A carboxy-terminal peptide from p47-phox is a substrate for phosphorylation by protein kinase C and by a neutrophil protein kinase.
Agonist-activated phosphorylation of neutrophil proteins including p47-phox, a cytosolic component of the respiratory burst oxidase, has been implicated in the signal transduction cascade which leads to activation of the superoxide generating respiratory burst. We have previously reported (J. Biol. Chem. 265, 17550-59) that in a cell-free activation system consisting of cytosol plus plasma membrane from human neutrophils, diacylglycerol acts synergistically with an anionic amphiphile such as sodium dodecyl sulfate (SDS) to augment superoxide generation and assembly of the oxidase, and that p47 phosphorylation can occur under these conditions. Herein, we show that a peptide corresponding to a carboxy terminal sequence of p47-phox is a substrate for phosphorylation both by purified protein kinase C (a mixture of alpha, beta, and gamma forms) and by a distinct kinase or kinases present in neutrophil cytosol. Based on its activator requirements, the neutrophil kinase differs from classical protein kinase C, but may be a protein kinase C variant, based on inhibition by a protein kinase C peptide. Although in the cell-free system phosphorylation occurs under conditions which are similar to those for activation of superoxide generation, phosphorylation is not required for activation (1). Rather, protein assembly or aggregation which occurs under activation conditions may also promote phosphorylation.
Uhlinger
D J
DJ
Department of Biochemistry, Emory University School of Medicine, Atlanta, GA 30322.
Perry
D K
DK
D000255:Adenosine Triphosphate / Q000378:metabolism; D000470:Alkaloids / Q000494:pharmacology; D002118:Calcium / Q000494:pharmacology; D003600:Cytosol / Q000201:enzymology; D004789:Enzyme Activation; D006801:Humans; D009252:NADPH Dehydrogenase / Q000378:metabolism*; D019255:NADPH Oxidases; D009504:Neutrophils / Q000201:enzymology*; D010446:Peptide Fragments / Q000378:metabolism*; D010718:Phosphatidylserines / Q000494:pharmacology; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011494:Protein Kinases / Q000378:metabolism*; D019311:Staurosporine; D013379:Substrate Specificity; D013481:Superoxides / Q000378:metabolism
D000470:Alkaloids; D010446:Peptide Fragments; D010718:Phosphatidylserines; D010750:Phosphoproteins; D013481:Superoxides; D000255:Adenosine Triphosphate; D019255:NADPH Oxidases; C060505:neutrophil cytosolic factor 1; D009252:NADPH Dehydrogenase; D011494:Protein Kinases; D011493:Protein Kinase C; D019311:Staurosporine; D002118:Calcium