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. 1990 Aug;64(8):3581–3589. doi: 10.1128/jvi.64.8.3581-3589.1990

Mutagenic analysis of the v-crk oncogene: requirement for SH2 and SH3 domains and correlation between increased cellular phosphotyrosine and transformation.

B J Mayer 1, H Hanafusa 1
PMCID: PMC249650  PMID: 1695251

Abstract

We have constructed a series of mutants with deletion, linker insertion, and point mutations in the v-crk oncogene of avian sarcoma virus CT10. The v-crk gene contains no apparent catalytic domain, but does contain two blocks of homology to putative regulatory domains, termed SH2 and SH3, found in a variety of proteins implicated in signal transduction. Infection with CT10 causes a dramatic increase in the level of tyrosine phosphorylation of several cellular proteins. We found that mutation of either the SH2 or SH3 domain of v-crk reduced or eliminated transforming activity, whereas mutation of regions outside the conserved domains had no effect. Deletion of amino-terminal gag sequences caused a partial loss of transforming activity and a change in subcellular distribution of the crk protein. In all cases, there was an absolute correlation between increased cellular phosphotyrosine and 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. Bristol A., Hall S. M., Kriz R. W., Stahl M. L., Fan Y. S., Byers M. G., Eddy R. L., Shows T. B., Knopf J. L. Phospholipase C-148: chromosomal location and deletion mapping of functional domains. Cold Spring Harb Symp Quant Biol. 1988;53(Pt 2):915–920. doi: 10.1101/sqb.1988.053.01.105. [DOI] [PubMed] [Google Scholar]
  2. Broek D., Toda T., Michaeli T., Levin L., Birchmeier C., Zoller M., Powers S., Wigler M. The S. cerevisiae CDC25 gene product regulates the RAS/adenylate cyclase pathway. Cell. 1987 Mar 13;48(5):789–799. doi: 10.1016/0092-8674(87)90076-6. [DOI] [PubMed] [Google Scholar]
  3. Brugge J. S., Darrow D. Analysis of the catalytic domain of phosphotransferase activity of two avian sarcoma virus-transforming proteins. J Biol Chem. 1984 Apr 10;259(7):4550–4557. [PubMed] [Google Scholar]
  4. Cooper J. A., Hunter T. Changes in protein phosphorylation in Rous sarcoma virus-transformed chicken embryo cells. Mol Cell Biol. 1981 Feb;1(2):165–178. doi: 10.1128/mcb.1.2.165. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Cross F. R., Garber E. A., Hanafusa H. N-terminal deletions in Rous sarcoma virus p60src: effects on tyrosine kinase and biological activities and on recombination in tissue culture with the cellular src gene. Mol Cell Biol. 1985 Oct;5(10):2789–2795. doi: 10.1128/mcb.5.10.2789. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Cross F. R., Hanafusa H. Local mutagenesis of Rous sarcoma virus: the major sites of tyrosine and serine phosphorylation of pp60src are dispensable for transformation. Cell. 1983 Sep;34(2):597–607. doi: 10.1016/0092-8674(83)90392-6. [DOI] [PubMed] [Google Scholar]
  7. Daley G. Q., McLaughlin J., Witte O. N., Baltimore D. The CML-specific P210 bcr/abl protein, unlike v-abl, does not transform NIH/3T3 fibroblasts. Science. 1987 Jul 31;237(4814):532–535. doi: 10.1126/science.2440107. [DOI] [PubMed] [Google Scholar]
  8. Drubin D. G., Mulholland J., Zhu Z. M., Botstein D. Homology of a yeast actin-binding protein to signal transduction proteins and myosin-I. Nature. 1990 Jan 18;343(6255):288–290. doi: 10.1038/343288a0. [DOI] [PubMed] [Google Scholar]
  9. Emori Y., Homma Y., Sorimachi H., Kawasaki H., Nakanishi O., Suzuki K., Takenawa T. A second type of rat phosphoinositide-specific phospholipase C containing a src-related sequence not essential for phosphoinositide-hydrolyzing activity. J Biol Chem. 1989 Dec 25;264(36):21885–21890. [PubMed] [Google Scholar]
  10. Foster D. A., Shibuya M., Hanafusa H. Activation of the transformation potential of the cellular fps gene. Cell. 1985 Aug;42(1):105–115. doi: 10.1016/s0092-8674(85)80106-9. [DOI] [PubMed] [Google Scholar]
  11. Hamaguchi M., Grandori C., Hanafusa H. Phosphorylation of cellular proteins in Rous sarcoma virus-infected cells: analysis by use of anti-phosphotyrosine antibodies. Mol Cell Biol. 1988 Aug;8(8):3035–3042. doi: 10.1128/mcb.8.8.3035. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Hamaguchi M., Hanafusa H. Localization of major potential substrates of p60v-src kinase in the plasma membrane matrix fraction. Oncogene Res. 1989;4(1):29–37. [PubMed] [Google Scholar]
  13. Hanafusa H. Rapid transformation of cells by Rous sarcoma virus. Proc Natl Acad Sci U S A. 1969 Jun;63(2):318–325. doi: 10.1073/pnas.63.2.318. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Iba H., Takeya T., Cross F. R., Hanafusa T., Hanafusa H. Rous sarcoma virus variants that carry the cellular src gene instead of the viral src gene cannot transform chicken embryo fibroblasts. Proc Natl Acad Sci U S A. 1984 Jul;81(14):4424–4428. doi: 10.1073/pnas.81.14.4424. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Kornbluth S., Cross F. R., Harbison M., Hanafusa H. Transformation of chicken embryo fibroblasts and tumor induction by the middle T antigen of polyomavirus carried in an avian retroviral vector. Mol Cell Biol. 1986 May;6(5):1545–1551. doi: 10.1128/mcb.6.5.1545. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Lomax K. J., Leto T. L., Nunoi H., Gallin J. I., Malech H. L. Recombinant 47-kilodalton cytosol factor restores NADPH oxidase in chronic granulomatous disease. Science. 1989 Jul 28;245(4916):409–412. doi: 10.1126/science.2547247. [DOI] [PubMed] [Google Scholar]
  17. Marshall M. S., Hill W. S., Ng A. S., Vogel U. S., Schaber M. D., Scolnick E. M., Dixon R. A., Sigal I. S., Gibbs J. B. A C-terminal domain of GAP is sufficient to stimulate ras p21 GTPase activity. EMBO J. 1989 Apr;8(4):1105–1110. doi: 10.1002/j.1460-2075.1989.tb03480.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Mayer B. J., Hamaguchi M., Hanafusa H. A novel viral oncogene with structural similarity to phospholipase C. Nature. 1988 Mar 17;332(6161):272–275. doi: 10.1038/332272a0. [DOI] [PubMed] [Google Scholar]
  19. Mayer B. J., Hamaguchi M., Hanafusa H. Characterization of p47gag-crk, a novel oncogene product with sequence similarity to a putative modulatory domain of protein-tyrosine kinases and phospholipase C. Cold Spring Harb Symp Quant Biol. 1988;53(Pt 2):907–914. doi: 10.1101/sqb.1988.053.01.104. [DOI] [PubMed] [Google Scholar]
  20. Mayer B. J., Hanafusa H. Association of the v-crk oncogene product with phosphotyrosine-containing proteins and protein kinase activity. Proc Natl Acad Sci U S A. 1990 Apr;87(7):2638–2642. doi: 10.1073/pnas.87.7.2638. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Neckameyer W. S., Wang L. H. Molecular cloning and characterization of avian sarcoma virus UR2 and comparison of its transforming sequence with those of other avian sarcoma viruses. J Virol. 1984 Jun;50(3):914–921. doi: 10.1128/jvi.50.3.914-921.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. 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]
  23. Pawson T. Non-catalytic domains of cytoplasmic protein-tyrosine kinases: regulatory elements in signal transduction. Oncogene. 1988 Nov;3(5):491–495. [PubMed] [Google Scholar]
  24. 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]
  25. Prywes R., Hoag J., Rosenberg N., Baltimore D. Protein stabilization explains the gag requirement for transformation of lymphoid cells by Abelson murine leukemia virus. J Virol. 1985 Apr;54(1):123–132. doi: 10.1128/jvi.54.1.123-132.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Rodaway A. R., Sternberg M. J., Bentley D. L. Similarity in membrane proteins. Nature. 1989 Dec 7;342(6250):624–624. doi: 10.1038/342624a0. [DOI] [PubMed] [Google Scholar]
  27. Sadowski I., Stone J. C., Pawson T. A noncatalytic domain conserved among cytoplasmic protein-tyrosine kinases modifies the kinase function and transforming activity of Fujinami sarcoma virus P130gag-fps. Mol Cell Biol. 1986 Dec;6(12):4396–4408. doi: 10.1128/mcb.6.12.4396. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Stahl M. L., Ferenz C. R., Kelleher K. L., Kriz R. W., Knopf J. L. Sequence similarity of phospholipase C with the non-catalytic region of src. Nature. 1988 Mar 17;332(6161):269–272. doi: 10.1038/332269a0. [DOI] [PubMed] [Google Scholar]
  29. Suh P. G., Ryu S. H., Moon K. H., Suh H. W., Rhee S. G. Inositol phospholipid-specific phospholipase C: complete cDNA and protein sequences and sequence homology to tyrosine kinase-related oncogene products. Proc Natl Acad Sci U S A. 1988 Aug;85(15):5419–5423. doi: 10.1073/pnas.85.15.5419. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Towler D. A., Gordon J. I., Adams S. P., Glaser L. The biology and enzymology of eukaryotic protein acylation. Annu Rev Biochem. 1988;57:69–99. doi: 10.1146/annurev.bi.57.070188.000441. [DOI] [PubMed] [Google Scholar]
  31. Trahey M., Wong G., Halenbeck R., Rubinfeld B., Martin G. A., Ladner M., Long C. M., Crosier W. J., Watt K., Koths K. Molecular cloning of two types of GAP complementary DNA from human placenta. Science. 1988 Dec 23;242(4886):1697–1700. doi: 10.1126/science.3201259. [DOI] [PubMed] [Google Scholar]
  32. Tsuchie H., Chang C. H., Yoshida M., Vogt P. K. A newly isolated avian sarcoma virus, ASV-1, carries the crk oncogene. Oncogene. 1989 Nov;4(11):1281–1284. [PubMed] [Google Scholar]
  33. Vogel U. S., Dixon R. A., Schaber M. D., Diehl R. E., Marshall M. S., Scolnick E. M., Sigal I. S., Gibbs J. B. Cloning of bovine GAP and its interaction with oncogenic ras p21. Nature. 1988 Sep 1;335(6185):90–93. doi: 10.1038/335090a0. [DOI] [PubMed] [Google Scholar]
  34. 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]
  35. Wigler M., Pellicer A., Silverstein S., Axel R., Urlaub G., Chasin L. DNA-mediated transfer of the adenine phosphoribosyltransferase locus into mammalian cells. Proc Natl Acad Sci U S A. 1979 Mar;76(3):1373–1376. doi: 10.1073/pnas.76.3.1373. [DOI] [PMC free article] [PubMed] [Google Scholar]

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