Involvement of protein kinase D in Fc gamma-receptor activation of the NADPH oxidase in neutrophils.
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.
Davidson-Moncada
Jan K
JK
Centre for Molecular Medicine, Department of Medicine, University College London, The Rayne Institute, 5 University Street, London WC1E 6JJ, UK.
Lopez-Lluch
Guillermo
G
Segal
Anthony W
AW
Dekker
Lodewijk V
LV
D002227:Carbazoles / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D015536:Down-Regulation; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D007074:Immunoglobulin G / Q000378:metabolism; D007211:Indoles / Q000494:pharmacology; D008301:Maleimides / Q000494:pharmacology; D008856:Microscopy, Fluorescence; D019255:NADPH Oxidases / Q000378:metabolism*; D009504:Neutrophils / Q000201:enzymology* / Q000378:metabolism; D016376:Oligonucleotides, Antisense / Q000494:pharmacology; D010100:Oxygen / Q000378:metabolism; D010101:Oxygen Consumption; D010587:Phagocytosis; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D017452:Receptors, IgG / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D015398:Signal Transduction; D013211:Staphylococcus aureus / Q000378:metabolism
D002227:Carbazoles; D004791:Enzyme Inhibitors; D007074:Immunoglobulin G; D007211:Indoles; D008301:Maleimides; D016376:Oligonucleotides, Antisense; D010750:Phosphoproteins; D017452:Receptors, IgG; D011994:Recombinant Proteins; C085072:neutrophil cytosol factor 40K; C081021:Go 6976; D019255:NADPH Oxidases; C060505:neutrophil cytosolic factor 1; C089212:protein kinase D; D011493:Protein Kinase C; C070515:bisindolylmaleimide I; D010100:Oxygen; C064758:Ro 31-8220