Cytosolic phospholipase A2 (cPLA2)–generated arachidonic acid (AA) has been shown to be an essential requirement for the activation of NADPH oxidase, in addition to its being the major enzyme involved in the formation of eicosanoid at the nuclear membranes. The mechanism by which cPLA2 regulates NADPH oxidase activity is not known, particularly since the NADPH oxidase complex is localized in the plasma membranes of stimulated cells. The present study is the first to demonstrate that upon stimulation cPLA2 is transiently recruited to the plasma membranes by a functional NADPH oxidase in neutrophils and in granulocyte-like PLB-985 cells. Coimmunoprecipitation experiments and double labeling immunofluorescence analysis demonstrated the unique colocalization of cPLA2 and the NADPH oxidase in plasma membranes of stimulated cells, in correlation with the kinetic burst of superoxide production. A specific affinity in vitro binding was detected between GST-p47phox or GST-p67phox and cPLA2 in lysates of stimulated cells. The association between these two enzymes provides the molecular basis for AA released by cPLA2 to activate the assembled NADPH oxidase. The ability of cPLA2 to regulate two different functions in the same cells (superoxide generation and eicosanoid production) is achieved by a novel dual subcellular localization of cPLA2 to different targets.

We have previously demonstrated a physical interaction between cytosolic phospholipase A 2 ␣ (cPLA 2 ) and the assembled NADPH oxidase on plasma membranes following neutrophil stimulation. The aim of the present study was to define the exact binding sites between these two enzymes. Here we show, based on blot overlay experiments, Förster resonance energy transfer analysis and studies in neutrophils from patients with chronic granulomatous disease deficient in p67 phox or p47 phox , that cPLA 2 specifically binds to p47 phox and that p47 phox is sufficient to anchor cPLA 2 to the assembled oxidase on the plasma membranes upon stimulation. Blot overlay and affinity binding experiments using subfragments of cPLA 2 and p47 phox demonstrated that the cPLA 2 -C2 domain and the p47 phox -PX domain interact to form a complex that is resistant to high salt. Computational docking was used to identify hydrophobic peptides within these two domains that inhibited the association between the two enzymes and NADPH oxidase activity in electro-permeabilized neutrophils. These results were used in new docking computations that produced an interaction model. Based on this model, cPLA 2 -C2 domain mutations were designed to explore its interaction p47 phox in neutrophil lysates. The triple mutant F35A/M38A/L39A of the cPLA 2 -C2 domain caused a slight inhibition of the affinity binding to p47 phox , whereas the single mutant I67A was highly effective. The double mutant M59A/H115A of the p47 phox -PX domain caused a significant inhibition of the affinity binding to cPLA 2 . Thus, Ile 67 of the cPLA 2 -C2 domain is identified as a critical, centrally positioned residue in a hydrophobic interaction in the p47 phox -PX domain.

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