Skip to main content
NCBI home page
The EMBO Journal logoLink to The EMBO Journal
. 1987 Jan;6(1):153–160. doi: 10.1002/j.1460-2075.1987.tb04733.x

Regulation of SV40 DNA replication by phosphorylation of T antigen.

I J Mohr, B Stillman, Y Gluzman
PMCID: PMC553371  PMID: 3034573

Abstract

The role of phosphorylation in regulating the biochemical properties of SV40 large T antigen has been examined. Treatment of purified T antigen with calf intestinal alkaline phosphatase resulted in the removal of 80% of the 32P label. This partially dephosphorylated T antigen displayed an increase in its ability to support DNA replication in vitro. This increase in replication activity was paralleled by an activation of specific DNA binding to site II, a necessary element within the origin of SV40 DNA replication. In contrast, the ATPase activity of dephosphorylated T antigen remained unchanged. These results demonstrate that DNA replication is regulated by phosphorylation of an origin specific DNA binding protein.

Full text

PDF
153

Images in this article

154Fig. 1.
on p.154
156Fig. 3.
on p.156

Selected References

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

  1. Adelstein R. S., Eisenberg E. Regulation and kinetics of the actin-myosin-ATP interaction. Annu Rev Biochem. 1980;49:921–956. doi: 10.1146/annurev.bi.49.070180.004421. [DOI] [PubMed] [Google Scholar]
  2. Ariga H., Sugano S. Initiation of simian virus 40 DNA replication in vitro. J Virol. 1983 Nov;48(2):481–491. doi: 10.1128/jvi.48.2.481-491.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. 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]
  4. Bishop J. M. Cellular oncogenes and retroviruses. Annu Rev Biochem. 1983;52:301–354. doi: 10.1146/annurev.bi.52.070183.001505. [DOI] [PubMed] [Google Scholar]
  5. Bowers G. N., Jr, McComb R. B. A continuous spectrophotometric method for measuring the activity of serum alkaline phosphatase. Clin Chem. 1966 Feb;12(2):70–89. [PubMed] [Google Scholar]
  6. 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]
  7. Bradley M. K., Hudson J., Villanueva M. S., Livingston D. M. Specific in vitro adenylylation of the simian virus 40 large tumor antigen. Proc Natl Acad Sci U S A. 1984 Nov;81(21):6574–6578. doi: 10.1073/pnas.81.21.6574. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. 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]
  9. Chou J. Y., Martin R. G. DNA infectivity and the induction of host DNA synthesis with temperature-sensitive mutants of simian virus 40. J Virol. 1975 Jan;15(1):145–150. doi: 10.1128/jvi.15.1.145-150.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Clark R., Lane D. P., Tjian R. Use of monoclonal antibodies as probes of simian virus 40 T antigen ATPase activity. J Biol Chem. 1981 Nov 25;256(22):11854–11858. [PubMed] [Google Scholar]
  11. Clertant P., Gaudray P., May E., Cuzin F. The nucleotide binding site detected by affinity labeling in the large T proteins of polyoma and SV40 viruses is distinct from their ATPase catalytic site. J Biol Chem. 1984 Dec 25;259(24):15196–15203. [PubMed] [Google Scholar]
  12. Cohen P. The role of protein phosphorylation in neural and hormonal control of cellular activity. Nature. 1982 Apr 15;296(5858):613–620. doi: 10.1038/296613a0. [DOI] [PubMed] [Google Scholar]
  13. DeLucia A. L., Lewton B. A., Tjian R., Tegtmeyer P. Topography of simian virus 40 A protein-DNA complexes: arrangement of pentanucleotide interaction sites at the origin of replication. J Virol. 1983 Apr;46(1):143–150. doi: 10.1128/jvi.46.1.143-150.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Duceman B. W., Rose K. M., Jacob S. T. Activation of purified hepatoma RNA polymerase I by homologous protein kinase NII. J Biol Chem. 1981 Nov 10;256(21):10755–10758. [PubMed] [Google Scholar]
  15. Durban E., Goodenough M., Mills J., Busch H. Topoisomerase I phosphorylation in vitro and in rapidly growing Novikoff hepatoma cells. EMBO J. 1985 Nov;4(11):2921–2926. doi: 10.1002/j.1460-2075.1985.tb04024.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Efstratiadis A., Vournakis J. N., Donis-Keller H., Chaconas G., Dougall D. K., Kafatos F. C. End labeling of enzymatically decapped mRNA. Nucleic Acids Res. 1977 Dec;4(12):4165–4174. doi: 10.1093/nar/4.12.4165. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. 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]
  18. Galanti N., Jonak G. J., Soprano K. J., Floros J., Kaczmarek L., Weissman S., Reddy V. B., Tilghman S. M., Baserga R. Characterization and biological activity of cloned simian virus 40 DNA fragments. J Biol Chem. 1981 Jun 25;256(12):6469–6474. [PubMed] [Google Scholar]
  19. Gerace L., Blobel G. The nuclear envelope lamina is reversibly depolymerized during mitosis. Cell. 1980 Jan;19(1):277–287. doi: 10.1016/0092-8674(80)90409-2. [DOI] [PubMed] [Google Scholar]
  20. Gerard R. D., Gluzman Y. New host cell system for regulated simian virus 40 DNA replication. Mol Cell Biol. 1985 Nov;5(11):3231–3240. doi: 10.1128/mcb.5.11.3231. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Giacherio D., Hager L. P. A poly(dT)-stimulated ATPase activity associated with simian virus 40 large T antigen. J Biol Chem. 1979 Sep 10;254(17):8113–8116. [PubMed] [Google Scholar]
  22. 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]
  23. Goldman N., Brown M., Khoury G. Modification of SV40 T antigen by poly ADP-ribosylation. Cell. 1981 May;24(2):567–572. doi: 10.1016/0092-8674(81)90347-0. [DOI] [PubMed] [Google Scholar]
  24. 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]
  25. Huganir R. L., Delcour A. H., Greengard P., Hess G. P. Phosphorylation of the nicotinic acetylcholine receptor regulates its rate of desensitization. Nature. 1986 Jun 19;321(6072):774–776. doi: 10.1038/321774a0. [DOI] [PubMed] [Google Scholar]
  26. Jarvis D. L., Butel J. S. Modification of simian virus 40 large tumor antigen by glycosylation. Virology. 1985 Mar;141(2):173–189. doi: 10.1016/0042-6822(85)90250-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Kalderon D., Smith A. E. In vitro mutagenesis of a putative DNA binding domain of SV40 large-T. Virology. 1984 Nov;139(1):109–137. doi: 10.1016/0042-6822(84)90334-9. [DOI] [PubMed] [Google Scholar]
  28. 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]
  29. Klockmann U., Deppert W. Acylated simian virus 40 large T-antigen: a new subclass associated with a detergent-resistant lamina of the plasma membrane. EMBO J. 1983;2(7):1151–1157. doi: 10.1002/j.1460-2075.1983.tb01560.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Lane D. P., Crawford L. V. T antigen is bound to a host protein in SV40-transformed cells. Nature. 1979 Mar 15;278(5701):261–263. doi: 10.1038/278261a0. [DOI] [PubMed] [Google Scholar]
  31. Lebkowski J. S., Clancy S., Calos M. P. Simian virus 40 replication in adenovirus-transformed human cells antagonizes gene expression. Nature. 1985 Sep 12;317(6033):169–171. doi: 10.1038/317169a0. [DOI] [PubMed] [Google Scholar]
  32. Lewis E. D., Manley J. L. Repression of simian virus 40 early transcription by viral DNA replication in human 293 cells. Nature. 1985 Sep 12;317(6033):172–175. doi: 10.1038/317172a0. [DOI] [PubMed] [Google Scholar]
  33. Li J. J., Kelly T. J. Simian virus 40 DNA replication in vitro. Proc Natl Acad Sci U S A. 1984 Nov;81(22):6973–6977. doi: 10.1073/pnas.81.22.6973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Li J. J., Kelly T. J. Simian virus 40 DNA replication in vitro: specificity of initiation and evidence for bidirectional replication. Mol Cell Biol. 1985 Jun;5(6):1238–1246. doi: 10.1128/mcb.5.6.1238. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Margolskee R. F., Nathans D. Simian virus 40 mutant T antigens with relaxed specificity for the nucleotide sequence at the viral DNA origin of replication. J Virol. 1984 Feb;49(2):386–393. doi: 10.1128/jvi.49.2.386-393.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. May P., May E., Bordé J. Stimulation of cellular RNA synthesis in mouse-kidney cell cultures infected with SV40 virus. Exp Cell Res. 1976 Jul;100(2):433–436. doi: 10.1016/0014-4827(76)90175-0. [DOI] [PubMed] [Google Scholar]
  37. 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]
  38. 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]
  39. Miake-Lye R., Kirschner M. W. Induction of early mitotic events in a cell-free system. Cell. 1985 May;41(1):165–175. doi: 10.1016/0092-8674(85)90071-6. [DOI] [PubMed] [Google Scholar]
  40. Myers R. M., Williams R. C., Tjian R. Oligomeric structure of a simian virus 40 T antigen in free form and bound to DNA. J Mol Biol. 1981 Jun 5;148(4):347–353. doi: 10.1016/0022-2836(81)90180-7. [DOI] [PubMed] [Google Scholar]
  41. Ottaviano Y., Gerace L. Phosphorylation of the nuclear lamins during interphase and mitosis. J Biol Chem. 1985 Jan 10;260(1):624–632. [PubMed] [Google Scholar]
  42. 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]
  43. Pöckl E., Wintersberger E. Increased rate of RNA synthesis: early reaction of primary mouse kidney cells to infection with polyoma virus of simian virus 40. J Virol. 1980 Jul;35(1):8–19. doi: 10.1128/jvi.35.1.8-19.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. 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]
  45. 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]
  46. 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]
  47. Scheidtmann K. H., Schickedanz J., Walter G., Lanford R. E., Butel J. S. Differential phosphorylation of cytoplasmic and nuclear variants of simian virus 40 large T antigen encoded by simian virus 40-adenovirus 7 hybrid viruses. J Virol. 1984 May;50(2):636–640. doi: 10.1128/jvi.50.2.636-640.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Shaw S. B., Tegtmeyer P. Binding of dephosphorylated A protein to SV40 DNA. Virology. 1981 Nov;115(1):88–96. doi: 10.1016/0042-6822(81)90091-x. [DOI] [PubMed] [Google Scholar]
  49. Shichi H., Somers R. L. Light-dependent phosphorylation of rhodopsin. Purification and properties of rhodopsin kinase. J Biol Chem. 1978 Oct 10;253(19):7040–7046. [PubMed] [Google Scholar]
  50. 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]
  51. 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]
  52. Simmons D. T., Chou W., Rodgers K. Phosphorylation downregulates the DNA-binding activity of simian virus 40 T antigen. J Virol. 1986 Dec;60(3):888–894. doi: 10.1128/jvi.60.3.888-894.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. 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]
  54. 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]
  55. Stahl H., Dröge P., Knippers R. DNA helicase activity of SV40 large tumor antigen. EMBO J. 1986 Aug;5(8):1939–1944. doi: 10.1002/j.1460-2075.1986.tb04447.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Stillman B. W., Gluzman Y. Replication and supercoiling of simian virus 40 DNA in cell extracts from human cells. Mol Cell Biol. 1985 Aug;5(8):2051–2060. doi: 10.1128/mcb.5.8.2051. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. 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]
  58. 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]
  59. Tegtmeyer P., Schwartz M., Collins J. K., Rundell K. Regulation of tumor antigen synthesis by simain virus 40 gene A. J Virol. 1975 Jul;16(1):168–178. doi: 10.1128/jvi.16.1.168-178.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  60. 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]
  61. Tjian R., Fey G., Graessmann A. Biological activity of purified simian virus 40 T antigen proteins. Proc Natl Acad Sci U S A. 1978 Mar;75(3):1279–1283. doi: 10.1073/pnas.75.3.1279. [DOI] [PMC free article] [PubMed] [Google Scholar]
  62. Tjian R., Robbins A. Enzymatic activities associated with a purified simian virus 40 T antigen-related protein. Proc Natl Acad Sci U S A. 1979 Feb;76(2):610–614. doi: 10.1073/pnas.76.2.610. [DOI] [PMC free article] [PubMed] [Google Scholar]
  63. 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]
  64. Van Heuverswyn H., Cole C., Berg P., Fiers W. Nucleotide sequence analysis of two simian virus 40 mutants with deletions in the region coding for the carboxyl terminus of the T antigen. J Virol. 1979 Jun;30(3):936–941. doi: 10.1128/jvi.30.3.936-941.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  65. Wobbe C. R., Dean F. B., Murakami Y., Weissbach L., Hurwitz J. Simian virus 40 DNA replication in vitro: study of events preceding elongation of chains. Proc Natl Acad Sci U S A. 1986 Jul;83(13):4612–4616. doi: 10.1073/pnas.83.13.4612. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES