Skip to main content
Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1988 Jun;8(6):2435–2441. doi: 10.1128/mcb.8.6.2435

The first seven amino acids encoded by the v-src oncogene act as a myristylation signal: lysine 7 is a critical determinant.

J M Kaplan 1, G Mardon 1, J M Bishop 1, H E Varmus 1
PMCID: PMC363442  PMID: 2841581

Abstract

The transforming protein of Rous sarcoma virus, pp60v-src, is covalently coupled to myristic acid by an amide linkage to glycine 2. Myristylation promotes the association of pp60v-src with cellular membranes, and this subcellular location is essential for transforming activity. The findings presented here, in conjunction with the previous reports of others, imply that the seventh amino acid encoded by v-src might be important in the myristylation reaction. Replacement of lysine 7 by asparagine greatly reduced the myristylation, membrane association, and transforming activity of pp60v-src. In contrast, substitution of arginine at residue 7 had no effect on any of these properties of pp60v-src. Addition of amino acids 1 to 7 encoded by v-src was sufficient to cause myristylation of a src-pyruvate kinase fusion protein. We conclude that the recognition sequence for myristylation of pp60v-src comprises amino acids 1 to 7 and that lysine 7 is a critical component of this sequence.

Full text

PDF
2435

Images in this article

Selected References

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

  1. Aitken A., Cohen P., Santikarn S., Williams D. H., Calder A. G., Smith A., Klee C. B. Identification of the NH2-terminal blocking group of calcineurin B as myristic acid. FEBS Lett. 1982 Dec 27;150(2):314–318. doi: 10.1016/0014-5793(82)80759-x. [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. 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. 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]
  5. 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]
  6. Buss J. E., Sefton B. M. Myristic acid, a rare fatty acid, is the lipid attached to the transforming protein of Rous sarcoma virus and its cellular homolog. J Virol. 1985 Jan;53(1):7–12. doi: 10.1128/jvi.53.1.7-12.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Carr S. A., Biemann K., Shoji S., Parmelee D. C., Titani K. n-Tetradecanoyl is the NH2-terminal blocking group of the catalytic subunit of cyclic AMP-dependent protein kinase from bovine cardiac muscle. Proc Natl Acad Sci U S A. 1982 Oct;79(20):6128–6131. doi: 10.1073/pnas.79.20.6128. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Chow M., Newman J. F., Filman D., Hogle J. M., Rowlands D. J., Brown F. Myristylation of picornavirus capsid protein VP4 and its structural significance. Nature. 1987 Jun 11;327(6122):482–486. doi: 10.1038/327482a0. [DOI] [PubMed] [Google Scholar]
  9. 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]
  10. Courtneidge S. A., Levinson A. D., Bishop J. M. The protein encoded by the transforming gene of avian sarcoma virus (pp60src) and a homologous protein in normal cells (pp60proto-src) are associated with the plasma membrane. Proc Natl Acad Sci U S A. 1980 Jul;77(7):3783–3787. doi: 10.1073/pnas.77.7.3783. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. 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]
  12. Garber E. A., Hanafusa H. NH2-terminal sequences of two src proteins that cause aberrant transformation. Proc Natl Acad Sci U S A. 1987 Jan;84(1):80–84. doi: 10.1073/pnas.84.1.80. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Graham F. L., van der Eb A. J. A new technique for the assay of infectivity of human adenovirus 5 DNA. Virology. 1973 Apr;52(2):456–467. doi: 10.1016/0042-6822(73)90341-3. [DOI] [PubMed] [Google Scholar]
  14. Henderson L. E., Krutzsch H. C., Oroszlan S. Myristyl amino-terminal acylation of murine retrovirus proteins: an unusual post-translational proteins modification. Proc Natl Acad Sci U S A. 1983 Jan;80(2):339–343. doi: 10.1073/pnas.80.2.339. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Kalderon D., Roberts B. L., Richardson W. D., Smith A. E. A short amino acid sequence able to specify nuclear location. Cell. 1984 Dec;39(3 Pt 2):499–509. doi: 10.1016/0092-8674(84)90457-4. [DOI] [PubMed] [Google Scholar]
  16. Kamps M. P., Buss J. E., Sefton B. M. Mutation of NH2-terminal glycine of p60src prevents both myristoylation and morphological transformation. Proc Natl Acad Sci U S A. 1985 Jul;82(14):4625–4628. doi: 10.1073/pnas.82.14.4625. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Katamine S., Notario V., Rao C. D., Miki T., Cheah M. S., Tronick S. R., Robbins K. C. Primary structure of the human fgr proto-oncogene product p55c-fgr. Mol Cell Biol. 1988 Jan;8(1):259–266. doi: 10.1128/mcb.8.1.259. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Krueger J. G., Garber E. A., Goldberg A. R., Hanafusa H. Changes in amino-terminal sequences of pp60src lead to decreased membrane association and decreased in vivo tumorigenicity. Cell. 1982 Apr;28(4):889–896. doi: 10.1016/0092-8674(82)90068-x. [DOI] [PubMed] [Google Scholar]
  19. Krueger J. G., Garber E. A., Goldberg A. R. Subcellular localization of pp60src in RSV-transformed cells. Curr Top Microbiol Immunol. 1983;107:51–124. [PubMed] [Google Scholar]
  20. 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]
  21. Lee J. S., Varmus H. E., Bishop J. M. Virus-specific messenger RNAs in permissive cells infected by avian sarcoma virus. J Biol Chem. 1979 Aug 25;254(16):8015–8022. [PubMed] [Google Scholar]
  22. 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]
  23. Magee A. I., Courtneidge S. A. Two classes of fatty acid acylated proteins exist in eukaryotic cells. EMBO J. 1985 May;4(5):1137–1144. doi: 10.1002/j.1460-2075.1985.tb03751.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Marchildon G. A., Casnellie J. E., Walsh K. A., Krebs E. G. Covalently bound myristate in a lymphoma tyrosine protein kinase. Proc Natl Acad Sci U S A. 1984 Dec;81(24):7679–7682. doi: 10.1073/pnas.81.24.7679. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Marth J. D., Peet R., Krebs E. G., Perlmutter R. M. A lymphocyte-specific protein-tyrosine kinase gene is rearranged and overexpressed in the murine T cell lymphoma LSTRA. Cell. 1985 Dec;43(2 Pt 1):393–404. doi: 10.1016/0092-8674(85)90169-2. [DOI] [PubMed] [Google Scholar]
  26. McIlhinney R. A., Pelly S. J., Chadwick J. K., Cowley G. P. Studies on the attachment of myristic and palmitic acid to cell proteins in human squamous carcinoma cell lines: evidence for two pathways. EMBO J. 1985 May;4(5):1145–1152. doi: 10.1002/j.1460-2075.1985.tb03752.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Olson E. N., Spizz G. Fatty acylation of cellular proteins. Temporal and subcellular differences between palmitate and myristate acylation. J Biol Chem. 1986 Feb 15;261(5):2458–2466. [PubMed] [Google Scholar]
  28. Olson E. N., Towler D. A., Glaser L. Specificity of fatty acid acylation of cellular proteins. J Biol Chem. 1985 Mar 25;260(6):3784–3790. [PubMed] [Google Scholar]
  29. Oppermann H., Levinson A. D., Varmus H. E. The structure and protein kinase activity of proteins encoded by nonconditional mutants and back mutants in the sec gene of avian sarcoma virus. Virology. 1981 Jan 15;108(1):47–70. doi: 10.1016/0042-6822(81)90526-2. [DOI] [PubMed] [Google Scholar]
  30. Ozols J., Carr S. A., Strittmatter P. Identification of the NH2-terminal blocking group of NADH-cytochrome b5 reductase as myristic acid and the complete amino acid sequence of the membrane-binding domain. J Biol Chem. 1984 Nov 10;259(21):13349–13354. [PubMed] [Google Scholar]
  31. Pellman D., Garber E. A., Cross F. R., Hanafusa H. An N-terminal peptide from p60src can direct myristylation and plasma membrane localization when fused to heterologous proteins. 1985 Mar 28-Apr 3Nature. 314(6009):374–377. doi: 10.1038/314374a0. [DOI] [PubMed] [Google Scholar]
  32. Pellman D., Garber E. A., Cross F. R., Hanafusa H. Fine structural mapping of a critical NH2-terminal region of p60src. Proc Natl Acad Sci U S A. 1985 Mar;82(6):1623–1627. doi: 10.1073/pnas.82.6.1623. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Persing D. H., Varmus H. E., Ganem D. The preS1 protein of hepatitis B virus is acylated at its amino terminus with myristic acid. J Virol. 1987 May;61(5):1672–1677. doi: 10.1128/jvi.61.5.1672-1677.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Quintrell N., Lebo R., Varmus H., Bishop J. M., Pettenati M. J., Le Beau M. M., Diaz M. O., Rowley J. D. Identification of a human gene (HCK) that encodes a protein-tyrosine kinase and is expressed in hemopoietic cells. Mol Cell Biol. 1987 Jun;7(6):2267–2275. doi: 10.1128/mcb.7.6.2267. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Roebroek A. J., Schalken J. A., Onnekink C., Bloemers H. P., Van de Ven W. J. Structure of the feline c-fes/fps proto-oncogene: genesis of a retroviral oncogene. J Virol. 1987 Jun;61(6):2009–2016. doi: 10.1128/jvi.61.6.2009-2016.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Rohrschneider L. R. Adhesion plaques of Rous sarcoma virus-transformed cells contain the src gene product. Proc Natl Acad Sci U S A. 1980 Jun;77(6):3514–3518. doi: 10.1073/pnas.77.6.3514. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Schultz A. M., Henderson L. E., Oroszlan S., Garber E. A., Hanafusa H. Amino terminal myristylation of the protein kinase p60src, a retroviral transforming protein. Science. 1985 Jan 25;227(4685):427–429. doi: 10.1126/science.3917576. [DOI] [PubMed] [Google Scholar]
  38. 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]
  39. Seeburg P. H., Colby W. W., Capon D. J., Goeddel D. V., Levinson A. D. Biological properties of human c-Ha-ras1 genes mutated at codon 12. Nature. 1984 Nov 1;312(5989):71–75. doi: 10.1038/312071a0. [DOI] [PubMed] [Google Scholar]
  40. Semba K., Nishizawa M., Miyajima N., Yoshida M. C., Sukegawa J., Yamanashi Y., Sasaki M., Yamamoto T., Toyoshima K. yes-related protooncogene, syn, belongs to the protein-tyrosine kinase family. Proc Natl Acad Sci U S A. 1986 Aug;83(15):5459–5463. doi: 10.1073/pnas.83.15.5459. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Shtivelman E., Lifshitz B., Gale R. P., Roe B. A., Canaani E. Alternative splicing of RNAs transcribed from the human abl gene and from the bcr-abl fused gene. Cell. 1986 Oct 24;47(2):277–284. doi: 10.1016/0092-8674(86)90450-2. [DOI] [PubMed] [Google Scholar]
  42. Streuli C. H., Griffin B. E. Myristic acid is coupled to a structural protein of polyoma virus and SV40. Nature. 1987 Apr 9;326(6113):619–622. doi: 10.1038/326619a0. [DOI] [PubMed] [Google Scholar]
  43. Sukegawa J., Semba K., Yamanashi Y., Nishizawa M., Miyajima N., Yamamoto T., Toyoshima K. Characterization of cDNA clones for the human c-yes gene. Mol Cell Biol. 1987 Jan;7(1):41–47. doi: 10.1128/mcb.7.1.41. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. TEMIN H. M., RUBIN H. Characteristics of an assay for Rous sarcoma virus and Rous sarcoma cells in tissue culture. Virology. 1958 Dec;6(3):669–688. doi: 10.1016/0042-6822(58)90114-4. [DOI] [PubMed] [Google Scholar]
  45. 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]
  46. Towler D. A., Adams S. P., Eubanks S. R., Towery D. S., Jackson-Machelski E., Glaser L., Gordon J. I. Myristoyl CoA:protein N-myristoyltransferase activities from rat liver and yeast possess overlapping yet distinct peptide substrate specificities. J Biol Chem. 1988 Feb 5;263(4):1784–1790. [PubMed] [Google Scholar]
  47. Towler D. A., Adams S. P., Eubanks S. R., Towery D. S., Jackson-Machelski E., Glaser L., Gordon J. I. Purification and characterization of yeast myristoyl CoA:protein N-myristoyltransferase. Proc Natl Acad Sci U S A. 1987 May;84(9):2708–2712. doi: 10.1073/pnas.84.9.2708. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Towler D. A., Eubanks S. R., Towery D. S., Adams S. P., Glaser L. Amino-terminal processing of proteins by N-myristoylation. Substrate specificity of N-myristoyl transferase. J Biol Chem. 1987 Jan 25;262(3):1030–1036. [PubMed] [Google Scholar]
  49. Towler D., Glaser L. Protein fatty acid acylation: enzymatic synthesis of an N-myristoylglycyl peptide. Proc Natl Acad Sci U S A. 1986 May;83(9):2812–2816. doi: 10.1073/pnas.83.9.2812. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Varmus H. E., Quintrell N., Wyke J. Revertants of an ASV-transformed rat cell line have lost the complete provius or sustained mutations in src. Virology. 1981 Jan 15;108(1):28–46. doi: 10.1016/0042-6822(81)90525-0. [DOI] [PubMed] [Google Scholar]
  51. Wilcox C., Hu J. S., Olson E. N. Acylation of proteins with myristic acid occurs cotranslationally. Science. 1987 Nov 27;238(4831):1275–1278. doi: 10.1126/science.3685978. [DOI] [PubMed] [Google Scholar]
  52. Yamanashi Y., Fukushige S., Semba K., Sukegawa J., Miyajima N., Matsubara K., Yamamoto T., Toyoshima K. The yes-related cellular gene lyn encodes a possible tyrosine kinase similar to p56lck. Mol Cell Biol. 1987 Jan;7(1):237–243. doi: 10.1128/mcb.7.1.237. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Young J. C., Martin G. S. Cellular localization of c-fps gene product NCP98. J Virol. 1984 Dec;52(3):913–918. doi: 10.1128/jvi.52.3.913-918.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Molecular and Cellular Biology are provided here courtesy of Taylor & Francis

RESOURCES