Skip to main content
NCBI home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1991 Jan 15;88(2):598–602. doi: 10.1073/pnas.88.2.598

Overexpression of protein kinase C beta 1 enhances phospholipase D activity and diacylglycerol formation in phorbol ester-stimulated rat fibroblasts.

J K Pai 1, J A Pachter 1, I B Weinstein 1, W R Bishop 1
PMCID: PMC50859  PMID: 1988955

Abstract

We are using a Rat-6 fibroblast cell line that stably overexpresses the beta 1 isozyme of protein kinase C (PKC) to study regulation of phospholipid hydrolysis by PKC. Stimulation of control (R6-C1) or overexpressing (R6-PKC3) cells with phorbol ester results in an increase in diacylglycerol (DAG) mass with no increase in inositol phosphates, indicating that DAG is not formed by inositol phospholipid breakdown. A more dramatic DAG increase occurs in R6-PKC3 cells (4.0-fold over basal) compared to R6-C1 cells (1.5-fold over basal). To further define the source of DAG, phosphatidylcholine (PC) pools were labeled with [3H]myristic acid or with [3H]- or [32P]alkyllyso-PC and formation of labeled phosphatidylethanol, an unambiguous marker of phospholipase D activation, was monitored. Phorbol ester-stimulated phosphatidylethanol formation is 5-fold greater in the R6-PKC3 cell line. Formation of radiolabeled phosphatidic acid (PA) is also enhanced by PKC overexpression. In cells double-labeled with [3H]- and [32P]-alkyl-lysoPC, the 3H/32P ratio of PA and PC are identical 15 min after stimulation, suggesting that a phospholipase D mechanism predominates. In support of this, the PA phosphohydrolase inhibitor propranolol decreased phorbol 12-myristate 13-acetate-stimulated DAG formation by 72%. Increases in DAG and phosphatidylethanol were inhibited by the PKC inhibitors K252a and staurosporine. These results indicate that phospholipase D is regulated by the action of PKC. Enhanced phospholipase D activity may contribute to the growth abnormalities seen in PKC-overexpressing cells.

Full text

PDF
598

Selected References

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

  1. AMES B. N., DUBIN D. T. The role of polyamines in the neutralization of bacteriophage deoxyribonucleic acid. J Biol Chem. 1960 Mar;235:769–775. [PubMed] [Google Scholar]
  2. Augert G., Bocckino S. B., Blackmore P. F., Exton J. H. Hormonal stimulation of diacylglycerol formation in hepatocytes. Evidence for phosphatidylcholine breakdown. J Biol Chem. 1989 Dec 25;264(36):21689–21698. [PubMed] [Google Scholar]
  3. 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]
  4. Ben-Av P., Liscovitch M. Phospholipase D activation by the mitogens platelet-derived growth factor and 12-O-tetradecanoylphorbol 13-acetate in NIH-3T3 cells. FEBS Lett. 1989 Dec 18;259(1):64–66. doi: 10.1016/0014-5793(89)81495-4. [DOI] [PubMed] [Google Scholar]
  5. Berridge M. J., Dawson R. M., Downes C. P., Heslop J. P., Irvine R. F. Changes in the levels of inositol phosphates after agonist-dependent hydrolysis of membrane phosphoinositides. Biochem J. 1983 May 15;212(2):473–482. doi: 10.1042/bj2120473. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Besterman J. M., Duronio V., Cuatrecasas P. Rapid formation of diacylglycerol from phosphatidylcholine: a pathway for generation of a second messenger. Proc Natl Acad Sci U S A. 1986 Sep;83(18):6785–6789. doi: 10.1073/pnas.83.18.6785. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Billah M. M., Eckel S., Mullmann T. J., Egan R. W., Siegel M. I. Phosphatidylcholine hydrolysis by phospholipase D determines phosphatidate and diglyceride levels in chemotactic peptide-stimulated human neutrophils. Involvement of phosphatidate phosphohydrolase in signal transduction. J Biol Chem. 1989 Oct 15;264(29):17069–17077. [PubMed] [Google Scholar]
  8. Billah M. M., Pai J. K., Mullmann T. J., Egan R. W., Siegel M. I. Regulation of phospholipase D in HL-60 granulocytes. Activation by phorbol esters, diglyceride, and calcium ionophore via protein kinase- independent mechanisms. J Biol Chem. 1989 May 25;264(15):9069–9076. [PubMed] [Google Scholar]
  9. Bishop W. R., August J., Petrin J. M., Pai J. K. Regulation of sn-1,2-diacylglycerol second-messenger formation in thrombin-stimulated human platelets. Potentiation by protein kinase C inhibitors. Biochem J. 1990 Jul 15;269(2):465–473. doi: 10.1042/bj2690465. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Blackmore P. F., Exton J. H. Studies on the hepatic calcium-mobilizing activity of aluminum fluoride and glucagon. Modulation by cAMP and phorbol myristate acetate. J Biol Chem. 1986 Aug 25;261(24):11056–11063. [PubMed] [Google Scholar]
  11. Bocckino S. B., Blackmore P. F., Wilson P. B., Exton J. H. Phosphatidate accumulation in hormone-treated hepatocytes via a phospholipase D mechanism. J Biol Chem. 1987 Nov 5;262(31):15309–15315. [PubMed] [Google Scholar]
  12. Cabot M. C., Welsh C. J., Zhang Z. C., Cao H. T., Chabbott H., Lebowitz M. Vasopressin, phorbol diesters and serum elicit choline glycerophospholipid hydrolysis and diacylglycerol formation in nontransformed cells: transformed derivatives do not respond. Biochim Biophys Acta. 1988 Mar 4;959(1):46–57. doi: 10.1016/0005-2760(88)90148-8. [DOI] [PubMed] [Google Scholar]
  13. Exton J. H. Signaling through phosphatidylcholine breakdown. J Biol Chem. 1990 Jan 5;265(1):1–4. [PubMed] [Google Scholar]
  14. Gschwendt M., Leibersperger H., Marks F. Differentiative action of K252a on protein kinase C and a calcium-unresponsive, phorbol ester/phospholipid-activated protein kinase. Biochem Biophys Res Commun. 1989 Nov 15;164(3):974–982. doi: 10.1016/0006-291x(89)91765-8. [DOI] [PubMed] [Google Scholar]
  15. Hockberger P., Toselli M., Swandulla D., Lux H. D. A diacylglycerol analogue reduces neuronal calcium currents independently of protein kinase C activation. Nature. 1989 Mar 23;338(6213):340–342. doi: 10.1038/338340a0. [DOI] [PubMed] [Google Scholar]
  16. Housey G. M., Johnson M. D., Hsiao W. L., O'Brian C. A., Murphy J. P., Kirschmeier P., Weinstein I. B. Overproduction of protein kinase C causes disordered growth control in rat fibroblasts. Cell. 1988 Feb 12;52(3):343–354. doi: 10.1016/s0092-8674(88)80027-8. [DOI] [PubMed] [Google Scholar]
  17. Hsiao W. L., Housey G. M., Johnson M. D., Weinstein I. B. Cells that overproduce protein kinase C are more susceptible to transformation by an activated H-ras oncogene. Mol Cell Biol. 1989 Jun;9(6):2641–2647. doi: 10.1128/mcb.9.6.2641. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Irving H. R., Exton J. H. Phosphatidylcholine breakdown in rat liver plasma membranes. Roles of guanine nucleotides and P2-purinergic agonists. J Biol Chem. 1987 Mar 15;262(8):3440–3443. [PubMed] [Google Scholar]
  19. Liscovitch M., Amsterdam A. Gonadotropin-releasing hormone activates phospholipase D in ovarian granulosa cells. Possible role in signal transduction. J Biol Chem. 1989 Jul 15;264(20):11762–11767. [PubMed] [Google Scholar]
  20. Liscovitch M. Phosphatidylethanol biosynthesis in ethanol-exposed NG108-15 neuroblastoma X glioma hybrid cells. Evidence for activation of a phospholipase D phosphatidyl transferase activity by protein kinase C. J Biol Chem. 1989 Jan 25;264(3):1450–1456. [PubMed] [Google Scholar]
  21. Martin T. W., Feldman D. R., Goldstein K. E., Wagner J. R. Long-term phorbol ester treatment dissociates phospholipase D activation from phosphoinositide hydrolysis and prostacyclin synthesis in endothelial cells stimulated with bradykinin. Biochem Biophys Res Commun. 1989 Nov 30;165(1):319–326. doi: 10.1016/0006-291x(89)91072-3. [DOI] [PubMed] [Google Scholar]
  22. Martinson E. A., Goldstein D., Brown J. H. Muscarinic receptor activation of phosphatidylcholine hydrolysis. Relationship to phosphoinositide hydrolysis and diacylglycerol metabolism. J Biol Chem. 1989 Sep 5;264(25):14748–14754. [PubMed] [Google Scholar]
  23. Matozaki T., Williams J. A. Multiple sources of 1,2-diacylglycerol in isolated rat pancreatic acini stimulated by cholecystokinin. Involvement of phosphatidylinositol bisphosphate and phosphatidylcholine hydrolysis. J Biol Chem. 1989 Sep 5;264(25):14729–14734. [PubMed] [Google Scholar]
  24. Moolenaar W. H., Kruijer W., Tilly B. C., Verlaan I., Bierman A. J., de Laat S. W. Growth factor-like action of phosphatidic acid. Nature. 1986 Sep 11;323(6084):171–173. doi: 10.1038/323171a0. [DOI] [PubMed] [Google Scholar]
  25. Mufson R. A., Okin E., Weinstein I. B. Phorbol esters stimulate the rapid release of choline from prelabelled cells. Carcinogenesis. 1981;2(11):1095–1102. doi: 10.1093/carcin/2.11.1095. [DOI] [PubMed] [Google Scholar]
  26. Muir J. G., Murray A. W. Bombesin and phorbol ester stimulate phosphatidylcholine hydrolysis by phospholipase C: evidence for a role of protein kinase C. J Cell Physiol. 1987 Mar;130(3):382–391. doi: 10.1002/jcp.1041300311. [DOI] [PubMed] [Google Scholar]
  28. Pachter J. A., Law G. J., Dannies P. S. Bombesin stimulates inositol polyphosphate production in GH4C1 pituitary tumor cells: comparison with TRH. Biochem Biophys Res Commun. 1988 Jul 29;154(2):654–659. doi: 10.1016/0006-291x(88)90189-1. [DOI] [PubMed] [Google Scholar]
  29. Pai J. K., Siegel M. I., Egan R. W., Billah M. M. Activation of phospholipase D by chemotactic peptide in HL-60 granulocytes. Biochem Biophys Res Commun. 1988 Jan 15;150(1):355–364. doi: 10.1016/0006-291x(88)90528-1. [DOI] [PubMed] [Google Scholar]
  30. Pai J. K., Siegel M. I., Egan R. W., Billah M. M. Phospholipase D catalyzes phospholipid metabolism in chemotactic peptide-stimulated HL-60 granulocytes. J Biol Chem. 1988 Sep 5;263(25):12472–12477. [PubMed] [Google Scholar]
  31. Persons D. A., Wilkison W. O., Bell R. M., Finn O. J. Altered growth regulation and enhanced tumorigenicity of NIH 3T3 fibroblasts transfected with protein kinase C-I cDNA. Cell. 1988 Feb 12;52(3):447–458. doi: 10.1016/s0092-8674(88)80037-0. [DOI] [PubMed] [Google Scholar]
  32. Pfeilschifter J., Ochsner M., Whitebread S., De Gasparo M. Down-regulation of protein kinase C potentiates angiotensin II-stimulated polyphosphoinositide hydrolysis in vascular smooth-muscle cells. Biochem J. 1989 Aug 15;262(1):285–291. doi: 10.1042/bj2620285. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. 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]
  34. Qian Z., Drewes L. R. Muscarinic acetylcholine receptor regulates phosphatidylcholine phospholipase D in canine brain. J Biol Chem. 1989 Dec 25;264(36):21720–21724. [PubMed] [Google Scholar]
  35. Rhee S. G., Suh P. G., Ryu S. H., Lee S. Y. Studies of inositol phospholipid-specific phospholipase C. Science. 1989 May 5;244(4904):546–550. doi: 10.1126/science.2541501. [DOI] [PubMed] [Google Scholar]
  36. Rittenhouse S. E., Sasson J. P. Mass changes in myoinositol trisphosphate in human platelets stimulated by thrombin. Inhibitory effects of phorbol ester. J Biol Chem. 1985 Jul 25;260(15):8657–8660. [PubMed] [Google Scholar]
  37. 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]
  38. Takuwa N., Takuwa Y., Rasmussen H. A tumour promoter, 12-O-tetradecanoylphorbol 13-acetate, increases cellular 1,2-diacylglycerol content through a mechanism other than phosphoinositide hydrolysis in Swiss-mouse 3T3 fibroblasts. Biochem J. 1987 May 1;243(3):647–653. doi: 10.1042/bj2430647. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Tettenborn C. S., Mueller G. C. 12-O-tetradecanoylphorbol-13-acetate activates phosphatidylethanol and phosphatidylglycerol synthesis by phospholipase D in cell lysates. Biochem Biophys Res Commun. 1988 Aug 30;155(1):249–255. doi: 10.1016/s0006-291x(88)81076-3. [DOI] [PubMed] [Google Scholar]
  40. Uhing R. J., Prpic V., Hollenbach P. W., Adams D. O. Involvement of protein kinase C in platelet-activating factor-stimulated diacylglycerol accumulation in murine peritoneal macrophages. J Biol Chem. 1989 Jun 5;264(16):9224–9230. [PubMed] [Google Scholar]
  41. Yu C. L., Tsai M. H., Stacey D. W. Cellular ras activity and phospholipid metabolism. Cell. 1988 Jan 15;52(1):63–71. doi: 10.1016/0092-8674(88)90531-4. [DOI] [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES