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Mol Cell Biol. 1984 January; 4(1): 77–85.
PMCID: PMC368660

Biochemical characterization of a 34-kilodalton normal cellular substrate of pp60v-src and an associated 6-kilodalton protein.

Abstract

Transformation of fibroblasts by several retroviruses that produce transforming gene products associated with protein kinase activity results in the phosphorylation of a normal cellular protein with an Mr of 34,000 (the 34K protein). Evidence is presented here that, as extracted from chicken embryo fibroblasts, this protein exists in two forms that differ both in their elution from hydroxylapatite and in their native molecular weight. The form that eluted from hydroxylapatite at 210 to 295 mM potassium phosphate displayed a native molecular weight of 30,000 to 40,000, whereas the form that eluted at 320 to 440 mM displayed a native molecular weight of 60,000 to 70,000. The latter form copurified with a low-molecular-weight protein with an approximate Mr of 6,000 (6K). Both forms of 34K were completely separable from malate dehydrogenase activity. Phosphorylated 34K, isolated from Rous sarcoma virus-transformed cells, was also present in two forms; hence, in the cell neither form serves as a preferential substrate for pp60v-src. We found that the expression of 34K differed greatly in various avian tissues. In particular, it was present in the highest concentration in cultured fibroblasts and in very low concentration in brain tissue. Its expression in this tissue seems to be controlled at the level of transcription, since 34K mRNA in brain tissue was barely detectable. The expression of 6K was similar to that of 34K.

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Selected References

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  • Amini S, Kaji A. Association of pp36, a phosphorylated form of the presumed target protein for the src protein of Rous sarcoma virus, with the membrane of chicken cells transformed by Rous sarcoma virus. Proc Natl Acad Sci U S A. 1983 Feb;80(4):960–964. [PubMed]
  • Arrigo AP, Darlix JL, Spahr PF. A cellular protein phosphorylated by the avian sarcoma virus transforming gene product is associated with ribonucleoprotein particles. EMBO J. 1983;2(3):309–315. [PubMed]
  • Burnette WN. "Western blotting": electrophoretic transfer of proteins from sodium dodecyl sulfate--polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A. Anal Biochem. 1981 Apr;112(2):195–203. [PubMed]
  • Chamberlain JP. Fluorographic detection of radioactivity in polyacrylamide gels with the water-soluble fluor, sodium salicylate. Anal Biochem. 1979 Sep 15;98(1):132–135. [PubMed]
  • Collett MS, Erikson E, Erikson RL. Structural analysis of the avian sarcoma virus transforming protein: sites of phosphorylation. J Virol. 1979 Feb;29(2):770–781. [PMC free article] [PubMed]
  • Cooper JA, Hunter T. Changes in protein phosphorylation in Rous sarcoma virus-transformed chicken embryo cells. Mol Cell Biol. 1981 Feb;1(2):165–178. [PMC free article] [PubMed]
  • Cooper JA, Hunter T. Discrete primary locations of a tyrosine-protein kinase and of three proteins that contain phosphotyrosine in virally transformed chick fibroblasts. J Cell Biol. 1982 Aug;94(2):287–296. [PMC free article] [PubMed]
  • Cooper JA, Hunter T. Identification and characterization of cellular targets for tyrosine protein kinases. J Biol Chem. 1983 Jan 25;258(2):1108–1115. [PubMed]
  • Cooper J, Nakamura KD, Hunter T, Weber MJ. Phosphotyrosine-containing proteins and expression of transformation parameters in cells infected with partial transformation mutants of Rous sarcoma virus. J Virol. 1983 Apr;46(1):15–28. [PMC free article] [PubMed]
  • Cooper JA, Reiss NA, Schwartz RJ, Hunter T. Three glycolytic enzymes are phosphorylated at tyrosine in cells transformed by Rous sarcoma virus. Nature. 1983 Mar 17;302(5905):218–223. [PubMed]
  • Courtneidge S, Ralston R, Alitalo K, Bishop JM. Subcellular location of an abundant substrate (p36) for tyrosine-specific protein kinases. Mol Cell Biol. 1983 Mar;3(3):340–350. [PMC free article] [PubMed]
  • Erikson E, Collett MS, Erikson RL. In vitro synthesis of a functional avian sarcoma virus transforming-gene product. Nature. 1978 Aug 31;274(5674):919–921. [PubMed]
  • Erikson E, Cook R, Miller GJ, Erikson RL. The same normal cell protein is phosphorylated after transformation by avian sarcoma viruses with unrelated transforming genes. Mol Cell Biol. 1981 Jan;1(1):43–50. [PMC free article] [PubMed]
  • Erikson E, Erikson RL. Identification of a cellular protein substrate phosphorylated by the avian sarcoma virus-transforming gene product. Cell. 1980 Oct;21(3):829–836. [PubMed]
  • Erikson RL. Studies on the RNA from avian myeloblastosis virus. Virology. 1969 Jan;37(1):124–131. [PubMed]
  • Erikson RL, Purchio AF, Erikson E, Collett MS, Brugge JS. Molecular events in cells transformed by Rous Sarcoma virus. J Cell Biol. 1980 Nov;87(2 Pt 1):319–325. [PMC free article] [PubMed]
  • Greenberg ME, Edelman GM. Comparison of the 34,000-Da pp60src substrate and a 38,000-Da phosphoprotein identified by monoclonal antibodies. J Biol Chem. 1983 Jul 10;258(13):8497–8502. [PubMed]
  • Greenberg ME, Edelman GM. The 34 kd pp60src substrate is located at the inner face of the plasma membrane. Cell. 1983 Jul;33(3):767–779. [PubMed]
  • Hunter T. Protein phosphorylated by the RSV transforming function. Cell. 1980 Dec;22(3):647–648. [PubMed]
  • Kessler SW. Rapid isolation of antigens from cells with a staphylococcal protein A-antibody adsorbent: parameters of the interaction of antibody-antigen complexes with protein A. J Immunol. 1975 Dec;115(6):1617–1624. [PubMed]
  • Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. [PubMed]
  • LOWRY OH, ROSEBROUGH NJ, FARR AL, RANDALL RJ. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed]
  • Martinez R, Nakamura KD, Weber MJ. Identification of phosphotyrosine-containing proteins in untransformed and Rous sarcoma virus-transformed chicken embryo fibroblasts. Mol Cell Biol. 1982 Jun;2(6):653–665. [PMC free article] [PubMed]
  • Nigg EA, Cooper JA, Hunter T. Immunofluorescent localization of a 39,000-dalton substrate of tyrosine protein kinases to the cytoplasmic surface of the plasma membrane. J Cell Biol. 1983 Jun;96(6):1601–1609. [PMC free article] [PubMed]
  • Oakley BR, Kirsch DR, Morris NR. A simplified ultrasensitive silver stain for detecting proteins in polyacrylamide gels. Anal Biochem. 1980 Jul 1;105(2):361–363. [PubMed]
  • O'Farrell PZ, Goodman HM, O'Farrell PH. High resolution two-dimensional electrophoresis of basic as well as acidic proteins. Cell. 1977 Dec;12(4):1133–1141. [PubMed]
  • Pelham HR, Jackson RJ. An efficient mRNA-dependent translation system from reticulocyte lysates. Eur J Biochem. 1976 Aug 1;67(1):247–256. [PubMed]
  • Radke K, Gilmore T, Martin GS. Transformation by Rous sarcoma virus: a cellular substrate for transformation-specific protein phosphorylation contains phosphotyrosine. Cell. 1980 Oct;21(3):821–828. [PubMed]
  • Radke K, Martin GS. 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. [PubMed]
  • Rübsamen H, Saltenberger K, Friis RR, Eigenbrodt E. Cytosolic malic dehydrogenase activity is associated with a putative substrate for the transforming gene product of Rous sarcoma virus. Proc Natl Acad Sci U S A. 1982 Jan;79(2):228–232. [PubMed]
  • Sefton BM, Hunter T, Ball EH, Singer SJ. Vinculin: a cytoskeletal target of the transforming protein of Rous sarcoma virus. Cell. 1981 Apr;24(1):165–174. [PubMed]
  • Sefton BM, Hunter T, Cooper JA. Some lymphoid cell lines transformed by Abelson murine leukemia virus lack a major 36,000-dalton tyrosine protein kinase substrate. Mol Cell Biol. 1983 Jan;3(1):56–63. [PMC free article] [PubMed]

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