Cytosolic Phospholipase A2α Is Targeted to the p47 -PX Domain of the Assembled NADPH Oxidase via a Novel Binding Site in Its C2 Domain

Abstract

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.

p47 Phox Homology Domain Regulates Plasma Membrane but Not Phagosome Neutrophil NADPH Oxidase Activation

“…Because of insensitivity to p47 phox PX domain mutations affecting phosphoinositide binding, interaction with moesin was proposed to mediate p47 phox plasma membrane translocation after insulin-like growth factor-1 stimulation in COS cells (29), although there was no direct evidence for this conclusion. The PX domain of p47 phox also binds to cytosolic phospholipase A 2 ␣, which is required for maximal response to fMLF or PMA stimulation in neutrophils or PLB-985 granulocytes (64). However, the interaction of p47 phox and cytosolic phospholipase A 2 ␣ involves the face of the p47 phox PX domain opposite the phosphoinositide binding pocket (64).…”

“…The PX domain of p47 phox also binds to cytosolic phospholipase A 2 ␣, which is required for maximal response to fMLF or PMA stimulation in neutrophils or PLB-985 granulocytes (64). However, the interaction of p47 phox and cytosolic phospholipase A 2 ␣ involves the face of the p47 phox PX domain opposite the phosphoinositide binding pocket (64).…”

Phospholipase A2 Enzymes: Physical Structure, Biological Function, Disease Implication, Chemical Inhibition, and Therapeutic Intervention

“…258 GIVA PLA 2 is targeted to the p47phox-PX domain of the assembled NADPH oxidase via a novel binding site in its C2 domain. 259 …”

Phospholipases A2 and Inflammatory Responses in the Central Nervous System

“…In human synovial cells, IL-1 β and TNF α mediated the increase in PLA2 activity and the release of AA, and this inflammatory event was linked to superoxide production through activation of NADPH oxidase (Chenevier-Gobeaux et al 2007). Studies with neutrophils demonstrated a complex formation between the C2 domain of cPLA2 with the p47 phox and PX domain of NADPH oxidase (Shmelzer et al 2008). In human tracheal smooth muscle cells, cigarette smoke extract was shown to induce cPLA2 expression and activation involving NADPH oxidase, MAPKs, AP-1, and NF- κ B (Cheng et al 2009).…”