Skip to main content
NCBI home page
Journal of Virology logoLink to Journal of Virology
. 1987 May;61(5):1593–1601. doi: 10.1128/jvi.61.5.1593-1601.1987

Forms of pp60v-src isolated from Rous sarcoma virus-transformed cells.

M S Collett, S K Belzer
PMCID: PMC254140  PMID: 2437320

Abstract

It has previously been shown that an electrophoretic variant form of the Rous sarcoma virus transforming protein, pp60v-src, exists in src-transformed cells. This variant, which was readily observed in vanadate-treated cells, was characterized as possessing extensive amino-terminal domain phosphotyrosine modification. Its appearance was further correlated with increased src-specific protein kinase activity. In this study, we used a src-specific monoclonal antibody (MAb) to resolve immunologic forms of pp60v-src. The MAb was able to distinguish between two populations of typical lower-band pp60v-src and was unreactive with the electrophoretic variant upper-band pp60v-src species. Using serial immunoprecipitations, we resolved four populations of pp60v-src: src protein either immunoreactive or unreactive with the MAb from both untreated and vanadate-treated transformed cells. The pp60v-src in each fraction displayed a distinct phosphoamino acid composition and tryptic phosphopeptide profile. However, analysis of their tyrosyl kinase specific activities showed that the immunologically resolved populations of pp60v-src from a given culture did not differ. Both pp60v-src fractions from vanadate-treated cells exhibited similar kinase specific activities, which were greatly enhanced over those of enzyme preparations from untreated cells. Since the MAb-reactive pp60v-src fraction from vanadate-treated cells lacked the electrophoretic variant upper-band pp60v-src species yet still possessed enhanced enzymatic specific activity, the initially stated correlation between the appearance of the electrophoretic variant src form and increased src kinase activity breaks down. These results suggest that yet to be defined modifications of the src protein may be involved in its functional regulation.

Full text

PDF
1593

Images in this article

1595Image
on p.1595
1596Image
on p.1596
1597Image
on p.1597
1598Image
on p.1598

Selected References

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

  1. 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]
  2. Bolen J. B., Thiele C. J., Israel M. A., Yonemoto W., Lipsich L. A., Brugge J. S. Enhancement of cellular src gene product associated tyrosyl kinase activity following polyoma virus infection and transformation. Cell. 1984 Oct;38(3):767–777. doi: 10.1016/0092-8674(84)90272-1. [DOI] [PubMed] [Google Scholar]
  3. Brown D. J., Gordon J. A. The stimulation of pp60v-src kinase activity by vanadate in intact cells accompanies a new phosphorylation state of the enzyme. J Biol Chem. 1984 Aug 10;259(15):9580–9586. [PubMed] [Google Scholar]
  4. Brugge J. S., Collett M. S., Siddiqui A., Marczynska B., Deinhardt F., Erikson R. L. Detection of the viral sarcoma gene product in cells infected with various strains of avian sarcoma virus and of a related protein in uninfected chicken cells. J Virol. 1979 Mar;29(3):1196–1203. doi: 10.1128/jvi.29.3.1196-1203.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Brugge J. S., Cotton P. C., Queral A. E., Barrett J. N., Nonner D., Keane R. W. Neurones express high levels of a structurally modified, activated form of pp60c-src. Nature. 1985 Aug 8;316(6028):554–557. doi: 10.1038/316554a0. [DOI] [PubMed] [Google Scholar]
  6. 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]
  7. Collett M. S., Belzer S. K., Kamp L. E. Enzymatic characteristics of pp60v-src isolated from vanadium-treated transformed cells. J Cell Biochem. 1984;26(2):95–106. doi: 10.1002/jcb.240260205. [DOI] [PubMed] [Google Scholar]
  8. Collett M. S., Belzer S. K., Purchio A. F. Structurally and functionally modified forms of pp60v-src in Rous sarcoma virus-transformed cell lysates. Mol Cell Biol. 1984 Jul;4(7):1213–1220. doi: 10.1128/mcb.4.7.1213. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Collett M. S., Brugge J. S., Erikson R. L. Characterization of a normal avian cell protein related to the avian sarcoma virus transforming gene product. Cell. 1978 Dec;15(4):1363–1369. doi: 10.1016/0092-8674(78)90061-2. [DOI] [PubMed] [Google Scholar]
  10. 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]
  11. 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]
  12. Collett M. S., Wells S. K., Purchio A. F. Physical modification of purified Rous sarcoma virus pp60v-src protein after incubation with ATP/Mg2+. Virology. 1983 Jul 30;128(2):285–297. doi: 10.1016/0042-6822(83)90256-8. [DOI] [PubMed] [Google Scholar]
  13. Cross F. R., Hanafusa H. Local mutagenesis of Rous sarcoma virus: the major sites of tyrosine and serine phosphorylation of pp60src are dispensable for transformation. Cell. 1983 Sep;34(2):597–607. doi: 10.1016/0092-8674(83)90392-6. [DOI] [PubMed] [Google Scholar]
  14. 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]
  15. 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]
  16. Feramisco J. R., Burridge K. A rapid purification of alpha-actinin, filamin, and a 130,000-dalton protein from smooth muscle. J Biol Chem. 1980 Feb 10;255(3):1194–1199. [PubMed] [Google Scholar]
  17. Gentry L. E., Rohrschneider L. R., Casnellie J. E., Krebs E. G. Antibodies to a defined region of pp60src neutralize the tyrosine-specific kinase activity. J Biol Chem. 1983 Sep 25;258(18):11219–11228. [PubMed] [Google Scholar]
  18. Gilmer T. M., Erikson R. L. Development of anti-pp60src serum with antigen produced in Escherichia coli. J Virol. 1983 Jan;45(1):462–465. doi: 10.1128/jvi.45.1.462-465.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Highfield P. E., Rafield L. F., Gilmer T. M., Parsons J. T. Molecular cloning of avian sarcoma virus closed circular DNA: structural and biological characterization of three recombinant clones. J Virol. 1980 Oct;36(1):271–279. doi: 10.1128/jvi.36.1.271-279.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Iba H., Cross F. R., Garber E. A., Hanafusa H. Low level of cellular protein phosphorylation by nontransforming overproduced p60c-src. Mol Cell Biol. 1985 May;5(5):1058–1066. doi: 10.1128/mcb.5.5.1058. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Ito S., Richert N., Pastan I. Phospholipids stimulate phosphorylation of vinculin by the tyrosine-specific protein kinase of Rous sarcoma virus. Proc Natl Acad Sci U S A. 1982 Aug;79(15):4628–4631. doi: 10.1073/pnas.79.15.4628. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Keegan K., Collett M. S. Use of bacterial expression cloning to define the amino acid sequences of antigenic determinants on the G2 glycoprotein of Rift Valley fever virus. J Virol. 1986 May;58(2):263–270. doi: 10.1128/jvi.58.2.263-270.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Lipsich L. A., Lewis A. J., Brugge J. S. Isolation of monoclonal antibodies that recognize the transforming proteins of avian sarcoma viruses. J Virol. 1983 Nov;48(2):352–360. doi: 10.1128/jvi.48.2.352-360.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Parker R. C., Varmus H. E., Bishop J. M. Expression of v-src and chicken c-src in rat cells demonstrates qualitative differences between pp60v-src and pp60c-src. Cell. 1984 May;37(1):131–139. doi: 10.1016/0092-8674(84)90308-8. [DOI] [PubMed] [Google Scholar]
  25. Parsons S. J., McCarley D. J., Ely C. M., Benjamin D. C., Parsons J. T. Isolation and partial characterization of a monoclonal antibody to the Rous sarcoma virus transforming protein pp60src. J Virol. 1983 Mar;45(3):1190–1194. doi: 10.1128/jvi.45.3.1190-1194.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Parsons S. J., McCarley D. J., Ely C. M., Benjamin D. C., Parsons J. T. Monoclonal antibodies to Rous sarcoma virus pp60src react with enzymatically active cellular pp60src of avian and mammalian origin. J Virol. 1984 Aug;51(2):272–282. doi: 10.1128/jvi.51.2.272-282.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Parsons S. J., McCarley D. J., Raymond V. W., Parsons J. T. Localization of conserved and nonconserved epitopes within the Rous sarcoma virus-encoded src protein. J Virol. 1986 Sep;59(3):755–758. doi: 10.1128/jvi.59.3.755-758.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Patschinsky T., Hunter T., Esch F. S., Cooper J. A., Sefton B. M. Analysis of the sequence of amino acids surrounding sites of tyrosine phosphorylation. Proc Natl Acad Sci U S A. 1982 Feb;79(4):973–977. doi: 10.1073/pnas.79.4.973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Patschinsky T., Hunter T., Sefton B. M. Phosphorylation of the transforming protein of Rous sarcoma virus: direct demonstration of phosphorylation of serine 17 and identification of an additional site of tyrosine phosphorylation in p60v-src of Prague Rous sarcoma virus. J Virol. 1986 Jul;59(1):73–81. doi: 10.1128/jvi.59.1.73-81.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. 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]
  31. Purchio A. F., Wells S. K., Collett M. S. Increase in the phosphotransferase specific activity of purified Rous sarcoma virus pp60v-src protein after incubation with ATP plus Mg2+. Mol Cell Biol. 1983 Sep;3(9):1589–1597. doi: 10.1128/mcb.3.9.1589. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Ralston R., Bishop J. M. The product of the protooncogene c-src is modified during the cellular response to platelet-derived growth factor. Proc Natl Acad Sci U S A. 1985 Dec;82(23):7845–7849. doi: 10.1073/pnas.82.23.7845. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Resh M. D., Erikson R. L. Highly specific antibody to Rous sarcoma virus src gene product recognizes a novel population of pp60v-src and pp60c-src molecules. J Cell Biol. 1985 Feb;100(2):409–417. doi: 10.1083/jcb.100.2.409. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Russell D. R., Bennett G. N. Construction and analysis of in vivo activity of E. coli promoter hybrids and promoter mutants that alter the -35 to -10 spacing. Gene. 1982 Dec;20(2):231–243. doi: 10.1016/0378-1119(82)90042-7. [DOI] [PubMed] [Google Scholar]
  35. Schneider C., Newman R. A., Sutherland D. R., Asser U., Greaves M. F. A one-step purification of membrane proteins using a high efficiency immunomatrix. J Biol Chem. 1982 Sep 25;257(18):10766–10769. [PubMed] [Google Scholar]
  36. Sefton B. M., Hunter T. Tyrosine protein kinases. Adv Cyclic Nucleotide Protein Phosphorylation Res. 1984;18:195–226. [PubMed] [Google Scholar]
  37. Sefton B. M., Walter G. Antiserum specific for the carboxy terminus of the transforming protein of Rous sarcoma virus. J Virol. 1982 Nov;44(2):467–474. doi: 10.1128/jvi.44.2.467-474.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Shalloway D., Coussens P. M., Yaciuk P. Overexpression of the c-src protein does not induce transformation of NIH 3T3 cells. Proc Natl Acad Sci U S A. 1984 Nov;81(22):7071–7075. doi: 10.1073/pnas.81.22.7071. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Shealy D. J., Erikson R. L. Human cells contain two forms of pp60c-src. Nature. 1981 Oct 22;293(5834):666–669. doi: 10.1038/293666a0. [DOI] [PubMed] [Google Scholar]
  40. 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]
  41. Snyder M. A., Bishop J. M. A mutation at the major phosphotyrosine in pp60v-src alters oncogenic potential. Virology. 1984 Jul 30;136(2):375–386. doi: 10.1016/0042-6822(84)90174-0. [DOI] [PubMed] [Google Scholar]
  42. 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]
  43. Tamura T., Bauer H., Birr C., Pipkorn R. Antibodies against synthetic peptides as a tool for functional analysis of the transforming protein pp60src. Cell. 1983 Sep;34(2):587–596. doi: 10.1016/0092-8674(83)90391-4. [DOI] [PubMed] [Google Scholar]
  44. Watson R. J., Weis J. H., Salstrom J. S., Enquist L. W. Bacterial synthesis of herpes simplex virus types 1 and 2 glycoprotein D antigens. J Invest Dermatol. 1984 Jul;83(1 Suppl):102s–111s. doi: 10.1111/1523-1747.ep12281828. [DOI] [PubMed] [Google Scholar]
  45. Weis J. H., Enquist L. W., Salstrom J. S., Watson R. J. An immunologically active chimaeric protein containing herpes simplex virus type 1 glycoprotein D. Nature. 1983 Mar 3;302(5903):72–74. doi: 10.1038/302072a0. [DOI] [PubMed] [Google Scholar]
  46. Wells S. K., Collett M. S. Specific proteolytic fragmentation of p60v-src in transformed cell lysates. J Virol. 1983 Jul;47(1):253–258. doi: 10.1128/jvi.47.1.253-258.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Wong T. W., Goldberg A. R. Synthetic peptide fragment of src gene product inhibits the src protein kinase and crossreacts immunologically with avian onc kinases and cellular phosphoproteins. Proc Natl Acad Sci U S A. 1981 Dec;78(12):7412–7416. doi: 10.1073/pnas.78.12.7412. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Yonemoto W., Jarvis-Morar M., Brugge J. S., Bolen J. B., Israel M. A. Tyrosine phosphorylation within the amino-terminal domain of pp60c-src molecules associated with polyoma virus middle-sized tumor antigen. Proc Natl Acad Sci U S A. 1985 Jul;82(14):4568–4572. doi: 10.1073/pnas.82.14.4568. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES