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. 1983 Sep;3(9):1589–1597. doi: 10.1128/mcb.3.9.1589

Increase in the phosphotransferase specific activity of purified Rous sarcoma virus pp60v-src protein after incubation with ATP plus Mg2+.

A F Purchio, S K Wells, M S Collett
PMCID: PMC370012  PMID: 6314123

Abstract

pp60v-src, the product of the Rous sarcoma virus src gene, was partially purified by immunoaffinity chromatography from extracts of Rous sarcoma virus-transformed field vole cells. Incubation of this preparation with ATP plus Mg2+ and subsequent repurification by chromatography on hexylamine-agarose resulted in a net increase in the specific activity of the src protein kinase. This increase in phosphotransferase activity was detected by using a variety of substrates including casein, tubulin, and a 34,000-dalton protein presumed to be an in vivo target substrate of pp60v-src. In all cases, the phosphorylation was at tyrosine residues, and the kinase activity was inhibited by preincubation of the enzyme with immunoglobulin G prepared from tumor-bearing rabbit sera. The implications of these results for the regulation and control of pp60v-src-associated kinase activity are discussed.

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Selected References

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

  1. Brugge J. S., Darrow D. Rous sarcoma virus-induced phosphorylation of a 50,000-molecular weight cellular protein. Nature. 1982 Jan 21;295(5846):250–253. doi: 10.1038/295250a0. [DOI] [PubMed] [Google Scholar]
  2. Brugge J. S., Erikson R. L. Identification of a transformation-specific antigen induced by an avian sarcoma virus. Nature. 1977 Sep 22;269(5626):346–348. doi: 10.1038/269346a0. [DOI] [PubMed] [Google Scholar]
  3. Chamberlain J. P. Fluorographic detection of radioactivity in polyacrylamide gels with the water-soluble fluor, sodium salicylate. Anal Biochem. 1979 Sep 15;98(1):132–135. doi: 10.1016/0003-2697(79)90716-4. [DOI] [PubMed] [Google Scholar]
  4. Collett M. S., Erikson E., Erikson R. L. Structural analysis of the avian sarcoma virus transforming protein: sites of phosphorylation. J Virol. 1979 Feb;29(2):770–781. doi: 10.1128/jvi.29.2.770-781.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Collett M. S., Erikson R. L. Protein kinase activity associated with the avian sarcoma virus src gene product. Proc Natl Acad Sci U S A. 1978 Apr;75(4):2021–2024. doi: 10.1073/pnas.75.4.2021. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Collett M. S., Purchio A. F., Erikson R. L. Avian sarcoma virus-transforming protein, pp60src shows protein kinase activity specific for tyrosine. Nature. 1980 May 15;285(5761):167–169. doi: 10.1038/285167a0. [DOI] [PubMed] [Google Scholar]
  7. Erikson E., Erikson R. L. Identification of a cellular protein substrate phosphorylated by the avian sarcoma virus-transforming gene product. Cell. 1980 Oct;21(3):829–836. doi: 10.1016/0092-8674(80)90446-8. [DOI] [PubMed] [Google Scholar]
  8. Erikson R. I., Collett M. S., Erikson E., Purchio A. F., Brugge J. S. Protein phosphorylation mediated by partially purified avian sarcoma virus transforming-gene product. Cold Spring Harb Symp Quant Biol. 1980;44(Pt 2):907–917. doi: 10.1101/sqb.1980.044.01.098. [DOI] [PubMed] [Google Scholar]
  9. Erikson R. L., Collett M. S., Erikson E., Purchio A. F. Evidence that the avian sarcoma virus transforming gene product is a cyclic AMP-independent protein kinase. Proc Natl Acad Sci U S A. 1979 Dec;76(12):6260–6264. doi: 10.1073/pnas.76.12.6260. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Gilmer T. M., Erikson R. L. Rous sarcoma virus transforming protein, p60src, expressed in E. coli, functions as a protein kinase. Nature. 1981 Dec 24;294(5843):771–773. doi: 10.1038/294771a0. [DOI] [PubMed] [Google Scholar]
  11. Gilmer T. M., Parsons J. T., Erikson R. L. Construction of plasmids for expression of Rous sarcoma virus transforming protein, p60src, in Escherichia coli. Proc Natl Acad Sci U S A. 1982 Apr;79(7):2152–2156. doi: 10.1073/pnas.79.7.2152. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Gilmore T. D., Radke K., Martin G. S. Tyrosine phosphorylation of a 50K cellular polypeptide associated with the Rous sarcoma virus transforming protein pp60src. Mol Cell Biol. 1982 Feb;2(2):199–206. doi: 10.1128/mcb.2.2.199. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Hunter T., Sefton B. M. Transforming gene product of Rous sarcoma virus phosphorylates tyrosine. Proc Natl Acad Sci U S A. 1980 Mar;77(3):1311–1315. doi: 10.1073/pnas.77.3.1311. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  15. Levinson A. D., Oppermann H., Levintow L., Varmus H. E., Bishop J. M. Evidence that the transforming gene of avian sarcoma virus encodes a protein kinase associated with a phosphoprotein. Cell. 1978 Oct;15(2):561–572. doi: 10.1016/0092-8674(78)90024-7. [DOI] [PubMed] [Google Scholar]
  16. Levinson A. D., Oppermann H., Varmus H. E., Bishop J. M. The purified product of the transforming gene of avian sarcoma virus phosphorylates tyrosine. J Biol Chem. 1980 Dec 25;255(24):11973–11980. [PubMed] [Google Scholar]
  17. McGrath J. P., Levinson A. D. Bacterial expression of an enzymatically active protein encoded by RSV src gene. Nature. 1982 Feb 4;295(5848):423–425. doi: 10.1038/295423a0. [DOI] [PubMed] [Google Scholar]
  18. Oakley B. R., Kirsch D. R., Morris N. R. A simplified ultrasensitive silver stain for detecting proteins in polyacrylamide gels. Anal Biochem. 1980 Jul 1;105(2):361–363. doi: 10.1016/0003-2697(80)90470-4. [DOI] [PubMed] [Google Scholar]
  19. Oppermann H., Levinson W., Bishop J. M. A cellular protein that associates with the transforming protein of Rous sarcoma virus is also a heat-shock protein. Proc Natl Acad Sci U S A. 1981 Feb;78(2):1067–1071. doi: 10.1073/pnas.78.2.1067. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Purchio A. F., Erikson E., Brugge J. S., Erikson R. L. Identification of a polypeptide encoded by the avian sarcoma virus src gene. Proc Natl Acad Sci U S A. 1978 Mar;75(3):1567–1571. doi: 10.1073/pnas.75.3.1567. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Purchio A. F. Evidence the pp60src, the product of the Rous sarcoma virus src gene, undergoes autophosphorylation. J Virol. 1982 Jan;41(1):1–7. doi: 10.1128/jvi.41.1.1-7.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Radke K., Gilmore T., Martin G. S. Transformation by Rous sarcoma virus: a cellular substrate for transformation-specific protein phosphorylation contains phosphotyrosine. Cell. 1980 Oct;21(3):821–828. doi: 10.1016/0092-8674(80)90445-6. [DOI] [PubMed] [Google Scholar]
  23. Radke K., Martin G. S. Transformation by Rous sarcoma virus: effects of src gene expression on the synthesis and phosphorylation of cellular polypeptides. Proc Natl Acad Sci U S A. 1979 Oct;76(10):5212–5216. doi: 10.1073/pnas.76.10.5212. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Richert N. D., Blithe D. L., Pastan I. Properties of the src kinase purified from Rous sarcoma virus-induced rat tumors. J Biol Chem. 1982 Jun 25;257(12):7143–7150. [PubMed] [Google Scholar]
  25. Sefton B. M., Patschinsky T., Berdot C., Hunter T., Elliott T. Phosphorylation and metabolism of the transforming protein of Rous sarcoma virus. J Virol. 1982 Mar;41(3):813–820. doi: 10.1128/jvi.41.3.813-820.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Smart J. E., Oppermann H., Czernilofsky A. P., Purchio A. F., Erikson R. L., Bishop J. M. Characterization of sites for tyrosine phosphorylation in the transforming protein of Rous sarcoma virus (pp60v-src) and its normal cellular homologue (pp60c-src). Proc Natl Acad Sci U S A. 1981 Oct;78(10):6013–6017. doi: 10.1073/pnas.78.10.6013. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Snyder M. A., Bishop J. M., Colby W. W., Levinson A. D. Phosphorylation of tyrosine-416 is not required for the transforming properties and kinase activity of pp60v-src. Cell. 1983 Mar;32(3):891–901. doi: 10.1016/0092-8674(83)90074-0. [DOI] [PubMed] [Google Scholar]

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