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
. 1986 Jun;83(12):4228–4232. doi: 10.1073/pnas.83.12.4228

Activation of the transforming potential of p60c-src by a single amino acid change.

J B Levy, H Iba, H Hanafusa
PMCID: PMC323705  PMID: 2424022

Abstract

Previous work showed that overexpression of the cellular src (c-src) gene does not cause transformation of chicken cells in culture. However, viral stocks isolated from cells transfected with Rous sarcoma virus DNA containing the c-src gene in place of the viral src gene did occasionally produce foci. Virus obtained from these foci were highly transforming and appeared to arise via spontaneous mutation in the c-src-containing viral populations. The p60 proteins of the transforming mutant src viruses were found to have higher levels of in vitro tyrosine kinase activity than the levels observed with the parental viruses. In this study, we have molecularly cloned the src DNA sequences of two transforming mutant src viruses. When compared to the DNA sequence of the parental c-src viruses, the mutant viruses contain single point mutations that result in single amino acid changes in the src gene products (p60 proteins). Both amino acid changes reside in the tyrosine kinase domain of the protein. The mutation detected in one virus involves replacement of the normal Glu-378 in p60c-src by Gly, whereas the p60 of the other transforming virus has Phe instead of the normal Ile-441. Our data indicate that when p60c-src is expressed at elevated levels in a retroviral context, a single amino acid change in its primary sequence can activate the kinase activity of this protein and cause cellular transformation.

Full text

PDF
4228

Images in this article

4230Image
on p.4230
4230Image
on p.4230
4231Image
on p.4231
4231Image
on p.4231

Selected References

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

  1. Benton W. D., Davis R. W. Screening lambdagt recombinant clones by hybridization to single plaques in situ. Science. 1977 Apr 8;196(4286):180–182. doi: 10.1126/science.322279. [DOI] [PubMed] [Google Scholar]
  2. Blattner F. R., Blechl A. E., Denniston-Thompson K., Faber H. E., Richards J. E., Slightom J. L., Tucker P. W., Smithies O. Cloning human fetal gamma globin and mouse alpha-type globin DNA: preparation and screening of shotgun collections. Science. 1978 Dec 22;202(4374):1279–1284. doi: 10.1126/science.725603. [DOI] [PubMed] [Google Scholar]
  3. Bos J. L., Toksoz D., Marshall C. J., Verlaan-de Vries M., Veeneman G. H., van der Eb A. J., van Boom J. H., Janssen J. W., Steenvoorden A. C. Amino-acid substitutions at codon 13 of the N-ras oncogene in human acute myeloid leukaemia. 1985 Jun 27-Jul 3Nature. 315(6022):726–730. doi: 10.1038/315726a0. [DOI] [PubMed] [Google Scholar]
  4. 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]
  5. Collett M. S., Erikson E., Purchio A. F., Brugge J. S., Erikson R. L. A normal cell protein similar in structure and function to the avian sarcoma virus transforming gene product. Proc Natl Acad Sci U S A. 1979 Jul;76(7):3159–3163. doi: 10.1073/pnas.76.7.3159. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Cooper J. A., Esch F. S., Taylor S. S., Hunter T. Phosphorylation sites in enolase and lactate dehydrogenase utilized by tyrosine protein kinases in vivo and in vitro. J Biol Chem. 1984 Jun 25;259(12):7835–7841. [PubMed] [Google Scholar]
  7. 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]
  8. 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]
  9. Fasano O., Aldrich T., Tamanoi F., Taparowsky E., Furth M., Wigler M. Analysis of the transforming potential of the human H-ras gene by random mutagenesis. Proc Natl Acad Sci U S A. 1984 Jul;81(13):4008–4012. doi: 10.1073/pnas.81.13.4008. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Garber E. A., Cross F. R., Hanafusa H. Processing of p60v-src to its myristylated membrane-bound form. Mol Cell Biol. 1985 Oct;5(10):2781–2788. doi: 10.1128/mcb.5.10.2781. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Hanafusa H., Halpern C. C., Buchhagen D. L., Kawai S. Recovery of avian sarcoma virus from tumors induced by transformation-defective mutants. J Exp Med. 1977 Dec 1;146(6):1735–1747. doi: 10.1084/jem.146.6.1735. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. 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]
  13. 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]
  14. 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]
  15. Jakobovits E. B., Majors J. E., Varmus H. E. Hormonal regulation of the Rous sarcoma virus src gene via a heterologous promoter defines a threshold dose for cellular transformation. Cell. 1984 Oct;38(3):757–765. doi: 10.1016/0092-8674(84)90271-x. [DOI] [PubMed] [Google Scholar]
  16. Karess R. E., Hanafusa H. Viral and cellular src genes contribute to the structure of recovered avian sarcoma virus transforming protein. Cell. 1981 Apr;24(1):155–164. doi: 10.1016/0092-8674(81)90511-0. [DOI] [PubMed] [Google Scholar]
  17. Karess R. E., Hayward W. S., Hanafusa H. Cellular information in the genome of recovered avian sarcoma virus directs the synthesis of transforming protein. Proc Natl Acad Sci U S A. 1979 Jul;76(7):3154–3158. doi: 10.1073/pnas.76.7.3154. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Leder P., Tiemeier D., Enquist L. EK2 derivatives of bacteriophage lambda useful in the cloning of DNA from higher organisms: the lambdagtWES system. Science. 1977 Apr 8;196(4286):175–177. doi: 10.1126/science.322278. [DOI] [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. 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]
  21. Messing J., Vieira J. A new pair of M13 vectors for selecting either DNA strand of double-digest restriction fragments. Gene. 1982 Oct;19(3):269–276. doi: 10.1016/0378-1119(82)90016-6. [DOI] [PubMed] [Google Scholar]
  22. Oppermann H., Levinson A. D., Varmus H. E., Levintow L., Bishop J. M. Uninfected vertebrate cells contain a protein that is closely related to the product of the avian sarcoma virus transforming gene (src). Proc Natl Acad Sci U S A. 1979 Apr;76(4):1804–1808. doi: 10.1073/pnas.76.4.1804. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Parker R. C., Varmus H. E., Bishop J. M. Cellular homologue (c-src) of the transforming gene of Rous sarcoma virus: isolation, mapping, and transcriptional analysis of c-src and flanking regions. Proc Natl Acad Sci U S A. 1981 Sep;78(9):5842–5846. doi: 10.1073/pnas.78.9.5842. [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. Reddy E. P., Reynolds R. K., Santos E., Barbacid M. A point mutation is responsible for the acquisition of transforming properties by the T24 human bladder carcinoma oncogene. Nature. 1982 Nov 11;300(5888):149–152. doi: 10.1038/300149a0. [DOI] [PubMed] [Google Scholar]
  26. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Schwartz D. E., Tizard R., Gilbert W. Nucleotide sequence of Rous sarcoma virus. Cell. 1983 Mar;32(3):853–869. doi: 10.1016/0092-8674(83)90071-5. [DOI] [PubMed] [Google Scholar]
  28. 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]
  29. Shalloway D., Zelenetz A. D., Cooper G. M. Molecular cloning and characterization of the chicken gene homologous to the transforming gene of Rous sarcoma virus. Cell. 1981 May;24(2):531–541. doi: 10.1016/0092-8674(81)90344-5. [DOI] [PubMed] [Google Scholar]
  30. Shibuya M., Hanafusa H., Balduzzi P. C. Cellular sequences related to three new onc genes of avian sarcoma virus (fps, yes, and ros) and their expression in normal and transformed cells. J Virol. 1982 Apr;42(1):143–152. doi: 10.1128/jvi.42.1.143-152.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Spector D. H., Varmus H. E., Bishop J. M. Nucleotide sequences related to the transforming gene of avian sarcoma virus are present in DNA of uninfected vertebrates. Proc Natl Acad Sci U S A. 1978 Sep;75(9):4102–4106. doi: 10.1073/pnas.75.9.4102. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Stehelin D., Varmus H. E., Bishop J. M., Vogt P. K. DNA related to the transforming gene(s) of avian sarcoma viruses is present in normal avian DNA. Nature. 1976 Mar 11;260(5547):170–173. doi: 10.1038/260170a0. [DOI] [PubMed] [Google Scholar]
  33. Tabin C. J., Bradley S. M., Bargmann C. I., Weinberg R. A., Papageorge A. G., Scolnick E. M., Dhar R., Lowy D. R., Chang E. H. Mechanism of activation of a human oncogene. Nature. 1982 Nov 11;300(5888):143–149. doi: 10.1038/300143a0. [DOI] [PubMed] [Google Scholar]
  34. Takeya T., Feldman R. A., Hanafusa H. DNA sequence of the viral and cellular src gene of chickens. 1. Complete nucleotide sequence of an EcoRI fragment of recovered avian sarcoma virus which codes for gp37 and pp60src. J Virol. 1982 Oct;44(1):1–11. doi: 10.1128/jvi.44.1.1-11.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. 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]
  36. 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]
  37. 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]
  38. Taparowsky E., Suard Y., Fasano O., Shimizu K., Goldfarb M., Wigler M. Activation of the T24 bladder carcinoma transforming gene is linked to a single amino acid change. Nature. 1982 Dec 23;300(5894):762–765. doi: 10.1038/300762a0. [DOI] [PubMed] [Google Scholar]
  39. Thomas P. S. Hybridization of denatured RNA and small DNA fragments transferred to nitrocellulose. Proc Natl Acad Sci U S A. 1980 Sep;77(9):5201–5205. doi: 10.1073/pnas.77.9.5201. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. 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]
  41. Yuasa Y., Srivastava S. K., Dunn C. Y., Rhim J. S., Reddy E. P., Aaronson S. A. Acquisition of transforming properties by alternative point mutations within c-bas/has human proto-oncogene. Nature. 1983 Jun 30;303(5920):775–779. doi: 10.1038/303775a0. [DOI] [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