Skip to main content
PMC home page
Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1989 Apr;9(4):1651–1658. doi: 10.1128/mcb.9.4.1651

Phosphatidylinositol kinase activity associates with viral p60src protein.

Y Fukui 1, H Hanafusa 1
PMCID: PMC362583  PMID: 2542773

Abstract

Immunoprecipitates of p60v-src proteins from chicken embryo fibroblasts infected with Rous sarcoma virus were assayed for phosphatidylinositol (PI) kinase activity in the absence of detergents. The product of the PI kinase reaction, phosphatidylinositol monophosphate (PIP), migrated slightly slower than did the authentic phosphatidylinositol-4-monophosphate marker in thin-layer chromatography and was indistinguishable from phosphatidylinositol-3-monophosphate produced by PI kinase type I. Furthermore, the deacylated product comigrated with glycerophosphoinositol-3-phosphate in high-performance liquid chromatography. Both sucrose gradient fractionation and the heat stability of PI kinase activity from cells infected with temperature-sensitive mutants suggest that the PI kinase activity is not intrinsic to p60v-src but is a property of another molecule complexed with p60v-src. All transforming variants of p60src were associated with PI kinase activity, whereas this enzyme activity was hardly detectable in immunoprecipitates from cells infected with nontransforming viruses encoding p60c-src or an enzymatically inactive variant. However, PI kinase activity was found in p60src immunoprecipitates from cells infected with nonmyristylated, nontransforming mutants as well as temperature-sensitive mutants at the nonpermissive temperature, which indicated that simple association of PI kinase activity with p60src is not sufficient for cell transformation.

Full text

PDF
1655

Images in this article

1653Image
on p.1653
1653Image
on p.1653
1653Image
on p.1653
1654Image
on p.1654
1655Image
on p.1655
1656Image
on p.1656

Selected References

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

  1. Avemann K., Knippers R., Koller T., Sogo J. M. Camptothecin, a specific inhibitor of type I DNA topoisomerase, induces DNA breakage at replication forks. Mol Cell Biol. 1988 Aug;8(8):3026–3034. doi: 10.1128/mcb.8.8.3026. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Berridge M. J. Inositol trisphosphate and diacylglycerol as second messengers. Biochem J. 1984 Jun 1;220(2):345–360. doi: 10.1042/bj2200345. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Berridge M. J., Irvine R. F. Inositol trisphosphate, a novel second messenger in cellular signal transduction. Nature. 1984 Nov 22;312(5992):315–321. doi: 10.1038/312315a0. [DOI] [PubMed] [Google Scholar]
  4. Calothy G., Poirier F., Dambrine G., Mignatti P., Combes P., Pessac B. Expression of viral oncogenes in differentiating chick embryo neuroretinal cells infected with avian tumor viruses. Cold Spring Harb Symp Quant Biol. 1980;44(Pt 2):983–990. doi: 10.1101/sqb.1980.044.01.106. [DOI] [PubMed] [Google Scholar]
  5. Courtneidge S. A., Heber A. An 81 kd protein complexed with middle T antigen and pp60c-src: a possible phosphatidylinositol kinase. Cell. 1987 Sep 25;50(7):1031–1037. doi: 10.1016/0092-8674(87)90169-3. [DOI] [PubMed] [Google Scholar]
  6. 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]
  7. Cross F. R., Garber E. A., Pellman D., Hanafusa H. A short sequence in the p60src N terminus is required for p60src myristylation and membrane association and for cell transformation. Mol Cell Biol. 1984 Sep;4(9):1834–1842. doi: 10.1128/mcb.4.9.1834. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Fry M. J., Gebhardt A., Parker P. J., Foulkes J. G. Phosphatidylinositol turnover and transformation of cells by Abelson murine leukaemia virus. EMBO J. 1985 Dec 1;4(12):3173–3178. doi: 10.1002/j.1460-2075.1985.tb04061.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Garber E. A., Krueger J. G., Hanafusa H., Goldberg A. R. Temperature-sensitive membrane association of pp60src in tsNY68-infected cells correlates with increased tyrosine phosphorylation of membrane-associated proteins. Virology. 1983 Apr 15;126(1):73–86. doi: 10.1016/0042-6822(83)90462-2. [DOI] [PubMed] [Google Scholar]
  10. 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]
  11. 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]
  12. Ito Y., Spurr N., Griffin B. E. Middle T antigen as primary inducer of full expression of the phenotype of transformation by polyoma virus. J Virol. 1980 Jul;35(1):219–232. doi: 10.1128/jvi.35.1.219-232.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Jackowski S., Rettenmier C. W., Sherr C. J., Rock C. O. A guanine nucleotide-dependent phosphatidylinositol 4,5-diphosphate phospholipase C in cells transformed by the v-fms and v-fes oncogenes. J Biol Chem. 1986 Apr 15;261(11):4978–4985. [PubMed] [Google Scholar]
  14. Kaplan D. R., Whitman M., Schaffhausen B., Pallas D. C., White M., Cantley L., Roberts T. M. Common elements in growth factor stimulation and oncogenic transformation: 85 kd phosphoprotein and phosphatidylinositol kinase activity. Cell. 1987 Sep 25;50(7):1021–1029. doi: 10.1016/0092-8674(87)90168-1. [DOI] [PubMed] [Google Scholar]
  15. Kato J. Y., Takeya T., Grandori C., Iba H., Levy J. B., Hanafusa H. Amino acid substitutions sufficient to convert the nontransforming p60c-src protein to a transforming protein. Mol Cell Biol. 1986 Dec;6(12):4155–4160. doi: 10.1128/mcb.6.12.4155. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Kato M., Kawai S., Takenawa T. Altered signal transduction in erbB-transformed cells. Implication of enhanced inositol phospholipid metabolism in erbB-induced transformation. J Biol Chem. 1987 Apr 25;262(12):5696–5704. [PubMed] [Google Scholar]
  17. Kawai S., Hanafusa H. The effects of reciprocal changes in temperature on the transformed state of cells infected with a rous sarcoma virus mutant. Virology. 1971 Nov;46(2):470–479. doi: 10.1016/0042-6822(71)90047-x. [DOI] [PubMed] [Google Scholar]
  18. Koch M. A., Diringer H. A difference in the breakdown of phosphatidylinositol in normal and SV40 transformed mouse fibroblasts. Biochem Biophys Res Commun. 1973 Nov 16;55(2):305–311. doi: 10.1016/0006-291x(73)91088-7. [DOI] [PubMed] [Google Scholar]
  19. 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]
  20. Levy J. B., Iba H., Hanafusa H. Activation of the transforming potential of p60c-src by a single amino acid change. Proc Natl Acad Sci U S A. 1986 Jun;83(12):4228–4232. doi: 10.1073/pnas.83.12.4228. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Lipsich L. A., Lewis A. J., Brugge J. S. Isolation of monoclonal antibodies that recognize the transforming proteins of avian sarcoma viruses. J Virol. 1983 Nov;48(2):352–360. doi: 10.1128/jvi.48.2.352-360.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Macara I. G., Marinetti G. V., Balduzzi P. C. Transforming protein of avian sarcoma virus UR2 is associated with phosphatidylinositol kinase activity: possible role in tumorigenesis. Proc Natl Acad Sci U S A. 1984 May;81(9):2728–2732. doi: 10.1073/pnas.81.9.2728. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Mayer B. J., Jove R., Krane J. F., Poirier F., Calothy G., Hanafusa H. Genetic lesions involved in temperature sensitivity of the src gene products of four Rous sarcoma virus mutants. J Virol. 1986 Dec;60(3):858–867. doi: 10.1128/jvi.60.3.858-867.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Nishizawa M., Mayer B. J., Takeya T., Yamamoto T., Toyoshima K., Hanafusa H., Kawai S. Two independent mutations are required for temperature-sensitive cell transformation by a Rous sarcoma virus temperature-sensitive mutant. J Virol. 1985 Dec;56(3):743–749. doi: 10.1128/jvi.56.3.743-749.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Nishizuka Y. The role of protein kinase C in cell surface signal transduction and tumour promotion. Nature. 1984 Apr 19;308(5961):693–698. doi: 10.1038/308693a0. [DOI] [PubMed] [Google Scholar]
  26. Parsons S. J., McCarley D. J., Ely C. M., Benjamin D. C., Parsons J. T. Monoclonal antibodies to Rous sarcoma virus pp60src react with enzymatically active cellular pp60src of avian and mammalian origin. J Virol. 1984 Aug;51(2):272–282. doi: 10.1128/jvi.51.2.272-282.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Piwnica-Worms H., Kaplan D. R., Whitman M., Roberts T. M. Retrovirus shuttle vector for study of kinase activities of pp60c-src synthesized in vitro and overproduced in vivo. Mol Cell Biol. 1986 Jun;6(6):2033–2040. doi: 10.1128/mcb.6.6.2033. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Rozengurt E. Early signals in the mitogenic response. Science. 1986 Oct 10;234(4773):161–166. doi: 10.1126/science.3018928. [DOI] [PubMed] [Google Scholar]
  29. Snyder M. A., Bishop J. M., McGrath J. P., Levinson A. D. A mutation at the ATP-binding site of pp60v-src abolishes kinase activity, transformation, and tumorigenicity. Mol Cell Biol. 1985 Jul;5(7):1772–1779. doi: 10.1128/mcb.5.7.1772. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Sugano S., Hanafusa H. Phosphatidylinositol kinase activity in virus-transformed and nontransformed cells. Mol Cell Biol. 1985 Sep;5(9):2399–2404. doi: 10.1128/mcb.5.9.2399. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Sugimoto Y., Erikson R. L. Phosphatidylinositol kinase activities in normal and Rous sarcoma virus-transformed cells. Mol Cell Biol. 1985 Nov;5(11):3194–3198. doi: 10.1128/mcb.5.11.3194. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Sugimoto Y., Whitman M., Cantley L. C., Erikson R. L. Evidence that the Rous sarcoma virus transforming gene product phosphorylates phosphatidylinositol and diacylglycerol. Proc Natl Acad Sci U S A. 1984 Apr;81(7):2117–2121. doi: 10.1073/pnas.81.7.2117. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Whitman M., Downes C. P., Keeler M., Keller T., Cantley L. Type I phosphatidylinositol kinase makes a novel inositol phospholipid, phosphatidylinositol-3-phosphate. Nature. 1988 Apr 14;332(6165):644–646. doi: 10.1038/332644a0. [DOI] [PubMed] [Google Scholar]
  34. Whitman M., Kaplan D., Roberts T., Cantley L. Evidence for two distinct phosphatidylinositol kinases in fibroblasts. Implications for cellular regulation. Biochem J. 1987 Oct 1;247(1):165–174. doi: 10.1042/bj2470165. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Molecular and Cellular Biology are provided here courtesy of Taylor & Francis

RESOURCES