Reciprocal interactions between protein kinase C and components of the NADPH oxidase complex may regulate superoxide production by neutrophils stimulated with a phorbol ester.
The 47-kDa subunit of the NADPH oxidase system (p47-phox) of neutrophils undergoes an association with proteins in the Triton X-100-insoluble fraction upon stimulation of the cells with 4 beta-phorbol 12-myristate 13-acetate. This fraction contains the assembled oxidase that catalyzes the generation of superoxide by stimulated cells. In this paper, we report that the addition of an inhibitor of protein kinases (1-(5-isoquinolinylsulfonyl-2-methylpiperazine) to neutrophils that are already stimulated results in the dissociation of p47-phox from this fraction. Antagonists of type 1 and 2A protein phosphatases (calyculin A, okadaic acid) prevented this phenomenon. In contrast, norokadanone, an inactive analog of okadaic acid, did not affect this response. These observations are correlated with previous studies on the phosphorylation of p47-phox and superoxide release. In addition, we show that protein kinase C (PKC) also undergoes an extensive redistribution to the Triton X-100-insoluble fraction in 4 beta-phorbol 12-myristate 13-acetate-stimulated cells, the extent of which is diminished significantly in neutrophils from chronic granulomatous disease patients who lack either p47-phox or cytochrome b558. These studies strongly indicate that PKC and type 1 and/or 2A protein phosphatases are involved in a continuous phosphorylation reaction that maintains the oxidase in the assembled/active state. Moreover, components of the oxidase may target and facilitate the translocation of PKC to a cellular site in close apposition to the oxidase.
Curnutte
J T
JT
Department of Molecular and Experimental Medicine, Scripps Research Institute, La Jolla, California 92037.
Erickson
R W
RW
Ding
J
J
Badwey
J A
JA
D000595:Amino Acid Sequence; D001692:Biological Transport; D003902:Detergents; D006105:Granulomatous Disease, Chronic / Q000276:immunology / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism*; D009252:NADPH Dehydrogenase / Q000378:metabolism; D019255:NADPH Oxidases; D009504:Neutrophils / Q000187:drug effects / Q000276:immunology / Q000378:metabolism*; D017830:Octoxynol; D010100:Oxygen / Q000378:metabolism; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors; D010750:Phosphoproteins / Q000378:metabolism; D011493:Protein Kinase C / Q000378:metabolism*; D047428:Protein Kinase Inhibitors; D013481:Superoxides / Q000378:metabolism*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*
D003902:Detergents; D010750:Phosphoproteins; D047428:Protein Kinase Inhibitors; D013481:Superoxides; D017830:Octoxynol; D009247:NADH, NADPH Oxidoreductases; D019255:NADPH Oxidases; C060505:neutrophil cytosolic factor 1; D009252:NADPH Dehydrogenase; D011493:Protein Kinase C; D010749:Phosphoprotein Phosphatases; D013755:Tetradecanoylphorbol Acetate; D010100:Oxygen