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. 2000 Dec 1;352(Pt 2):491–499. doi: 10.1042/0264-6021:3520491

Phospholipase D in rat myometrium: occurrence of a membrane-bound ARF6 (ADP-ribosylation factor 6)-regulated activity controlled by betagamma subunits of heterotrimeric G-proteins.

H Le Stunff 1, L Dokhac 1, S Bourgoin 1, M F Bader 1, S Harbon 1
PMCID: PMC1221481  PMID: 11085943

Abstract

Both protein kinase C and protein tyrosine kinases have been shown to be involved in phospholipase D (PLD) activation in intact rat myometrium [Le Stunff, Dokhac and Harbon (2000) J. Pharmacol. Exp. Ther. 292, 629-637]. In this study we assessed the involvement of monomeric G-proteins in PLD activation in a cell-free system derived from myometrial tissue. Both the PLD1 and PLD2 isoforms were detected. Two forms of PLD activity, essentially membrane-bound, were found in myometrial preparations. One form was stimulated by oleate and insensitive to guanosine 5'-[gamma-thio] triphosphate (GTP[S]). The second required ammonium sulphate to be detected and was stimulated by GTP[S]. ADP-ribosylation factors (ARF1 and ARF6) and RhoA were immunodetected in myometrial preparations. ARF1 and RhoA were present in the membrane and cytosolic fractions whereas ARF6 was detected exclusively in the membrane fraction. A synthetic myristoylated peptide corresponding to the N-terminal domain of ARF6 [myrARF6((2-13))] totally abolished PLD activation in the presence of ammonium sulphate and GTP[S], whereas myrARF1((2-17)) and the inhibitory GDP/GTP-exchange factor, Rho GDI, did not. These data are consistent with a membrane-bound ARF6-regulated PLD activity. Finally, the stimulation of PLD by ARF6 was inhibited by AlF(-)(4) and this inhibition was counteracted by the fusion protein glutathione S-transferase-beta-adrenergic receptor kinase 1 (495-689) and by the QEHA peptide (from adenylate cyclase ACII), which act as G-protein betagamma-subunit scavengers. It is concluded that G-protein subunits betagamma are involved in a pathway modulating PLD activation by ARF6, illustrating cross-talk between heterotrimeric and monomeric G-proteins.

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Selected References

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  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. Banno Y., Tamiya-Koizumi K., Oshima H., Morikawa A., Yoshida S., Nozawa Y. Nuclear ADP-ribosylation factor (ARF)- and oleate-dependent phospholipase D (PLD) in rat liver cells. Increases of ARF-dependent PLD activity in regenerating liver cells. J Biol Chem. 1997 Feb 21;272(8):5208–5213. doi: 10.1074/jbc.272.8.5208. [DOI] [PubMed] [Google Scholar]
  3. Bigay J., Deterre P., Pfister C., Chabre M. Fluoride complexes of aluminium or beryllium act on G-proteins as reversibly bound analogues of the gamma phosphate of GTP. EMBO J. 1987 Oct;6(10):2907–2913. doi: 10.1002/j.1460-2075.1987.tb02594.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bourgoin S., Harbour D., Desmarais Y., Takai Y., Beaulieu A. Low molecular weight GTP-binding proteins in HL-60 granulocytes. Assessment of the role of ARF and of a 50-kDa cytosolic protein in phospholipase D activation. J Biol Chem. 1995 Feb 17;270(7):3172–3178. doi: 10.1074/jbc.270.7.3172. [DOI] [PubMed] [Google Scholar]
  5. Bowman E. P., Uhlinger D. J., Lambeth J. D. Neutrophil phospholipase D is activated by a membrane-associated Rho family small molecular weight GTP-binding protein. J Biol Chem. 1993 Oct 15;268(29):21509–21512. [PubMed] [Google Scholar]
  6. Caumont A. S., Galas M. C., Vitale N., Aunis D., Bader M. F. Regulated exocytosis in chromaffin cells. Translocation of ARF6 stimulates a plasma membrane-associated phospholipase D. J Biol Chem. 1998 Jan 16;273(3):1373–1379. doi: 10.1074/jbc.273.3.1373. [DOI] [PubMed] [Google Scholar]
  7. Chen J., DeVivo M., Dingus J., Harry A., Li J., Sui J., Carty D. J., Blank J. L., Exton J. H., Stoffel R. H. A region of adenylyl cyclase 2 critical for regulation by G protein beta gamma subunits. Science. 1995 May 26;268(5214):1166–1169. doi: 10.1126/science.7761832. [DOI] [PubMed] [Google Scholar]
  8. Cockcroft S. Phospholipase D: regulation by GTPases and protein kinase C and physiological relevance. Prog Lipid Res. 1996 Dec;35(4):345–370. doi: 10.1016/s0163-7827(96)00009-4. [DOI] [PubMed] [Google Scholar]
  9. Colley W. C., Sung T. C., Roll R., Jenco J., Hammond S. M., Altshuller Y., Bar-Sagi D., Morris A. J., Frohman M. A. Phospholipase D2, a distinct phospholipase D isoform with novel regulatory properties that provokes cytoskeletal reorganization. Curr Biol. 1997 Mar 1;7(3):191–201. doi: 10.1016/s0960-9822(97)70090-3. [DOI] [PubMed] [Google Scholar]
  10. Colombo M. I., Inglese J., D'Souza-Schorey C., Beron W., Stahl P. D. Heterotrimeric G proteins interact with the small GTPase ARF. Possibilities for the regulation of vesicular traffic. J Biol Chem. 1995 Oct 13;270(41):24564–24571. doi: 10.1074/jbc.270.41.24564. [DOI] [PubMed] [Google Scholar]
  11. Dai J., Williams S. A., Ziegelhöffer A., Panagia V. Structure-activity relationship of the effect of cis-unsaturated fatty acids on heart sarcolemmal phospholipase D activity. Prostaglandins Leukot Essent Fatty Acids. 1995 Feb-Mar;52(2-3):167–171. doi: 10.1016/0952-3278(95)90017-9. [DOI] [PubMed] [Google Scholar]
  12. Exton J. H. Regulation of phospholipase D. Biochim Biophys Acta. 1999 Jul 30;1439(2):121–133. doi: 10.1016/s1388-1981(99)00089-x. [DOI] [PubMed] [Google Scholar]
  13. Franco M., Paris S., Chabre M. The small G-protein ARF1GDP binds to the Gt beta gamma subunit of transducin, but not to Gt alpha GDP-Gt beta gamma. FEBS Lett. 1995 Apr 10;362(3):286–290. doi: 10.1016/0014-5793(95)00258-b. [DOI] [PubMed] [Google Scholar]
  14. Frohman M. A., Morris A. J. Phospholipase D structure and regulation. Chem Phys Lipids. 1999 Apr;98(1-2):127–140. doi: 10.1016/s0009-3084(99)00025-0. [DOI] [PubMed] [Google Scholar]
  15. Gaits F., Li R. Y., Bigay J., Ragab A., Ragab-Thomas M. F., Chap H. G-protein beta gamma subunits mediate specific phosphorylation of the protein-tyrosine phosphatase SH-PTP1 induced by lysophosphatidic acid. J Biol Chem. 1996 Aug 16;271(33):20151–20155. [PubMed] [Google Scholar]
  16. Galas M. C., Helms J. B., Vitale N., Thiersé D., Aunis D., Bader M. F. Regulated exocytosis in chromaffin cells. A potential role for a secretory granule-associated ARF6 protein. J Biol Chem. 1997 Jan 31;272(5):2788–2793. doi: 10.1074/jbc.272.5.2788. [DOI] [PubMed] [Google Scholar]
  17. Gibbs T. C., Meier K. E. Expression and regulation of phospholipase D isoforms in mammalian cell lines. J Cell Physiol. 2000 Jan;182(1):77–87. doi: 10.1002/(SICI)1097-4652(200001)182:1<77::AID-JCP9>3.0.CO;2-B. [DOI] [PubMed] [Google Scholar]
  18. Hammond S. M., Altshuller Y. M., Sung T. C., Rudge S. A., Rose K., Engebrecht J., Morris A. J., Frohman M. A. Human ADP-ribosylation factor-activated phosphatidylcholine-specific phospholipase D defines a new and highly conserved gene family. J Biol Chem. 1995 Dec 15;270(50):29640–29643. doi: 10.1074/jbc.270.50.29640. [DOI] [PubMed] [Google Scholar]
  19. Hancock J. F., Hall A. A novel role for RhoGDI as an inhibitor of GAP proteins. EMBO J. 1993 May;12(5):1915–1921. doi: 10.1002/j.1460-2075.1993.tb05840.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Houle M. G., Bourgoin S. Regulation of phospholipase D by phosphorylation-dependent mechanisms. Biochim Biophys Acta. 1999 Jul 30;1439(2):135–149. doi: 10.1016/s1388-1981(99)00090-6. [DOI] [PubMed] [Google Scholar]
  21. Houle M. G., Kahn R. A., Naccache P. H., Bourgoin S. ADP-ribosylation factor translocation correlates with potentiation of GTP gamma S-stimulated phospholipase D activity in membrane fractions of HL-60 cells. J Biol Chem. 1995 Sep 29;270(39):22795–22800. doi: 10.1074/jbc.270.39.22795. [DOI] [PubMed] [Google Scholar]
  22. Kahn R. A. Fluoride is not an activator of the smaller (20-25 kDa) GTP-binding proteins. J Biol Chem. 1991 Aug 25;266(24):15595–15597. [PubMed] [Google Scholar]
  23. Kasai T., Ohguchi K., Nakashima S., Ito Y., Naganawa T., Kondo N., Nozawa Y. Increased activity of oleate-dependent type phospholipase D during actinomycin D-induced apoptosis in Jurkat T cells. J Immunol. 1998 Dec 15;161(12):6469–6474. [PubMed] [Google Scholar]
  24. Kim J. H., Kim Y., Lee S. D., Lopez I., Arnold R. S., Lambeth J. D., Suh P. G., Ryu S. H. Selective activation of phospholipase D2 by unsaturated fatty acid. FEBS Lett. 1999 Jul 2;454(1-2):42–46. doi: 10.1016/s0014-5793(99)00745-0. [DOI] [PubMed] [Google Scholar]
  25. Kwak J. Y., Uhlinger D. J. Downregulation of phospholipase D by protein kinase A in a cell-free system of human neutrophils. Biochem Biophys Res Commun. 2000 Jan 7;267(1):305–310. doi: 10.1006/bbrc.1999.1941. [DOI] [PubMed] [Google Scholar]
  26. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  27. Le Stunff H., Dokhac L., Harbon S. The roles of protein kinase C and tyrosine kinases in mediating endothelin-1-stimulated phospholipase D activity in rat myometrium: differential inhibition by ceramides and cyclic AMP. J Pharmacol Exp Ther. 2000 Feb;292(2):629–637. [PubMed] [Google Scholar]
  28. Lee S. Y., Yeo E. J., Choi M. U. Phospholipase D activity in L1210 cells: a model for oleate-activated phospholipase D in intact mammalian cells. Biochem Biophys Res Commun. 1998 Mar 27;244(3):825–831. doi: 10.1006/bbrc.1998.8348. [DOI] [PubMed] [Google Scholar]
  29. Li L., Fleming N. Aluminum fluoride inhibition of cabbage phospholipase D by a phosphate-mimicking mechanism. FEBS Lett. 1999 Nov 12;461(1-2):1–5. doi: 10.1016/s0014-5793(99)01414-3. [DOI] [PubMed] [Google Scholar]
  30. Li L., Fleming N. Aluminum fluoride inhibits phospholipase D activation by a GTP-binding protein-independent mechanism. FEBS Lett. 1999 Sep 24;458(3):419–423. doi: 10.1016/s0014-5793(99)01196-5. [DOI] [PubMed] [Google Scholar]
  31. Liu S. Y., Tappia P. S., Dai J., Williams S. A., Panagia V. Phospholipase A2-mediated activation of phospholipase D in rat heart sarcolemma. J Mol Cell Cardiol. 1998 Jun;30(6):1203–1214. doi: 10.1006/jmcc.1998.0685. [DOI] [PubMed] [Google Scholar]
  32. Mackay D. J., Hall A. Rho GTPases. J Biol Chem. 1998 Aug 14;273(33):20685–20688. doi: 10.1074/jbc.273.33.20685. [DOI] [PubMed] [Google Scholar]
  33. Marcil J., Harbour D., Naccache P. H., Bourgoin S. Human phospholipase D1 can be tyrosine-phosphorylated in HL-60 granulocytes. J Biol Chem. 1997 Aug 15;272(33):20660–20664. doi: 10.1074/jbc.272.33.20660. [DOI] [PubMed] [Google Scholar]
  34. Marshansky V., Bourgoin S., Londoño I., Bendayan M., Vinay P. Identification of ADP-ribosylation factor-6 in brush-border membrane and early endosomes of human kidney proximal tubules. Electrophoresis. 1997 Mar-Apr;18(3-4):538–547. doi: 10.1002/elps.1150180334. [DOI] [PubMed] [Google Scholar]
  35. Massenburg D., Han J. S., Liyanage M., Patton W. A., Rhee S. G., Moss J., Vaughan M. Activation of rat brain phospholipase D by ADP-ribosylation factors 1,5, and 6: separation of ADP-ribosylation factor-dependent and oleate-dependent enzymes. Proc Natl Acad Sci U S A. 1994 Nov 22;91(24):11718–11722. doi: 10.1073/pnas.91.24.11718. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Meier K. E., Gibbs T. C., Knoepp S. M., Ella K. M. Expression of phospholipase D isoforms in mammalian cells. Biochim Biophys Acta. 1999 Jul 30;1439(2):199–213. doi: 10.1016/s1388-1981(99)00095-5. [DOI] [PubMed] [Google Scholar]
  37. Mhaouty-Kodja S., Bouet-Alard R., Limon-Boulez I., Maltier J. P., Legrand C. Molecular diversity of adenylyl cyclases in human and rat myometrium. Correlation with global adenylyl cyclase activity during mid- and term pregnancy. J Biol Chem. 1997 Dec 5;272(49):31100–31106. doi: 10.1074/jbc.272.49.31100. [DOI] [PubMed] [Google Scholar]
  38. Millar C. A., Powell K. A., Hickson G. R., Bader M. F., Gould G. W. Evidence for a role for ADP-ribosylation factor 6 in insulin-stimulated glucose transporter-4 (GLUT4) trafficking in 3T3-L1 adipocytes. J Biol Chem. 1999 Jun 18;274(25):17619–17625. doi: 10.1074/jbc.274.25.17619. [DOI] [PubMed] [Google Scholar]
  39. Mizuno T., Kaibuchi K., Ando S., Musha T., Hiraoka K., Takaishi K., Asada M., Nunoi H., Matsuda I., Takai Y. Regulation of the superoxide-generating NADPH oxidase by a small GTP-binding protein and its stimulatory and inhibitory GDP/GTP exchange proteins. J Biol Chem. 1992 May 25;267(15):10215–10218. [PubMed] [Google Scholar]
  40. Moss J., Vaughan M. Molecules in the ARF orbit. J Biol Chem. 1998 Aug 21;273(34):21431–21434. doi: 10.1074/jbc.273.34.21431. [DOI] [PubMed] [Google Scholar]
  41. Mukherjee S., Gurevich V. V., Jones J. C., Casanova J. E., Frank S. R., Maizels E. T., Bader M. F., Kahn R. A., Palczewski K., Aktories K. The ADP ribosylation factor nucleotide exchange factor ARNO promotes beta-arrestin release necessary for luteinizing hormone/choriogonadotropin receptor desensitization. Proc Natl Acad Sci U S A. 2000 May 23;97(11):5901–5906. doi: 10.1073/pnas.100127097. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Nakamura S., Shimooku K., Akisue T., Jinnai H., Hitomi T., Kiyohara Y., Ogino C., Yoshida K., Nishizuka Y. Mammalian phospholipase D: activation by ammonium sulfate and nucleotides. Proc Natl Acad Sci U S A. 1995 Dec 19;92(26):12319–12322. doi: 10.1073/pnas.92.26.12319. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Naze S., Le Stunff H., Dokhac L., Thomas G., Harbon S. Activation of phospholipase D by endothelin-1 in rat myometrium. Role of calcium and protein kinase C. J Pharmacol Exp Ther. 1997 Apr;281(1):15–23. [PubMed] [Google Scholar]
  44. Okamura S., Yamashita S. Purification and characterization of phosphatidylcholine phospholipase D from pig lung. J Biol Chem. 1994 Dec 9;269(49):31207–31213. [PubMed] [Google Scholar]
  45. Palmier B., Leiber D., Harbon S. Pervanadate mediated an increased generation of inositol phosphates and tension in rat myometrium. Activation and phosphorylation of phospholipase C-gamma 1. Biol Reprod. 1996 Jun;54(6):1383–1389. doi: 10.1095/biolreprod54.6.1383. [DOI] [PubMed] [Google Scholar]
  46. Park J. B., Kim J. H., Kim Y., Ha S. H., Yoo J. S., Du G., Frohman M. A., Suh P. G., Ryu S. H. Cardiac phospholipase D2 localizes to sarcolemmal membranes and is inhibited by alpha-actinin in an ADP-ribosylation factor-reversible manner. J Biol Chem. 2000 Jul 14;275(28):21295–21301. doi: 10.1074/jbc.M002463200. [DOI] [PubMed] [Google Scholar]
  47. Pitcher J. A., Inglese J., Higgins J. B., Arriza J. L., Casey P. J., Kim C., Benovic J. L., Kwatra M. M., Caron M. G., Lefkowitz R. J. Role of beta gamma subunits of G proteins in targeting the beta-adrenergic receptor kinase to membrane-bound receptors. Science. 1992 Aug 28;257(5074):1264–1267. doi: 10.1126/science.1325672. [DOI] [PubMed] [Google Scholar]
  48. Sung T. C., Altshuller Y. M., Morris A. J., Frohman M. A. Molecular analysis of mammalian phospholipase D2. J Biol Chem. 1999 Jan 1;274(1):494–502. doi: 10.1074/jbc.274.1.494. [DOI] [PubMed] [Google Scholar]
  49. Welsh C. F., Moss J., Vaughan M. Isolation of recombinant ADP-ribosylation factor 6, an approximately 20-kDa guanine nucleotide-binding protein, in an activated GTP-bound state. J Biol Chem. 1994 Jun 3;269(22):15583–15587. [PubMed] [Google Scholar]
  50. Yang C. Z., Heimberg H., D'Souza-Schorey C., Mueckler M. M., Stahl P. D. Subcellular distribution and differential expression of endogenous ADP-ribosylation factor 6 in mammalian cells. J Biol Chem. 1998 Feb 13;273(7):4006–4011. doi: 10.1074/jbc.273.7.4006. [DOI] [PubMed] [Google Scholar]
  51. van Blitterswijk W. J., Hilkmann H., de Widt J., van der Bend R. L. Phospholipid metabolism in bradykinin-stimulated human fibroblasts. II. Phosphatidylcholine breakdown by phospholipases C and D; involvement of protein kinase C. J Biol Chem. 1991 Jun 5;266(16):10344–10350. [PubMed] [Google Scholar]

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