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. 1990 Jul;64(7):3337–3349. doi: 10.1128/jvi.64.7.3337-3349.1990

Delineation of functional determinants in the transforming protein of Fujinami sarcoma virus.

K A Johnson 1, J C Stone 1
PMCID: PMC249574  PMID: 2352326

Abstract

We analyzed linker insertion mutations throughout the 3' region of the v-fps gene of Fujinami sarcoma virus to identify tyrosine kinase transforming protein (P130gag-fps) determinants that are important for catalysis and transforming activity and, in particular, to define residues that participate in substrate selection. Mutations that encode kinase-active, transformation-defective v-fps alleles were recovered, defining sites in the transforming protein that may normally facilitate kinase-substrate interaction. Additionally, one region within the catalytic domain of the transforming protein (amino acid residues 1012 to 1020) that tolerates peptide insertions without loss of transforming activity was discovered, although the insertion mutations in this region of v-fps exhibited qualitatively abnormal transforming function. Transformed rat cell lines that express these mutations displayed unusual phenotypes, including giant cells and cells with an extremely fusiform shape. Furthermore, the insertion mutations in this region were temperature sensitive, transformed cells assumed a flat morphology, cellular protein phosphotyrosine was reduced, and the kinase activity of the transforming protein was decreased when cells were incubated at 40.5 degrees C. Point mutations that specify the ancestral chicken c-fps sequence in the insertion-tolerant region were also introduced into v-fps. These back mutations led to a modest decrease in kinase activity, decreased tumorigenic potential in chickens, and an unexpected increase in transforming activity in rat cells. These results indicate that the insertion-tolerant region of P130gag-fps influences the biologic activity and thermostability of the kinase.

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Selected References

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

  1. Anderson D. D., Beckmann R. P., Harms E. H., Nakamura K., Weber M. J. Biological properties of "partial" transformation mutants of Rous sarcoma virus and characterization of their pp60src kinase. J Virol. 1981 Jan;37(1):445–458. doi: 10.1128/jvi.37.1.445-458.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Ariizumi K., Shibuya M. Construction and biological analysis of deletion mutants of Fujinami sarcoma virus: 5'-fps sequence has a role in the transforming activity. J Virol. 1985 Sep;55(3):660–669. doi: 10.1128/jvi.55.3.660-669.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Besmer P., Murphy J. E., George P. C., Qiu F. H., Bergold P. J., Lederman L., Snyder H. W., Jr, Brodeur D., Zuckerman E. E., Hardy W. D. A new acute transforming feline retrovirus and relationship of its oncogene v-kit with the protein kinase gene family. Nature. 1986 Apr 3;320(6061):415–421. doi: 10.1038/320415a0. [DOI] [PubMed] [Google Scholar]
  4. Blair D. G., Oskarsson M. K., Seth A., Dunn K. J., Dean M., Zweig M., Tainsky M. A., Vande Woude G. F. Analysis of the transforming potential of the human homolog of mos. Cell. 1986 Aug 29;46(5):785–794. doi: 10.1016/0092-8674(86)90354-5. [DOI] [PubMed] [Google Scholar]
  5. Brooks-Wilson A. R., Ball E., Pawson T. The myristylation signal of p60v-src functionally complements the N-terminal fps-specific region of P130gag-fps. Mol Cell Biol. 1989 May;9(5):2214–2219. doi: 10.1128/mcb.9.5.2214. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Chen L. H., Hatada E., Wheatley W., Lee W. H. Single amino acid substitution, from Glu1025 to Asp, of the fps oncogenic protein causes temperature sensitivity in transformation and kinase activity. Virology. 1986 Nov;155(1):106–119. doi: 10.1016/0042-6822(86)90172-8. [DOI] [PubMed] [Google Scholar]
  7. 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]
  8. Coussens P. M., Cooper J. A., Hunter T., Shalloway D. Restriction of the in vitro and in vivo tyrosine protein kinase activities of pp60c-src relative to pp60v-src. Mol Cell Biol. 1985 Oct;5(10):2753–2763. doi: 10.1128/mcb.5.10.2753. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. 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]
  10. 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]
  11. Enquist L. W., Vande Woude G. F., Wagner M., Smiley J. R., Summers W. C. Construction and characterization of a recombinant plasmid encoding the gene for the thymidine kinase of Herpes simplex type 1 virus. Gene. 1979 Nov;7(3-4):335–342. doi: 10.1016/0378-1119(79)90052-0. [DOI] [PubMed] [Google Scholar]
  12. Escobedo J. A., Williams L. T. A PDGF receptor domain essential for mitogenesis but not for many other responses to PDGF. Nature. 1988 Sep 1;335(6185):85–87. doi: 10.1038/335085a0. [DOI] [PubMed] [Google Scholar]
  13. Foster D. A., Hanafusa H. A fps gene without gag gene sequences transforms cells in culture and induces tumors in chickens. J Virol. 1983 Dec;48(3):744–751. doi: 10.1128/jvi.48.3.744-751.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Gluzman Y. SV40-transformed simian cells support the replication of early SV40 mutants. Cell. 1981 Jan;23(1):175–182. doi: 10.1016/0092-8674(81)90282-8. [DOI] [PubMed] [Google Scholar]
  15. Hampe A., Gobet M., Sherr C. J., Galibert F. Nucleotide sequence of the feline retroviral oncogene v-fms shows unexpected homology with oncogenes encoding tyrosine-specific protein kinases. Proc Natl Acad Sci U S A. 1984 Jan;81(1):85–89. doi: 10.1073/pnas.81.1.85. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Hanks S. K., Quinn A. M., Hunter T. The protein kinase family: conserved features and deduced phylogeny of the catalytic domains. Science. 1988 Jul 1;241(4861):42–52. doi: 10.1126/science.3291115. [DOI] [PubMed] [Google Scholar]
  17. Huang C. C., Hammond C., Bishop J. M. Nucleotide sequence and topography of chicken c-fps. Genesis of a retroviral oncogene encoding a tyrosine-specific protein kinase. J Mol Biol. 1985 Jan 20;181(2):175–186. doi: 10.1016/0022-2836(85)90083-x. [DOI] [PubMed] [Google Scholar]
  18. 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]
  19. Ingman-Baker J., Hinze E., Levy J. G., Pawson T. Monoclonal antibodies to the transforming protein of Fujinami avian sarcoma virus discriminate between different fps-encoded proteins. J Virol. 1984 May;50(2):572–578. doi: 10.1128/jvi.50.2.572-578.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. 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]
  21. Kipreos E. T., Lee G. J., Wang J. Y. Isolation of temperature-sensitive tyrosine kinase mutants of v-abl oncogene by screening with antibodies for phosphotyrosine. Proc Natl Acad Sci U S A. 1987 Mar;84(5):1345–1349. doi: 10.1073/pnas.84.5.1345. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Koch C. A., Moran M., Sadowski I., Pawson T. The common src homology region 2 domain of cytoplasmic signaling proteins is a positive effector of v-fps tyrosine kinase function. Mol Cell Biol. 1989 Oct;9(10):4131–4140. doi: 10.1128/mcb.9.10.4131. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Lathe R., Kieny M. P., Skory S., Lecocq J. P. Linker tailing: unphosphorylated linker oligonucleotides for joining DNA termini. DNA. 1984;3(2):173–182. doi: 10.1089/dna.1984.3.173. [DOI] [PubMed] [Google Scholar]
  24. Mulligan R. C., Berg P. Selection for animal cells that express the Escherichia coli gene coding for xanthine-guanine phosphoribosyltransferase. Proc Natl Acad Sci U S A. 1981 Apr;78(4):2072–2076. doi: 10.1073/pnas.78.4.2072. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Ng M., Privalsky M. L. Structural domains of the avian erythroblastosis virus erbB protein required for fibroblast transformation: dissection by in-frame insertional mutagenesis. J Virol. 1986 May;58(2):542–553. doi: 10.1128/jvi.58.2.542-553.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Norrander J., Kempe T., Messing J. Construction of improved M13 vectors using oligodeoxynucleotide-directed mutagenesis. Gene. 1983 Dec;26(1):101–106. doi: 10.1016/0378-1119(83)90040-9. [DOI] [PubMed] [Google Scholar]
  27. Pawson T., Guyden J., Kung T. H., Radke K., Gilmore T., Martin G. S. A strain of Fujinami sarcoma virus which is temperature-sensitive in protein phosphorylation and cellular transformation. Cell. 1980 Dec;22(3):767–775. doi: 10.1016/0092-8674(80)90553-x. [DOI] [PubMed] [Google Scholar]
  28. Reddy E. P., Smith M. J., Srinivasan A. Nucleotide sequence of Abelson murine leukemia virus genome: structural similarity of its transforming gene product to other onc gene products with tyrosine-specific kinase activity. Proc Natl Acad Sci U S A. 1983 Jun;80(12):3623–3627. doi: 10.1073/pnas.80.12.3623. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Roebroek A. J., Schalken J. A., Onnekink C., Bloemers H. P., Van de Ven W. J. Structure of the feline c-fes/fps proto-oncogene: genesis of a retroviral oncogene. J Virol. 1987 Jun;61(6):2009–2016. doi: 10.1128/jvi.61.6.2009-2016.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. 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]
  31. Sealy L., Privalsky M. L., Moscovici G., Moscovici C., Bishop J. M. Site-specific mutagenesis of avian erythroblastosis virus: erb-B is required for oncogenicity. Virology. 1983 Oct 15;130(1):155–178. doi: 10.1016/0042-6822(83)90125-3. [DOI] [PubMed] [Google Scholar]
  32. Severinsson L., Ek B., Mellström K., Claesson-Welsh L., Heldin C. H. Deletion of the kinase insert sequence of the platelet-derived growth factor beta-receptor affects receptor kinase activity and signal transduction. Mol Cell Biol. 1990 Feb;10(2):801–809. doi: 10.1128/mcb.10.2.801. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Shibuya M., Hanafusa H. Nucleotide sequence of Fujinami sarcoma virus: evolutionary relationship of its transforming gene with transforming genes of other sarcoma viruses. Cell. 1982 Oct;30(3):787–795. doi: 10.1016/0092-8674(82)90283-5. [DOI] [PubMed] [Google Scholar]
  34. Stone J. C., Atkinson T., Smith M., Pawson T. Identification of functional regions in the transforming protein of Fujinami sarcoma virus by in-phase insertion mutagenesis. Cell. 1984 Jun;37(2):549–558. doi: 10.1016/0092-8674(84)90385-4. [DOI] [PubMed] [Google Scholar]
  35. Stone J. C., Pawson T. Correspondence between immunological and functional domains in the transforming protein of Fujinami sarcoma virus. J Virol. 1985 Sep;55(3):721–727. doi: 10.1128/jvi.55.3.721-727.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. TEMIN H. M., RUBIN H. Characteristics of an assay for Rous sarcoma virus and Rous sarcoma cells in tissue culture. Virology. 1958 Dec;6(3):669–688. doi: 10.1016/0042-6822(58)90114-4. [DOI] [PubMed] [Google Scholar]
  37. Takeya T., Feldman R. A., Hanafusa H. DNA sequence of the viral and cellular src gene of chickens. 1. Complete nucleotide sequence of an EcoRI fragment of recovered avian sarcoma virus which codes for gp37 and pp60src. J Virol. 1982 Oct;44(1):1–11. doi: 10.1128/jvi.44.1.1-11.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Taylor G. R., Reedijk M., Rothwell V., Rohrschneider L., Pawson T. The unique insert of cellular and viral fms protein tyrosine kinase domains is dispensable for enzymatic and transforming activities. EMBO J. 1989 Jul;8(7):2029–2037. doi: 10.1002/j.1460-2075.1989.tb03611.x. [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. Yarden Y., Escobedo J. A., Kuang W. J., Yang-Feng T. L., Daniel T. O., Tremble P. M., Chen E. Y., Ando M. E., Harkins R. N., Francke U. Structure of the receptor for platelet-derived growth factor helps define a family of closely related growth factor receptors. Nature. 1986 Sep 18;323(6085):226–232. doi: 10.1038/323226a0. [DOI] [PubMed] [Google Scholar]
  41. Zoller M. J., Smith M. Oligonucleotide-directed mutagenesis of DNA fragments cloned into M13 vectors. Methods Enzymol. 1983;100:468–500. doi: 10.1016/0076-6879(83)00074-9. [DOI] [PubMed] [Google Scholar]

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