Skip to main content
NCBI home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1993 Sep 1;90(17):8113–8117. doi: 10.1073/pnas.90.17.8113

Mitosis-specific phosphorylation of polyomavirus middle-sized tumor antigen and its role during cell transformation.

L Pérez 1, A Paasinen 1, B Schnierle 1, S Käch 1, M Senften 1, K Ballmer-Hofer 1
PMCID: PMC47298  PMID: 7690142

Abstract

Transformation of cells in culture by polyomavirus is mediated by one of its early gene products, middle-sized tumor antigen (MTAg). This protein forms multiple complexes with cellular enzymes such as tyrosine kinases (pp60c-src), a phosphatidylinositol 3-kinase, and phosphatase 2A. Association with MTAg leads to the activation of pp60c-src through interference with phosphorylation at Tyr-527, a site negatively regulating src kinase activity. MTAg abrogates mitosis-specific activation of pp60c-src, resulting in constitutive high kinase activity of the enzyme throughout all phases of the cell cycle. Here we report that MTAg is transiently modified during mitosis, resulting in an increase in its apparent molecular size on SDS/acrylamide gels. Similarly, MTAg isolated from interphase cells and phosphorylated by the cell cycle-regulated serine/threonine kinase p34cdc2 in vitro has increased molecular mass. The large molecular mass form of the protein can be converted to the authentic 56-kDa form upon dephosphorylation by potato acid phosphatase. Two putative phosphorylation sites for a cdc2-like kinase were identified as Thr-160 and -291, respectively. Conversion of Thr-160 to Ala resulted in a transformation-defective mutant protein that was still capable of associating with pp60c-src, phosphatidylinositol 3-kinase, and phosphatase 2A, while the corresponding mutant in position 291 was wild type with respect to all parameters measured so far. These data suggest that phosphorylation by p34cdc2 or a related cell cycle-regulated kinase modulates the interaction of MTAg with cellular targets that are crucial for cell transformation.

Full text

PDF
8113

Images in this article

8114Fig. 1
on p.8114
8115Fig. 2
on p.8115
8115Fig. 3
on p.8115
8116Fig. 4
on p.8116
8116Fig. 5
on p.8116

Selected References

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

  1. Bagrodia S., Chackalaparampil I., Kmiecik T. E., Shalloway D. Altered tyrosine 527 phosphorylation and mitotic activation of p60c-src. Nature. 1991 Jan 10;349(6305):172–175. doi: 10.1038/349172a0. [DOI] [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. 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]
  4. 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]
  5. Cantley L. C., Auger K. R., Carpenter C., Duckworth B., Graziani A., Kapeller R., Soltoff S. Oncogenes and signal transduction. Cell. 1991 Jan 25;64(2):281–302. doi: 10.1016/0092-8674(91)90639-g. [DOI] [PubMed] [Google Scholar]
  6. Cartwright C. A., Kaplan P. L., Cooper J. A., Hunter T., Eckhart W. Altered sites of tyrosine phosphorylation in pp60c-src associated with polyomavirus middle tumor antigen. Mol Cell Biol. 1986 May;6(5):1562–1570. doi: 10.1128/mcb.6.5.1562. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Chackalaparampil I., Shalloway D. Altered phosphorylation and activation of pp60c-src during fibroblast mitosis. Cell. 1988 Mar 25;52(6):801–810. doi: 10.1016/0092-8674(88)90422-9. [DOI] [PubMed] [Google Scholar]
  8. 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]
  9. Cheng S. H., Piwnica-Worms H., Harvey R. W., Roberts T. M., Smith A. E. The carboxy terminus of pp60c-src is a regulatory domain and is involved in complex formation with the middle-T antigen of polyomavirus. Mol Cell Biol. 1988 Apr;8(4):1736–1747. doi: 10.1128/mcb.8.4.1736. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Cohen B., Yoakim M., Piwnica-Worms H., Roberts T. M., Schaffhausen B. S. Tyrosine phosphorylation is a signal for the trafficking of pp85, an 85-kDa phosphorylated polypeptide associated with phosphatidylinositol kinase activity. Proc Natl Acad Sci U S A. 1990 Jun;87(12):4458–4462. doi: 10.1073/pnas.87.12.4458. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Cooper J. A., Gould K. L., Cartwright C. A., Hunter T. Tyr527 is phosphorylated in pp60c-src: implications for regulation. Science. 1986 Mar 21;231(4744):1431–1434. doi: 10.1126/science.2420005. [DOI] [PubMed] [Google Scholar]
  12. Courtneidge S. A. Activation of the pp60c-src kinase by middle T antigen binding or by dephosphorylation. EMBO J. 1985 Jun;4(6):1471–1477. doi: 10.1002/j.1460-2075.1985.tb03805.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. 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]
  14. Dilworth S. M. Cell alterations induced by the large T-antigens of SV40 and polyoma virus. Semin Cancer Biol. 1990 Dec;1(6):407–414. [PubMed] [Google Scholar]
  15. Dilworth S. M., Griffin B. E. Monoclonal antibodies against polyoma virus tumor antigens. Proc Natl Acad Sci U S A. 1982 Feb;79(4):1059–1063. doi: 10.1073/pnas.79.4.1059. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Druker B. J., Ling L. E., Cohen B., Roberts T. M., Schaffhausen B. S. A completely transformation-defective point mutant of polyomavirus middle T antigen which retains full associated phosphatidylinositol kinase activity. J Virol. 1990 Sep;64(9):4454–4461. doi: 10.1128/jvi.64.9.4454-4461.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Druker B. J., Sibert L., Roberts T. M. Polyomavirus middle T-antigen NPTY mutants. J Virol. 1992 Oct;66(10):5770–5776. doi: 10.1128/jvi.66.10.5770-5776.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Fanning E. Simian virus 40 large T antigen: the puzzle, the pieces, and the emerging picture. J Virol. 1992 Mar;66(3):1289–1293. doi: 10.1128/jvi.66.3.1289-1293.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Jans D. A., Ackermann M. J., Bischoff J. R., Beach D. H., Peters R. p34cdc2-mediated phosphorylation at T124 inhibits nuclear import of SV-40 T antigen proteins. J Cell Biol. 1991 Dec;115(5):1203–1212. doi: 10.1083/jcb.115.5.1203. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Kaech S., Covic L., Wyss A., Ballmer-Hofer K. Association of p60c-src with polyoma virus middle-T antigen abrogating mitosis-specific activation. Nature. 1991 Apr 4;350(6317):431–433. doi: 10.1038/350431a0. [DOI] [PubMed] [Google Scholar]
  21. 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]
  22. Kemp B. E., Pearson R. B. Protein kinase recognition sequence motifs. Trends Biochem Sci. 1990 Sep;15(9):342–346. doi: 10.1016/0968-0004(90)90073-k. [DOI] [PubMed] [Google Scholar]
  23. 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]
  24. 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]
  25. Labbé J. C., Capony J. P., Caput D., Cavadore J. C., Derancourt J., Kaghad M., Lelias J. M., Picard A., Dorée M. MPF from starfish oocytes at first meiotic metaphase is a heterodimer containing one molecule of cdc2 and one molecule of cyclin B. EMBO J. 1989 Oct;8(10):3053–3058. doi: 10.1002/j.1460-2075.1989.tb08456.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Levine A. J. The p53 protein and its interactions with the oncogene products of the small DNA tumor viruses. Virology. 1990 Aug;177(2):419–426. doi: 10.1016/0042-6822(90)90505-l. [DOI] [PubMed] [Google Scholar]
  27. Ling L. E., Druker B. J., Cantley L. C., Roberts T. M. Transformation-defective mutants of polyomavirus middle T antigen associate with phosphatidylinositol 3-kinase (PI 3-kinase) but are unable to maintain wild-type levels of PI 3-kinase products in intact cells. J Virol. 1992 Mar;66(3):1702–1708. doi: 10.1128/jvi.66.3.1702-1708.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Markland W., Smith A. E. Mutants of polyomavirus middle-T antigen. Biochim Biophys Acta. 1987 Nov 25;907(3):299–321. doi: 10.1016/0304-419x(87)90011-4. [DOI] [PubMed] [Google Scholar]
  29. Matthews J. T., Benjamin T. L. 12-O-tetradecanoylphorbol-13-acetate stimulates phosphorylation of the 58,000-Mr form of polyomavirus middle T antigen in vivo: implications for a possible role of protein kinase C in middle T function. J Virol. 1986 May;58(2):239–246. doi: 10.1128/jvi.58.2.239-246.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Moreno S., Nurse P. Substrates for p34cdc2: in vivo veritas? Cell. 1990 May 18;61(4):549–551. doi: 10.1016/0092-8674(90)90463-o. [DOI] [PubMed] [Google Scholar]
  31. Morgan D. O., Kaplan J. M., Bishop J. M., Varmus H. E. Mitosis-specific phosphorylation of p60c-src by p34cdc2-associated protein kinase. Cell. 1989 Jun 2;57(5):775–786. doi: 10.1016/0092-8674(89)90792-7. [DOI] [PubMed] [Google Scholar]
  32. Muser J., Kaech S., Moroni C., Ballmer-Hofer K. Stimulation of pp60c-src kinase activity in FDC-P1 cells by polyoma middle-T antigen and hematopoietic growth factors. Oncogene. 1989 Dec;4(12):1433–1439. [PubMed] [Google Scholar]
  33. Pallas D. C., Shahrik L. K., Martin B. L., Jaspers S., Miller T. B., Brautigan D. L., Roberts T. M. Polyoma small and middle T antigens and SV40 small t antigen form stable complexes with protein phosphatase 2A. Cell. 1990 Jan 12;60(1):167–176. doi: 10.1016/0092-8674(90)90726-u. [DOI] [PubMed] [Google Scholar]
  34. Pelech S. L., Sanghera J. S. Mitogen-activated protein kinases: versatile transducers for cell signaling. Trends Biochem Sci. 1992 Jun;17(6):233–238. doi: 10.1016/s0968-0004(00)80005-5. [DOI] [PubMed] [Google Scholar]
  35. Prives C. The replication functions of SV40 T antigen are regulated by phosphorylation. Cell. 1990 Jun 1;61(5):735–738. doi: 10.1016/0092-8674(90)90179-i. [DOI] [PubMed] [Google Scholar]
  36. Raptis L., Bell J., Whitfield J. F. Protein kinase C increases the activity of the polyoma virus middle T antigen-associated phosphatidylinositol kinase. Biochem Biophys Res Commun. 1988 Jul 15;154(1):306–311. doi: 10.1016/0006-291x(88)90685-7. [DOI] [PubMed] [Google Scholar]
  37. Raptis L., Boynton A. L., Whitfield J. F. Protein kinase C promotes the phosphorylation of immunoprecipitated middle T antigen from polyoma-transformed cells. Biochem Biophys Res Commun. 1986 May 14;136(3):995–1000. doi: 10.1016/0006-291x(86)90431-6. [DOI] [PubMed] [Google Scholar]
  38. Shenoy S., Choi J. K., Bagrodia S., Copeland T. D., Maller J. L., Shalloway D. Purified maturation promoting factor phosphorylates pp60c-src at the sites phosphorylated during fibroblast mitosis. Cell. 1989 Jun 2;57(5):763–774. doi: 10.1016/0092-8674(89)90791-5. [DOI] [PubMed] [Google Scholar]
  39. Whitman M., Kaplan D. R., Schaffhausen B., Cantley L., Roberts T. M. Association of phosphatidylinositol kinase activity with polyoma middle-T competent for transformation. Nature. 1985 May 16;315(6016):239–242. doi: 10.1038/315239a0. [DOI] [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES