Skip to main content
NCBI home page
Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1986 Dec;6(12):4155–4160. doi: 10.1128/mcb.6.12.4155

Amino acid substitutions sufficient to convert the nontransforming p60c-src protein to a transforming protein.

J Y Kato, T Takeya, C Grandori, H Iba, J B Levy, H Hanafusa
PMCID: PMC367194  PMID: 2432397

Abstract

We have previously shown that Rous sarcoma virus variants that carry the cellular homolog (c-src) of the viral src gene (v-src) do not transform chicken embryo fibroblasts. We also have shown that replacement of sequences upstream or downstream from the BglI site of the cellular src gene with the corresponding regions of v-src restored transforming activity to the hybrid genes. Since there are only six amino acid changes between p60c-src and p60v-src within the sequences upstream from BglI, we constructed chimeric molecules involving v-src and c-src to determine the effect of each amino acid substitution on the biological activities of the gene product. We found that the change from Thr to Ile at position 338 or the replacement of a fragment of c-src containing Gly-63, Arg-95, and Thr-96 with a corresponding fragment of v-src containing Asp-63, Trp-95, and Ile-96 converted p60c-src into a transforming protein by the criteria of focus formation, anchorage-independent growth, and tumor formation in newborn chickens. These mutations also resulted in elevation of the protein kinase activity of p60c-src.

Full text

PDF
4155

Images in this article

4157Image
on p.4157
4158Image
on p.4158
4158Image
on p.4158

Selected References

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

  1. 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]
  2. Brugge J. S., Darrow D. Analysis of the catalytic domain of phosphotransferase activity of two avian sarcoma virus-transforming proteins. J Biol Chem. 1984 Apr 10;259(7):4550–4557. [PubMed] [Google Scholar]
  3. Bryant D., Parsons J. T. Site-directed mutagenesis of the src gene of Rous sarcoma virus: construction and characterization of a deletion mutant temperature sensitive for transformation. J Virol. 1982 Nov;44(2):683–691. doi: 10.1128/jvi.44.2.683-691.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Cooper J. A., Hunter T. Changes in protein phosphorylation in Rous sarcoma virus-transformed chicken embryo cells. Mol Cell Biol. 1981 Feb;1(2):165–178. doi: 10.1128/mcb.1.2.165. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Courtneidge S. A., Smith A. E. Polyoma virus transforming protein associates with the product of the c-src cellular gene. Nature. 1983 Jun 2;303(5916):435–439. doi: 10.1038/303435a0. [DOI] [PubMed] [Google Scholar]
  6. Coussens P. M., Cooper J. A., Hunter T., Shalloway D. Restriction of the in vitro and in vivo tyrosine protein kinase activities of pp60c-src relative to pp60v-src. Mol Cell Biol. 1985 Oct;5(10):2753–2763. doi: 10.1128/mcb.5.10.2753. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Cross F. R., Garber E. A., Hanafusa H. N-terminal deletions in Rous sarcoma virus p60src: effects on tyrosine kinase and biological activities and on recombination in tissue culture with the cellular src gene. Mol Cell Biol. 1985 Oct;5(10):2789–2795. doi: 10.1128/mcb.5.10.2789. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Cross F. R., Garber E. A., Pellman D., Hanafusa H. A short sequence in the p60src N terminus is required for p60src myristylation and membrane association and for cell transformation. Mol Cell Biol. 1984 Sep;4(9):1834–1842. doi: 10.1128/mcb.4.9.1834. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. 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]
  10. Czernilofsky A. P., Levinson A. D., Varmus H. E., Bishop J. M., Tischer E., Goodman H. Corrections to the nucleotide sequence of the src gene of Rous sarcoma virus. Nature. 1983 Feb 24;301(5902):736–738. doi: 10.1038/301736b0. [DOI] [PubMed] [Google Scholar]
  11. Davis R. L., Konopka J. B., Witte O. N. Activation of the c-abl oncogene by viral transduction or chromosomal translocation generates altered c-abl proteins with similar in vitro kinase properties. Mol Cell Biol. 1985 Jan;5(1):204–213. doi: 10.1128/mcb.5.1.204. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. 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]
  13. Foster D. A., Shibuya M., Hanafusa H. Activation of the transformation potential of the cellular fps gene. Cell. 1985 Aug;42(1):105–115. doi: 10.1016/s0092-8674(85)80106-9. [DOI] [PubMed] [Google Scholar]
  14. Hanafusa H. Rapid transformation of cells by Rous sarcoma virus. Proc Natl Acad Sci U S A. 1969 Jun;63(2):318–325. doi: 10.1073/pnas.63.2.318. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. 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]
  16. Iba H., Takeya T., Cross F. R., Hanafusa T., Hanafusa H. Rous sarcoma virus variants that carry the cellular src gene instead of the viral src gene cannot transform chicken embryo fibroblasts. Proc Natl Acad Sci U S A. 1984 Jul;81(14):4424–4428. doi: 10.1073/pnas.81.14.4424. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Konopka J. B., Watanabe S. M., Witte O. N. An alteration of the human c-abl protein in K562 leukemia cells unmasks associated tyrosine kinase activity. Cell. 1984 Jul;37(3):1035–1042. doi: 10.1016/0092-8674(84)90438-0. [DOI] [PubMed] [Google Scholar]
  18. Lerner T. L., Hanafusa H. DNA sequence of the Bryan high-titer strain of Rous sarcoma virus: extent of env deletion and possible genealogical relationship with other viral strains. J Virol. 1984 Feb;49(2):549–556. doi: 10.1128/jvi.49.2.549-556.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Levinson A. D., Courtneidge S. A., Bishop J. M. Structural and functional domains of the Rous sarcoma virus transforming protein (pp60src). Proc Natl Acad Sci U S A. 1981 Mar;78(3):1624–1628. doi: 10.1073/pnas.78.3.1624. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Levy J. B., Iba H., Hanafusa H. Activation of the transforming potential of p60c-src by a single amino acid change. Proc Natl Acad Sci U S A. 1986 Jun;83(12):4228–4232. doi: 10.1073/pnas.83.12.4228. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. 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]
  22. Neckameyer W. S., Wang L. H. Nucleotide sequence of avian sarcoma virus UR2 and comparison of its transforming gene with other members of the tyrosine protein kinase oncogene family. J Virol. 1985 Mar;53(3):879–884. doi: 10.1128/jvi.53.3.879-884.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. 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]
  24. Poirier F., Calothy G., Karess R. E., Erikson E., Hanafusa H. Role of p60src kinase activity in the induction of neuroretinal cell proliferation by rous sarcoma virus. J Virol. 1982 Jun;42(3):780–789. doi: 10.1128/jvi.42.3.780-789.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Shtivelman E., Lifshitz B., Gale R. P., Canaani E. Fused transcript of abl and bcr genes in chronic myelogenous leukaemia. Nature. 1985 Jun 13;315(6020):550–554. doi: 10.1038/315550a0. [DOI] [PubMed] [Google Scholar]
  26. Sudol M., Lerner T. L., Hanafusa H. Polymerase-defective mutant of the Bryan high-titer strain of Rous sarcoma virus. Nucleic Acids Res. 1986 Mar 11;14(5):2391–2405. doi: 10.1093/nar/14.5.2391. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Takeya T., Hanafusa H. DNA sequence of the viral and cellular src gene of chickens. II. Comparison of the src genes of two strains of avian sarcoma virus and of the cellular homolog. J Virol. 1982 Oct;44(1):12–18. doi: 10.1128/jvi.44.1.12-18.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Takeya T., Hanafusa H., Junghans R. P., Ju G., Skalka A. M. Comparison between the viral transforming gene (src) of recovered avian sarcoma virus and its cellular homolog. Mol Cell Biol. 1981 Nov;1(11):1024–1037. doi: 10.1128/mcb.1.11.1024. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Takeya T., Hanafusa H. Structure and sequence of the cellular gene homologous to the RSV src gene and the mechanism for generating the transforming virus. Cell. 1983 Mar;32(3):881–890. doi: 10.1016/0092-8674(83)90073-9. [DOI] [PubMed] [Google Scholar]
  30. Wigler M., Pellicer A., Silverstein S., Axel R., Urlaub G., Chasin L. DNA-mediated transfer of the adenine phosphoribosyltransferase locus into mammalian cells. Proc Natl Acad Sci U S A. 1979 Mar;76(3):1373–1376. doi: 10.1073/pnas.76.3.1373. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. 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 Molecular and Cellular Biology are provided here courtesy of Taylor & Francis

RESOURCES