title: Differential requirement for classic and novel PKC isoforms in respiratory burst and phagocytosis in RAW 264.7 cells.
abstract: The binding of Ab (IgG)-opsonized particles by FcgammaRs on macrophages results in phagocytosis of the particles and generation of a respiratory burst. Both IgG-stimulated phagocytosis and respiratory burst involve activation of protein kinase C (PKC). However, the specific PKC isoforms required for these responses have yet to be identified. We have studied the involvement of PKC isoforms in IgG-mediated phagocytosis and respiratory burst in the mouse macrophage-like cell line, RAW 264.7. Like primary monocyte/macrophages, their IgG-mediated phagocytosis was calcium independent and diacylglycerol sensitive, consistent with novel PKC activation. Respiratory burst in these cells was Ca2+ dependent and inhibited by staurosporine and calphostin C as well as by the classic PKC-selective inhibitors Gö 6976 and CGP 41251, suggesting that classic PKC is required. In contrast, phagocytosis was blocked by general PKC inhibitors but not by the classic PKC-specific drugs. RAW 264.7 cells expressed PKCs alpha, betaI, delta, epsilon, and zeta. Subcellular fractionation demonstrated that PKCs alpha, delta, and epsilon translocate to membranes during phagocytosis. In Ca2+-depleted cells, only novel PKCs delta and epsilon increased in membranes, and the time course of their translocation was consistent with phagosome formation. Confocal microscopy of cells transfected with green fluorescent protein-conjugated PKC alpha or epsilon confirmed that these isoforms translocated to the forming phagosome in Ca-replete cells, but only PKC epsilon colocalized with phagosomes in Ca2+-depleted cells. Taken together, these results suggest that the classic PKC alpha mediates IgG-stimulated respiratory burst in macrophages, whereas the novel PKCs delta and/or epsilon are necessary for phagocytosis.
authors.affiliation: Center for Cell Biology and Cancer Research, Department of Biochemistry, Albany Medical College, Albany, NY 12208, USA.
authors.forename: E C
authors.initials: EC
authors.lastname: Larsen
authors.forename: J A
authors.initials: JA
authors.lastname: DiGennaro
authors.forename: N
authors.initials: N
authors.lastname: Saito
authors.forename: S
authors.initials: S
authors.lastname: Mehta
authors.forename: D J
authors.initials: DJ
authors.lastname: Loegering
authors.forename: J E
authors.initials: JE
authors.lastname: Mazurkiewicz
authors.forename: M R
authors.initials: MR
authors.lastname: Lennartz
mesh_terms: D000818:Animals; D001692:Biological Transport / Q000276:immunology; D002118:Calcium / Q000378:metabolism / Q000502:physiology; D002460:Cell Line; D002462:Cell Membrane / Q000201:enzymology / Q000276:immunology / Q000378:metabolism; D004075:Diglycerides / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D049452:Green Fluorescent Proteins; D007425:Intracellular Membranes / Q000201:enzymology / Q000276:immunology; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008264:Macrophages / Q000187:drug effects / Q000201:enzymology* / Q000276:immunology / Q000378:metabolism; D051379:Mice; D009281:Naphthalenes / Q000494:pharmacology; D010587:Phagocytosis / Q000187:drug effects / Q000276:immunology*; D010588:Phagosomes / Q000201:enzymology / Q000276:immunology; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D064546:Protein Kinase C beta; D051571:Protein Kinase C-alpha; D051745:Protein Kinase C-delta; D051744:Protein Kinase C-epsilon; D016897:Respiratory Burst / Q000187:drug effects / Q000276:immunology*; D019311:Staurosporine / Q000031:analogs & derivatives* / Q000494:pharmacology
chemical_list: C011439:1,2-diacylglycerol; D004075:Diglycerides; D004791:Enzyme Inhibitors; D007527:Isoenzymes; D008164:Luminescent Proteins; D009281:Naphthalenes; D049452:Green Fluorescent Proteins; C497259:Prkcd protein, mouse; C497256:Prkce protein, mouse; C496630:Prkca protein, mouse; D011493:Protein Kinase C; D064546:Protein Kinase C beta; D051571:Protein Kinase C-alpha; D051745:Protein Kinase C-delta; D051744:Protein Kinase C-epsilon; D019311:Staurosporine; C058819:calphostin C; C059539:midostaurin; D002118:Calcium