Skip to main content
NCBI home page
Journal of Virology logoLink to Journal of Virology
. 1992 Jul;66(7):4315–4324. doi: 10.1128/jvi.66.7.4315-4324.1992

Host range mutants of v-src: alterations in kinase activity and substrate interactions.

E C Liebl 1, L J England 1, J E DeClue 1, G S Martin 1
PMCID: PMC241237  PMID: 1534851

Abstract

Host range mutants of Schmidt-Ruppin v-src that transform chicken embryo fibroblasts (CEF) but not Rat-2 cells were generated previously by linker insertion-deletion mutagenesis (J. E. DeClue and G. S. Martin, J. Virol. 63:542-554, 1989). One of these mutants, SRX5, in which Tyr-416 is substituted by the sequence Ser Arg Asp, retained high levels of kinase activity in vitro and in vivo, both in CEF and in Rat-2 cells. Phosphorylation of p36 (the calpactin I heavy chain) was drastically reduced in cells expressing SRX5 src, suggesting that the phenotype of SRX5 results from an alteration in substrate recognition by the src kinase. Three mutants, SPX1, SHX13, and XD6, containing linker insertions or small deletions within the src homology 2 (SH2) region, induced reduced levels of kinase activity in both CEF and Rat-2 cells. However, the residual levels of kinase activity in Rat-2 cells were above the threshold at which wild-type pp60v-src transforms Rat-2 cells, indicating that the reduction in kinase activity was not sufficient to account for the failure to transform. Cells infected by these mutants exhibited reduced levels of phosphorylation of 120- and 62-kDa proteins. We have reported elsewhere (M. F. Moran, C. A. Koch, D. Anderson, C. Ellis, L. England, G. S. Martin, and T. Pawson, Proc. Natl. Acad. Sci. USA 87:8622-8626, 1990) that ras GTPase-activating protein GAP and associated protein p62 are not tyrosine phosphorylated in Rat-2 cells expressing SHX13 or XD6. The transformation defect in Rat-2 cells may result from the failure to phosphorylate those proteins. The fifth mutant, XD4, contains a deletion which removes all of the src homology 3 (SH3) and most of the SH2 sequences of src. The protein encoded by XD4 is active as a kinase when expressed in CEF, indicating that in CEF the SH2 and SH3 regions of v-src are not necessary for kinase activity and transformation. The XD4 src product is not tyrosine phosphorylated and is inactive as a kinase when expressed in Rat-2 cells. Thus, host cell factors can affect the tyrosine phosphorylation and activity of the v-src kinase in the absence of the SH2 and SH3 regions. These results indicate that the host-dependent transformation phenotype results from alterations in src kinase activity and substrate specificity.

Full text

PDF
4315

Images in this article

4317Image
on p.4317
4319Image
on p.4319
4320Image
on p.4320
4321Image
on p.4321
4321Image
on p.4321

Selected References

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

  1. BRYAN W. R. Enhancement of virus yield of Rous sarcomas. Acta Unio Int Contra Cancrum. 1959;15:764–767. [PubMed] [Google Scholar]
  2. Bouton A. H., Kanner S. B., Vines R. R., Wang H. C., Gibbs J. B., Parsons J. T. Transformation by pp60src or stimulation of cells with epidermal growth factor induces the stable association of tyrosine-phosphorylated cellular proteins with GTPase-activating protein. Mol Cell Biol. 1991 Feb;11(2):945–953. doi: 10.1128/mcb.11.2.945. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bryant D., Parsons J. T. Site-directed mutagenesis of the src gene of Rous sarcoma virus: construction and characterization of a deletion mutant temperature sensitive for transformation. J Virol. 1982 Nov;44(2):683–691. doi: 10.1128/jvi.44.2.683-691.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Cantley L. C., Auger K. R., Carpenter C., Duckworth B., Graziani A., Kapeller R., Soltoff S. Oncogenes and signal transduction. Cell. 1991 Jan 25;64(2):281–302. doi: 10.1016/0092-8674(91)90639-g. [DOI] [PubMed] [Google Scholar]
  5. Cooper J. A., Hunter T. Regulation of cell growth and transformation by tyrosine-specific protein kinases: the search for important cellular substrate proteins. Curr Top Microbiol Immunol. 1983;107:125–161. doi: 10.1007/978-3-642-69075-4_4. [DOI] [PubMed] [Google Scholar]
  6. Cooper J., Nakamura K. D., Hunter T., Weber M. J. 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. doi: 10.1128/jvi.46.1.15-28.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Davis S., Lu M. L., Lo S. H., Lin S., Butler J. A., Druker B. J., Roberts T. M., An Q., Chen L. B. Presence of an SH2 domain in the actin-binding protein tensin. Science. 1991 May 3;252(5006):712–715. doi: 10.1126/science.1708917. [DOI] [PubMed] [Google Scholar]
  8. DeClue J. E., Martin G. S. Linker insertion-deletion mutagenesis of the v-src gene: isolation of host- and temperature-dependent mutants. J Virol. 1989 Feb;63(2):542–554. doi: 10.1128/jvi.63.2.542-554.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. DeClue J. E., Sadowski I., Martin G. S., Pawson T. A conserved domain regulates interactions of the v-fps protein-tyrosine kinase with the host cell. Proc Natl Acad Sci U S A. 1987 Dec;84(24):9064–9068. doi: 10.1073/pnas.84.24.9064. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Ellis C., Moran M., McCormick F., Pawson T. Phosphorylation of GAP and GAP-associated proteins by transforming and mitogenic tyrosine kinases. Nature. 1990 Jan 25;343(6256):377–381. doi: 10.1038/343377a0. [DOI] [PubMed] [Google Scholar]
  11. Ferrell J. E., Jr, Martin G. S. Identification of a 42-kilodalton phosphotyrosyl protein as a serine(threonine) protein kinase by renaturation. Mol Cell Biol. 1990 Jun;10(6):3020–3026. doi: 10.1128/mcb.10.6.3020. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Ferrell J. E., Jr, Martin G. S. Tyrosine-specific protein phosphorylation is regulated by glycoprotein IIb-IIIa in platelets. Proc Natl Acad Sci U S A. 1989 Apr;86(7):2234–2238. doi: 10.1073/pnas.86.7.2234. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Foster R., Martin G. S. A mutation in the catalytic domain of pp60v-src is responsible for the host- and temperature-dependent phenotype of the Rous sarcoma virus mutant tsLA33-1. Virology. 1992 Mar;187(1):145–155. doi: 10.1016/0042-6822(92)90303-7. [DOI] [PubMed] [Google Scholar]
  14. Fukui Y., O'Brien M. C., Hanafusa H. Deletions in the SH2 domain of p60v-src prevent association with the detergent-insoluble cellular matrix. Mol Cell Biol. 1991 Mar;11(3):1207–1213. doi: 10.1128/mcb.11.3.1207. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Hirai H., Varmus H. E. Mutations in src homology regions 2 and 3 of activated chicken c-src that result in preferential transformation of mouse or chicken cells. Proc Natl Acad Sci U S A. 1990 Nov;87(21):8592–8596. doi: 10.1073/pnas.87.21.8592. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Hirai H., Varmus H. E. Site-directed mutagenesis of the SH2- and SH3-coding domains of c-src produces varied phenotypes, including oncogenic activation of p60c-src. Mol Cell Biol. 1990 Apr;10(4):1307–1318. doi: 10.1128/mcb.10.4.1307. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Hughes S. H., Greenhouse J. J., Petropoulos C. J., Sutrave P. Adaptor plasmids simplify the insertion of foreign DNA into helper-independent retroviral vectors. J Virol. 1987 Oct;61(10):3004–3012. doi: 10.1128/jvi.61.10.3004-3012.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Hunter E. Biological techniques for avian sarcoma viruses. Methods Enzymol. 1979;58:379–393. doi: 10.1016/s0076-6879(79)58153-1. [DOI] [PubMed] [Google Scholar]
  19. Hunter T. Cooperation between oncogenes. Cell. 1991 Jan 25;64(2):249–270. doi: 10.1016/0092-8674(91)90637-e. [DOI] [PubMed] [Google Scholar]
  20. Jakobovits E. B., Majors J. E., Varmus H. E. Hormonal regulation of the Rous sarcoma virus src gene via a heterologous promoter defines a threshold dose for cellular transformation. Cell. 1984 Oct;38(3):757–765. doi: 10.1016/0092-8674(84)90271-x. [DOI] [PubMed] [Google Scholar]
  21. Jhappan C., Vande Woude G. F., Robins T. S. Transduction of host cellular sequences by a retroviral shuttle vector. J Virol. 1986 Nov;60(2):750–753. doi: 10.1128/jvi.60.2.750-753.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Kamps M. P., Sefton B. M. Identification of multiple novel polypeptide substrates of the v-src, v-yes, v-fps, v-ros, and v-erb-B oncogenic tyrosine protein kinases utilizing antisera against phosphotyrosine. Oncogene. 1988 Apr;2(4):305–315. [PubMed] [Google Scholar]
  23. Kanner S. B., Reynolds A. B., Wang H. C., Vines R. R., Parsons J. T. The SH2 and SH3 domains of pp60src direct stable association with tyrosine phosphorylated proteins p130 and p110. EMBO J. 1991 Jul;10(7):1689–1698. doi: 10.1002/j.1460-2075.1991.tb07693.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Kawai S., Nishizawa M. New procedure for DNA transfection with polycation and dimethyl sulfoxide. Mol Cell Biol. 1984 Jun;4(6):1172–1174. doi: 10.1128/mcb.4.6.1172. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Kmiecik T. E., Shalloway D. Activation and suppression of pp60c-src transforming ability by mutation of its primary sites of tyrosine phosphorylation. Cell. 1987 Apr 10;49(1):65–73. doi: 10.1016/0092-8674(87)90756-2. [DOI] [PubMed] [Google Scholar]
  26. Knighton D. R., Zheng J. H., Ten Eyck L. F., Ashford V. A., Xuong N. H., Taylor S. S., Sowadski J. M. Crystal structure of the catalytic subunit of cyclic adenosine monophosphate-dependent protein kinase. Science. 1991 Jul 26;253(5018):407–414. doi: 10.1126/science.1862342. [DOI] [PubMed] [Google Scholar]
  27. Knighton D. R., Zheng J. H., Ten Eyck L. F., Xuong N. H., Taylor S. S., Sowadski J. M. Structure of a peptide inhibitor bound to the catalytic subunit of cyclic adenosine monophosphate-dependent protein kinase. Science. 1991 Jul 26;253(5018):414–420. doi: 10.1126/science.1862343. [DOI] [PubMed] [Google Scholar]
  28. Kypta R. M., Goldberg Y., Ulug E. T., Courtneidge S. A. Association between the PDGF receptor and members of the src family of tyrosine kinases. Cell. 1990 Aug 10;62(3):481–492. doi: 10.1016/0092-8674(90)90013-5. [DOI] [PubMed] [Google Scholar]
  29. Mann R., Mulligan R. C., Baltimore D. Construction of a retrovirus packaging mutant and its use to produce helper-free defective retrovirus. Cell. 1983 May;33(1):153–159. doi: 10.1016/0092-8674(83)90344-6. [DOI] [PubMed] [Google Scholar]
  30. Moran M. F., Koch C. A., Anderson D., Ellis C., England L., Martin G. S., Pawson T. Src homology region 2 domains direct protein-protein interactions in signal transduction. Proc Natl Acad Sci U S A. 1990 Nov;87(21):8622–8626. doi: 10.1073/pnas.87.21.8622. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Parsons J. T., Weber M. J. Genetics of src: structure and functional organization of a protein tyrosine kinase. Curr Top Microbiol Immunol. 1989;147:79–127. doi: 10.1007/978-3-642-74697-0_3. [DOI] [PubMed] [Google Scholar]
  32. 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]
  33. 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]
  34. Radke K., Gilmore T., Martin G. S. Transformation by Rous sarcoma virus: a cellular substrate for transformation-specific protein phosphorylation contains phosphotyrosine. Cell. 1980 Oct;21(3):821–828. doi: 10.1016/0092-8674(80)90445-6. [DOI] [PubMed] [Google Scholar]
  35. Raymond V. W., Parsons J. T. Identification of an amino terminal domain required for the transforming activity of the Rous sarcoma virus src protein. Virology. 1987 Oct;160(2):400–410. doi: 10.1016/0042-6822(87)90011-0. [DOI] [PubMed] [Google Scholar]
  36. Schieven G., Martin G. S. Nonenzymatic phosphorylation of tyrosine and serine by ATP is catalyzed by manganese but not magnesium. J Biol Chem. 1988 Oct 25;263(30):15590–15593. [PubMed] [Google Scholar]
  37. Snyder M. A., Bishop J. M., Colby W. W., Levinson A. D. Phosphorylation of tyrosine-416 is not required for the transforming properties and kinase activity of pp60v-src. Cell. 1983 Mar;32(3):891–901. doi: 10.1016/0092-8674(83)90074-0. [DOI] [PubMed] [Google Scholar]
  38. Sudol M., Lerner T. L., Hanafusa H. Polymerase-defective mutant of the Bryan high-titer strain of Rous sarcoma virus. Nucleic Acids Res. 1986 Mar 11;14(5):2391–2405. doi: 10.1093/nar/14.5.2391. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Topp W. C. Normal rat cell lines deficient in nuclear thymidine kinase. Virology. 1981 Aug;113(1):408–411. doi: 10.1016/0042-6822(81)90168-9. [DOI] [PubMed] [Google Scholar]
  40. Verderame M. F., Kaplan J. M., Varmus H. E. A mutation in v-src that removes a single conserved residue in the SH-2 domain of pp60v-src restricts transformation in a host-dependent manner. J Virol. 1989 Jan;63(1):338–348. doi: 10.1128/jvi.63.1.338-348.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Wendler P. A., Boschelli F. Src homology 2 domain deletion mutants of p60v-src do not phosphorylate cellular proteins of 120-150 kDa. Oncogene. 1989 Feb;4(2):231–236. [PubMed] [Google Scholar]
  42. Young J. C., Liebl E., Martin G. S. A host-dependent temperature-sensitive mutant of Rous sarcoma virus: evidence for host factors affecting transformation. Virology. 1988 Oct;166(2):561–572. doi: 10.1016/0042-6822(88)90527-2. [DOI] [PubMed] [Google Scholar]

Articles from Journal of Virology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES