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. 1985 Sep;4(9):2321–2327. doi: 10.1002/j.1460-2075.1985.tb03933.x

Phosphorylation of polyoma middle T antigen and cellular proteins in purified plasma membranes of polyoma virus-infected cells.

K Ballmer-Hofer, T L Benjamin
PMCID: PMC554504  PMID: 2416563

Abstract

We have studied phosphorylation carried out by purified plasma membranes from polyoma virus-infected cells. When isolated membranes are incubated with [gamma-32P]ATP, polyoma virus middle T antigen (mT) becomes phosphorylated on tyrosine. Partial proteolysis mapping shows the same pattern as previously noted for mT labeled in immune complexes. Membranes labeled in vitro were also extracted and immunoprecipitated with anti-T or anti-src antibody. With either antibody, both mT and pp60c-src were brought down and shown to be labeled on tyrosine. The mT of an hr-t mutant (NG59) showed only a trace amount of labeling in membranes under the same conditions. Proteins from infected and uninfected cell membranes labeled in vitro were separated on two-dimensional gels. An acidic 40-kd phosphoprotein was labeled in uninfected cell membranes, but was not seen using membranes from wild-type virus-infected cells. Neither NG59, which encodes a defective but membrane-associated mT, nor a mutant encoding a truncated mT that fails to associate with membranes, alters the level of the 40-kd phosphoprotein in membranes labeled in vitro. These results suggest that mT, acting through pp60c-src and possibly other cellular kinases and phosphatases, can affect cell protein phosphorylation as part of the transformation process.

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Selected References

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

  1. Bendig M. M., Thomas T., Folk W. R. Viable deletion mutant in the medium and large T-antigen-coding sequences of the polyoma virus genome. J Virol. 1980 Mar;33(3):1215–1220. doi: 10.1128/jvi.33.3.1215-1220.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Benjamin T. L. Host range mutants of polyoma virus. Proc Natl Acad Sci U S A. 1970 Sep;67(1):394–399. doi: 10.1073/pnas.67.1.394. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Benjamin T. L. The hr-t gene of polyoma virus. Biochim Biophys Acta. 1982 Dec 21;695(2):69–95. doi: 10.1016/0304-419x(82)90018-x. [DOI] [PubMed] [Google Scholar]
  4. Bolen J. B., Israel M. A. Middle tumor antigen of polyomavirus transformation-defective mutant NG59 is associated with pp60c-src. J Virol. 1985 Jan;53(1):114–119. doi: 10.1128/jvi.53.1.114-119.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. 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]
  6. Carmichael G. G., Benjamin T. L. Identification of DNA sequence changes leading to loss of transforming ability in polyoma virus. J Biol Chem. 1980 Jan 10;255(1):230–235. [PubMed] [Google Scholar]
  7. 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]
  8. Carmichael G., Schaffhausen B. S., Mandel G., Liang T. J., Benjamin T. L. Transformation by polyoma virus is drastically reduced by substitution of phenylalanine for tyrosine at residue 315 of middle-sized tumor antigen. Proc Natl Acad Sci U S A. 1984 Feb;81(3):679–683. doi: 10.1073/pnas.81.3.679. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. 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]
  10. Cooper J. A., Hunter T. Changes in protein phosphorylation in Rous sarcoma virus-transformed chicken embryo cells. Mol Cell Biol. 1981 Feb;1(2):165–178. doi: 10.1128/mcb.1.2.165. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Cooper J. A., Hunter T. Discrete primary locations of a tyrosine-protein kinase and of three proteins that contain phosphotyrosine in virally transformed chick fibroblasts. J Cell Biol. 1982 Aug;94(2):287–296. doi: 10.1083/jcb.94.2.287. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Cooper J. A., Reiss N. A., Schwartz R. J., Hunter T. Three glycolytic enzymes are phosphorylated at tyrosine in cells transformed by Rous sarcoma virus. Nature. 1983 Mar 17;302(5905):218–223. doi: 10.1038/302218a0. [DOI] [PubMed] [Google Scholar]
  13. Courtneidge S. A., Bishop J. M. Transit of pp60v-src to the plasma membrane. Proc Natl Acad Sci U S A. 1982 Dec;79(23):7117–7121. doi: 10.1073/pnas.79.23.7117. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Courtneidge S. A., Levinson A. D., Bishop J. M. The protein encoded by the transforming gene of avian sarcoma virus (pp60src) and a homologous protein in normal cells (pp60proto-src) are associated with the plasma membrane. Proc Natl Acad Sci U S A. 1980 Jul;77(7):3783–3787. doi: 10.1073/pnas.77.7.3783. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. 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]
  16. Courtneidge S. A., Smith A. E. The complex of polyoma virus middle-T antigen and pp60c-src. EMBO J. 1984 Mar;3(3):585–591. doi: 10.1002/j.1460-2075.1984.tb01852.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Eckhart W. Complementation between temperature-sensitive (ts) and host range nontransforming (hr-t) mutants of polyoma virus. Virology. 1977 Apr;77(2):589–597. doi: 10.1016/0042-6822(77)90484-6. [DOI] [PubMed] [Google Scholar]
  18. Eckhart W., Hutchinson M. A., Hunter T. An activity phosphorylating tyrosine in polyoma T antigen immunoprecipitates. Cell. 1979 Dec;18(4):925–933. doi: 10.1016/0092-8674(79)90205-8. [DOI] [PubMed] [Google Scholar]
  19. Feunteun J., Sompayrac L., Fluck M., Benjamin T. Localization of gene functions in polyoma virus DNA. Proc Natl Acad Sci U S A. 1976 Nov;73(11):4169–4173. doi: 10.1073/pnas.73.11.4169. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Fluck M. M., Staneloni R. J., Benjamin T. L. Hr-t and ts-a: two early gene functions of polyoma virus. Virology. 1977 Apr;77(2):610–624. doi: 10.1016/0042-6822(77)90486-x. [DOI] [PubMed] [Google Scholar]
  21. Gallis B., Bornstein P., Brautigan D. L. Tyrosylprotein kinase and phosphatase activities in membrane vesicles from normal and Rous sarcoma virus-transformed rat cells. Proc Natl Acad Sci U S A. 1981 Nov;78(11):6689–6693. doi: 10.1073/pnas.78.11.6689. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Hattori J., Carmichael G. G., Benjamin T. L. DNA sequence alterations in Hr-t deletion mutants of polyoma virus. Cell. 1979 Mar;16(3):505–513. doi: 10.1016/0092-8674(79)90025-4. [DOI] [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. Leis J. F., Kaplan N. O. An acid phosphatase in the plasma membranes of human astrocytoma showing marked specificity toward phosphotyrosine protein. Proc Natl Acad Sci U S A. 1982 Nov;79(21):6507–6511. doi: 10.1073/pnas.79.21.6507. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. 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]
  26. O'Farrell P. H. High resolution two-dimensional electrophoresis of proteins. J Biol Chem. 1975 May 25;250(10):4007–4021. [PMC free article] [PubMed] [Google Scholar]
  27. O'Farrell P. Z., Goodman H. M., O'Farrell P. H. High resolution two-dimensional electrophoresis of basic as well as acidic proteins. Cell. 1977 Dec;12(4):1133–1141. doi: 10.1016/0092-8674(77)90176-3. [DOI] [PubMed] [Google Scholar]
  28. Oostra B. A., Harvey R., Ely B. K., Markham A. F., Smith A. E. Transforming activity of polyoma virus middle-T antigen probed by site-directed mutagenesis. Nature. 1983 Aug 4;304(5925):456–459. doi: 10.1038/304456a0. [DOI] [PubMed] [Google Scholar]
  29. Radke K., Carter V. C., Moss P., Dehazya P., Schliwa M., Martin G. S. Membrane association of a 36,000-dalton substrate for tyrosine phosphorylation in chicken embryo fibroblasts transformed by avian sarcoma viruses. J Cell Biol. 1983 Nov;97(5 Pt 1):1601–1611. doi: 10.1083/jcb.97.5.1601. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Schaffhausen B. S., Benjamin T. L. Phosphorylation of polyoma T antigens. Cell. 1979 Dec;18(4):935–946. doi: 10.1016/0092-8674(79)90206-x. [DOI] [PubMed] [Google Scholar]
  31. Schaffhausen B. S., Dorai H., Arakere G., Benjamin T. L. Polyoma virus middle T antigen: relationship to cell membranes and apparent lack of ATP-binding activity. Mol Cell Biol. 1982 Oct;2(10):1187–1198. doi: 10.1128/mcb.2.10.1187. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Schaffhausen B. S., Silver J. E., Benjamin T. L. Tumor antigen(s) in cell productively infected by wild-type polyoma virus and mutant NG-18. Proc Natl Acad Sci U S A. 1978 Jan;75(1):79–83. doi: 10.1073/pnas.75.1.79. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Schaffhausen B., Benjamin T. L. Comparison of phosphorylation of two polyoma virus middle T antigens in vivo and in vitro. J Virol. 1981 Oct;40(1):184–196. doi: 10.1128/jvi.40.1.184-196.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Schaffhausen B., Benjamin T. L., Lodge J., Kaplan D., Roberts T. M. Expression of polyoma early gene products in E. coli. Nucleic Acids Res. 1985 Jan 25;13(2):501–519. doi: 10.1093/nar/13.2.501. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Segawa K., Ito Y. Differential subcellular localization of in vivo-phosphorylated and nonphosphorylated middle-sized tumor antigen of polyoma virus and its relationship to middle-sized tumor antigen phosphorylating activity in vitro. Proc Natl Acad Sci U S A. 1982 Nov;79(22):6812–6816. doi: 10.1073/pnas.79.22.6812. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Smith A. E., Smith R., Griffin B., Fried M. Protein kinase activity associated with polyoma virus middle T antigen in vitro. Cell. 1979 Dec;18(4):915–924. doi: 10.1016/0092-8674(79)90204-6. [DOI] [PubMed] [Google Scholar]
  37. Staneloni R. J., Fluck M. M., Benjamin T. L. Host range selection of transformation-defective hr-t mutants of polyoma virus. Virology. 1977 Apr;77(2):598–609. doi: 10.1016/0042-6822(77)90485-8. [DOI] [PubMed] [Google Scholar]
  38. 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]
  39. Thom D., Powell A. J., Lloyd C. W., Rees D. A. Rapid isolation of plasma membranes in high yield from cultured fibroblasts. Biochem J. 1977 Nov 15;168(2):187–194. doi: 10.1042/bj1680187. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. 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]
  41. Witt D. P., Gordon J. A. Specific dephosphorylation of membrane proteins in Rous sarcoma virus-transformed chick embryo fibroblasts. Nature. 1980 Sep 18;287(5779):241–244. doi: 10.1038/287241a0. [DOI] [PubMed] [Google Scholar]

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