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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
. 1986 Sep;83(18):6785–6789. doi: 10.1073/pnas.83.18.6785

Rapid formation of diacylglycerol from phosphatidylcholine: a pathway for generation of a second messenger.

J M Besterman, V Duronio, P Cuatrecasas
PMCID: PMC386594  PMID: 3462727

Abstract

The classic pathway for agonist-induced generation of diacylglycerol is via activation of a phospholipase C-mediated hydrolysis of the "phosphoinositides." We now report findings from a variety of cell types, which indicate that tumor-promoting phorbol diesters, serum, and platelet-derived growth factor activate within seconds the hydrolysis of phosphatidylcholine, as detected by the formation of diacylglycerol and phosphocholine. It is known that phorbol diesters do not stimulate hydrolysis of the phosphoinositides. Yet, in cells prelabeled with either [14C]oleate or [32P]orthophosphate, addition of the tumor promoter phorbol dibutyrate (PBt2) resulted in the rapid generation of both diacylglycerol and phosphatidate in a time- and dose-dependent manner. The fatty acid composition of the phosphatidate most resembled the fatty acid profile of phosphatidylcholine from the same cell type. Taken together, these findings suggested a role for protein kinase C in the generation of diacylglycerol (and phosphatidate) from phosphatidylcholine. To define further the pathways involved, the metabolism of cellular phosphatidylcholine was studied. In cells prelabeled with [3H]choline, addition of PBt2, but not 4 alpha-phorbol, stimulated the formation of intracellular phosphocholine within 45 sec. Furthermore, addition of platelet-derived growth factor (PDGF) or serum to "serum-starved" cells prelabeled with [3H]choline resulted in increased levels of intracellular phosphocholine within 15-30 sec. Thus, the data suggest that agonists that stimulate protein kinase C either directly (e.g., PBt2) or indirectly via activation of phosphoinositide hydrolysis (e.g., PDGF and serum) may stimulate degradation of phosphatidylcholine by phospholipase C in intact cells. However, prior down-regulation of protein kinase C by prolonged pretreatment of cells with PBt2 almost totally abolished subsequent stimulation of phosphatidylcholine degradation by PBt2 but only partially attenuated subsequent stimulation by PDGF and serum. These observations suggest that PDGF and serum act, at least partially, through a protein kinase C-independent mechanism. Lastly, the size of the cellular choline and CDP-choline pools were shown to be small and relatively insensitive to agonist addition, as compared to the size and behavior of the phosphocholine pool. Thus, the rapidly increased levels of phosphocholine (and diacylglycerol) arising in response to agonist addition appear to be derived directly from phosphatidylcholine by a phospholipase C-mediated mechanism.

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Selected References

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

  1. Ballester R., Rosen O. M. Fate of immunoprecipitable protein kinase C in GH3 cells treated with phorbol 12-myristate 13-acetate. J Biol Chem. 1985 Dec 5;260(28):15194–15199. [PubMed] [Google Scholar]
  2. 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]
  3. Besterman J. M., Watson S. P., Cuatrecasas P. Lack of association of epidermal growth factor-, insulin-, and serum-induced mitogenesis with stimulation of phosphoinositide degradation in BALB/c 3T3 fibroblasts. J Biol Chem. 1986 Jan 15;261(2):723–727. [PubMed] [Google Scholar]
  4. Billah M. M., Lapetina E. G. Formation of lysophosphatidylinositol in platelets stimulated with thrombin or ionophore A23187. J Biol Chem. 1982 May 10;257(9):5196–5200. [PubMed] [Google Scholar]
  5. 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]
  6. 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]
  7. Brock T. A., Rittenhouse S. E., Powers C. W., Ekstein L. S., Gimbrone M. A., Jr, Alexander R. W. Phorbol ester and 1-oleoyl-2-acetylglycerol inhibit angiotensin activation of phospholipase C in cultured vascular smooth muscle cells. J Biol Chem. 1985 Nov 15;260(26):14158–14162. [PubMed] [Google Scholar]
  8. Burgess G. M., Godfrey P. P., McKinney J. S., Berridge M. J., Irvine R. F., Putney J. W., Jr The second messenger linking receptor activation to internal Ca release in liver. Nature. 1984 May 3;309(5963):63–66. doi: 10.1038/309063a0. [DOI] [PubMed] [Google Scholar]
  9. Coughlin S. R., Lee W. M., Williams P. W., Giels G. M., Williams L. T. c-myc gene expression is stimulated by agents that activate protein kinase C and does not account for the mitogenic effect of PDGF. Cell. 1985 Nov;43(1):243–251. doi: 10.1016/0092-8674(85)90029-7. [DOI] [PubMed] [Google Scholar]
  10. Di Virgilio F., Lew D. P., Pozzan T. Protein kinase C activation of physiological processes in human neutrophils at vanishingly small cytosolic Ca2+ levels. Nature. 1984 Aug 23;310(5979):691–693. doi: 10.1038/310691a0. [DOI] [PubMed] [Google Scholar]
  11. Drust D. S., Martin T. F. Protein kinase C translocates from cytosol to membrane upon hormone activation: effects of thyrotropin-releasing hormone in GH3 cells. Biochem Biophys Res Commun. 1985 Apr 30;128(2):531–537. doi: 10.1016/0006-291x(85)90079-8. [DOI] [PubMed] [Google Scholar]
  12. Grove R. I., Schimmel S. D. Effects of 12-O-tetradecanoylphorbol 13-acetate on glycerolipid metabolism in cultured myoblasts. Biochim Biophys Acta. 1982 May 13;711(2):272–280. doi: 10.1016/0005-2760(82)90036-4. [DOI] [PubMed] [Google Scholar]
  13. Guy G. R., Murray A. W. Tumor promoter stimulation of phosphatidylcholine turnover in HeLa cells. Cancer Res. 1982 May;42(5):1980–1985. [PubMed] [Google Scholar]
  14. Harris C. E., Staley D., Conn P. M. Diacylglycerols and protein kinase C. Potential amplifying mechanism for Ca2+-mediated gonadotropin-releasing hormone-stimulated luteinizing hormone release. Mol Pharmacol. 1985 May;27(5):532–536. [PubMed] [Google Scholar]
  15. Hughes B. P., Rye K. A., Pickford L. B., Barritt G. J., Chalmers A. H. A transient increase in diacylglycerols is associated with the action of vasopressin on hepatocytes. Biochem J. 1984 Sep 1;222(2):535–540. doi: 10.1042/bj2220535. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Joseph S. K., Thomas A. P., Williams R. J., Irvine R. F., Williamson J. R. myo-Inositol 1,4,5-trisphosphate. A second messenger for the hormonal mobilization of intracellular Ca2+ in liver. J Biol Chem. 1984 Mar 10;259(5):3077–3081. [PubMed] [Google Scholar]
  17. Kaibuchi K., Takai Y., Sawamura M., Hoshijima M., Fujikura T., Nishizuka Y. Synergistic functions of protein phosphorylation and calcium mobilization in platelet activation. J Biol Chem. 1983 Jun 10;258(11):6701–6704. [PubMed] [Google Scholar]
  18. Kanoh H., Kondoh H., Ono T. Diacylglycerol kinase from pig brain. Purification and phospholipid dependencies. J Biol Chem. 1983 Feb 10;258(3):1767–1774. [PubMed] [Google Scholar]
  19. Kishimoto A., Takai Y., Mori T., Kikkawa U., Nishizuka Y. Activation of calcium and phospholipid-dependent protein kinase by diacylglycerol, its possible relation to phosphatidylinositol turnover. J Biol Chem. 1980 Mar 25;255(6):2273–2276. [PubMed] [Google Scholar]
  20. Labarca R., Janowsky A., Patel J., Paul S. M. Phorbol esters inhibit agonist-induced [3H] inositol-1-phosphate accumulation in rat hippocampal slices. Biochem Biophys Res Commun. 1984 Sep 17;123(2):703–709. doi: 10.1016/0006-291x(84)90286-9. [DOI] [PubMed] [Google Scholar]
  21. Lapetina E. G., Cuatrecasas P. Stimulation of phosphatidic acid production in platelets precedes the formation of arachidonate and parallels the release of serotonin. Biochim Biophys Acta. 1979 May 25;573(2):394–402. doi: 10.1016/0005-2760(79)90072-9. [DOI] [PubMed] [Google Scholar]
  22. Majerus P. W., Neufeld E. J., Wilson D. B. Production of phosphoinositide-derived messengers. Cell. 1984 Jul;37(3):701–703. doi: 10.1016/0092-8674(84)90405-7. [DOI] [PubMed] [Google Scholar]
  23. May W. S., Jacobs S., Cuatrecasas P. Association of phorbol ester-induced hyperphosphorylation and reversible regulation of transferrin membrane receptors in HL60 cells. Proc Natl Acad Sci U S A. 1984 Apr;81(7):2016–2020. doi: 10.1073/pnas.81.7.2016. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. May W. S., Jr, Sahyoun N., Wolf M., Cuatrecasas P. Role of intracellular calcium mobilization in the regulation of protein kinase C-mediated membrane processes. Nature. 1985 Oct 10;317(6037):549–551. doi: 10.1038/317549a0. [DOI] [PubMed] [Google Scholar]
  25. May W. S., Lapetina E. G., Cuatrecasas P. Intracellular activation of protein kinase C and regulation of the surface transferrin receptor by diacylglycerol is a spontaneously reversible process that is associated with rapid formation of phosphatidic acid. Proc Natl Acad Sci U S A. 1986 Mar;83(5):1281–1284. doi: 10.1073/pnas.83.5.1281. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Mori T., Takai Y., Yu B., Takahashi J., Nishizuka Y., Fujikura T. Specificity of the fatty acyl moieties of diacylglycerol for the activation of calcium-activated, phospholipid-dependent protein kinase. J Biochem. 1982 Feb;91(2):427–431. doi: 10.1093/oxfordjournals.jbchem.a133714. [DOI] [PubMed] [Google Scholar]
  27. 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]
  28. Nishizuka Y. Turnover of inositol phospholipids and signal transduction. Science. 1984 Sep 21;225(4668):1365–1370. doi: 10.1126/science.6147898. [DOI] [PubMed] [Google Scholar]
  29. Prentki M., Biden T. J., Janjic D., Irvine R. F., Berridge M. J., Wollheim C. B. Rapid mobilization of Ca2+ from rat insulinoma microsomes by inositol-1,4,5-trisphosphate. Nature. 1984 Jun 7;309(5968):562–564. doi: 10.1038/309562a0. [DOI] [PubMed] [Google Scholar]
  30. Rittenhouse-Simmons S. Production of diglyceride from phosphatidylinositol in activated human platelets. J Clin Invest. 1979 Apr;63(4):580–587. doi: 10.1172/JCI109339. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Rodriguez-Pena A., Rozengurt E. Disappearance of Ca2+-sensitive, phospholipid-dependent protein kinase activity in phorbol ester-treated 3T3 cells. Biochem Biophys Res Commun. 1984 May 16;120(3):1053–1059. doi: 10.1016/s0006-291x(84)80213-2. [DOI] [PubMed] [Google Scholar]
  32. Streb H., Irvine R. F., Berridge M. J., Schulz I. Release of Ca2+ from a nonmitochondrial intracellular store in pancreatic acinar cells by inositol-1,4,5-trisphosphate. Nature. 1983 Nov 3;306(5938):67–69. doi: 10.1038/306067a0. [DOI] [PubMed] [Google Scholar]
  33. Takai Y., Kishimoto A., Kikkawa U., Mori T., Nishizuka Y. Unsaturated diacylglycerol as a possible messenger for the activation of calcium-activated, phospholipid-dependent protein kinase system. Biochem Biophys Res Commun. 1979 Dec 28;91(4):1218–1224. doi: 10.1016/0006-291x(79)91197-5. [DOI] [PubMed] [Google Scholar]
  34. Watson S. P., Lapetina E. G. 1,2-Diacylglycerol and phorbol ester inhibit agonist-induced formation of inositol phosphates in human platelets: possible implications for negative feedback regulation of inositol phospholipid hydrolysis. Proc Natl Acad Sci U S A. 1985 May;82(9):2623–2626. doi: 10.1073/pnas.82.9.2623. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Wolf M., Cuatrecasas P., Sahyoun N. Interaction of protein kinase C with membranes is regulated by Ca2+, phorbol esters, and ATP. J Biol Chem. 1985 Dec 15;260(29):15718–15722. [PubMed] [Google Scholar]
  36. Wolf M., LeVine H., 3rd, May W. S., Jr, Cuatrecasas P., Sahyoun N. A model for intracellular translocation of protein kinase C involving synergism between Ca2+ and phorbol esters. Nature. 1985 Oct 10;317(6037):546–549. doi: 10.1038/317546a0. [DOI] [PubMed] [Google Scholar]
  37. Wolf R. A., Gross R. W. Identification of neutral active phospholipase C which hydrolyzes choline glycerophospholipids and plasmalogen selective phospholipase A2 in canine myocardium. J Biol Chem. 1985 Jun 25;260(12):7295–7303. [PubMed] [Google Scholar]
  38. Yavin E. Regulation of phospholipid metabolism in differentiating cells from rat brain cerebral hemispheres in culture. Patterns of acetylcholine phosphocholine, and choline phosphoglycerides labeling from (methyl-14C)choline. J Biol Chem. 1976 Mar 10;251(5):1392–1397. [PubMed] [Google Scholar]

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