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. 2000 Nov 1;351(Pt 3):613–619.

Insulin-induced phospholipase D1 and phospholipase D2 activity in human embryonic kidney-293 cells mediated by the phospholipase C gamma and protein kinase C alpha signalling cascade.

R Slaaby 1, G Du 1, Y M Altshuller 1, M A Frohman 1, K Seedorf 1
PMCID: PMC1221400  PMID: 11042115

Abstract

Phospholipase D (PLD)1 is quiescent in vitro and in vivo until stimulated by classical protein kinase C (PKC) isoforms, ADP-ribosylation factor or Rho family members. By contrast, PLD2 has high basal activity, and the mechanisms involved in agonist-induced activation of PLD2 are poorly understood. Using transiently transfected human embryonic kidney (HEK)-293 cells as a model system, we report in the present study that PLD2 overexpressed in HEK-293 cells exhibits regulatory properties similar to PLD1 when stimulated in response to insulin and phorbol ester. Co-expression of PLD1 or PLD2 with PKC alpha results in constitutive activation of both PLD isoforms, which cannot be further stimulated by insulin. Co-expression of PLD1 with phospholipase C (PLC)gamma has the same effect, while co-expression of PLD2 with PLC gamma allows PLD2 activity to be stimulated in an insulin-dependent manner. The PKC-specific inhibitors bisindolylmaleimide and Gö 6976 abolish insulin-induced PLD2 activation in HEK-293 cells co-expressing the insulin receptor, PLC gamma and PLD2, confirming that not only PLD1, but PLD2 as well, is regulated in a PKC-dependent manner. Finally, we provide evidence that PKC alpha is constitutively associated with PLD2. In summary, we demonstrate that insulin treatment results in activation of both PLD1 and PLD2 in appropriate cell types when the appropriate upstream intermediate signalling components, i.e. PKC alpha and PLC gamma, are expressed at sufficient levels.

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Selected References

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  1. Bosch R. R., Smeets R. L., Sleutels F., Patel A. M., Emst-de Vries S. E., Joep J., de Pont H. H., Willems P. H. Concerted action of cytosolic Ca2+ and protein kinase C in receptor-mediated phospholipase D activation in Chinese hamster ovary cells expressing the cholecystokinin-A receptor. Biochem J. 1999 Jan 15;337(Pt 2):263–268. [PMC free article] [PubMed] [Google Scholar]
  2. Chen C., Okayama H. High-efficiency transformation of mammalian cells by plasmid DNA. Mol Cell Biol. 1987 Aug;7(8):2745–2752. doi: 10.1128/mcb.7.8.2745. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. 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]
  4. Czarny M., Lavie Y., Fiucci G., Liscovitch M. Localization of phospholipase D in detergent-insoluble, caveolin-rich membrane domains. Modulation by caveolin-1 expression and caveolin-182-101. J Biol Chem. 1999 Jan 29;274(5):2717–2724. doi: 10.1074/jbc.274.5.2717. [DOI] [PubMed] [Google Scholar]
  5. Exton J. H. Phosphatidylcholine breakdown and signal transduction. Biochim Biophys Acta. 1994 Apr 14;1212(1):26–42. doi: 10.1016/0005-2760(94)90186-4. [DOI] [PubMed] [Google Scholar]
  6. 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]
  7. Frohman M. A., Sung T. C., Morris A. J. Mammalian phospholipase D structure and regulation. Biochim Biophys Acta. 1999 Jul 30;1439(2):175–186. doi: 10.1016/s1388-1981(99)00093-1. [DOI] [PubMed] [Google Scholar]
  8. Gorman C. M., Gies D., McCray G., Huang M. The human cytomegalovirus major immediate early promoter can be trans-activated by adenovirus early proteins. Virology. 1989 Aug;171(2):377–385. doi: 10.1016/0042-6822(89)90605-3. [DOI] [PubMed] [Google Scholar]
  9. 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]
  10. Hammond S. M., Jenco J. M., Nakashima S., Cadwallader K., Gu Q., Cook S., Nozawa Y., Prestwich G. D., Frohman M. A., Morris A. J. Characterization of two alternately spliced forms of phospholipase D1. Activation of the purified enzymes by phosphatidylinositol 4,5-bisphosphate, ADP-ribosylation factor, and Rho family monomeric GTP-binding proteins and protein kinase C-alpha. J Biol Chem. 1997 Feb 7;272(6):3860–3868. doi: 10.1074/jbc.272.6.3860. [DOI] [PubMed] [Google Scholar]
  11. Hitomi T., Yanagi S., Inatome R., Yamamura H. Cross-linking of the B cell receptor induces activation of phospholipase D through Syk, Btk and phospholipase C-gamma2. FEBS Lett. 1999 Feb 26;445(2-3):371–374. doi: 10.1016/s0014-5793(99)00153-2. [DOI] [PubMed] [Google Scholar]
  12. Jenco J. M., Rawlingson A., Daniels B., Morris A. J. Regulation of phospholipase D2: selective inhibition of mammalian phospholipase D isoenzymes by alpha- and beta-synucleins. Biochemistry. 1998 Apr 7;37(14):4901–4909. doi: 10.1021/bi972776r. [DOI] [PubMed] [Google Scholar]
  13. Karnam P., Standaert M. L., Galloway L., Farese R. V. Activation and translocation of Rho (and ADP ribosylation factor) by insulin in rat adipocytes. Apparent involvement of phosphatidylinositol 3-kinase. J Biol Chem. 1997 Mar 7;272(10):6136–6140. doi: 10.1074/jbc.272.10.6136. [DOI] [PubMed] [Google Scholar]
  14. Kayali A. G., Eichhorn J., Haruta T., Morris A. J., Nelson J. G., Vollenweider P., Olefsky J. M., Webster N. J. Association of the insulin receptor with phospholipase C-gamma (PLCgamma) in 3T3-L1 adipocytes suggests a role for PLCgamma in metabolic signaling by insulin. J Biol Chem. 1998 May 29;273(22):13808–13818. doi: 10.1074/jbc.273.22.13808. [DOI] [PubMed] [Google Scholar]
  15. Kim J. H., Lee S. D., Han J. M., Lee T. G., Kim Y., Park J. B., Lambeth J. D., Suh P. G., Ryu S. H. Activation of phospholipase D1 by direct interaction with ADP-ribosylation factor 1 and RalA. FEBS Lett. 1998 Jul 3;430(3):231–235. doi: 10.1016/s0014-5793(98)00661-9. [DOI] [PubMed] [Google Scholar]
  16. Kim Y., Han J. M., Park J. B., Lee S. D., Oh Y. S., Chung C., Lee T. G., Kim J. H., Park S. K., Yoo J. S. Phosphorylation and activation of phospholipase D1 by protein kinase C in vivo: determination of multiple phosphorylation sites. Biochemistry. 1999 Aug 10;38(32):10344–10351. doi: 10.1021/bi990579h. [DOI] [PubMed] [Google Scholar]
  17. Kodaki T., Yamashita S. Cloning, expression, and characterization of a novel phospholipase D complementary DNA from rat brain. J Biol Chem. 1997 Apr 25;272(17):11408–11413. doi: 10.1074/jbc.272.17.11408. [DOI] [PubMed] [Google Scholar]
  18. Lee T. G., Park J. B., Lee S. D., Hong S., Kim J. H., Kim Y., Yi K. S., Bae S., Hannun Y. A., Obeid L. M. Phorbol myristate acetate-dependent association of protein kinase C alpha with phospholipase D1 in intact cells. Biochim Biophys Acta. 1997 Aug 16;1347(2-3):199–204. doi: 10.1016/s0005-2760(97)00083-0. [DOI] [PubMed] [Google Scholar]
  19. Liang J. O., Kornfeld S. Comparative activity of ADP-ribosylation factor family members in the early steps of coated vesicle formation on rat liver Golgi membranes. J Biol Chem. 1997 Feb 14;272(7):4141–4148. doi: 10.1074/jbc.272.7.4141. [DOI] [PubMed] [Google Scholar]
  20. Lopez I., Arnold R. S., Lambeth J. D. Cloning and initial characterization of a human phospholipase D2 (hPLD2). ADP-ribosylation factor regulates hPLD2. J Biol Chem. 1998 May 22;273(21):12846–12852. doi: 10.1074/jbc.273.21.12846. [DOI] [PubMed] [Google Scholar]
  21. Min D. S., Kim E. G., Exton J. H. Involvement of tyrosine phosphorylation and protein kinase C in the activation of phospholipase D by H2O2 in Swiss 3T3 fibroblasts. J Biol Chem. 1998 Nov 6;273(45):29986–29994. doi: 10.1074/jbc.273.45.29986. [DOI] [PubMed] [Google Scholar]
  22. Morris A. J., Frohman M. A., Engebrecht J. Measurement of phospholipase D activity. Anal Biochem. 1997 Oct 1;252(1):1–9. doi: 10.1006/abio.1997.2299. [DOI] [PubMed] [Google Scholar]
  23. Rizzo M. A., Shome K., Vasudevan C., Stolz D. B., Sung T. C., Frohman M. A., Watkins S. C., Romero G. Phospholipase D and its product, phosphatidic acid, mediate agonist-dependent raf-1 translocation to the plasma membrane and the activation of the mitogen-activated protein kinase pathway. J Biol Chem. 1999 Jan 8;274(2):1131–1139. doi: 10.1074/jbc.274.2.1131. [DOI] [PubMed] [Google Scholar]
  24. Sciorra V. A., Rudge S. A., Prestwich G. D., Frohman M. A., Engebrecht J., Morris A. J. Identification of a phosphoinositide binding motif that mediates activation of mammalian and yeast phospholipase D isoenzymes. EMBO J. 1999 Nov 1;18(21):5911–5921. doi: 10.1093/emboj/18.21.5911. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Seedorf K., Shearman M., Ullrich A. Rapid and long-term effects on protein kinase C on receptor tyrosine kinase phosphorylation and degradation. J Biol Chem. 1995 Aug 11;270(32):18953–18960. doi: 10.1074/jbc.270.32.18953. [DOI] [PubMed] [Google Scholar]
  26. Shome K., Nie Y., Romero G. ADP-ribosylation factor proteins mediate agonist-induced activation of phospholipase D. J Biol Chem. 1998 Nov 13;273(46):30836–30841. doi: 10.1074/jbc.273.46.30836. [DOI] [PubMed] [Google Scholar]
  27. Shome K., Vasudevan C., Romero G. ARF proteins mediate insulin-dependent activation of phospholipase D. Curr Biol. 1997 Jun 1;7(6):387–396. doi: 10.1016/s0960-9822(06)00186-2. [DOI] [PubMed] [Google Scholar]
  28. Slaaby R., Jensen T., Hansen H. S., Frohman M. A., Seedorf K. PLD2 complexes with the EGF receptor and undergoes tyrosine phosphorylation at a single site upon agonist stimulation. J Biol Chem. 1998 Dec 11;273(50):33722–33727. doi: 10.1074/jbc.273.50.33722. [DOI] [PubMed] [Google Scholar]
  29. Steed P. M., Nagar S., Wennogle L. P. Phospholipase D regulation by a physical interaction with the actin-binding protein gelsolin. Biochemistry. 1996 Apr 23;35(16):5229–5237. doi: 10.1021/bi952370j. [DOI] [PubMed] [Google Scholar]
  30. 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]
  31. Sung T. C., Roper R. L., Zhang Y., Rudge S. A., Temel R., Hammond S. M., Morris A. J., Moss B., Engebrecht J., Frohman M. A. Mutagenesis of phospholipase D defines a superfamily including a trans-Golgi viral protein required for poxvirus pathogenicity. EMBO J. 1997 Aug 1;16(15):4519–4530. doi: 10.1093/emboj/16.15.4519. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Sung T. C., Zhang Y., Morris A. J., Frohman M. A. Structural analysis of human phospholipase D1. J Biol Chem. 1999 Feb 5;274(6):3659–3666. doi: 10.1074/jbc.274.6.3659. [DOI] [PubMed] [Google Scholar]
  33. Voss M., Weernink P. A., Haupenthal S., Möller U., Cool R. H., Bauer B., Camonis J. H., Jakobs K. H., Schmidt M. Phospholipase D stimulation by receptor tyrosine kinases mediated by protein kinase C and a Ras/Ral signaling cascade. J Biol Chem. 1999 Dec 3;274(49):34691–34698. doi: 10.1074/jbc.274.49.34691. [DOI] [PubMed] [Google Scholar]
  34. Yamada K., Avignon A., Standaert M. L., Cooper D. R., Spencer B., Farese R. V. Effects of insulin on the translocation of protein kinase C-theta and other protein kinase C isoforms in rat skeletal muscles. Biochem J. 1995 May 15;308(Pt 1):177–180. doi: 10.1042/bj3080177. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Yamazaki M., Zhang Y., Watanabe H., Yokozeki T., Ohno S., Kaibuchi K., Shibata H., Mukai H., Ono Y., Frohman M. A. Interaction of the small G protein RhoA with the C terminus of human phospholipase D1. J Biol Chem. 1999 Mar 5;274(10):6035–6038. doi: 10.1074/jbc.274.10.6035. [DOI] [PubMed] [Google Scholar]
  36. Yeo E. J., Exton J. H. Stimulation of phospholipase D by epidermal growth factor requires protein kinase C activation in Swiss 3T3 cells. J Biol Chem. 1995 Feb 24;270(8):3980–3988. doi: 10.1074/jbc.270.8.3980. [DOI] [PubMed] [Google Scholar]
  37. Yeo E. J., Kazlauskas A., Exton J. H. Activation of phospholipase C-gamma is necessary for stimulation of phospholipase D by platelet-derived growth factor. J Biol Chem. 1994 Nov 11;269(45):27823–27826. [PubMed] [Google Scholar]
  38. Zhang Y., Altshuller Y. M., Hammond S. M., Hayes F., Morris A. J., Frohman M. A. Loss of receptor regulation by a phospholipase D1 mutant unresponsive to protein kinase C. EMBO J. 1999 Nov 15;18(22):6339–6348. doi: 10.1093/emboj/18.22.6339. [DOI] [PMC free article] [PubMed] [Google Scholar]

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