Arachidonic acid and phosphorylation synergistically induce a conformational change of p47(phox) to activate the phagocyte NADPH oxidase

The superoxide-producing phagocyte NADPH oxidase can be activated by arachi donic acid (AA) or by phosphorylation of p47(phox) under cell-free conditio ns. The molecular mechanism underlying the activation, however, has remaine d largely unknown. Here we demonstrate that All at high concentrations (50- 100 mu M), induces direct interaction between the oxidase factors p47(phox) and p22(phox) parallel with superoxide production. The interaction, being required for the oxidase activation, is mediated via the Src homology 3 (SH 3) domains of p47(phox) (p47-(SH3)(2)), which are intramolecularly masked i n a resting state. We also show that AA disrupts complexation of p47-(SH3)( 2) with its intramolecular target fragment (amino acids 286-340) without af fecting association of p47-(SH3)(2) with p22(phox), indicating that the dis ruption plays a crucial role in the induced interaction with p22(phox). Pho sphorylation of p47(phox) by protein kinase C partially replaces the effect s of AA; treatment of the SH3 target fragment with PKC in vitro results in a completely impaired interaction with p47-(SH3)(2), and the same treatment of the full-length p47(phox) leads to both interaction with p22(phox) and oxidase activation without AA, but to a lesser extent. Furthermore, phospho rylated p47(phox) effectively binds to p22(phox) and activates the oxidase in the presence of AA at low concentrations (1-5 mu M), where an unphosphor ylated protein only slightly supports superoxide production. Thus AA, at hi gh concentrations, fully induces the interaction of p47(phox) with p22(phox ) by itself, whereas, at low concentrations, AA synergizes with phosphoryla tion of p47(phox) to facilitate the interaction, thereby activating the NAD PH oxidase.