Skip to main content
PMC home page
The EMBO Journal logoLink to The EMBO Journal
. 1990 Dec;9(12):3907–3912. doi: 10.1002/j.1460-2075.1990.tb07611.x

Evidence for a role of phosphatidylcholine-hydrolysing phospholipase C in the regulation of protein kinase C by ras and src oncogenes.

I Diaz-Laviada 1, P Larrodera 1, M T Diaz-Meco 1, M E Cornet 1, P H Guddal 1, T Johansen 1, J Moscat 1
PMCID: PMC552161  PMID: 2123453

Abstract

The products of ras and src oncogenes are thought to be important components in pathways regulating cell proliferation and differentiation. In fibroblasts transformed by these oncogenes, increased diacylglycerol levels have been found which most probably arise from activation of the turnover of phosphatidylcholine. Diacylglycerol is a key activator of protein kinase C whose role in cell growth and transformation has been proposed. We demonstrate here by using immunochemical techniques that transformation by ras or src oncogenes is associated with permanent translocation of protein kinase C to the cytoplasmic membrane. However, no down-regulation of the enzyme is observed despite its permanent activation in these transformants. Importantly, the lack of down-regulation observed in ras and src transformed cell lines is mimicked by chronic treatment of NIH 3T3 fibroblasts with exogenous Bacillus cereus phosphatidylcholine-hydrolysing phospholipase C, but not with phorbol myristate acetate or exogenous Bacillus thuringiensis phosphatidylinositol-hydrolysing phospholipase C. These results strongly suggest that diacylglycerol derived from phosphatidylcholine but not from phosphoinositide turnover is responsible for the atypical regulation of protein kinase C in cell lines transformed by ras and src oncogenes.

Full text

PDF
3907

Images in this article

3909Fig. 1.
on p.3909
3909Fig. 2.
on p.3909
3910Fig. 3.
on p.3910

Selected References

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

  1. BLIGH E. G., DYER W. J. A rapid method of total lipid extraction and purification. Can J Biochem Physiol. 1959 Aug;37(8):911–917. doi: 10.1139/o59-099. [DOI] [PubMed] [Google Scholar]
  2. Barbacid M. ras genes. Annu Rev Biochem. 1987;56:779–827. doi: 10.1146/annurev.bi.56.070187.004023. [DOI] [PubMed] [Google Scholar]
  3. Berridge M. J., Irvine R. F. Inositol phosphates and cell signalling. Nature. 1989 Sep 21;341(6239):197–205. doi: 10.1038/341197a0. [DOI] [PubMed] [Google Scholar]
  4. 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]
  5. Bishop J. M. The molecular genetics of cancer. Science. 1987 Jan 16;235(4786):305–311. doi: 10.1126/science.3541204. [DOI] [PubMed] [Google Scholar]
  6. Blackshear P. J., Witters L. A., Girard P. R., Kuo J. F., Quamo S. N. Growth factor-stimulated protein phosphorylation in 3T3-L1 cells. Evidence for protein kinase C-dependent and -independent pathways. J Biol Chem. 1985 Oct 25;260(24):13304–13315. [PubMed] [Google Scholar]
  7. Bocckino S. B., Blackmore P. F., Exton J. H. Stimulation of 1,2-diacylglycerol accumulation in hepatocytes by vasopressin, epinephrine, and angiotensin II. J Biol Chem. 1985 Nov 15;260(26):14201–14207. [PubMed] [Google Scholar]
  8. Delli Bovi P., Curatola A. M., Kern F. G., Greco A., Ittmann M., Basilico C. An oncogene isolated by transfection of Kaposi's sarcoma DNA encodes a growth factor that is a member of the FGF family. Cell. 1987 Aug 28;50(5):729–737. doi: 10.1016/0092-8674(87)90331-x. [DOI] [PubMed] [Google Scholar]
  9. Doolittle R. F., Hunkapiller M. W., Hood L. E., Devare S. G., Robbins K. C., Aaronson S. A., Antoniades H. N. Simian sarcoma virus onc gene, v-sis, is derived from the gene (or genes) encoding a platelet-derived growth factor. Science. 1983 Jul 15;221(4607):275–277. doi: 10.1126/science.6304883. [DOI] [PubMed] [Google Scholar]
  10. Exton J. H. Signaling through phosphatidylcholine breakdown. J Biol Chem. 1990 Jan 5;265(1):1–4. [PubMed] [Google Scholar]
  11. Farrar W. L., Anderson W. B. Interleukin-2 stimulates association of protein kinase C with plasma membrane. Nature. 1985 May 16;315(6016):233–235. doi: 10.1038/315233a0. [DOI] [PubMed] [Google Scholar]
  12. Fearon C. W., Tashjian A. H., Jr Ionomycin inhibits thyrotropin-releasing hormone-induced translocation of protein kinase C in GH4C1 pituitary cells. J Biol Chem. 1987 Jul 15;262(20):9515–9520. [PubMed] [Google Scholar]
  13. Fleischman L. F., Chahwala S. B., Cantley L. ras-transformed cells: altered levels of phosphatidylinositol-4,5-bisphosphate and catabolites. Science. 1986 Jan 24;231(4736):407–410. doi: 10.1126/science.3001936. [DOI] [PubMed] [Google Scholar]
  14. Halsey D. L., Girard P. R., Kuo J. F., Blackshear P. J. Protein kinase C in fibroblasts. Characteristics of its intracellular location during growth and after exposure to phorbol esters and other mitogens. J Biol Chem. 1987 Feb 15;262(5):2234–2243. [PubMed] [Google Scholar]
  15. Hirota K., Hirota T., Aguilera G., Catt K. J. Hormone-induced redistribution of calcium-activated phospholipid-dependent protein kinase in pituitary gonadotrophs. J Biol Chem. 1985 Mar 25;260(6):3243–3246. [PubMed] [Google Scholar]
  16. Ito T., Tanaka T., Yoshida T., Onoda K., Ohta H., Hagiwara M., Itoh Y., Ogura M., Saito H., Hidaka H. Immunocytochemical evidence for translocation of protein kinase C in human megakaryoblastic leukemic cells: synergistic effects of Ca2+ and activators of protein kinase C on the plasma membrane association. J Cell Biol. 1988 Sep;107(3):929–937. doi: 10.1083/jcb.107.3.929. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Johansen T., Holm T., Guddal P. H., Sletten K., Haugli F. B., Little C. Cloning and sequencing of the gene encoding the phosphatidylcholine-preferring phospholipase C of Bacillus cereus. Gene. 1988 May 30;65(2):293–304. doi: 10.1016/0378-1119(88)90466-0. [DOI] [PubMed] [Google Scholar]
  18. Kaplan P. L., Anderson M., Ozanne B. Transforming growth factor(s) production enables cells to grow in the absence of serum: an autocrine system. Proc Natl Acad Sci U S A. 1982 Jan;79(2):485–489. doi: 10.1073/pnas.79.2.485. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Kraft A. S., Anderson W. B. Phorbol esters increase the amount of Ca2+, phospholipid-dependent protein kinase associated with plasma membrane. Nature. 1983 Feb 17;301(5901):621–623. doi: 10.1038/301621a0. [DOI] [PubMed] [Google Scholar]
  20. Lacal J. C., Moscat J., Aaronson S. A. Novel source of 1,2-diacylglycerol elevated in cells transformed by Ha-ras oncogene. Nature. 1987 Nov 19;330(6145):269–272. doi: 10.1038/330269a0. [DOI] [PubMed] [Google Scholar]
  21. Larrodera P., Cornet M. E., Diaz-Meco M. T., Lopez-Barahona M., Diaz-Laviada I., Guddal P. H., Johansen T., Moscat J. Phospholipase C-mediated hydrolysis of phosphatidylcholine is an important step in PDGF-stimulated DNA synthesis. Cell. 1990 Jun 15;61(6):1113–1120. doi: 10.1016/0092-8674(90)90074-o. [DOI] [PubMed] [Google Scholar]
  22. Little C. Phospholipase C. Biochem Soc Trans. 1989 Apr;17(2):271–273. doi: 10.1042/bst0170271. [DOI] [PubMed] [Google Scholar]
  23. Lloyd A. C., Paterson H. F., Morris J. D., Hall A., Marshall C. J. p21H-ras-induced morphological transformation and increases in c-myc expression are independent of functional protein kinase C. EMBO J. 1989 Apr;8(4):1099–1104. doi: 10.1002/j.1460-2075.1989.tb03479.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Lopez-Barahona M., Kaplan P. L., Cornet M. E., Diaz-Meco M. T., Larrodera P., Diaz-Laviada I., Municio A. M., Moscat J. Kinetic evidence of a rapid activation of phosphatidylcholine hydrolysis by Ki-ras oncogene. Possible involvement in late steps of the mitogenic cascade. J Biol Chem. 1990 Jun 5;265(16):9022–9026. [PubMed] [Google Scholar]
  25. Martin T. F., Hsieh K. P., Porter B. W. The sustained second phase of hormone-stimulated diacylglycerol accumulation does not activate protein kinase C in GH3 cells. J Biol Chem. 1990 May 5;265(13):7623–7631. [PubMed] [Google Scholar]
  26. Morris J. D., Price B., Lloyd A. C., Self A. J., Marshall C. J., Hall A. Scrape-loading of Swiss 3T3 cells with ras protein rapidly activates protein kinase C in the absence of phosphoinositide hydrolysis. Oncogene. 1989 Jan;4(1):27–31. [PubMed] [Google Scholar]
  27. Moscat J., Cornet M. E., Diaz-Meco M. T., Larrodera P., Lopez-Alanon D., Lopez-Barahona M. Activation of phosphatidylcholine-specific phospholipase C in cell growth and oncogene transformation. Biochem Soc Trans. 1989 Dec;17(6):988–991. doi: 10.1042/bst0170988. [DOI] [PubMed] [Google Scholar]
  28. Moscat J., Fleming T. P., Molloy C. J., Lopez-Barahona M., Aaronson S. A. The calcium signal for Balb/MK keratinocyte terminal differentiation induces sustained alterations in phosphoinositide metabolism without detectable protein kinase C activation. J Biol Chem. 1989 Jul 5;264(19):11228–11235. [PubMed] [Google Scholar]
  29. Myrnes B. J., Little C. A simple purification scheme yielding crystalline phospholipase C from Bacillus cereus. Acta Chem Scand B. 1980;34(5):375–377. doi: 10.3891/acta.chem.scand.34b-0375. [DOI] [PubMed] [Google Scholar]
  30. Nishizuka Y. Studies and perspectives of protein kinase C. Science. 1986 Jul 18;233(4761):305–312. doi: 10.1126/science.3014651. [DOI] [PubMed] [Google Scholar]
  31. Nishizuka Y. The molecular heterogeneity of protein kinase C and its implications for cellular regulation. Nature. 1988 Aug 25;334(6184):661–665. doi: 10.1038/334661a0. [DOI] [PubMed] [Google Scholar]
  32. Ozanne B., Fulton R. J., Kaplan P. L. Kirsten murine sarcoma virus transformed cell lines and a spontaneously transformed rat cell-line produce transforming factors. J Cell Physiol. 1980 Oct;105(1):163–180. doi: 10.1002/jcp.1041050118. [DOI] [PubMed] [Google Scholar]
  33. Pessin M. S., Baldassare J. J., Raben D. M. Molecular species analysis of mitogen-stimulated 1,2-diglycerides in fibroblasts. Comparison of alpha-thrombin, epidermal growth factor, and platelet-derived growth factor. J Biol Chem. 1990 May 15;265(14):7959–7966. [PubMed] [Google Scholar]
  34. Preiss J., Loomis C. R., Bishop W. R., Stein R., Niedel J. E., Bell R. M. Quantitative measurement of sn-1,2-diacylglycerols present in platelets, hepatocytes, and ras- and sis-transformed normal rat kidney cells. J Biol Chem. 1986 Jul 5;261(19):8597–8600. [PubMed] [Google Scholar]
  35. Price B. D., Morris J. D., Marshall C. J., Hall A. Stimulation of phosphatidylcholine hydrolysis, diacylglycerol release, and arachidonic acid production by oncogenic ras is a consequence of protein kinase C activation. J Biol Chem. 1989 Oct 5;264(28):16638–16643. [PubMed] [Google Scholar]
  36. Rose-John S., Dietrich A., Marks F. Molecular cloning of mouse protein kinase C (PKC) cDNA from Swiss 3T3 fibroblasts. Gene. 1988 Dec 30;74(2):465–471. doi: 10.1016/0378-1119(88)90179-5. [DOI] [PubMed] [Google Scholar]
  37. Sherr C. J., Rettenmier C. W., Sacca R., Roussel M. F., Look A. T., Stanley E. R. The c-fms proto-oncogene product is related to the receptor for the mononuclear phagocyte growth factor, CSF-1. Cell. 1985 Jul;41(3):665–676. doi: 10.1016/s0092-8674(85)80047-7. [DOI] [PubMed] [Google Scholar]
  38. Shih T. Y., Weeks M. O., Young H. A., Scolnick E. M. p21 of Kirsten murine sarcoma virus is thermolabile in a viral mutant temperature sensitive for the maintenance of transformation. J Virol. 1979 Aug;31(2):546–546. doi: 10.1128/jvi.31.2.546-546.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Slivka S. R., Meier K. E., Insel P. A. Alpha 1-adrenergic receptors promote phosphatidylcholine hydrolysis in MDCK-D1 cells. A mechanism for rapid activation of protein kinase C. J Biol Chem. 1988 Sep 5;263(25):12242–12246. [PubMed] [Google Scholar]
  40. Stein R. B., Tai J. Y., Scolnick E. M. Molecular cloning of the temperature-sensitive 371 Kirsten murine sarcoma virus and expression in Escherichia coli of the mutant and wild-type viral Kirsten ras p21 proteins. J Virol. 1986 Nov;60(2):782–786. doi: 10.1128/jvi.60.2.782-786.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Ullrich A., Coussens L., Hayflick J. S., Dull T. J., Gray A., Tam A. W., Lee J., Yarden Y., Libermann T. A., Schlessinger J. Human epidermal growth factor receptor cDNA sequence and aberrant expression of the amplified gene in A431 epidermoid carcinoma cells. 1984 May 31-Jun 6Nature. 309(5967):418–425. doi: 10.1038/309418a0. [DOI] [PubMed] [Google Scholar]
  42. Waterfield M. D., Scrace G. T., Whittle N., Stroobant P., Johnsson A., Wasteson A., Westermark B., Heldin C. H., Huang J. S., Deuel T. F. Platelet-derived growth factor is structurally related to the putative transforming protein p28sis of simian sarcoma virus. Nature. 1983 Jul 7;304(5921):35–39. doi: 10.1038/304035a0. [DOI] [PubMed] [Google Scholar]
  43. Weinberger C., Thompson C. C., Ong E. S., Lebo R., Gruol D. J., Evans R. M. The c-erb-A gene encodes a thyroid hormone receptor. Nature. 1986 Dec 18;324(6098):641–646. doi: 10.1038/324641a0. [DOI] [PubMed] [Google Scholar]
  44. Wolfman A., Macara I. G. Elevated levels of diacylglycerol and decreased phorbol ester sensitivity in ras-transformed fibroblasts. Nature. 1987 Jan 22;325(6102):359–361. doi: 10.1038/325359a0. [DOI] [PubMed] [Google Scholar]
  45. Wolfman A., Wingrove T. G., Blackshear P. J., Macara I. G. Down-regulation of protein kinase C and of an endogenous 80-kDa substrate in transformed fibroblasts. J Biol Chem. 1987 Dec 5;262(34):16546–16552. [PubMed] [Google Scholar]
  46. Woodgett J. R., Hunter T. Immunological evidence for two physiological forms of protein kinase C. Mol Cell Biol. 1987 Jan;7(1):85–96. doi: 10.1128/mcb.7.1.85. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Yoshida T., Miyagawa K., Odagiri H., Sakamoto H., Little P. F., Terada M., Sugimura T. Genomic sequence of hst, a transforming gene encoding a protein homologous to fibroblast growth factors and the int-2-encoded protein. Proc Natl Acad Sci U S A. 1987 Oct;84(20):7305–7309. doi: 10.1073/pnas.84.20.7305. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Young S., Parker P. J., Ullrich A., Stabel S. Down-regulation of protein kinase C is due to an increased rate of degradation. Biochem J. 1987 Jun 15;244(3):775–779. doi: 10.1042/bj2440775. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from The EMBO Journal are provided here courtesy of Nature Publishing Group

RESOURCES