Activation of the leukocyte NADPH oxidase by protein kinase C in a partially recombinant cell-free system.
The leukocyte NADPH oxidase is an enzyme present in phagocytes and B lymphocytes that when activated catalyzes the production of O-2 from oxygen at the expense of NADPH. A correlation between the activation of the oxidase and the phosphorylation of p47(PHOX), a cytosolic oxidase component, is well recognized in whole cells, and direct evidence for a relationship between the phosphorylation of this oxidase component and the activation of the oxidase has been obtained in a number of cell-free systems containing neutrophil membrane and cytosol. Using superoxide dismutase-inhibitable cytochrome c reduction to quantify O-2 production, we now show that p47(PHOX) phosphorylated by protein kinase C activates the NADPH oxidase not only in a cell-free system containing neutrophil membrane and cytosol, but also in a system in which the cytosol is replaced by the recombinant proteins p67(PHOX), Rac2, and phosphorylated p47(PHOX), suggesting that neutrophil plasma membrane plus those three cytosolic proteins are both necessary and sufficient for oxidase activation. In both the cytosol-containing and recombinant cell-free systems, however, activation by SDS yielded greater rates of O-2 production than activation by protein kinase C-phosphorylated p47(PHOX), indicating that a system that employs protein kinase C-phosphorylated p47(PHOX) as the sole activating agent, although more physiological than the SDS-activated system, is nevertheless incomplete.
Lopes
L R
LR
Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, California 92037, USA.
Hoyal
C R
CR
Knaus
U G
UG
Babior
B M
BM
D002474:Cell-Free System; D003573:Cytochrome b Group / Q000378:metabolism; D003574:Cytochrome c Group / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D004789:Enzyme Activation; D019204:GTP-Binding Proteins / Q000378:metabolism; D006153:Guanosine Diphosphate / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D006801:Humans; D007962:Leukocytes / Q000201:enzymology*; D019255:NADPH Oxidases / Q000378:metabolism*; D009504:Neutrophils / Q000201:enzymology / Q000378:metabolism; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism*; D017382:Reactive Oxygen Species / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D012967:Sodium Dodecyl Sulfate / Q000494:pharmacology; D020744:rac GTP-Binding Proteins
D003573:Cytochrome b Group; D003574:Cytochrome c Group; D010750:Phosphoproteins; D017382:Reactive Oxygen Species; D011994:Recombinant Proteins; C079789:neutrophil cytosol factor 67K; D006153:Guanosine Diphosphate; D012967:Sodium Dodecyl Sulfate; D006160:Guanosine Triphosphate; D019255:NADPH Oxidases; C060505:neutrophil cytosolic factor 1; D011493:Protein Kinase C; D019204:GTP-Binding Proteins; D020744:rac GTP-Binding Proteins