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. 1990 Oct;64(10):5163–5166. doi: 10.1128/jvi.64.10.5163-5166.1990

Myristylation of pp60c-src is not required for complex formation with polyomavirus middle-T antigen.

A Wyss 1, S Kaech 1, K Ballmer-Hofer 1
PMCID: PMC248010  PMID: 2168992

Abstract

Middle-T antigen (middle-T), the transforming gene product of polyomavirus, associates with several cellular tyrosine kinases, such as pp60c-src. Complex formation leads to kinase activation and is essential for cell transformation. Middle-T-associated as well as uncomplexed pp60c-src is predominantly found in the plasma membrane. We transfected mouse 3T3 fibroblasts with a mutated c-src gene (2Ac-src), allowing the expression of a protein containing alanine instead of glycine in position 2 of the primary translation product. Contrary to the wild-type c-src gene product, pp60c-src(2A) was not myristylated and accumulated in the cytoplasm instead of being transferred to cellular membranes. The mutant protein was able to associate with middle-T and was activated similarly to the wild-type c-src gene product. Both wild-type and 2A mutant protein were membrane associated upon complex formation with middle-T. This finding suggests that the putative carboxy-terminal membrane anchor sequence of middle-T is sufficient to hold middle-T-associated pp60c-src(2A) in the plasma membrane.

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Selected References

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

  1. Ballmer-Hofer K., Benjamin T. L. Phosphorylation of polyoma middle T antigen and cellular proteins in purified plasma membranes of polyoma virus-infected cells. EMBO J. 1985 Sep;4(9):2321–2327. doi: 10.1002/j.1460-2075.1985.tb03933.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Ballmer-Hofer K., Mandel G., Faller D. V., Roberts T. M., Benjamin T. L. Expression of influenza hemagglutinin-polyoma T-antigen fusion proteins in a rat embryo fibroblast cell line. Virus Res. 1987 Jan;6(4):345–361. doi: 10.1016/0168-1702(87)90066-9. [DOI] [PubMed] [Google Scholar]
  3. Bolen J. B., Thiele C. J., Israel M. A., Yonemoto W., Lipsich L. A., Brugge J. S. Enhancement of cellular src gene product associated tyrosyl kinase activity following polyoma virus infection and transformation. Cell. 1984 Oct;38(3):767–777. doi: 10.1016/0092-8674(84)90272-1. [DOI] [PubMed] [Google Scholar]
  4. Buss J. E., Kamps M. P., Gould K., Sefton B. M. The absence of myristic acid decreases membrane binding of p60src but does not affect tyrosine protein kinase activity. J Virol. 1986 May;58(2):468–474. doi: 10.1128/jvi.58.2.468-474.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Carmichael G. G., Schaffhausen B. S., Dorsky D. I., Oliver D. B., Benjamin T. L. Carboxy terminus of polyoma middle-sized tumor antigen is required for attachment to membranes, associated protein kinase activities, and cell transformation. Proc Natl Acad Sci U S A. 1982 Jun;79(11):3579–3583. doi: 10.1073/pnas.79.11.3579. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Cheng S. H., Harvey R., Espino P. C., Semba K., Yamamoto T., Toyoshima K., Smith A. E. Peptide antibodies to the human c-fyn gene product demonstrate pp59c-fyn is capable of complex formation with the middle-T antigen of polyomavirus. EMBO J. 1988 Dec 1;7(12):3845–3855. doi: 10.1002/j.1460-2075.1988.tb03270.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Cleveland D. W., Fischer S. G., Kirschner M. W., Laemmli U. K. Peptide mapping by limited proteolysis in sodium dodecyl sulfate and analysis by gel electrophoresis. J Biol Chem. 1977 Feb 10;252(3):1102–1106. [PubMed] [Google Scholar]
  8. Collett M. S., Erikson E., Erikson R. L. Structural analysis of the avian sarcoma virus transforming protein: sites of phosphorylation. J Virol. 1979 Feb;29(2):770–781. doi: 10.1128/jvi.29.2.770-781.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Cooper J. A., Esch F. S., Taylor S. S., Hunter T. Phosphorylation sites in enolase and lactate dehydrogenase utilized by tyrosine protein kinases in vivo and in vitro. J Biol Chem. 1984 Jun 25;259(12):7835–7841. [PubMed] [Google Scholar]
  10. Courtneidge S. A., Read M., Wilson J. B., Fried M. Cytoplasmic interaction between pp60c-src and a truncated polyoma virus middle T antigen. Oncogene Res. 1989;4(1):75–80. [PubMed] [Google Scholar]
  11. Courtneidge S. A., Smith A. E. Polyoma virus transforming protein associates with the product of the c-src cellular gene. Nature. 1983 Jun 2;303(5916):435–439. doi: 10.1038/303435a0. [DOI] [PubMed] [Google Scholar]
  12. Cross F. R., Garber E. A., Pellman D., Hanafusa H. A short sequence in the p60src N terminus is required for p60src myristylation and membrane association and for cell transformation. Mol Cell Biol. 1984 Sep;4(9):1834–1842. doi: 10.1128/mcb.4.9.1834. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Cuzin F. The polyoma virus oncogenes. Coordinated functions of three distinct proteins in the transformation of rodent cells in culture. Biochim Biophys Acta. 1984 Apr 5;781(3):193–204. doi: 10.1016/0167-4781(84)90084-8. [DOI] [PubMed] [Google Scholar]
  14. Dilworth S. M., Hansson H. A., Darnfors C., Bjursell G., Streuli C. H., Griffin B. E. Subcellular localisation of the middle and large T-antigens of polyoma virus. EMBO J. 1986 Mar;5(3):491–499. doi: 10.1002/j.1460-2075.1986.tb04238.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Donoghue D. J., Anderson C., Hunter T., Kaplan P. L. Transmission of the polyoma virus middle T gene as the oncogene of a murine retrovirus. Nature. 1984 Apr 19;308(5961):748–750. doi: 10.1038/308748a0. [DOI] [PubMed] [Google Scholar]
  16. Hamaguchi M., Hanafusa H. Association of p60src with Triton X-100-resistant cellular structure correlates with morphological transformation. Proc Natl Acad Sci U S A. 1987 Apr;84(8):2312–2316. doi: 10.1073/pnas.84.8.2312. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Kaplan D. R., Pallas D. C., Morgan W., Schaffhausen B., Roberts T. M. Mechanisms of transformation by polyoma virus middle T antigen. Biochim Biophys Acta. 1989 Feb;948(3):345–364. doi: 10.1016/0304-419x(89)90006-1. [DOI] [PubMed] [Google Scholar]
  18. Kornbluth S., Sudol M., Hanafusa H. Association of the polyomavirus middle-T antigen with c-yes protein. Nature. 1987 Jan 8;325(7000):171–173. doi: 10.1038/325171a0. [DOI] [PubMed] [Google Scholar]
  19. Kypta R. M., Hemming A., Courtneidge S. A. Identification and characterization of p59fyn (a src-like protein tyrosine kinase) in normal and polyoma virus transformed cells. EMBO J. 1988 Dec 1;7(12):3837–3844. doi: 10.1002/j.1460-2075.1988.tb03269.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Lee F., Mulligan R., Berg P., Ringold G. Glucocorticoids regulate expression of dihydrofolate reductase cDNA in mouse mammary tumour virus chimaeric plasmids. Nature. 1981 Nov 19;294(5838):228–232. doi: 10.1038/294228a0. [DOI] [PubMed] [Google Scholar]
  21. Levinson A. D., Courtneidge S. A., Bishop J. M. Structural and functional domains of the Rous sarcoma virus transforming protein (pp60src). Proc Natl Acad Sci U S A. 1981 Mar;78(3):1624–1628. doi: 10.1073/pnas.78.3.1624. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Linder M. E., Burr J. G. Nonmyristoylated p60v-src fails to phosphorylate proteins of 115-120 kDa in chicken embryo fibroblasts. Proc Natl Acad Sci U S A. 1988 Apr;85(8):2608–2612. doi: 10.1073/pnas.85.8.2608. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Lipsich L. A., Lewis A. J., Brugge J. S. Isolation of monoclonal antibodies that recognize the transforming proteins of avian sarcoma viruses. J Virol. 1983 Nov;48(2):352–360. doi: 10.1128/jvi.48.2.352-360.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Loeb D. M., Woolford J., Beemon K. pp60c-src has less affinity for the detergent-insoluble cellular matrix than do pp60v-src and other viral protein-tyrosine kinases. J Virol. 1987 Aug;61(8):2420–2427. doi: 10.1128/jvi.61.8.2420-2427.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Markland W., Cheng S. H., Oostra B. A., Smith A. E. In vitro mutagenesis of the putative membrane-binding domain of polyomavirus middle-T antigen. J Virol. 1986 Jul;59(1):82–89. doi: 10.1128/jvi.59.1.82-89.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Parsons S. J., McCarley D. J., Ely C. M., Benjamin D. C., Parsons J. T. Monoclonal antibodies to Rous sarcoma virus pp60src react with enzymatically active cellular pp60src of avian and mammalian origin. J Virol. 1984 Aug;51(2):272–282. doi: 10.1128/jvi.51.2.272-282.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Pellman D., Garber E. A., Cross F. R., Hanafusa H. Fine structural mapping of a critical NH2-terminal region of p60src. Proc Natl Acad Sci U S A. 1985 Mar;82(6):1623–1627. doi: 10.1073/pnas.82.6.1623. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Piwnica-Worms H., Kaplan D. R., Whitman M., Roberts T. M. Retrovirus shuttle vector for study of kinase activities of pp60c-src synthesized in vitro and overproduced in vivo. Mol Cell Biol. 1986 Jun;6(6):2033–2040. doi: 10.1128/mcb.6.6.2033. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Raptis L., Bolen J. B. Polyomavirus transforms rat F111 and mouse NIH 3T3 cells by different mechanisms. J Virol. 1989 Feb;63(2):753–758. doi: 10.1128/jvi.63.2.753-758.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Reynolds A. B., Roesel D. J., Kanner S. B., Parsons J. T. Transformation-specific tyrosine phosphorylation of a novel cellular protein in chicken cells expressing oncogenic variants of the avian cellular src gene. Mol Cell Biol. 1989 Feb;9(2):629–638. doi: 10.1128/mcb.9.2.629. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Schuh S. M., Brugge J. S. Investigation of factors that influence phosphorylation of pp60c-src on tyrosine 527. Mol Cell Biol. 1988 Jun;8(6):2465–2471. doi: 10.1128/mcb.8.6.2465. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Schultz A. M., Henderson L. E., Oroszlan S., Garber E. A., Hanafusa H. Amino terminal myristylation of the protein kinase p60src, a retroviral transforming protein. Science. 1985 Jan 25;227(4685):427–429. doi: 10.1126/science.3917576. [DOI] [PubMed] [Google Scholar]
  33. Shalloway D., Coussens P. M., Yaciuk P. Overexpression of the c-src protein does not induce transformation of NIH 3T3 cells. Proc Natl Acad Sci U S A. 1984 Nov;81(22):7071–7075. doi: 10.1073/pnas.81.22.7071. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Templeton D., Eckhart W. Mutation causing premature termination of the polyoma virus medium T antigen blocks cell transformation. J Virol. 1982 Mar;41(3):1014–1024. doi: 10.1128/jvi.41.3.1014-1024.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Templeton D., Voronova A., Eckhart W. Construction and expression of a recombinant DNA gene encoding a polyomavirus middle-size tumor antigen with the carboxyl terminus of the vesicular stomatitis virus glycoprotein G. Mol Cell Biol. 1984 Feb;4(2):282–289. doi: 10.1128/mcb.4.2.282. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Treisman R., Novak U., Favaloro J., Kamen R. Transformation of rat cells by an altered polyoma virus genome expressing only the middle-T protein. Nature. 1981 Aug 13;292(5824):595–600. doi: 10.1038/292595a0. [DOI] [PubMed] [Google Scholar]
  37. Wells S. K., Collett M. S. Specific proteolytic fragmentation of p60v-src in transformed cell lysates. J Virol. 1983 Jul;47(1):253–258. doi: 10.1128/jvi.47.1.253-258.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]

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