Phosphatidic acid and diacylglycerol directly activate NADPH oxidase by interacting with enzyme components

The enzyme NADPH oxidase is regulated by phospholipase D in intact neutroph ils and is activated by phosphatidic acid (PA) plus diacylglycerol (DG) in cell-free systems. We showed previously that cell-free NADPH oxidase activa tion by these lipids involves both protein kinase-dependent and -independen t pathways. Here we demonstrate that only the protein kinase-independent pa thway is operative in a cell-free system of purified and recombinant NADPH oxidase components. Activation by PA + DG was ATP-independent and unaffecte d by the protein kinase inhibitor staurosporine, indicating the lack of pro tein kinase involvement. Both PA and DG were required for optimal activatio n to occur. The drug R59949 reduced activation of NADPH oxidase by either a rachidonic acid or PA + DG, with IC50 values of 46 and 25 CCM, respectively . The optimal concentration of arachidonic acid or PA + DG for oxidase acti vation was shifted to the right with R59949, indicating interference of the drug with the interaction of lipid activators and enzyme components. R5994 9 inhibited the lipid-induced aggregation/sedimentation of oxidase componen ts p47(phox) and p67(phox), suggesting a disruption of the lipid-mediated a ssembly process. The direct effects of R59949 on NADPH oxidase activation c omplicate its use as a "specific" inhibitor of DG kinase, We conclude that the protein kinase-independent pathway of NADPH oxidase activation by PA an d DG involves direct interaction with NADPH oxidase components. Thus, NADPH oxidase proteins are functional targets for these lipid messengers in the neutrophil.