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. 1988 Apr;85(8):2608–2612. doi: 10.1073/pnas.85.8.2608

Nonmyristoylated p60v-src fails to phosphorylate proteins of 115-120 kDa in chicken embryo fibroblasts.

M E Linder 1, J G Burr 1
PMCID: PMC280047  PMID: 2451829

Abstract

We have used anti-phosphotyrosine antibodies to identify a large number of tyrosine phosphoproteins in Rous sarcoma virus (RSV)-transformed chicken embryo fibroblasts. Most of these proteins were found in the 100,000 X g supernatant when cells were separated into soluble and particulate fractions; however, one group of proteins, of 115-120 kDa, was found in the particulate fraction. The phosphotyrosine content of the 115- to 120-kDa proteins was greatly reduced in chicken embryo fibroblasts infected with mutants of RSV (NY314 and SD10) encoding nonmyristoylated forms of the viral src gene product that do not associate with cellular membranes. Since RSV mutants NY314 and SD10 do not transform cells, phosphorylation of this group of 115- to 120-kDa membrane proteins may be related to the process of transformation.

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Selected References

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

  1. Brown D. J., Gordon J. A. The stimulation of pp60v-src kinase activity by vanadate in intact cells accompanies a new phosphorylation state of the enzyme. J Biol Chem. 1984 Aug 10;259(15):9580–9586. [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. Collett M. S., Belzer S. K., Purchio A. F. Structurally and functionally modified forms of pp60v-src in Rous sarcoma virus-transformed cell lysates. Mol Cell Biol. 1984 Jul;4(7):1213–1220. doi: 10.1128/mcb.4.7.1213. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Comoglio P. M., Di Renzo M. F., Tarone G., Giancotti F. G., Naldini L., Marchisio P. C. Detection of phosphotyrosine-containing proteins in the detergent-insoluble fraction of RSV-transformed fibroblasts by azobenzene phosphonate antibodies. EMBO J. 1984 Mar;3(3):483–489. doi: 10.1002/j.1460-2075.1984.tb01834.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. 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]
  8. Di Renzo M. F., Ferracini R., Naldini L., Giordano S., Comoglio P. M. Immunological detection of proteins phosphorylated at tyrosine in cells stimulated by growth factors or transformed by retroviral-oncogene-coded tyrosine kinases. Eur J Biochem. 1986 Jul 15;158(2):383–391. doi: 10.1111/j.1432-1033.1986.tb09765.x. [DOI] [PubMed] [Google Scholar]
  9. Ek B., Heldin C. H. Use of an antiserum against phosphotyrosine for the identification of phosphorylated components in human fibroblasts stimulated by platelet-derived growth factor. J Biol Chem. 1984 Sep 10;259(17):11145–11152. [PubMed] [Google Scholar]
  10. Frackelton A. R., Jr, Ross A. H., Eisen H. N. Characterization and use of monoclonal antibodies for isolation of phosphotyrosyl proteins from retrovirus-transformed cells and growth factor-stimulated cells. Mol Cell Biol. 1983 Aug;3(8):1343–1352. doi: 10.1128/mcb.3.8.1343. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. 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]
  12. Glenney J. Phospholipid-dependent Ca2+ binding by the 36-kDa tyrosine kinase substrate (calpactin) and its 33-kDa core. J Biol Chem. 1986 Jun 5;261(16):7247–7252. [PubMed] [Google Scholar]
  13. 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]
  14. Huhn R. D., Posner M. R., Rayter S. I., Foulkes J. G., Frackelton A. R., Jr Cell lines and peripheral blood leukocytes derived from individuals with chronic myelogenous leukemia display virtually identical proteins phosphorylated on tyrosine residues. Proc Natl Acad Sci U S A. 1987 Jul;84(13):4408–4412. doi: 10.1073/pnas.84.13.4408. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Hultquist D. E., Moyer R. W., Boyer P. D. The preparation and characterization of 1-phosphohistidine and 3-phosphohistidine. Biochemistry. 1966 Jan;5(1):322–331. doi: 10.1021/bi00865a041. [DOI] [PubMed] [Google Scholar]
  16. Hunter T., Cooper J. A. Protein-tyrosine kinases. Annu Rev Biochem. 1985;54:897–930. doi: 10.1146/annurev.bi.54.070185.004341. [DOI] [PubMed] [Google Scholar]
  17. 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]
  18. 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]
  19. Klarlund J. K. Transformation of cells by an inhibitor of phosphatases acting on phosphotyrosine in proteins. Cell. 1985 Jul;41(3):707–717. doi: 10.1016/s0092-8674(85)80051-9. [DOI] [PubMed] [Google Scholar]
  20. 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]
  21. Morla A. O., Wang J. Y. Protein tyrosine phosphorylation in the cell cycle of BALB/c 3T3 fibroblasts. Proc Natl Acad Sci U S A. 1986 Nov;83(21):8191–8195. doi: 10.1073/pnas.83.21.8191. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. 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]
  23. Piwnica-Worms H., Saunders K. B., Roberts T. M., Smith A. E., Cheng S. H. Tyrosine phosphorylation regulates the biochemical and biological properties of pp60c-src. Cell. 1987 Apr 10;49(1):75–82. doi: 10.1016/0092-8674(87)90757-4. [DOI] [PubMed] [Google Scholar]
  24. Rein A., Rubin H. Effects of local cell concentrations upon the growth of chick embryo cells in tissue culture. Exp Cell Res. 1968 Mar;49(3):666–678. doi: 10.1016/0014-4827(68)90213-9. [DOI] [PubMed] [Google Scholar]
  25. Resh M. D., Erikson R. L. Highly specific antibody to Rous sarcoma virus src gene product recognizes a novel population of pp60v-src and pp60c-src molecules. J Cell Biol. 1985 Feb;100(2):409–417. doi: 10.1083/jcb.100.2.409. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Ross A. H., Baltimore D., Eisen H. N. Phosphotyrosine-containing proteins isolated by affinity chromatography with antibodies to a synthetic hapten. Nature. 1981 Dec 17;294(5842):654–656. doi: 10.1038/294654a0. [DOI] [PubMed] [Google Scholar]
  27. 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]
  28. Sefton B. M., Hunter T., Ball E. H., Singer S. J. Vinculin: a cytoskeletal target of the transforming protein of Rous sarcoma virus. Cell. 1981 Apr;24(1):165–174. doi: 10.1016/0092-8674(81)90512-2. [DOI] [PubMed] [Google Scholar]
  29. Sefton B. M., Hunter T., Beemon K., Eckhart W. Evidence that the phosphorylation of tyrosine is essential for cellular transformation by Rous sarcoma virus. Cell. 1980 Jul;20(3):807–816. doi: 10.1016/0092-8674(80)90327-x. [DOI] [PubMed] [Google Scholar]
  30. Seki J., Owada M. K., Sakato N., Fujio H. Direct identification of phosphotyrosine-containing proteins in some retrovirus-transformed cells by use of anti-phosphotyrosine antibody. Cancer Res. 1986 Feb;46(2):907–916. [PubMed] [Google Scholar]
  31. Swarup G., Cohen S., Garbers D. L. Inhibition of membrane phosphotyrosyl-protein phosphatase activity by vanadate. Biochem Biophys Res Commun. 1982 Aug;107(3):1104–1109. doi: 10.1016/0006-291x(82)90635-0. [DOI] [PubMed] [Google Scholar]
  32. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Wang J. Y. Isolation of antibodies for phosphotyrosine by immunization with a v-abl oncogene-encoded protein. Mol Cell Biol. 1985 Dec;5(12):3640–3643. doi: 10.1128/mcb.5.12.3640. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Yonemoto W., Filson A. J., Queral-Lustig A. E., Wang J. Y., Brugge J. S. Detection of phosphotyrosine-containing proteins in polyomavirus middle tumor antigen-transformed cells after treatment with a phosphotyrosine phosphatase inhibitor. Mol Cell Biol. 1987 Feb;7(2):905–913. doi: 10.1128/mcb.7.2.905. [DOI] [PMC free article] [PubMed] [Google Scholar]

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