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 neutrophi ls undergoes an association with proteins in the Triton X-100-insolubl e fraction upon stimulation of the cells with 4 beta-phorbol 12-myrist ate 13-acetate. This fraction contains the assembled oxidase that cata lyzes 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 alre ady stimulated results in the dissociation of p47-phox from this fract ion. Antagonists of type 1 and 2A protein phosphatases (calyculin A, o kadaic acid) prevented this phenomenon. In contrast, norokadanone, an inactive analog of okadaic acid, did not affect this response. These o bservations are correlated with previous studies on the phosphorylatio n of p47-phox and superoxide release. In addition, we show that protei n kinase C (PKC) also undergoes an extensive redistribution to the Tri ton 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 eithe r p47-phox or cytochrome b(558). These studies strongly indicate that PKC and type 1 and/or 2A protein phosphatases are involved in a contin uous phosphorylation reaction that maintains the oxidase in the assemb led/active state. Moreover, components of the oxidase may target and f acilitate the translocation of PKC to a cellular site in close apposit ion to the oxidase.