Skip to main content
PMC 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
. 1984 Nov;81(21):6574–6578. doi: 10.1073/pnas.81.21.6574

Specific in vitro adenylylation of the simian virus 40 large tumor antigen.

M K Bradley, J Hudson, M S Villanueva, D M Livingston
PMCID: PMC391972  PMID: 6093107

Abstract

Incubation of the simian virus 40 (SV40) large tumor antigen (T) from either transformed or lytically infected cells with adenosine [8-3H]-, [alpha-32P]-, or [alpha-[35S]thio]-triphosphate in the presence of Mg2+ resulted in its labeling as defined by the appearance of an intact, appropriately immunoreactive band in NaDodSO4/polyacrylamide gels. Radioactivity remained associated with the protein after boiling in buffer containing 3% NaDodSO4, and 2-mercaptoethanol as well as after heating in 0.1 M HCl, 0.1 M NH4OH, or hydroxylamine, but it was dissociated after incubation in 0.1 M NaOH at 37 degrees C. After limited boiling of gel-purified [alpha-32P] ATP + T complex in 5.6 M HCl, o-[32P]phosphoserine was released, and snake venom phosphodiesterase or 0.5 M piperidine treatment of such a complex resulted in the liberation of [alpha-32P]AMP. The reaction proceeded when either purified, soluble T or insoluble, specifically immunoprecipitated antigen was used as substrate. ATP and dATP were the preferred nucleotide substrates by comparison with the other six standard ribonucleoside or deoxynucleoside triphosphates. Partial tryptic digests of T + [alpha-32P]ATP complexes revealed the presence of a single labeled peptide of Mr approximately equal to 12 - 14 X 10(3), and after exhaustive digestion, there was a single radioactive spot in the fingerprint. These data indicate that T can be adenylylated at a specific seryl residue(s) in a limited portion of the protein surface. Furthermore, adenylylation appears to be reversible and to proceed by a pyrophosphorylytic mechanism, since the nucleotide was released from the protein following incubation of adenylylated T with Mg2+, sodium pyrophosphate, and poly(dT).

Full text

PDF
6574

Images in this article

6575Image
on p.6575
6575Image
on p.6575
6575Image
on p.6575
6576Image
on p.6576
6576Image
on p.6576
6576Image
on p.6576
6577Image
on p.6577
6577Image
on p.6577
6577Image
on p.6577
6577Image
on p.6577

Selected References

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

  1. Bergsma D. J., Olive D. M., Hartzell S. W., Subramanian K. N. Territorial limits and functional anatomy of the simian virus 40 replication origin. Proc Natl Acad Sci U S A. 1982 Jan;79(2):381–385. doi: 10.1073/pnas.79.2.381. [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. Challberg M. D., Desiderio S. V., Kelly T. J., Jr Adenovirus DNA replication in vitro: characterization of a protein covalently linked to nascent DNA strands. Proc Natl Acad Sci U S A. 1980 Sep;77(9):5105–5109. doi: 10.1073/pnas.77.9.5105. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Challberg M. D., Kelly T. J., Jr Adenovirus DNA replication in vitro: origin and direction of daughter strand synthesis. J Mol Biol. 1979 Dec 25;135(4):999–1012. doi: 10.1016/0022-2836(79)90524-2. [DOI] [PubMed] [Google Scholar]
  6. Champoux J. J. DNA is linked to the rat liver DNA nicking-closing enzyme by a phosphodiester bond to tyrosine. J Biol Chem. 1981 May 25;256(10):4805–4809. [PubMed] [Google Scholar]
  7. 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]
  8. Clertant P., Cuzin F. Covalent affinity labeling by periodate-oxidized [alpha-32P]ATP of the large-T proteins of polyoma and SV40 viruses. J Biol Chem. 1982 Jun 10;257(11):6300–6305. [PubMed] [Google Scholar]
  9. Desiderio S. V., Kelly T. J., Jr Structure of the linkage between adenovirus DNA and the 55,000 molecular weight terminal protein. J Mol Biol. 1981 Jan 15;145(2):319–337. doi: 10.1016/0022-2836(81)90208-4. [DOI] [PubMed] [Google Scholar]
  10. Desiderio S. V., Kelly T. J., Jr Structure of the linkage between adenovirus DNA and the 55,000 molecular weight terminal protein. J Mol Biol. 1981 Jan 15;145(2):319–337. doi: 10.1016/0022-2836(81)90208-4. [DOI] [PubMed] [Google Scholar]
  11. DiMaio D., Nathans D. Regulatory mutants of simian virus 40. Effect of mutations at a T antigen binding site on DNA replication and expression of viral genes. J Mol Biol. 1982 Apr 15;156(3):531–548. doi: 10.1016/0022-2836(82)90265-0. [DOI] [PubMed] [Google Scholar]
  12. Easterbrook-Smith S. B., Wallace J. C., Keech D. B. Pyruvate carboxylase: affinity labelling of the magnesium adenosine triphosphate binding site. Eur J Biochem. 1976 Feb 2;62(1):125–130. doi: 10.1111/j.1432-1033.1976.tb10105.x. [DOI] [PubMed] [Google Scholar]
  13. Eckhart W., Hutchinson M. A., Hunter T. An activity phosphorylating tyrosine in polyoma T antigen immunoprecipitates. Cell. 1979 Dec;18(4):925–933. doi: 10.1016/0092-8674(79)90205-8. [DOI] [PubMed] [Google Scholar]
  14. Fey G., Lewis J. B., Grodzicker T., Bothwell A. Characterization of a fused protein specified by the adenovirus type 2-simian virus 40 hybrid Ad2+ND1 dp2. J Virol. 1979 Apr;30(1):201–217. doi: 10.1128/jvi.30.1.201-217.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. García J. A., Peñalva M. A., Blanco L., Salas M. Template requirements for initiation of phage phi 29 DNA replication in vitro. Proc Natl Acad Sci U S A. 1984 Jan;81(1):80–84. doi: 10.1073/pnas.81.1.80. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. 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]
  17. Girshovich A. S., Bochkareva E. S., Kurtskhalia T. V., Pozdnyakov V. A., Ovchinnikov Y. A. Binding of GTP to elongation factor G by photoaffinity labeling. Methods Enzymol. 1979;60:726–745. doi: 10.1016/s0076-6879(79)60068-x. [DOI] [PubMed] [Google Scholar]
  18. Gluzman Y. SV40-transformed simian cells support the replication of early SV40 mutants. Cell. 1981 Jan;23(1):175–182. doi: 10.1016/0092-8674(81)90282-8. [DOI] [PubMed] [Google Scholar]
  19. 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]
  20. Griffin J. D., Spangler G., Livingston D. M. Enzymatic activities associated with the SV40 large T antigen. Cold Spring Harb Symp Quant Biol. 1980;44(Pt 1):113–122. doi: 10.1101/sqb.1980.044.01.013. [DOI] [PubMed] [Google Scholar]
  21. Griffin J. D., Spangler G., Livingston D. M. Protein kinase activity associated with simian virus 40 T antigen. Proc Natl Acad Sci U S A. 1979 Jun;76(6):2610–2614. doi: 10.1073/pnas.76.6.2610. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Gumport R. I., Lehman I. R. Structure of the DNA ligase-adenylate intermediate: lysine (epsilon-amino)-linked adenosine monophosphoramidate. Proc Natl Acad Sci U S A. 1971 Oct;68(10):2559–2563. doi: 10.1073/pnas.68.10.2559. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. 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]
  24. Hay R. T., DePamphilis M. L. Initiation of SV40 DNA replication in vivo: location and structure of 5' ends of DNA synthesized in the ori region. Cell. 1982 Apr;28(4):767–779. doi: 10.1016/0092-8674(82)90056-3. [DOI] [PubMed] [Google Scholar]
  25. Hermoso J. M., Salas M. Protein p3 is linked to the DNA of phage phi 29 through a phosphoester bond between serine and 5'-dAMP. Proc Natl Acad Sci U S A. 1980 Nov;77(11):6425–6428. doi: 10.1073/pnas.77.11.6425. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Ikeda J. E., Enomoto T., Hurwitz J. Replication of adenovirus DNA-protein complex with purified proteins. Proc Natl Acad Sci U S A. 1981 Feb;78(2):884–888. doi: 10.1073/pnas.78.2.884. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. 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]
  28. Khandjian E. W., Loche M., Darlix J. L., Cramer R., Türler H., Weil R. Simian virus 40 large tumor antigen: a "RNA binding protein"? Proc Natl Acad Sci U S A. 1982 Feb;79(4):1139–1143. doi: 10.1073/pnas.79.4.1139. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Kilton L. J., Bradley M., Mehta C., Livingston D. M. Rapid and sensitive quantitative immunoassay for the large simian virus 40 T antigen. J Virol. 1981 May;38(2):612–620. doi: 10.1128/jvi.38.2.612-620.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Malcolm A. D., Moffatt J. R. Intrasubunit nucleotide binding in ribonucleic acid polymerase. Biochem J. 1978 Oct 1;175(1):189–192. doi: 10.1042/bj1750189. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Monroy G., Spencer E., Hurwitz J. Characteristics of reactions catalyzed by purified guanylyltransferase from vaccinia virus. J Biol Chem. 1978 Jun 25;253(12):4490–4498. [PubMed] [Google Scholar]
  32. Peñalva M. A., Salas M. Initiation of phage phi 29 DNA replication in vitro: formation of a covalent complex between the terminal protein, p3, and 5'-dAMP. Proc Natl Acad Sci U S A. 1982 Sep;79(18):5522–5526. doi: 10.1073/pnas.79.18.5522. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Rothberg P. G., Harris T. J., Nomoto A., Wimmer E. O4-(5'-uridylyl)tyrosine is the bond between the genome-linked protein and the RNA of poliovirus. Proc Natl Acad Sci U S A. 1978 Oct;75(10):4868–4872. doi: 10.1073/pnas.75.10.4868. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. 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]
  35. 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]
  36. 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]
  37. Smith A. E., Smith R., Paucha E. Extraction and fingerprint analysis of simian virus 40 large and small T-antigens. J Virol. 1978 Oct;28(1):140–153. doi: 10.1128/jvi.28.1.140-153.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Spillman T., Giacherio D., Hager L. P. Single strand DNA binding of simian virus 40 tumor antigen. J Biol Chem. 1979 Apr 25;254(8):3100–3104. [PubMed] [Google Scholar]
  39. Stillman B. W., Lewis J. B., Chow L. T., Mathews M. B., Smart J. E. Identification of the gene and mRNA for the adenovirus terminal protein precursor. Cell. 1981 Feb;23(2):497–508. doi: 10.1016/0092-8674(81)90145-8. [DOI] [PubMed] [Google Scholar]
  40. Tenen D. G., Taylor T. S., Haines L. L., Bradley M. K., Martin R. G., Livingston D. M. Binding of simian virus 40 large T antigen from virus-infected monkey cells to wild-type and mutant viral replication origins. J Mol Biol. 1983 Aug 25;168(4):791–808. doi: 10.1016/s0022-2836(83)80075-8. [DOI] [PubMed] [Google Scholar]
  41. Thummel C., Tjian R., Hu S. L., Grodzicker T. Translational control of SV40 T antigen expressed from the adenovirus late promoter. Cell. 1983 Jun;33(2):455–464. doi: 10.1016/0092-8674(83)90427-0. [DOI] [PubMed] [Google Scholar]
  42. 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]
  43. Todaro G. J., Green H., Swift M. R. Susceptibility of human diploid fibroblast strains to transformation by SV40 virus. Science. 1966 Sep 9;153(3741):1252–1254. doi: 10.1126/science.153.3741.1252. [DOI] [PubMed] [Google Scholar]
  44. Van Roy F., Fransen L., Fiers W. Protein kinase activities in immune complexes of simian virus 40 large T-antigen and transformation-associated cellular p53 protein. Mol Cell Biol. 1984 Feb;4(2):232–239. doi: 10.1128/mcb.4.2.232. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Venkatesan S., Gershowitz A., Moss B. Purification and characterization of mRNA guanylyltransferase from HeLa cell nuclei. J Biol Chem. 1980 Apr 10;255(7):2829–2834. [PubMed] [Google Scholar]
  46. Wimmer E. Genome-linked proteins of viruses. Cell. 1982 Feb;28(2):199–201. doi: 10.1016/0092-8674(82)90335-x. [DOI] [PubMed] [Google Scholar]
  47. Zylicz M., LeBowitz J. H., McMacken R., Georgopoulos C. The dnaK protein of Escherichia coli possesses an ATPase and autophosphorylating activity and is essential in an in vitro DNA replication system. Proc Natl Acad Sci U S A. 1983 Nov;80(21):6431–6435. doi: 10.1073/pnas.80.21.6431. [DOI] [PMC free article] [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