Skip to main content
NCBI home page
Journal of Virology logoLink to Journal of Virology
. 1986 Dec;60(3):888–894. doi: 10.1128/jvi.60.3.888-894.1986

Phosphorylation downregulates the DNA-binding activity of simian virus 40 T antigen.

D T Simmons, W Chou, K Rodgers
PMCID: PMC253314  PMID: 3023678

Abstract

Proteolytic fragments of simian virus 40 tumor (T) antigen and T antigen that was dephosphorylated with alkaline phosphatase bound between 1.5 to 2 times more origin-containing simian virus 40 DNA than did intact T antigen in DNA saturation experiments. Kinetic experiments showed that these treatments also enhanced the rate at which T antigen bound to the DNA. The enhanced binding of T-antigen fragments correlated with the generation of DNA-binding fragments that lacked the NH2-terminal region. Dephosphorylation of T antigen in vitro resulted in the removal of phosphate groups from the NH2-terminal region as well as from the COOH-terminal region. To test the effects of dephosphorylation on the size of the protein, immunoaffinity-purified T antigen was subjected to sedimentation with and without prior treatment with alkaline phosphatase. Most of the purified protein sedimented as a monomer and no significant effect was observed after dephosphorylation, indicating that the enhanced DNA-binding activity was probably not due to the uncovering of additional binding sites buried specifically in oligomerized T antigen. Taken together, these results indicate that in vivo phosphorylation of the NH2-terminal region (residues 106 to 124) decreases the binding of the protein to the DNA origin. The effect is reversed by in vitro dephosphorylation or by proteolysis which removes the highly phosphorylated NH2-terminal arm of the polypeptide. We suggest that phosphorylation inactivates one of two distinct DNA-binding activities on the polypeptide chain perhaps corresponding to two separate regions in T antigen.

Full text

PDF
888

Images in this article

890Image
on p.890
891Image
on p.891

Selected References

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

  1. Baumann E. A., Hand R. Phosphorylation and dephosphorylation alter the structure of D2 hybrid T antigen. J Virol. 1982 Oct;44(1):78–87. doi: 10.1128/jvi.44.1.78-87.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bradley M. K., Griffin J. D., Livingston D. M. Relationship of oligomerization to enzymatic and DNA-binding properties of the SV40 large T antigen. Cell. 1982 Jan;28(1):125–134. doi: 10.1016/0092-8674(82)90382-8. [DOI] [PubMed] [Google Scholar]
  3. Brady J., Bolen J. B., Radonovich M., Salzman N., Khoury G. Stimulation of simian virus 40 late gene expression by simian virus 40 tumor antigen. Proc Natl Acad Sci U S A. 1984 Apr;81(7):2040–2044. doi: 10.1073/pnas.81.7.2040. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Burger C., Fanning E. Specific DNA binding activity of T antigen subclasses varies among different SV40-transformed cell lines. Virology. 1983 Apr 15;126(1):19–31. doi: 10.1016/0042-6822(83)90459-2. [DOI] [PubMed] [Google Scholar]
  5. Clark R., Peden K., Pipas J. M., Nathans D., Tjian R. Biochemical activities of T-antigen proteins encoded by simian virus 40 A gene deletion mutants. Mol Cell Biol. 1983 Feb;3(2):220–228. doi: 10.1128/mcb.3.2.220. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Fanning E., Nowak B., Burger C. Detection and characterization of multiple forms of simian virus 40 large T antigen. J Virol. 1981 Jan;37(1):92–102. doi: 10.1128/jvi.37.1.92-102.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Fanning E., Westphal K. H., Brauer D., Cörlin D. Subclasses of simian virus 40 large T antigen: differential binding of two subclasses of T antigen from productively infected cells to viral and cellular DNA. EMBO J. 1982;1(9):1023–1028. doi: 10.1002/j.1460-2075.1982.tb01290.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Gidoni D., Scheller A., Barnet B., Hantzopoulos P., Oren M., Prives C. Different forms of simian virus 40 large tumor antigen varying in their affinities for DNA. J Virol. 1982 May;42(2):456–466. doi: 10.1128/jvi.42.2.456-466.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Greenspan D. S., Carroll R. B. Complex of simian virus 40 large tumor antigen and 48,000-dalton host tumor antigen. Proc Natl Acad Sci U S A. 1981 Jan;78(1):105–109. doi: 10.1073/pnas.78.1.105. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Hansen U., Tenen D. G., Livingston D. M., Sharp P. A. T antigen repression of SV40 early transcription from two promoters. Cell. 1981 Dec;27(3 Pt 2):603–613. doi: 10.1016/0092-8674(81)90402-5. [DOI] [PubMed] [Google Scholar]
  11. Harlow E., Crawford L. V., Pim D. C., Williamson N. M. Monoclonal antibodies specific for simian virus 40 tumor antigens. J Virol. 1981 Sep;39(3):861–869. doi: 10.1128/jvi.39.3.861-869.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Keller J. M., Alwine J. C. Activation of the SV40 late promoter: direct effects of T antigen in the absence of viral DNA replication. Cell. 1984 Feb;36(2):381–389. doi: 10.1016/0092-8674(84)90231-9. [DOI] [PubMed] [Google Scholar]
  13. Levitsky K., Chandrasekaran K., Mora P. T., Simmons D. T. Immunoaffinity chromatography of a cellular tumor antigen from mouse neuroblastoma cells. Int J Cancer. 1983 Nov 15;32(5):597–602. doi: 10.1002/ijc.2910320513. [DOI] [PubMed] [Google Scholar]
  14. Livingston D. M., Henderson I. C., Hudson J. SV40 T antigen: partial purification and properties. Cold Spring Harb Symp Quant Biol. 1975;39(Pt 1):283–289. doi: 10.1101/sqb.1974.039.01.037. [DOI] [PubMed] [Google Scholar]
  15. McCormick F., Harlow E. Association of a murine 53,000-dalton phosphoprotein with simian virus 40 large-T antigen in transformed cells. J Virol. 1980 Apr;34(1):213–224. doi: 10.1128/jvi.34.1.213-224.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. McKay R. D. Binding of a simian virus 40 T antigen-related protein to DNA. J Mol Biol. 1981 Jan 25;145(3):471–488. doi: 10.1016/0022-2836(81)90540-4. [DOI] [PubMed] [Google Scholar]
  17. Morrison B., Kress M., Khoury G., Jay G. Simian virus 40 tumor antigen: isolation of the origin-specific DNA-binding domain. J Virol. 1983 Jul;47(1):106–114. doi: 10.1128/jvi.47.1.106-114.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Myers R. M., Rio D. C., Robbins A. K., Tjian R. SV40 gene expression is modulated by the cooperative binding of T antigen to DNA. Cell. 1981 Aug;25(2):373–384. doi: 10.1016/0092-8674(81)90056-8. [DOI] [PubMed] [Google Scholar]
  19. Myers R. M., Tjian R. Construction and analysis of simian virus 40 origins defective in tumor antigen binding and DNA replication. Proc Natl Acad Sci U S A. 1980 Nov;77(11):6491–6495. doi: 10.1073/pnas.77.11.6491. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Paucha E., Kalderon D., Harvey R. W., Smith A. E. Simian virus 40 origin DNA-binding domain on large T antigen. J Virol. 1986 Jan;57(1):50–64. doi: 10.1128/jvi.57.1.50-64.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Potter C. W., McLaughlin B. C., Oxford J. S. Simian virus 40-induced T and tumor antigens. J Virol. 1969 Nov;4(5):574–579. doi: 10.1128/jvi.4.5.574-579.1969. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Prives C., Beck Y., Gidoni D., Oren M., Shure H. DNA binding and sedimentation properties of SV40 T antigens synthesized in vivo and in vitro. Cold Spring Harb Symp Quant Biol. 1980;44(Pt 1):123–130. doi: 10.1101/sqb.1980.044.01.014. [DOI] [PubMed] [Google Scholar]
  23. Rio D. C., Tjian R. SV40 T antigen binding site mutations that affect autoregulation. Cell. 1983 Apr;32(4):1227–1240. doi: 10.1016/0092-8674(83)90305-7. [DOI] [PubMed] [Google Scholar]
  24. Rio D., Robbins A., Myers R., Tjian R. Regulation of simian virus 40 early transcription in vitro by a purified tumor antigen. Proc Natl Acad Sci U S A. 1980 Oct;77(10):5706–5710. doi: 10.1073/pnas.77.10.5706. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Scheidtmann K. H., Echle B., Walter G. Simian virus 40 large T antigen is phosphorylated at multiple sites clustered in two separate regions. J Virol. 1982 Oct;44(1):116–133. doi: 10.1128/jvi.44.1.116-133.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Scheidtmann K. H., Echle B., Walter G. Simian virus 40 large T antigen is phosphorylated at multiple sites clustered in two separate regions. J Virol. 1982 Oct;44(1):116–133. doi: 10.1128/jvi.44.1.116-133.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Scheidtmann K. H., Hardung M., Echle B., Walter G. DNA-binding activity of simian virus 40 large T antigen correlates with a distinct phosphorylation state. J Virol. 1984 Apr;50(1):1–12. doi: 10.1128/jvi.50.1.1-12.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Schwyzer M., Weil R., Frank G., Zuber H. Amino acid sequence analysis of fragments generated by partial proteolysis from large simian virus 40 tumor antigen. J Biol Chem. 1980 Jun 25;255(12):5627–5634. [PubMed] [Google Scholar]
  29. Shalloway D., Kleinberger T., Livingston D. M. Mapping of SV40 DNA replication origin region binding sites for the SV40 T antigen by protection against exonuclease III digestion. Cell. 1980 Jun;20(2):411–422. doi: 10.1016/0092-8674(80)90627-3. [DOI] [PubMed] [Google Scholar]
  30. Shortle D. R., Margolskee R. F., Nathans D. Mutational analysis of the simian virus 40 replicon: pseudorevertants of mutants with a defective replication origin. Proc Natl Acad Sci U S A. 1979 Dec;76(12):6128–6131. doi: 10.1073/pnas.76.12.6128. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Shortle D., Nathans D. Mutants of simian virus 40 with base substitutions at the origin of DNA replication. Cold Spring Harb Symp Quant Biol. 1979;43(Pt 2):663–668. doi: 10.1101/sqb.1979.043.01.074. [DOI] [PubMed] [Google Scholar]
  32. Shortle D., Nathans D. Regulatory mutants of simian virus 40: constructed mutants with base substitutions at the origin of DNA replication. J Mol Biol. 1979 Jul 15;131(4):801–817. doi: 10.1016/0022-2836(79)90202-x. [DOI] [PubMed] [Google Scholar]
  33. Simanis V., Lane D. P. An immunoaffinity purification procedure for SV40 large T antigen. Virology. 1985 Jul 15;144(1):88–100. doi: 10.1016/0042-6822(85)90308-3. [DOI] [PubMed] [Google Scholar]
  34. Simmons D. T. DNA-binding region of the simian virus 40 tumor antigen. J Virol. 1986 Mar;57(3):776–785. doi: 10.1128/jvi.57.3.776-785.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Simmons D. T., Martin M. A., Mora P. T., Chang C. Relationship among Tau antigens isolated from various lines of simian virus 40-transformed cells. J Virol. 1980 Jun;34(3):650–657. doi: 10.1128/jvi.34.3.650-657.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Simmons D. T. Stepwise phosphorylation of the NH2-terminal region of the simian virus 40 large T antigen. J Biol Chem. 1984 Jul 10;259(13):8633–8640. [PubMed] [Google Scholar]
  37. Tegtmeyer P., Rundell K., Collins J. K. Modification of simian virus 40 protein A. J Virol. 1977 Feb;21(2):647–657. doi: 10.1128/jvi.21.2.647-657.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Tegtmeyer P. Simian virus 40 deoxyribonucleic acid synthesis: the viral replicon. J Virol. 1972 Oct;10(4):591–598. doi: 10.1128/jvi.10.4.591-598.1972. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Tjian R. Protein-DNA interactions at the origin of simian virus 40 DNA replication. Cold Spring Harb Symp Quant Biol. 1979;43(Pt 2):655–661. doi: 10.1101/sqb.1979.043.01.073. [DOI] [PubMed] [Google Scholar]
  40. Tjian R. The binding site on SV40 DNA for a T antigen-related protein. Cell. 1978 Jan;13(1):165–179. doi: 10.1016/0092-8674(78)90147-2. [DOI] [PubMed] [Google Scholar]
  41. Van Roy F., Fransen L., Fiers W. Phosphorylation patterns of tumour antigens in cells lytically infected or transformed by simian virus 40. J Virol. 1981 Oct;40(1):28–44. doi: 10.1128/jvi.40.1.28-44.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES