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. 1988 Jun;62(6):1898–1906. doi: 10.1128/jvi.62.6.1898-1906.1988

A single point mutation has pleiotropic effects on pp60v-src function.

M J Welham 1, J A Wyke 1
PMCID: PMC253272  PMID: 3130493

Abstract

The Rous sarcoma virus mutant tsLA29 encodes a pp60v-src molecule that is temperature sensitive for both tyrosine kinase activity and its ability to locate at the cell periphery. The defect in localization appears to be due to a perturbation in events following complex dissociation, since the mutant enzyme shows a rapidly reversible association with the cytoskeleton when shifted between permissive and restrictive temperatures. Although tsLA29 pp60v-src differs from the wild type at three amino acid residues, studies with chimeric proteins show that only one of the mutations, an alanine-for-proline substitution at residue 507, accounts for all the temperature-sensitive characteristics. Moreover, a single second site mutation, at residue 427, can restore the wild phenotype. Cells infected with a chimeric virus encoding only the alanine substitution at position 507 have a conspicuously fusiform morphology, suggesting that this mutation also has subtle effects on pp60v-src function that are apparently compensated for by the other mutations in native tsLA29.

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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., Erikson E., Erikson R. L. The specific interaction of the Rous sarcoma virus transforming protein, pp60src, with two cellular proteins. Cell. 1981 Aug;25(2):363–372. doi: 10.1016/0092-8674(81)90055-6. [DOI] [PubMed] [Google Scholar]
  2. Brugge J. S. Interaction of the Rous sarcoma virus protein pp60src with the cellular proteins pp50 and pp90. Curr Top Microbiol Immunol. 1986;123:1–22. doi: 10.1007/978-3-642-70810-7_1. [DOI] [PubMed] [Google Scholar]
  3. Brugge J., Yonemoto W., Darrow D. Interaction between the Rous sarcoma virus transforming protein and two cellular phosphoproteins: analysis of the turnover and distribution of this complex. Mol Cell Biol. 1983 Jan;3(1):9–19. doi: 10.1128/mcb.3.1.9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bryant D. L., Parsons J. T. Amino acid alterations within a highly conserved region of the Rous sarcoma virus src gene product pp60src inactivate tyrosine protein kinase activity. Mol Cell Biol. 1984 May;4(5):862–866. doi: 10.1128/mcb.4.5.862. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Burr J. G., Dreyfuss G., Penman S., Buchanan J. M. Association of the src gene product of Rous sarcoma virus with cytoskeletal structures of chicken embryo fibroblasts. Proc Natl Acad Sci U S A. 1980 Jun;77(6):3484–3488. doi: 10.1073/pnas.77.6.3484. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Buss J. E., Kamps M. P., Gould K., Sefton B. M. The absence of myristic acid decreases membrane binding of p60src but does not affect tyrosine protein kinase activity. J Virol. 1986 May;58(2):468–474. doi: 10.1128/jvi.58.2.468-474.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Buss J. E., Kamps M. P., Sefton B. M. Myristic acid is attached to the transforming protein of Rous sarcoma virus during or immediately after synthesis and is present in both soluble and membrane-bound forms of the protein. Mol Cell Biol. 1984 Dec;4(12):2697–2704. doi: 10.1128/mcb.4.12.2697. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Calothy G., Laugier D., Cross F. R., Jove R., Hanafusa T., Hanafusa H. The membrane-binding domain and myristylation of p60v-src are not essential for stimulation of cell proliferation. J Virol. 1987 May;61(5):1678–1681. doi: 10.1128/jvi.61.5.1678-1681.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Chou P. Y., Fasman G. D. Empirical predictions of protein conformation. Annu Rev Biochem. 1978;47:251–276. doi: 10.1146/annurev.bi.47.070178.001343. [DOI] [PubMed] [Google Scholar]
  10. 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]
  11. Courtneidge S. A., Bishop J. M. Transit of pp60v-src to the plasma membrane. Proc Natl Acad Sci U S A. 1982 Dec;79(23):7117–7121. doi: 10.1073/pnas.79.23.7117. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. 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]
  13. 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]
  14. DeClue J. E., Martin G. S. Phosphorylation of talin at tyrosine in Rous sarcoma virus-transformed cells. Mol Cell Biol. 1987 Jan;7(1):371–378. doi: 10.1128/mcb.7.1.371. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Enrietto P. J., Payne L. N., Wyke J. A. Analysis of the pathogenicity of transformation defective partial deletion mutants of avian sarcoma virus: characterization of recovered viruses which encode novel src specific proteins. Virology. 1983 Jun;127(2):397–411. doi: 10.1016/0042-6822(83)90153-8. [DOI] [PubMed] [Google Scholar]
  16. Fincham V. J., Chiswell D. J., Wyke J. A. Mapping of nonconditional and conditional mutants in the src gene of Prague strain Rous sarcoma virus. Virology. 1982 Jan 15;116(1):72–83. doi: 10.1016/0042-6822(82)90404-4. [DOI] [PubMed] [Google Scholar]
  17. Fincham V. J., Wyke J. A. Localization of temperature-sensitive transformation mutations and back mutations in the Rous sarcoma virus src gene. J Virol. 1986 May;58(2):694–699. doi: 10.1128/jvi.58.2.694-699.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. 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]
  19. 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]
  20. Hamaguchi M., Hanafusa H. Association of p60src with Triton X-100-resistant cellular structure correlates with morphological transformation. Proc Natl Acad Sci U S A. 1987 Apr;84(8):2312–2316. doi: 10.1073/pnas.84.8.2312. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Hirst R., Horwitz A., Buck C., Rohrschneider L. Phosphorylation of the fibronectin receptor complex in cells transformed by oncogenes that encode tyrosine kinases. Proc Natl Acad Sci U S A. 1986 Sep;83(17):6470–6474. doi: 10.1073/pnas.83.17.6470. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Jove R., Garber E. A., Iba H., Hanafusa H. Biochemical properties of p60v-src mutants that induce different cell transformation parameters. J Virol. 1986 Dec;60(3):849–857. doi: 10.1128/jvi.60.3.849-857.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Jove R., Mayer B. J., Iba H., Laugier D., Poirier F., Calothy G., Hanafusa T., Hanafusa H. Genetic analysis of p60v-src domains involved in the induction of different cell transformation parameters. J Virol. 1986 Dec;60(3):840–848. doi: 10.1128/jvi.60.3.840-848.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Kamps M. P., Buss J. E., Sefton B. M. Rous sarcoma virus transforming protein lacking myristic acid phosphorylates known polypeptide substrates without inducing transformation. Cell. 1986 Apr 11;45(1):105–112. doi: 10.1016/0092-8674(86)90542-8. [DOI] [PubMed] [Google Scholar]
  25. Kawai S., Nishizawa M. New procedure for DNA transfection with polycation and dimethyl sulfoxide. Mol Cell Biol. 1984 Jun;4(6):1172–1174. doi: 10.1128/mcb.4.6.1172. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Kellie S., Patel B., Wigglesworth N. M., Critchley D. R., Wyke J. A. The use of Rous sarcoma virus transformation mutants with differing tyrosine kinase activities to study the relationships between vinculin phosphorylation, pp60v-src location and adhesion plaque integrity. Exp Cell Res. 1986 Jul;165(1):216–228. doi: 10.1016/0014-4827(86)90546-x. [DOI] [PubMed] [Google Scholar]
  27. Krueger J. G., Garber E. A., Chin S. S., Hanafusa H., Goldberg A. R. Size-variant pp60src proteins of recovered avian sarcoma viruses interact with adhesion plaques as peripheral membrane proteins: effects on cell transformation. Mol Cell Biol. 1984 Mar;4(3):454–467. doi: 10.1128/mcb.4.3.454. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Krueger J. G., Wang E., Goldberg A. R. Evidence that the src gene product of Rous sarcoma virus is membrane associated. Virology. 1980 Feb;101(1):25–40. doi: 10.1016/0042-6822(80)90480-8. [DOI] [PubMed] [Google Scholar]
  29. 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]
  30. 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]
  31. 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]
  32. Loenen W. A., Brammar W. J. A bacteriophage lambda vector for cloning large DNA fragments made with several restriction enzymes. Gene. 1980 Aug;10(3):249–259. doi: 10.1016/0378-1119(80)90054-2. [DOI] [PubMed] [Google Scholar]
  33. Mayer B. J., Jove R., Krane J. F., Poirier F., Calothy G., Hanafusa H. Genetic lesions involved in temperature sensitivity of the src gene products of four Rous sarcoma virus mutants. J Virol. 1986 Dec;60(3):858–867. doi: 10.1128/jvi.60.3.858-867.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. 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]
  35. Pasquale E. B., Maher P. A., Singer S. J. Talin is phosphorylated on tyrosine in chicken embryo fibroblasts transformed by Rous sarcoma virus. Proc Natl Acad Sci U S A. 1986 Aug;83(15):5507–5511. doi: 10.1073/pnas.83.15.5507. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. 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]
  37. 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]
  38. Schuh S., Yonemoto W., Brugge J., Bauer V. J., Riehl R. M., Sullivan W. P., Toft D. O. A 90,000-dalton binding protein common to both steroid receptors and the Rous sarcoma virus transforming protein, pp60v-src. J Biol Chem. 1985 Nov 15;260(26):14292–14296. [PubMed] [Google Scholar]
  39. 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]
  40. Sefton B. M. The viral tyrosine protein kinases. Curr Top Microbiol Immunol. 1986;123:39–72. doi: 10.1007/978-3-642-70810-7_3. [DOI] [PubMed] [Google Scholar]
  41. Stoker A. W., Bissell M. J. Development of avian sarcoma and leukosis virus-based vector-packaging cell lines. J Virol. 1988 Mar;62(3):1008–1015. doi: 10.1128/jvi.62.3.1008-1015.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Stoker A. W., Enrietto P. J., Wyke J. A. Functional domains of the pp60v-src protein as revealed by analysis of temperature-sensitive Rous sarcoma virus mutants. Mol Cell Biol. 1984 Aug;4(8):1508–1514. doi: 10.1128/mcb.4.8.1508. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Stoker A. W., Kellie S., Wyke J. A. Intracellular localization and processing of pp60v-src proteins expressed by two distinct temperature-sensitive mutants of Rous sarcoma virus. J Virol. 1986 Jun;58(3):876–883. doi: 10.1128/jvi.58.3.876-883.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. 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]
  45. Tato F., Beamand J. A., Wyke J. A. A mutant of Rous sarcoma virus with a thermolabile defect in the virus envelope. Virology. 1978 Jul 1;88(1):71–81. doi: 10.1016/0042-6822(78)90111-3. [DOI] [PubMed] [Google Scholar]
  46. Vennström B., Fanshier L., Moscovici C., Bishop J. M. Molecular cloning of the avian erythroblastosis virus genome and recovery of oncogenic virus by transfection of chicken cells. J Virol. 1980 Nov;36(2):575–585. doi: 10.1128/jvi.36.2.575-585.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. White M. K., Weber M. J. Transformation by the src oncogene alters glucose transport into rat and chicken cells by different mechanisms. Mol Cell Biol. 1988 Jan;8(1):138–144. doi: 10.1128/mcb.8.1.138. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Wyke J. A. Complementation of transforming functions by temperature-sensitive mutants of avian sarcoma virus. Virology. 1973 Jul;54(1):28–36. doi: 10.1016/0042-6822(73)90111-6. [DOI] [PubMed] [Google Scholar]
  49. Wyke J. A., Kurth R. Reversion of temperature sensitive transformation mutants of Rous sarcoma virus and its effect on the expression of tumour specific surface antigen. J Gen Virol. 1978 Sep;40(3):701–704. doi: 10.1099/0022-1317-40-3-701. [DOI] [PubMed] [Google Scholar]
  50. Wyke J. A., Stoker A. W. Genetic analysis of the form and function of the viral src oncogene product. Biochim Biophys Acta. 1987 Apr 20;907(1):47–69. doi: 10.1016/0304-419x(87)90018-7. [DOI] [PubMed] [Google Scholar]
  51. Wyke J. A. The selective isolation of temperature-sensitive mutants of Rous sarcoma virus. Virology. 1973 Apr;52(2):587–590. doi: 10.1016/0042-6822(73)90357-7. [DOI] [PubMed] [Google Scholar]

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