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. 1996 Jun;70(6):3887–3893. doi: 10.1128/jvi.70.6.3887-3893.1996

Mechanisms of simian virus 40 T-antigen activation by phosphorylation of threonine 124.

D McVey 1, B Woelker 1, P Tegtmeyer 1
PMCID: PMC190266  PMID: 8648725

Abstract

Previous studies have shown that phosphorylation of simian virus 40 (SV40) T antigen at threonine 124 enhances the binding of T antigen to the SV40 core origin of replication and the unwinding of the core origin DNA via hexamer-hexamer interactions. Here, we report that threonine 124 phosphorylation enhances the interaction of T-antigen amino acids 1 to 259 and 89 to 259 with the core origin of replication. Phosphorylation, therefore, activates the minimal DNA binding domain of T antigen even in the absence of domains required for hexamer formation. Activation is mediated by only one of three DNA binding elements in the minimal DNA binding domain of T antigen. This element, including amino acids 167, 215, and 219, enhances binding to the unique arrangement of four pentanucleotides in the core origin but not to other pentanucleotide arrangements found in ancillary regions of the SV40 origin of replication. Interestingly, the same four pentanucleotides in the core origin are necessary and sufficient for phosphorylation-enhanced DNA binding. Further, we show that phosphorylation of threonine 124 promotes the assembly of high-order complexes of the minimal DNA binding domain of T antigen with core origin DNA. We propose that phosphorylation induces conformational shifts in the minimal DNA binding domain of T antigen and thereby enhances interactions among T-antigen subunits oriented by core origin pentanucleotides. Similar subunit interactions would enhance both assembly of full-length T antigen into binary hexamer complexes and origin unwinding.

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Selected References

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  1. Arthur A. K., Höss A., Fanning E. Expression of simian virus 40 T antigen in Escherichia coli: localization of T-antigen origin DNA-binding domain to within 129 amino acids. J Virol. 1988 Jun;62(6):1999–2006. doi: 10.1128/jvi.62.6.1999-2006.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Borowiec J. A., Hurwitz J. Localized melting and structural changes in the SV40 origin of replication induced by T-antigen. EMBO J. 1988 Oct;7(10):3149–3158. doi: 10.1002/j.1460-2075.1988.tb03182.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bradley M. K., Smith T. F., Lathrop R. H., Livingston D. M., Webster T. A. Consensus topography in the ATP binding site of the simian virus 40 and polyomavirus large tumor antigens. Proc Natl Acad Sci U S A. 1987 Jun;84(12):4026–4030. doi: 10.1073/pnas.84.12.4026. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Cegielska A., Moarefi I., Fanning E., Virshup D. M. T-antigen kinase inhibits simian virus 40 DNA replication by phosphorylation of intact T antigen on serines 120 and 123. J Virol. 1994 Jan;68(1):269–275. doi: 10.1128/jvi.68.1.269-275.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Cegielska A., Shaffer S., Derua R., Goris J., Virshup D. M. Different oligomeric forms of protein phosphatase 2A activate and inhibit simian virus 40 DNA replication. Mol Cell Biol. 1994 Jul;14(7):4616–4623. doi: 10.1128/mcb.14.7.4616. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. 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]
  7. DeLucia A. L., Deb S., Partin K., Tegtmeyer P. Functional interactions of the simian virus 40 core origin of replication with flanking regulatory sequences. J Virol. 1986 Jan;57(1):138–144. doi: 10.1128/jvi.57.1.138-144.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Dean F. B., Bullock P., Murakami Y., Wobbe C. R., Weissbach L., Hurwitz J. Simian virus 40 (SV40) DNA replication: SV40 large T antigen unwinds DNA containing the SV40 origin of replication. Proc Natl Acad Sci U S A. 1987 Jan;84(1):16–20. doi: 10.1073/pnas.84.1.16. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Dodson M., Dean F. B., Bullock P., Echols H., Hurwitz J. Unwinding of duplex DNA from the SV40 origin of replication by T antigen. Science. 1987 Nov 13;238(4829):964–967. doi: 10.1126/science.2823389. [DOI] [PubMed] [Google Scholar]
  10. Fanning E., Knippers R. Structure and function of simian virus 40 large tumor antigen. Annu Rev Biochem. 1992;61:55–85. doi: 10.1146/annurev.bi.61.070192.000415. [DOI] [PubMed] [Google Scholar]
  11. 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]
  12. 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]
  13. Höss A., Moarefi I. F., Fanning E., Arthur A. K. The finger domain of simian virus 40 large T antigen controls DNA-binding specificity. J Virol. 1990 Dec;64(12):6291–6296. doi: 10.1128/jvi.64.12.6291-6296.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Johnson L. N. Glycogen phosphorylase: control by phosphorylation and allosteric effectors. FASEB J. 1992 Mar;6(6):2274–2282. doi: 10.1096/fasebj.6.6.1544539. [DOI] [PubMed] [Google Scholar]
  15. Kunkel T. A. Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc Natl Acad Sci U S A. 1985 Jan;82(2):488–492. doi: 10.1073/pnas.82.2.488. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Loeber G., Parsons R., Tegtmeyer P. The zinc finger region of simian virus 40 large T antigen. J Virol. 1989 Jan;63(1):94–100. doi: 10.1128/jvi.63.1.94-100.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Loeber G., Stenger J. E., Ray S., Parsons R. E., Anderson M. E., Tegtmeyer P. The zinc finger region of simian virus 40 large T antigen is needed for hexamer assembly and origin melting. J Virol. 1991 Jun;65(6):3167–3174. doi: 10.1128/jvi.65.6.3167-3174.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Mastrangelo I. A., Hough P. V., Wall J. S., Dodson M., Dean F. B., Hurwitz J. ATP-dependent assembly of double hexamers of SV40 T antigen at the viral origin of DNA replication. Nature. 1989 Apr 20;338(6217):658–662. doi: 10.1038/338658a0. [DOI] [PubMed] [Google Scholar]
  19. Mastrangelo I. A., Hough P. V., Wilson V. G., Wall J. S., Hainfeld J. F., Tegtmeyer P. Monomers through trimers of large tumor antigen bind in region I and monomers through tetramers bind in region II of simian virus 40 origin of replication DNA as stable structures in solution. Proc Natl Acad Sci U S A. 1985 Jun;82(11):3626–3630. doi: 10.1073/pnas.82.11.3626. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. 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]
  21. McVey D., Ray S., Gluzman Y., Berger L., Wildeman A. G., Marshak D. R., Tegtmeyer P. cdc2 phosphorylation of threonine 124 activates the origin-unwinding functions of simian virus 40 T antigen. J Virol. 1993 Sep;67(9):5206–5215. doi: 10.1128/jvi.67.9.5206-5215.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. McVey D., Strauss M., Gluzman Y. Properties of the DNA-binding domain of the simian virus 40 large T antigen. Mol Cell Biol. 1989 Dec;9(12):5525–5536. doi: 10.1128/mcb.9.12.5525. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Moarefi I. F., Small D., Gilbert I., Höpfner M., Randall S. K., Schneider C., Russo A. A., Ramsperger U., Arthur A. K., Stahl H. Mutation of the cyclin-dependent kinase phosphorylation site in simian virus 40 (SV40) large T antigen specifically blocks SV40 origin DNA unwinding. J Virol. 1993 Aug;67(8):4992–5002. doi: 10.1128/jvi.67.8.4992-5002.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Mohr I. J., Gluzman Y., Fairman M. P., Strauss M., McVey D., Stillman B., Gerard R. D. Production of simian virus 40 large tumor antigen in bacteria: altered DNA-binding specificity and dna-replication activity of underphosphorylated large tumor antigen. Proc Natl Acad Sci U S A. 1989 Sep;86(17):6479–6483. doi: 10.1073/pnas.86.17.6479. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Mohr I. J., Stillman B., Gluzman Y. Regulation of SV40 DNA replication by phosphorylation of T antigen. EMBO J. 1987 Jan;6(1):153–160. doi: 10.1002/j.1460-2075.1987.tb04733.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Parsons R. E., Stenger J. E., Ray S., Welker R., Anderson M. E., Tegtmeyer P. Cooperative assembly of simian virus 40 T-antigen hexamers on functional halves of the replication origin. J Virol. 1991 Jun;65(6):2798–2806. doi: 10.1128/jvi.65.6.2798-2806.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Parsons R., Anderson M. E., Tegtmeyer P. Three domains in the simian virus 40 core origin orchestrate the binding, melting, and DNA helicase activities of T antigen. J Virol. 1990 Feb;64(2):509–518. doi: 10.1128/jvi.64.2.509-518.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Parsons R., Tegtmeyer P. Spacing is crucial for coordination of domain functions within the simian virus 40 core origin of replication. J Virol. 1992 Apr;66(4):1933–1942. doi: 10.1128/jvi.66.4.1933-1942.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. 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]
  30. 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]
  31. Reynisdóttir I., Lorimer H. E., Friedman P. N., Wang E. H., Prives C. Phosphorylation and active ATP hydrolysis are not required for SV40 T antigen hexamer formation. J Biol Chem. 1993 Nov 25;268(33):24647–24654. [PubMed] [Google Scholar]
  32. Ryder K., Vakalopoulou E., Mertz R., Mastrangelo I., Hough P., Tegtmeyer P., Fanning E. Seventeen base pairs of region I encode a novel tripartite binding signal for SV40 T antigen. Cell. 1985 Sep;42(2):539–548. doi: 10.1016/0092-8674(85)90111-4. [DOI] [PubMed] [Google Scholar]
  33. Scheidtmann K. H., Virshup D. M., Kelly T. J. Protein phosphatase 2A dephosphorylates simian virus 40 large T antigen specifically at residues involved in regulation of DNA-binding activity. J Virol. 1991 Apr;65(4):2098–2101. doi: 10.1128/jvi.65.4.2098-2101.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Schneider J., Fanning E. Mutations in the phosphorylation sites of simian virus 40 (SV40) T antigen alter its origin DNA-binding specificity for sites I or II and affect SV40 DNA replication activity. J Virol. 1988 May;62(5):1598–1605. doi: 10.1128/jvi.62.5.1598-1605.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. 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]
  36. 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]
  37. Simmons D. T., Loeber G., Tegtmeyer P. Four major sequence elements of simian virus 40 large T antigen coordinate its specific and nonspecific DNA binding. J Virol. 1990 May;64(5):1973–1983. doi: 10.1128/jvi.64.5.1973-1983.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Simmons D. T., Wun-Kim K., Young W. Identification of simian virus 40 T-antigen residues important for specific and nonspecific binding to DNA and for helicase activity. J Virol. 1990 Oct;64(10):4858–4865. doi: 10.1128/jvi.64.10.4858-4865.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Stillman B., Gerard R. D., Guggenheimer R. A., Gluzman Y. T antigen and template requirements for SV40 DNA replication in vitro. EMBO J. 1985 Nov;4(11):2933–2939. doi: 10.1002/j.1460-2075.1985.tb04026.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Strauss M., Argani P., Mohr I. J., Gluzman Y. Studies on the origin-specific DNA-binding domain of simian virus 40 large T antigen. J Virol. 1987 Oct;61(10):3326–3330. doi: 10.1128/jvi.61.10.3326-3330.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. 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]
  42. Virshup D. M., Russo A. A., Kelly T. J. Mechanism of activation of simian virus 40 DNA replication by protein phosphatase 2A. Mol Cell Biol. 1992 Nov;12(11):4883–4895. doi: 10.1128/mcb.12.11.4883. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Wold M. S., Li J. J., Kelly T. J. Initiation of simian virus 40 DNA replication in vitro: large-tumor-antigen- and origin-dependent unwinding of the template. Proc Natl Acad Sci U S A. 1987 Jun;84(11):3643–3647. doi: 10.1073/pnas.84.11.3643. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Wright P. J., DeLucia A. L., Tegtmeyer P. Sequence-specific binding of simian virus 40 A protein to nonorigin and cellular DNA. Mol Cell Biol. 1984 Dec;4(12):2631–2638. doi: 10.1128/mcb.4.12.2631. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Wun-Kim K., Simmons D. T. Mapping of helicase and helicase substrate-binding domains on simian virus 40 large T antigen. J Virol. 1990 May;64(5):2014–2020. doi: 10.1128/jvi.64.5.2014-2020.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Wun-Kim K., Upson R., Young W., Melendy T., Stillman B., Simmons D. T. The DNA-binding domain of simian virus 40 tumor antigen has multiple functions. J Virol. 1993 Dec;67(12):7608–7611. doi: 10.1128/jvi.67.12.7608-7611.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]

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