Skip to main content
PMC home page
Biochemical Journal logoLink to Biochemical Journal
. 1988 Mar 1;250(2):485–491. doi: 10.1042/bj2500485

N-myristoylation of p60src. Identification of a myristoyl-CoA:glycylpeptide N-myristoyltransferase in rat tissues.

C J Glover 1, C Goddard 1, R L Felsted 1
PMCID: PMC1148882  PMID: 3128285

Abstract

A 16-residue synthetic peptide corresponding to the N-terminal sequence of p60src was used as the acyl acceptor in an assay for myristoyl-CoA:glycylpeptide N-myristoyltransferase in rat tissues. An additional C-terminal tyrosine amide was added to this peptide to facilitate radioiodination and enhance detectability. Reverse-phase h.p.l.c. enabled the simultaneous detection and quantification of the peptide substrate and its N-myristoylated product. N-Myristoyltransferase activity was highest in the brain with decreasing activities in lung, small intestine, kidney, heart, skeletal muscle and liver. Brain activity was distributed approximately equally between the 100,000 g pellet and supernatant fractions. The soluble enzyme exhibited a Kappm of 20 microM for the src peptide and an optimum between pH 7.0 and 7.5. Maximum N-acylating activity was seen with myristoyl (C14:0)-CoA with lower activities found with the C10:0-CoA and C12:0-CoA homologues. No activity was obtained with palmitoyl (C18:0)-CoA but this derivative inhibited N-myristoyltransferase activity greater than 50% at equimolar concentrations with myristoyl-CoA. With a decapeptide corresponding to the N-terminal sequence of the cyclic AMP-dependent protein kinase catalytic subunit as the acyl acceptor, the brain enzyme displayed a Kapp.m of 117 microM and was about 14-fold less catalytically effective than with the p60src acyl acceptor. Transferase activity was also seen with a 16-residue peptide corresponding to the N-terminal sequence of the HIV p17gag structural protein. Inhibition studies with shorter src peptide analogues indicated an enzyme specificity for the p60src acyl acceptor beyond 9 residues.

Full text

PDF
489

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. 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]
  3. 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]
  4. 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]
  5. 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]
  6. Corbin J. D., Sugden P. H., Lincoln T. M., Keely S. L. Compartmentalization of adenosine 3':5'-monophosphate and adenosine 3':5'-monophosphate-dependent protein kinase in heart tissue. J Biol Chem. 1977 Jun 10;252(11):3854–3861. [PubMed] [Google Scholar]
  7. Cotton P. C., Brugge J. S. Neural tissues express high levels of the cellular src gene product pp60c-src. Mol Cell Biol. 1983 Jun;3(6):1157–1162. doi: 10.1128/mcb.3.6.1157. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. 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]
  9. 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]
  10. Gallick G. E., Arlinghaus R. B. Incorporation of lipids into variants of Moloney sarcoma virus which produce gag-mos fusion proteins. Virology. 1984 Feb;133(1):228–232. doi: 10.1016/0042-6822(84)90444-6. [DOI] [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., Krueger J. G., Hanafusa H., Goldberg A. R. Only membrane-associated RSV src proteins have amino-terminally bound lipid. Nature. 1983 Mar 10;302(5904):161–163. doi: 10.1038/302161a0. [DOI] [PubMed] [Google Scholar]
  13. HUNTER W. M., GREENWOOD F. C. Preparation of iodine-131 labelled human growth hormone of high specific activity. Nature. 1962 May 5;194:495–496. doi: 10.1038/194495a0. [DOI] [PubMed] [Google Scholar]
  14. Jacobs C., Rübsamen H. Expression of pp60c-src protein kinase in adult and fetal human tissue: high activities in some sarcomas and mammary carcinomas. Cancer Res. 1983 Apr;43(4):1696–1702. [PubMed] [Google Scholar]
  15. 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]
  16. Keenan T. W., Morré D. J. Phospholipid class and fatty acid composition of golgi apparatus isolated from rat liver and comparison with other cell fractions. Biochemistry. 1970 Jan 6;9(1):19–25. doi: 10.1021/bi00803a003. [DOI] [PubMed] [Google Scholar]
  17. 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]
  18. 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]
  19. Krzyzek R. A., Mitchell R. L., Lau A. F., Faras A. J. Association of pp60src and src protein kinase activity with the plasma membrane of nonpermissive and permissive avian sarcoma virus-infected cells. J Virol. 1980 Dec;36(3):805–815. doi: 10.1128/jvi.36.3.805-815.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  21. Moya F., Silbert D. F., Glaser L. The relation of temperature and lipid composition to cell adhesion. Biochim Biophys Acta. 1979 Feb 2;550(3):485–499. doi: 10.1016/0005-2736(79)90151-2. [DOI] [PubMed] [Google Scholar]
  22. Nimmo H. G., Cohen P. Hormonal control of protein phosphorylation. Adv Cyclic Nucleotide Res. 1977;8:145–266. [PubMed] [Google Scholar]
  23. 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]
  24. 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]
  25. 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]
  26. Rosen N., Bolen J. B., Schwartz A. M., Cohen P., DeSeau V., Israel M. A. Analysis of pp60c-src protein kinase activity in human tumor cell lines and tissues. J Biol Chem. 1986 Oct 15;261(29):13754–13759. [PubMed] [Google Scholar]
  27. Schultz A. M., Copeland T. D., Mark G. E., Rapp U. R., Oroszlan S. Detection of the myristylated gag-raf transforming protein with raf-specific antipeptide sera. Virology. 1985 Oct 15;146(1):78–89. doi: 10.1016/0042-6822(85)90054-6. [DOI] [PubMed] [Google Scholar]
  28. 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]
  29. Schultz A., Oroszlan S. Myristylation of gag-onc fusion proteins in mammalian transforming retroviruses. Virology. 1984 Mar;133(2):431–437. doi: 10.1016/0042-6822(84)90409-4. [DOI] [PubMed] [Google Scholar]
  30. Tallant E. A., Wallace R. W., Cheung W. Y. Purification and radioimmunoassay of calmodulin-dependent protein phosphatase from bovine brain. Methods Enzymol. 1983;102:244–256. doi: 10.1016/s0076-6879(83)02025-x. [DOI] [PubMed] [Google Scholar]
  31. 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]
  32. 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]
  33. Towler D., Glaser L. Acylation of cellular proteins with endogenously synthesized fatty acids. Biochemistry. 1986 Feb 25;25(4):878–884. doi: 10.1021/bi00352a021. [DOI] [PubMed] [Google Scholar]
  34. 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]
  35. Wallace R. W., Tallant E. A., Cheung W. Y. High levels of a heat-labile calmodulin-binding protein (CaM-BP80) in bovine neostriatum. Biochemistry. 1980 Apr 29;19(9):1831–1837. doi: 10.1021/bi00550a016. [DOI] [PubMed] [Google Scholar]
  36. Willingham M. C., Jay G., Pastan I. Localization of the ASV src gene product to the plasma membrane of transformed cells by electron microscopic immunocytochemistry. Cell. 1979 Sep;18(1):125–134. doi: 10.1016/0092-8674(79)90361-1. [DOI] [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES