Skip to main content
NCBI home page
Journal of Virology logoLink to Journal of Virology
. 1995 Jun;69(6):3729–3736. doi: 10.1128/jvi.69.6.3729-3736.1995

Amino-terminal regions of polyomavirus middle T antigen are required for interactions with protein phosphatase 2A.

G M Glenn 1, W Eckhart 1
PMCID: PMC189089  PMID: 7538175

Abstract

Polyomavirus middle T antigen (MT) is the major transforming protein of the virus. It functions through interactions with a number of cellular proteins involved in cell proliferation. MT forms complexes with protein phosphatase 2A (PP2A), pp60c-src, phosphatidylinositol 3-kinase, and Shc. We introduced both deletion and point mutations into three regions of MT and examined their ability to associate with PP2A and pp60c-src. The first 25 amino acid residues of MT are required for association with PP2A and pp60c-src. Amino acids 105 to 111, comprising the sequence Cys-Arg-Met-Pro-Leu-Thr-Cys, is also required for complex formation between MT and PP2A. However, the sequence Asp-Lys-Gly-Gly (amino acids 44 to 47), also found in the B subunit of PP2A, is dispensable for complex formation between MT and PP2A. We find a strict correlation between the ability of MT to associate with PP2A and the ability of MT to associate with pp60c-src. One mutant, L5E, associates with a phosphatase other than PP2A, pp60c-src, and phosphatidylinositol 3-kinase in a manner similar to that of wild-type MT yet is reduced in its transforming ability on NIH 3T3 cells.

Full Text

The Full Text of this article is available as a PDF (452.0 KB).

Selected References

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

  1. Aiken C., Konner J., Landau N. R., Lenburg M. E., Trono D. Nef induces CD4 endocytosis: requirement for a critical dileucine motif in the membrane-proximal CD4 cytoplasmic domain. Cell. 1994 Mar 11;76(5):853–864. doi: 10.1016/0092-8674(94)90360-3. [DOI] [PubMed] [Google Scholar]
  2. 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]
  3. Campbell K. S., Ogris E., Burke B., Su W., Auger K. R., Druker B. J., Schaffhausen B. S., Roberts T. M., Pallas D. C. Polyoma middle tumor antigen interacts with SHC protein via the NPTY (Asn-Pro-Thr-Tyr) motif in middle tumor antigen. Proc Natl Acad Sci U S A. 1994 Jul 5;91(14):6344–6348. doi: 10.1073/pnas.91.14.6344. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Cartwright C. A., Hutchinson M. A., Eckhart W. Structural and functional modification of pp60c-src associated with polyoma middle tumor antigen from infected or transformed cells. Mol Cell Biol. 1985 Oct;5(10):2647–2652. doi: 10.1128/mcb.5.10.2647. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. 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]
  6. 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]
  7. Cohen P. The structure and regulation of protein phosphatases. Annu Rev Biochem. 1989;58:453–508. doi: 10.1146/annurev.bi.58.070189.002321. [DOI] [PubMed] [Google Scholar]
  8. Cook D. N., Hassell J. A. The amino terminus of polyomavirus middle T antigen is required for transformation. J Virol. 1990 May;64(5):1879–1887. doi: 10.1128/jvi.64.5.1879-1887.1990. [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. 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]
  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. Dilworth S. M., Brewster C. E., Jones M. D., Lanfrancone L., Pelicci G., Pelicci P. G. Transformation by polyoma virus middle T-antigen involves the binding and tyrosine phosphorylation of Shc. Nature. 1994 Jan 6;367(6458):87–90. doi: 10.1038/367087a0. [DOI] [PubMed] [Google Scholar]
  13. Dilworth S. M., Horner V. P. Novel monoclonal antibodies that differentiate between the binding of pp60c-src or protein phosphatase 2A by polyomavirus middle T antigen. J Virol. 1993 Apr;67(4):2235–2244. doi: 10.1128/jvi.67.4.2235-2244.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. 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]
  15. Glenn G. M., Eckhart W. Mutation of a cysteine residue in polyomavirus middle T antigen abolishes interactions with protein phosphatase 2A, pp60c-src, and phosphatidylinositol-3 kinase, activation of c-fos expression, and cellular transformation. J Virol. 1993 Apr;67(4):1945–1952. doi: 10.1128/jvi.67.4.1945-1952.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Grussenmeyer T., Scheidtmann K. H., Hutchinson M. A., Eckhart W., Walter G. Complexes of polyoma virus medium T antigen and cellular proteins. Proc Natl Acad Sci U S A. 1985 Dec;82(23):7952–7954. doi: 10.1073/pnas.82.23.7952. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Hendrix P., Mayer-Jackel R. E., Cron P., Goris J., Hofsteenge J., Merlevede W., Hemmings B. A. Structure and expression of a 72-kDa regulatory subunit of protein phosphatase 2A. Evidence for different size forms produced by alternative splicing. J Biol Chem. 1993 Jul 15;268(20):15267–15276. [PubMed] [Google Scholar]
  18. Horak I. D., Kawakami T., Gregory F., Robbins K. C., Bolen J. B. Association of p60fyn with middle tumor antigen in murine polyomavirus-transformed rat cells. J Virol. 1989 May;63(5):2343–2347. doi: 10.1128/jvi.63.5.2343-2347.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Jelinek M. A., Hassell J. A. Reversion of middle T antigen-transformed Rat-2 cells by Krev-1: implications for the role of p21c-ras in polyomavirus-mediated transformation. Oncogene. 1992 Sep;7(9):1687–1698. [PubMed] [Google Scholar]
  20. Kaplan P. L., Simon S., Cartwright C. A., Eckhart W. cDNA cloning with a retrovirus expression vector: generation of a pp60c-src cDNA clone. J Virol. 1987 May;61(5):1731–1734. doi: 10.1128/jvi.61.5.1731-1734.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. 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]
  22. Kunkel T. A., Roberts J. D., Zakour R. A. Rapid and efficient site-specific mutagenesis without phenotypic selection. Methods Enzymol. 1987;154:367–382. doi: 10.1016/0076-6879(87)54085-x. [DOI] [PubMed] [Google Scholar]
  23. 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]
  24. Letourneur F., Klausner R. D. A novel di-leucine motif and a tyrosine-based motif independently mediate lysosomal targeting and endocytosis of CD3 chains. Cell. 1992 Jun 26;69(7):1143–1157. doi: 10.1016/0092-8674(92)90636-q. [DOI] [PubMed] [Google Scholar]
  25. Markland W., Oostra B. A., Harvey R., Markham A. F., Colledge W. H., Smith A. E. Site-directed mutagenesis of polyomavirus middle-T antigen sequences encoding tyrosine 315 and tyrosine 250. J Virol. 1986 Aug;59(2):384–391. doi: 10.1128/jvi.59.2.384-391.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. 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]
  27. Mayer-Jaekel R. E., Hemmings B. A. Protein phosphatase 2A--a 'ménage à trois'. Trends Cell Biol. 1994 Aug;4(8):287–291. doi: 10.1016/0962-8924(94)90219-4. [DOI] [PubMed] [Google Scholar]
  28. Mungre S., Enderle K., Turk B., Porrás A., Wu Y. Q., Mumby M. C., Rundell K. Mutations which affect the inhibition of protein phosphatase 2A by simian virus 40 small-t antigen in vitro decrease viral transformation. J Virol. 1994 Mar;68(3):1675–1681. doi: 10.1128/jvi.68.3.1675-1681.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Pallas D. C., Cherington V., Morgan W., DeAnda J., Kaplan D., Schaffhausen B., Roberts T. M. Cellular proteins that associate with the middle and small T antigens of polyomavirus. J Virol. 1988 Nov;62(11):3934–3940. doi: 10.1128/jvi.62.11.3934-3940.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. 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]
  31. Pallas D. C., Weller W., Jaspers S., Miller T. B., Lane W. S., Roberts T. M. The third subunit of protein phosphatase 2A (PP2A), a 55-kilodalton protein which is apparently substituted for by T antigens in complexes with the 36- and 63-kilodalton PP2A subunits, bears little resemblance to T antigens. J Virol. 1992 Feb;66(2):886–893. doi: 10.1128/jvi.66.2.886-893.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Raptis L., Marcellus R., Corbley M. J., Krook A., Whitfield J., Anderson S. K., Haliotis T. Cellular ras gene activity is required for full neoplastic transformation by polyomavirus. J Virol. 1991 Oct;65(10):5203–5210. doi: 10.1128/jvi.65.10.5203-5210.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Rozakis-Adcock M., Fernley R., Wade J., Pawson T., Bowtell D. The SH2 and SH3 domains of mammalian Grb2 couple the EGF receptor to the Ras activator mSos1. Nature. 1993 May 6;363(6424):83–85. doi: 10.1038/363083a0. [DOI] [PubMed] [Google Scholar]
  34. Ruediger R., Hentz M., Fait J., Mumby M., Walter G. Molecular model of the A subunit of protein phosphatase 2A: interaction with other subunits and tumor antigens. J Virol. 1994 Jan;68(1):123–129. doi: 10.1128/jvi.68.1.123-129.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Ruediger R., Roeckel D., Fait J., Bergqvist A., Magnusson G., Walter G. Identification of binding sites on the regulatory A subunit of protein phosphatase 2A for the catalytic C subunit and for tumor antigens of simian virus 40 and polyomavirus. Mol Cell Biol. 1992 Nov;12(11):4872–4882. doi: 10.1128/mcb.12.11.4872. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. 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]
  37. Skolnik E. Y., Lee C. H., Batzer A., Vicentini L. M., Zhou M., Daly R., Myers M. J., Jr, Backer J. M., Ullrich A., White M. F. The SH2/SH3 domain-containing protein GRB2 interacts with tyrosine-phosphorylated IRS1 and Shc: implications for insulin control of ras signalling. EMBO J. 1993 May;12(5):1929–1936. doi: 10.1002/j.1460-2075.1993.tb05842.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Subramani S., Southern P. J. Analysis of gene expression using simian virus 40 vectors. Anal Biochem. 1983 Nov;135(1):1–15. doi: 10.1016/0003-2697(83)90723-6. [DOI] [PubMed] [Google Scholar]
  39. Trowbridge I. S., Collawn J. F., Hopkins C. R. Signal-dependent membrane protein trafficking in the endocytic pathway. Annu Rev Cell Biol. 1993;9:129–161. doi: 10.1146/annurev.cb.09.110193.001021. [DOI] [PubMed] [Google Scholar]
  40. Ulug E. T., Cartwright A. J., Courtneidge S. A. Characterization of the interaction of polyomavirus middle T antigen with type 2A protein phosphatase. J Virol. 1992 Mar;66(3):1458–1467. doi: 10.1128/jvi.66.3.1458-1467.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Walter G., Mumby M. Protein serine/threonine phosphatases and cell transformation. Biochim Biophys Acta. 1993 Aug 23;1155(2):207–226. doi: 10.1016/0304-419x(93)90005-w. [DOI] [PubMed] [Google Scholar]
  42. Walter G., Ruediger R., Slaughter C., Mumby M. Association of protein phosphatase 2A with polyoma virus medium tumor antigen. Proc Natl Acad Sci U S A. 1990 Apr;87(7):2521–2525. doi: 10.1073/pnas.87.7.2521. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. 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]
  44. de Wet J. R., Wood K. V., DeLuca M., Helinski D. R., Subramani S. Firefly luciferase gene: structure and expression in mammalian cells. Mol Cell Biol. 1987 Feb;7(2):725–737. doi: 10.1128/mcb.7.2.725. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Journal of Virology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES