Skip to main content
PMC home page
Biochemical Journal logoLink to Biochemical Journal
. 2002 Apr 1;363(Pt 1):95–103. doi: 10.1042/0264-6021:3630095

Involvement of protein kinase D in Fc gamma-receptor activation of the NADPH oxidase in neutrophils.

Jan K Davidson-Moncada 1, Guillermo Lopez-Lluch 1, Anthony W Segal 1, Lodewijk V Dekker 1
PMCID: PMC1222456  PMID: 11903052

Abstract

Protein kinases involved in the activation of the NADPH oxidase by Fc gamma receptors in neutrophils were studied. Of three different protein kinase C (PKC) inhibitors, Gö 6976 inhibited the NADPH oxidase completely, whereas bisindolylmaleimide I and Ro 31-8220 caused a 70-80% inhibition. Thus a Gö 6976-sensitive, bisindolylmaleimide I/Ro 31-8220-insensitive component contributes to NADPH oxidase activation induced by Fc gamma receptors. Down-regulation of PKC isotypes resulted in inhibition of Fc gamma-receptor-activated NADPH oxidase, but a down-regulation-insensitive component was still present. This component was sensitive to Gö 6976, but insensitive to Ro 31-8220. It has been shown previously that protein kinase D/PKC-mu (PKD) shows this same pharmacology in vitro. We show that PKD is present in neutrophils and that, in contrast with PKC isotypes, PKD is not down-regulated. Therefore PKD may participate in NADPH oxidase activation. To obtain direct evidence for this we adopted an antisense approach. Antisense PKD inhibited NADPH oxidase induced by Fc gamma-receptor stimulation by 50% and the Ro 31-8220-insensitive component in the activation was inhibited by antisense PKD. In vitro kinase assays showed that PKD is activated by presenting IgG-opsonized particles to neutrophils. Furthermore, PKD localizes to the area of particle intake in the cell and phosphorylates two of the three cytosolic components of the NADPH oxidase, p40(phox) and p47(phox). Taken together, these data indicate that Fc gamma receptors engage PKD in the regulation of the NADPH oxidase.

Full Text

The Full Text of this article is available as a PDF (264.1 KB).

Selected References

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

  1. Abedi H., Rozengurt E., Zachary I. Rapid activation of the novel serine/threonine protein kinase, protein kinase D by phorbol esters, angiotensin II and PDGF-BB in vascular smooth muscle cells. FEBS Lett. 1998 May 8;427(2):209–212. doi: 10.1016/s0014-5793(98)00427-x. [DOI] [PubMed] [Google Scholar]
  2. Aoshiba K., Yasui S., Hayashi M., Tamaoki J., Nagai A. Role of p38-mitogen-activated protein kinase in spontaneous apoptosis of human neutrophils. J Immunol. 1999 Feb 1;162(3):1692–1700. [PubMed] [Google Scholar]
  3. Baggiolini M., Wymann M. P. Turning on the respiratory burst. Trends Biochem Sci. 1990 Feb;15(2):69–72. doi: 10.1016/0968-0004(90)90179-f. [DOI] [PubMed] [Google Scholar]
  4. Benna J. E., Dang P. M., Gaudry M., Fay M., Morel F., Hakim J., Gougerot-Pocidalo M. A. Phosphorylation of the respiratory burst oxidase subunit p67(phox) during human neutrophil activation. Regulation by protein kinase C-dependent and independent pathways. J Biol Chem. 1997 Jul 4;272(27):17204–17208. doi: 10.1074/jbc.272.27.17204. [DOI] [PubMed] [Google Scholar]
  5. Bottomley M. J., Salim K., Panayotou G. Phospholipid-binding protein domains. Biochim Biophys Acta. 1998 Dec 8;1436(1-2):165–183. doi: 10.1016/s0005-2760(98)00141-6. [DOI] [PubMed] [Google Scholar]
  6. Bouin A. P., Grandvaux N., Vignais P. V., Fuchs A. p40(phox) is phosphorylated on threonine 154 and serine 315 during activation of the phagocyte NADPH oxidase. Implication of a protein kinase c-type kinase in the phosphorylation process. J Biol Chem. 1998 Nov 13;273(46):30097–30103. doi: 10.1074/jbc.273.46.30097. [DOI] [PubMed] [Google Scholar]
  7. Cabanis A., Gressier B., Brunet C., Dine T., Luyckx M., Cazin M., Cazin J. C. Effect of the protein kinase C inhibitor GF 109 203X on elastase release and respiratory burst of human neutrophils. Gen Pharmacol. 1996 Dec;27(8):1409–1414. doi: 10.1016/s0306-3623(96)00053-5. [DOI] [PubMed] [Google Scholar]
  8. Cooke E., Hallett M. B. The role of C-kinase in the physiological activation of the neutrophil oxidase. Evidence from using pharmacological manipulation of C-kinase activity in intact cells. Biochem J. 1985 Dec 1;232(2):323–327. doi: 10.1042/bj2320323. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Cox J. A., Jeng A. Y., Sharkey N. A., Blumberg P. M., Tauber A. I. Activation of the human neutrophil nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase by protein kinase C. J Clin Invest. 1985 Nov;76(5):1932–1938. doi: 10.1172/JCI112190. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Dekker L. V., Leitges M., Altschuler G., Mistry N., McDermott A., Roes J., Segal A. W. Protein kinase C-beta contributes to NADPH oxidase activation in neutrophils. Biochem J. 2000 Apr 1;347(Pt 1):285–289. [PMC free article] [PubMed] [Google Scholar]
  11. Dekker L. V., McIntyre P., Parker P. J. Mutagenesis of the regulatory domain of rat protein kinase C-eta. A molecular basis for restricted histone kinase activity. J Biol Chem. 1993 Sep 15;268(26):19498–19504. [PubMed] [Google Scholar]
  12. Dekker L. V., Parker P. J. Protein kinase C--a question of specificity. Trends Biochem Sci. 1994 Feb;19(2):73–77. doi: 10.1016/0968-0004(94)90038-8. [DOI] [PubMed] [Google Scholar]
  13. Dibbert B., Weber M., Nikolaizik W. H., Vogt P., Schöni M. H., Blaser K., Simon H. U. Cytokine-mediated Bax deficiency and consequent delayed neutrophil apoptosis: a general mechanism to accumulate effector cells in inflammation. Proc Natl Acad Sci U S A. 1999 Nov 9;96(23):13330–13335. doi: 10.1073/pnas.96.23.13330. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Dieterich S., Herget T., Link G., Böttinger H., Pfizenmaier K., Johannes F. J. In vitro activation and substrates of recombinant, baculovirus expressed human protein kinase C mu. FEBS Lett. 1996 Mar 4;381(3):183–187. doi: 10.1016/0014-5793(96)00116-0. [DOI] [PubMed] [Google Scholar]
  15. Ding J., Knaus U. G., Lian J. P., Bokoch G. M., Badwey J. A. The renaturable 69- and 63-kDa protein kinases that undergo rapid activation in chemoattractant-stimulated guinea pig neutrophils are p21-activated kinases. J Biol Chem. 1996 Oct 4;271(40):24869–24873. doi: 10.1074/jbc.271.40.24869. [DOI] [PubMed] [Google Scholar]
  16. El Benna J., Faust R. P., Johnson J. L., Babior B. M. Phosphorylation of the respiratory burst oxidase subunit p47phox as determined by two-dimensional phosphopeptide mapping. Phosphorylation by protein kinase C, protein kinase A, and a mitogen-activated protein kinase. J Biol Chem. 1996 Mar 15;271(11):6374–6378. doi: 10.1074/jbc.271.11.6374. [DOI] [PubMed] [Google Scholar]
  17. Epling-Burnette P. K., Zhong B., Bai F., Jiang K., Bailey R. D., Garcia R., Jove R., Djeu J. Y., Loughran T. P., Jr, Wei S. Cooperative regulation of Mcl-1 by Janus kinase/stat and phosphatidylinositol 3-kinase contribute to granulocyte-macrophage colony-stimulating factor-delayed apoptosis in human neutrophils. J Immunol. 2001 Jun 15;166(12):7486–7495. doi: 10.4049/jimmunol.166.12.7486. [DOI] [PubMed] [Google Scholar]
  18. Gennaro R., Florio C., Romeo D. Activation of protein kinase C in neutrophil cytoplasts. Localization of protein substrates and possible relationship with stimulus-response coupling. FEBS Lett. 1985 Jan 28;180(2):185–190. doi: 10.1016/0014-5793(85)81068-1. [DOI] [PubMed] [Google Scholar]
  19. Gennaro R., Florio C., Romeo D. Co-activation of protein kinase C and NADPH oxidase in the plasma membrane of neutrophil cytoplasts. Biochem Biophys Res Commun. 1986 Jan 14;134(1):305–312. doi: 10.1016/0006-291x(86)90563-2. [DOI] [PubMed] [Google Scholar]
  20. Gschwendt M., Dieterich S., Rennecke J., Kittstein W., Mueller H. J., Johannes F. J. Inhibition of protein kinase C mu by various inhibitors. Differentiation from protein kinase c isoenzymes. FEBS Lett. 1996 Aug 26;392(2):77–80. doi: 10.1016/0014-5793(96)00785-5. [DOI] [PubMed] [Google Scholar]
  21. Gschwendt M., Kittstein W., Johannes F. J. Differential effects of suramin on protein kinase C isoenzymes. A novel tool for discriminating protein kinase C activities. FEBS Lett. 1998 Jan 9;421(2):165–168. doi: 10.1016/s0014-5793(97)01530-5. [DOI] [PubMed] [Google Scholar]
  22. Hayashi A., Seki N., Hattori A., Kozuma S., Saito T. PKCnu, a new member of the protein kinase C family, composes a fourth subfamily with PKCmu. Biochim Biophys Acta. 1999 May 6;1450(1):99–106. doi: 10.1016/s0167-4889(99)00040-3. [DOI] [PubMed] [Google Scholar]
  23. Heyworth P. G., Segal A. W. Further evidence for the involvement of a phosphoprotein in the respiratory burst oxidase of human neutrophils. Biochem J. 1986 Nov 1;239(3):723–731. doi: 10.1042/bj2390723. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Hug H., Sarre T. F. Protein kinase C isoenzymes: divergence in signal transduction? Biochem J. 1993 Apr 15;291(Pt 2):329–343. doi: 10.1042/bj2910329. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Iglesias T., Rozengurt E. Protein kinase D activation by mutations within its pleckstrin homology domain. J Biol Chem. 1998 Jan 2;273(1):410–416. doi: 10.1074/jbc.273.1.410. [DOI] [PubMed] [Google Scholar]
  26. Iglesias T., Waldron R. T., Rozengurt E. Identification of in vivo phosphorylation sites required for protein kinase D activation. J Biol Chem. 1998 Oct 16;273(42):27662–27667. doi: 10.1074/jbc.273.42.27662. [DOI] [PubMed] [Google Scholar]
  27. Jacobson P. B., Kuchera S. L., Metz A., Schächtele C., Imre K., Schrier D. J. Anti-inflammatory properties of Gö 6850: a selective inhibitor of protein kinase C. J Pharmacol Exp Ther. 1995 Nov;275(2):995–1002. [PubMed] [Google Scholar]
  28. Jamora C., Yamanouye N., Van Lint J., Laudenslager J., Vandenheede J. R., Faulkner D. J., Malhotra V. Gbetagamma-mediated regulation of Golgi organization is through the direct activation of protein kinase D. Cell. 1999 Jul 9;98(1):59–68. doi: 10.1016/S0092-8674(00)80606-6. [DOI] [PubMed] [Google Scholar]
  29. Johannes F. J., Hausser A., Storz P., Truckenmüller L., Link G., Kawakami T., Pfizenmaier K. Bruton's tyrosine kinase (Btk) associates with protein kinase C mu. FEBS Lett. 1999 Nov 12;461(1-2):68–72. doi: 10.1016/s0014-5793(99)01424-6. [DOI] [PubMed] [Google Scholar]
  30. Johannes F. J., Prestle J., Dieterich S., Oberhagemann P., Link G., Pfizenmaier K. Characterization of activators and inhibitors of protein kinase C mu. Eur J Biochem. 1995 Jan 15;227(1-2):303–307. doi: 10.1111/j.1432-1033.1995.tb20389.x. [DOI] [PubMed] [Google Scholar]
  31. Johannes F. J., Prestle J., Eis S., Oberhagemann P., Pfizenmaier K. PKCu is a novel, atypical member of the protein kinase C family. J Biol Chem. 1994 Feb 25;269(8):6140–6148. [PubMed] [Google Scholar]
  32. Knaus U. G., Morris S., Dong H. J., Chernoff J., Bokoch G. M. Regulation of human leukocyte p21-activated kinases through G protein--coupled receptors. Science. 1995 Jul 14;269(5221):221–223. doi: 10.1126/science.7618083. [DOI] [PubMed] [Google Scholar]
  33. Korchak H. M., Rossi M. W., Kilpatrick L. E. Selective role for beta-protein kinase C in signaling for O-2 generation but not degranulation or adherence in differentiated HL60 cells. J Biol Chem. 1998 Oct 16;273(42):27292–27299. doi: 10.1074/jbc.273.42.27292. [DOI] [PubMed] [Google Scholar]
  34. Kramer I. M., Verhoeven A. J., van der Bend R. L., Weening R. S., Roos D. Purified protein kinase C phosphorylates a 47-kDa protein in control neutrophil cytoplasts but not in neutrophil cytoplasts from patients with the autosomal form of chronic granulomatous disease. J Biol Chem. 1988 Feb 15;263(5):2352–2357. [PubMed] [Google Scholar]
  35. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  36. López-Lluch G., Bird M. M., Canas B., Godovac-Zimmerman J., Ridley A., Segal A. W., Dekker L. V. Protein kinase C-delta C2-like domain is a binding site for actin and enables actin redistribution in neutrophils. Biochem J. 2001 Jul 1;357(Pt 1):39–47. doi: 10.1042/0264-6021:3570039. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Martiny-Baron G., Kazanietz M. G., Mischak H., Blumberg P. M., Kochs G., Hug H., Marmé D., Schächtele C. Selective inhibition of protein kinase C isozymes by the indolocarbazole Gö 6976. J Biol Chem. 1993 May 5;268(13):9194–9197. [PubMed] [Google Scholar]
  38. Matthews S. A., Pettit G. R., Rozengurt E. Bryostatin 1 induces biphasic activation of protein kinase D in intact cells. J Biol Chem. 1997 Aug 8;272(32):20245–20250. doi: 10.1074/jbc.272.32.20245. [DOI] [PubMed] [Google Scholar]
  39. McPhail L. C., Qualliotine-Mann D., Waite K. A. Cell-free activation of neutrophil NADPH oxidase by a phosphatidic acid-regulated protein kinase. Proc Natl Acad Sci U S A. 1995 Aug 15;92(17):7931–7935. doi: 10.1073/pnas.92.17.7931. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Newton A. C. Protein kinase C: structure, function, and regulation. J Biol Chem. 1995 Dec 1;270(48):28495–28498. doi: 10.1074/jbc.270.48.28495. [DOI] [PubMed] [Google Scholar]
  41. Ninomiya N., Hazeki K., Fukui Y., Seya T., Okada T., Hazeki O., Ui M. Involvement of phosphatidylinositol 3-kinase in Fc gamma receptor signaling. J Biol Chem. 1994 Sep 9;269(36):22732–22737. [PubMed] [Google Scholar]
  42. 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]
  43. Park J. W., Hoyal C. R., Benna J. E., Babior B. M. Kinase-dependent activation of the leukocyte NADPH oxidase in a cell-free system. Phosphorylation of membranes and p47(PHOX) during oxidase activation. J Biol Chem. 1997 Apr 25;272(17):11035–11043. doi: 10.1074/jbc.272.17.11035. [DOI] [PubMed] [Google Scholar]
  44. Pongracz J., Lord J. M. Superoxide production in human neutrophils: evidence for signal redundancy and the involvement of more than one PKC isoenzyme class. Biochem Biophys Res Commun. 1998 Jun 29;247(3):624–629. doi: 10.1006/bbrc.1998.8867. [DOI] [PubMed] [Google Scholar]
  45. Reeves E. P., Dekker L. V., Forbes L. V., Wientjes F. B., Grogan A., Pappin D. J., Segal A. W. Direct interaction between p47phox and protein kinase C: evidence for targeting of protein kinase C by p47phox in neutrophils. Biochem J. 1999 Dec 15;344(Pt 3):859–866. [PMC free article] [PubMed] [Google Scholar]
  46. Rennecke J., Johannes F. J., Richter K. H., Kittstein W., Marks F., Gschwendt M. Immunological demonstration of protein kinase C mu in murine tissues and various cell lines. Differential recognition of phosphorylated forms and lack of down-regulation upon 12-O-tetradecanoylphorphol-13-acetate treatment of cells. Eur J Biochem. 1996 Dec 1;242(2):428–432. doi: 10.1111/j.1432-1033.1996.0428r.x. [DOI] [PubMed] [Google Scholar]
  47. Rozengurt E., Sinnett-Smith J., Van Lint J., Valverde A. M. Protein kinase D (PKD): a novel target for diacylglycerol and phorbol esters. Mutat Res. 1995 Dec;333(1-2):153–160. doi: 10.1016/0027-5107(95)00141-7. [DOI] [PubMed] [Google Scholar]
  48. Segal A. W., Coade S. B. Kinetics of oxygen consumption by phagocytosing human neutrophils. Biochem Biophys Res Commun. 1978 Oct 16;84(3):611–617. doi: 10.1016/0006-291x(78)90749-0. [DOI] [PubMed] [Google Scholar]
  49. Segal A. W., Garcia R., Goldstone H., Cross A. R., Jones O. T. Cytochrome b-245 of neutrophils is also present in human monocytes, macrophages and eosinophils. Biochem J. 1981 Apr 15;196(1):363–367. doi: 10.1042/bj1960363. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Thelen M., Dewald B., Baggiolini M. Neutrophil signal transduction and activation of the respiratory burst. Physiol Rev. 1993 Oct;73(4):797–821. doi: 10.1152/physrev.1993.73.4.797. [DOI] [PubMed] [Google Scholar]
  51. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Twomey B., Muid R. E., Nixon J. S., Sedgwick A. D., Wilkinson S. E., Dale M. M. The effect of new potent selective inhibitors of protein kinase C on the neutrophil respiratory burst. Biochem Biophys Res Commun. 1990 Sep 28;171(3):1087–1092. doi: 10.1016/0006-291x(90)90795-o. [DOI] [PubMed] [Google Scholar]
  53. Valverde A. M., Sinnett-Smith J., Van Lint J., Rozengurt E. Molecular cloning and characterization of protein kinase D: a target for diacylglycerol and phorbol esters with a distinctive catalytic domain. Proc Natl Acad Sci U S A. 1994 Aug 30;91(18):8572–8576. doi: 10.1073/pnas.91.18.8572. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Van Lint J. V., Sinnett-Smith J., Rozengurt E. Expression and characterization of PKD, a phorbol ester and diacylglycerol-stimulated serine protein kinase. J Biol Chem. 1995 Jan 20;270(3):1455–1461. doi: 10.1074/jbc.270.3.1455. [DOI] [PubMed] [Google Scholar]
  55. Van Lint J., Ni Y., Valius M., Merlevede W., Vandenheede J. R. Platelet-derived growth factor stimulates protein kinase D through the activation of phospholipase Cgamma and protein kinase C. J Biol Chem. 1998 Mar 20;273(12):7038–7043. doi: 10.1074/jbc.273.12.7038. [DOI] [PubMed] [Google Scholar]
  56. Waldron R. T., Iglesias T., Rozengurt E. The pleckstrin homology domain of protein kinase D interacts preferentially with the eta isoform of protein kinase C. J Biol Chem. 1999 Apr 2;274(14):9224–9230. doi: 10.1074/jbc.274.14.9224. [DOI] [PubMed] [Google Scholar]
  57. Wenzel-Seifert K., Schächtele C., Seifert R. N-protein kinase C isoenzymes may be involved in the regulation of various neutrophil functions. Biochem Biophys Res Commun. 1994 May 16;200(3):1536–1543. doi: 10.1006/bbrc.1994.1625. [DOI] [PubMed] [Google Scholar]
  58. Wientjes F. B., Segal A. W. NADPH oxidase and the respiratory burst. Semin Cell Biol. 1995 Dec;6(6):357–365. doi: 10.1016/s1043-4682(05)80006-6. [DOI] [PubMed] [Google Scholar]
  59. Wilkinson S. E., Parker P. J., Nixon J. S. Isoenzyme specificity of bisindolylmaleimides, selective inhibitors of protein kinase C. Biochem J. 1993 Sep 1;294(Pt 2):335–337. doi: 10.1042/bj2940335. [DOI] [PMC free article] [PubMed] [Google Scholar]
  60. Yamamori T., Inanami O., Nagahata H., Cui Y., Kuwabara M. Roles of p38 MAPK, PKC and PI3-K in the signaling pathways of NADPH oxidase activation and phagocytosis in bovine polymorphonuclear leukocytes. FEBS Lett. 2000 Feb 11;467(2-3):253–258. doi: 10.1016/s0014-5793(00)01167-4. [DOI] [PubMed] [Google Scholar]
  61. Zugaza J. L., Sinnett-Smith J., Van Lint J., Rozengurt E. Protein kinase D (PKD) activation in intact cells through a protein kinase C-dependent signal transduction pathway. EMBO J. 1996 Nov 15;15(22):6220–6230. [PMC free article] [PubMed] [Google Scholar]
  62. Zugaza J. L., Waldron R. T., Sinnett-Smith J., Rozengurt E. Bombesin, vasopressin, endothelin, bradykinin, and platelet-derived growth factor rapidly activate protein kinase D through a protein kinase C-dependent signal transduction pathway. J Biol Chem. 1997 Sep 19;272(38):23952–23960. doi: 10.1074/jbc.272.38.23952. [DOI] [PubMed] [Google Scholar]
  63. el Benna J., Faust L. P., Babior B. M. The phosphorylation of the respiratory burst oxidase component p47phox during neutrophil activation. Phosphorylation of sites recognized by protein kinase C and by proline-directed kinases. J Biol Chem. 1994 Sep 23;269(38):23431–23436. [PubMed] [Google Scholar]
  64. el-Benna J., Park J. W., Ruedi J. M., Babior B. M. Cell-free activation of the respiratory burst oxidase by protein kinase C. Blood Cells Mol Dis. 1995;21(3):201–206. doi: 10.1006/bcmd.1995.0023. [DOI] [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES