Kinase-dependent change in the conformation of the leukocyte NADPH oxidase subunit p47phox.
The leukocyte NADPH oxidase of neutrophils is a membrane-bound enzyme that catalyzes the production of O2- from oxygen using NADPH as the electron donor. Dormant in resting neutrophils, the enzyme acquires catalytic activity when the cells are exposed to appropriate stimuli. During activation, the cytosolic oxidase components p47phox and p67phox migrate to the plasma membrane, where they associate with cytochrome b558, a membrane-integrated flavohemoprotein, to assemble the active oxidase. In whole cells and under certain circumstances in the cell-free system, the phosphorylation of p47phox mediates the activation process. It has been proposed that conformational changes in the protein structure of cytosolic factor p47phox may be an important part of the activation mechanism. The total protein steady-state intrinsic fluorescence (an emission maximum of 338 nm) exhibited by the tryptophan residues of p47phox was substantially decreased, reflecting on the conformational change that occurs when p47phox was phosphorylated with protein kinase C. We show here that the phosphorylation of p47phox by protein kinase A or mitogen-activated protein kinase, however, had little effect on the intrinsic fluorescence of p47phox. In addition, the present experiments indicate that in the mutant p47phoxS379A, only the single S-->A mutation appears to be a major importance for the function of p47phox, which is able to undergo the change in conformation that takes place when p47phox is phosphorylated by protein kinase C.
Park
J W
JW
Department of Biochemistry, College of Natural Sciences, Kyungpook National University, Taegu, Korea. parkjw@kyungpook.ac.kr
Park
H S
HS
Chang
Y M
YM
D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D004789:Enzyme Activation; D005033:Ethylmaleimide / Q000378:metabolism; D006801:Humans; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D009154:Mutation; D019255:NADPH Oxidases / Q000737:chemistry*; D018375:Neutrophil Activation; D009504:Neutrophils / Q000201:enzymology*; D010750:Phosphoproteins / Q000737:chemistry* / Q000235:genetics; D010766:Phosphorylation; D011487:Protein Conformation; D011493:Protein Kinase C / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry; D013050:Spectrometry, Fluorescence; D014364:Tryptophan / Q000737:chemistry
D010750:Phosphoproteins; D011994:Recombinant Proteins; D014364:Tryptophan; D019255:NADPH Oxidases; C060505:neutrophil cytosolic factor 1; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C; D020928:Mitogen-Activated Protein Kinases; D005033:Ethylmaleimide