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. 1985 Jan;82(2):321–324. doi: 10.1073/pnas.82.2.321

Purification of the Rous sarcoma virus src kinase by casein-agarose and tyrosine-agarose affinity chromatography.

Y Fukami, F Lipmann
PMCID: PMC397029  PMID: 2982151

Abstract

A simple and effective purification method for the src kinase, the transforming gene product of Rous sarcoma virus, has been developed by using affinity chromatography on casein-agarose and tyrosine-agarose columns. NaDodSO4/polyacrylamide gel electrophoresis and silver staining analysis showed that the purified kinase preparation was composed of a predominant polypeptide of 60,000-Da. In most of the preparations, however, three minor proteins (54,000, 52,000, and 15,000 Da) were also detected, and they were partially characterized. As one of the exogenous substrates, calmodulin was found to be phosphorylated on tyrosine by the purified src kinase.

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Selected References

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  1. Blithe D. L., Richert N. D., Pastan I. H. Purification of a tyrosine-specific protein kinase from Rous sarcoma virus-induced rat tumor. J Biol Chem. 1982 Jun 25;257(12):7135–7142. [PubMed] [Google Scholar]
  2. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  3. 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]
  4. 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]
  5. Cooper J. A., Reiss N. A., Schwartz R. J., Hunter T. Three glycolytic enzymes are phosphorylated at tyrosine in cells transformed by Rous sarcoma virus. Nature. 1983 Mar 17;302(5905):218–223. doi: 10.1038/302218a0. [DOI] [PubMed] [Google Scholar]
  6. Decker S., Lipmann F. Transport of D-glucose by membrane vesicles from normal and avian sarcoma virus-transformed chicken embryo fibroblasts. Proc Natl Acad Sci U S A. 1981 Sep;78(9):5358–5361. doi: 10.1073/pnas.78.9.5358. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Donner P., Bunte T., Owada M. K., Moelling K. Biochemical characterization of pp60src-associated protein kinase from avian sarcoma virus Schmidt-Ruppin strain. J Biol Chem. 1981 Aug 25;256(16):8786–8794. [PubMed] [Google Scholar]
  8. Erikson R. L., Collett M. S., Erikson E., Purchio A. F. Evidence that the avian sarcoma virus transforming gene product is a cyclic AMP-independent protein kinase. Proc Natl Acad Sci U S A. 1979 Dec;76(12):6260–6264. doi: 10.1073/pnas.76.12.6260. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Fukami Y., Lipmann F. Purification of a specific reversible tyrosine-O-phosphate phosphatase. Proc Natl Acad Sci U S A. 1982 Jul;79(14):4275–4279. doi: 10.1073/pnas.79.14.4275. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Fukami Y., Lipmann F. Reversal of Rous sarcoma-specific immunoglobulin phosphorylation on tyrosine (ADP as phosphate acceptor) catalyzed by the src gene kinase. Proc Natl Acad Sci U S A. 1983 Apr;80(7):1872–1876. doi: 10.1073/pnas.80.7.1872. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Gilmer T. M., Erikson R. L. Rous sarcoma virus transforming protein, p60src, expressed in E. coli, functions as a protein kinase. Nature. 1981 Dec 24;294(5843):771–773. doi: 10.1038/294771a0. [DOI] [PubMed] [Google Scholar]
  12. Glossmann H., Presek P., Eigenbrodt E. Association of the src-gene product of Rous sarcoma virus with a pyruvate-kinase inactivation factor. Mol Cell Endocrinol. 1981 Jul;23(1):49–63. doi: 10.1016/0303-7207(81)90116-7. [DOI] [PubMed] [Google Scholar]
  13. Hunter T., Sefton B. M. Transforming gene product of Rous sarcoma virus phosphorylates tyrosine. Proc Natl Acad Sci U S A. 1980 Mar;77(3):1311–1315. doi: 10.1073/pnas.77.3.1311. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Kakiuchi S., Sobue K., Yamazaki R., Kambayashi J., Sakon M., Kosaki G. Lack of tissue specificity of calmodulin: a rapid and high-yield purification method. FEBS Lett. 1981 Apr 20;126(2):203–207. doi: 10.1016/0014-5793(81)80242-6. [DOI] [PubMed] [Google Scholar]
  15. Klee C. B., Crouch T. H., Richman P. G. Calmodulin. Annu Rev Biochem. 1980;49:489–515. doi: 10.1146/annurev.bi.49.070180.002421. [DOI] [PubMed] [Google Scholar]
  16. 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]
  17. Levinson A. D., Oppermann H., Levintow L., Varmus H. E., Bishop J. M. Evidence that the transforming gene of avian sarcoma virus encodes a protein kinase associated with a phosphoprotein. Cell. 1978 Oct;15(2):561–572. doi: 10.1016/0092-8674(78)90024-7. [DOI] [PubMed] [Google Scholar]
  18. Levinson A. D., Oppermann H., Varmus H. E., Bishop J. M. The purified product of the transforming gene of avian sarcoma virus phosphorylates tyrosine. J Biol Chem. 1980 Dec 25;255(24):11973–11980. [PubMed] [Google Scholar]
  19. MITCHELL H. K., LUNAN K. D. TYROSINE-O-PHOSPHATE IN DROSOPHILA. Arch Biochem Biophys. 1964 Jul 20;106:219–222. doi: 10.1016/0003-9861(64)90179-1. [DOI] [PubMed] [Google Scholar]
  20. Maness P. F., Engeser H., Greenberg M. E., O'Farrell M., Gall W. E., Edelman G. M. Characterization of the protein kinase activity of avian sarcoma virus src gene product. Proc Natl Acad Sci U S A. 1979 Oct;76(10):5028–5032. doi: 10.1073/pnas.76.10.5028. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. McGrath J. P., Levinson A. D. Bacterial expression of an enzymatically active protein encoded by RSV src gene. Nature. 1982 Feb 4;295(5848):423–425. doi: 10.1038/295423a0. [DOI] [PubMed] [Google Scholar]
  22. Oakley B. R., Kirsch D. R., Morris N. R. A simplified ultrasensitive silver stain for detecting proteins in polyacrylamide gels. Anal Biochem. 1980 Jul 1;105(2):361–363. doi: 10.1016/0003-2697(80)90470-4. [DOI] [PubMed] [Google Scholar]
  23. 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]
  24. Purchio A. F. Evidence the pp60src, the product of the Rous sarcoma virus src gene, undergoes autophosphorylation. J Virol. 1982 Jan;41(1):1–7. doi: 10.1128/jvi.41.1.1-7.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Radke K., Martin G. S. Transformation by Rous sarcoma virus: effects of src gene expression on the synthesis and phosphorylation of cellular polypeptides. Proc Natl Acad Sci U S A. 1979 Oct;76(10):5212–5216. doi: 10.1073/pnas.76.10.5212. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. 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]
  27. 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]

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