosteric regulation of the epidermal growth factor receptor kinase. J Cell Biol. 1986 Dec;103(6 Pt 1):2067–2072. doi: 10.1083/jcb.103.6.2067. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Schlessinger J., Shechter Y., Willingham M. C., Pastan I. Direct visualization of binding, aggregation, and internalization of insulin and epidermal growth factor on living fibroblastic cells. Proc Natl Acad Sci U S A. 1978 Jun;75(6):2659–2663. doi: 10.1073/pnas.75.6.2659. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Schlessinger J. Signal transduction by allosteric receptor oligomerization. Trends Biochem Sci. 1988 Nov;13(11):443–447. doi: 10.1016/0968-0004(88)90219-8. [DOI] [PubMed] [Google Scholar]
  35. Scimeca J. C., Ballotti R., Alengrin F., Honegger A. M., Ullrich A., Schlessinger J., Obberghen E. V. Metabolic effects induced by epidermal growth factor (EGF) in cells expressing EGF receptor mutants. J Biol Chem. 1989 Apr 25;264(12):6831–6835. [PubMed] [Google Scholar]
  36. 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]
  37. Wahl M. I., Daniel T. O., Carpenter G. Antiphosphotyrosine recovery of phospholipase C activity after EGF treatment of A-431 cells. Science. 1988 Aug 19;241(4868):968–970. doi: 10.1126/science.2457254. [DOI] [PubMed] [Google Scholar]
  38. Walker F., Burgess A. W. Transmodulation of the epidermal-growth-factor receptor in permeabilized 3T3 cells. Biochem J. 1988 Nov 15;256(1):109–115. doi: 10.1042/bj2560109. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Wigler M., Sweet R., Sim G. K., Wold B., Pellicer A., Lacy E., Maniatis T., Silverstein S., Axel R. Transformation of mammalian cells with genes from procaryotes and eucaryotes. Cell. 1979 Apr;16(4):777–785. doi: 10.1016/0092-8674(79)90093-x. [DOI] [PubMed] [Google Scholar]
  40. Yarden Y., Schlessinger J. Epidermal growth factor induces rapid, reversible aggregation of the purified epidermal growth factor receptor. Biochemistry. 1987 Mar 10;26(5):1443–1451. doi: 10.1021/bi00379a035. [DOI] [PubMed] [Google Scholar]
  41. 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]
  42. Zidovetzki R., Yarden Y., Schlessinger J., Jovin T. M. Microaggregation of hormone-occupied epidermal growth factor receptors on plasma membrane preparations. EMBO J. 1986 Feb;5(2):247–250. doi: 10.1002/j.1460-2075.1986.tb04205.x. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Molecular and Cellular Biology are provided here courtesy of Taylor & Francis

RESOURCES