Skip to main content
The EMBO Journal logoLink to The EMBO Journal
. 1984 Jan;3(1):73–79. doi: 10.1002/j.1460-2075.1984.tb01763.x

Polyoma middle-sized T antigen can be phosphorylated on tyrosine at multiple sites in vitro.

T Hunter, M A Hutchinson, W Eckhart
PMCID: PMC557299  PMID: 6200324

Abstract

The polyoma middle-sized T antigen (MT antigen) is associated with a protein kinase activity which phosphorylates tyrosine residues in polyoma T antigens in vitro. We have studied the sites of tyrosine phosphorylation of MT antigens phosphorylated in immunoprecipitates or in soluble form after partial purification by immunoaffinity chromatography. By analyzing the amino acid sequences of tryptic peptides of MT antigen, and by analyzing deletion mutant MT antigens, we have identified two major sites of phosphorylation in MT antigen, tyrosines 250 and 315. Additional sites were phosphorylated under some conditions. A synthetic peptide (Glu.Glu.Glu.Glu.Tyr.Met.Pro.Met.Glu), corresponding to the sequence around tyrosine 315, was phosphorylated when added to immunoprecipitates containing MT antigen.

Full text

PDF
73

Images in this article

Selected References

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

  1. Baldwin G. S., Burgess A. W., Kemp B. E. Phosphorylation of a synthetic gastrin peptide by the tyrosine kinase of A431 cell membranes. Biochem Biophys Res Commun. 1982 Dec 15;109(3):656–663. doi: 10.1016/0006-291x(82)91990-8. [DOI] [PubMed] [Google Scholar]
  2. Baldwin G. S. Gastrin and the transforming protein of polyoma virus have evolved from a common ancestor. FEBS Lett. 1982 Jan 11;137(1):1–5. doi: 10.1016/0014-5793(82)80302-5. [DOI] [PubMed] [Google Scholar]
  3. Baldwin G. S., Knesel J., Monckton J. M. Phosphorylation of gastrin-17 by epidermal growth factor-stimulated tyrosine kinase. Nature. 1983 Feb 3;301(5899):435–437. doi: 10.1038/301435a0. [DOI] [PubMed] [Google Scholar]
  4. 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]
  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. 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]
  7. 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]
  8. Friedmann T., Esty A., LaPorte P., Deininger P. The nucleotide sequence and genome organization of the polyoma early region: extensive nucleotide and amino acid homology with SV40. Cell. 1979 Jul;17(3):715–724. doi: 10.1016/0092-8674(79)90278-2. [DOI] [PubMed] [Google Scholar]
  9. Hunter T., Sefton B. M. Transforming gene product of Rous sarcoma virus phosphorylates tyrosine. Proc Natl Acad Sci U S A. 1980 Mar;77(3):1311–1315. doi: 10.1073/pnas.77.3.1311. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Hunter T. Synthetic peptide substrates for a tyrosine protein kinase. J Biol Chem. 1982 May 10;257(9):4843–4848. [PubMed] [Google Scholar]
  11. Hutchinson M. A., Hunter T., Eckhart W. Characterization of T antigens in polyoma-infected and transformed cells. Cell. 1978 Sep;15(1):65–77. doi: 10.1016/0092-8674(78)90083-1. [DOI] [PubMed] [Google Scholar]
  12. Magnusson G., Nilsson M. G., Dilworth S. M., Smolar N. Characterization of polyoma mutants with altered middle and large T-antigens. J Virol. 1981 Sep;39(3):673–683. doi: 10.1128/jvi.39.3.673-683.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Mes A. M., Hassell J. A. Polyoma viral middle T-antigen is required for transformation. J Virol. 1982 May;42(2):621–629. doi: 10.1128/jvi.42.2.621-629.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Nilsson S. V., Tyndall C., Magnusson G. Deletion mapping of a short polyoma virus middle T antigen segment important for transformation. J Virol. 1983 Apr;46(1):284–287. doi: 10.1128/jvi.46.1.284-287.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. 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]
  16. Patschinsky T., Hunter T., Esch F. S., Cooper J. A., Sefton B. M. Analysis of the sequence of amino acids surrounding sites of tyrosine phosphorylation. Proc Natl Acad Sci U S A. 1982 Feb;79(4):973–977. doi: 10.1073/pnas.79.4.973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. 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]
  18. 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]
  19. Schaffhausen B., Benjamin T. L., Pike L., Casnellie J., Krebs E. Antibody to the nonapeptide Glu-Glu-Glu-Glu-Tyr-Met-Pro-Met-Glu is specific for polyoma middle T antigen and inhibits in vitro kinase activity. J Biol Chem. 1982 Nov 10;257(21):12467–12470. [PubMed] [Google Scholar]
  20. Schneider C., Newman R. A., Sutherland D. R., Asser U., Greaves M. F. A one-step purification of membrane proteins using a high efficiency immunomatrix. J Biol Chem. 1982 Sep 25;257(18):10766–10769. [PubMed] [Google Scholar]
  21. 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]
  22. 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]
  23. Smolar N., Griffin B. E. DNA sequences of polyoma virus early deletion mutants. J Virol. 1981 Jun;38(3):958–967. doi: 10.1128/jvi.38.3.958-967.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. 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]
  25. 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]
  26. Walter G., Hutchinson M. A., Hunter T., Eckhart W. Antibodies specific for the polyoma virus middle-size tumor antigen. Proc Natl Acad Sci U S A. 1981 Aug;78(8):4882–4886. doi: 10.1073/pnas.78.8.4882. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Walter G., Hutchinson M. A., Hunter T., Eckhart W. Purification of polyoma virus medium-size tumor antigen by immunoaffinity chromatography. Proc Natl Acad Sci U S A. 1982 Jul;79(13):4025–4029. doi: 10.1073/pnas.79.13.4025. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES