Cytosolic phospholipase A2α is targeted to the p47 phox-PX domain of the assembled NADPH oxidase via a novel binding site in its C2 domain

Zeev Shmelzer, Maria Karter, Miriam Eisenstein, Thomas L. Leto, Nurit Hadad, David Ben-Menahem, Daniel Gitler, Shirly Banani, Baruch Wolach, Meir Rotem, Rachel Levy*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review


We have previously demonstrated a physical interaction between cytosolic phospholipase A2α (cPLA2) 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 p67phox or p47phox, that cPLA 2 specifically binds to p47phox and that p47 phox is sufficient to anchor cPLA2 to the assembled oxidase on the plasma membranes upon stimulation. Blot overlay and affinity binding experiments using subfragments of cPLA2 and p47 phox demonstrated that the cPLA2-C2 domain and the p47phox-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, cPLA2-C2 domain mutations were designed to explore its interaction p47phox in neutrophil lysates. The triple mutant F35A/M38A/L39A of the cPLA2-C2 domain caused a slight inhibition of the affinity binding to p47phox, whereas the single mutant I67A was highly effective. The double mutant M59A/H115A of the p47phox-PX domain caused a significant inhibition of the affinity binding to cPLA2. Thus, Ile67 of the cPLA2-C2 domain is identified as a critical, centrally positioned residue in a hydrophobic interaction in the p47phox-PX domain.

Original languageEnglish
Pages (from-to)31898-31908
Number of pages11
JournalJournal of Biological Chemistry
Issue number46
StatePublished - 14 Nov 2008
Externally publishedYes


FundersFunder number
National Institute of Allergy and Infectious DiseasesZIAAI000614


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