Cooperation of p40(phox) with p47(phox) for Nox2-based NADPH oxidase activation during Fcγ receptor (FcγR)-mediated phagocytosis: mechanism for acquisition of p40(phox) phosphatidylinositol 3-phosphate (PI(3)P) binding.

During activation of the phagocyte (Nox2-based) NADPH oxidase, the cytoplasmic Phox complex (p47(phox)-p67(phox)-p40(phox)) translocates and associates with the membrane-spanning flavocytochrome b(558). It is unclear where (in cytoplasm or on membranes), when (before or after assembly), and how p40(phox) acquires its PI(3)P-binding capabilities. We demonstrated that in addition to conformational changes induced by H(2)O(2) in the cytoplasm, p40(phox) acquires PI(3)P-binding through direct or indirect membrane targeting. We also found that p40(phox) is essential when p47(phox) is partially phosphorylated during FcγR-mediated oxidase activation; however, p40(phox) is less critical when p47(phox) is adequately phosphorylated, using phosphorylation-mimicking mutants in HEK293(Nox2/FcγRIIa) and RAW264.7(p40/p47KD) cells. Moreover, PI binding to p47(phox) is less important when the autoinhibitory PX-PB1 domain interaction in p40(phox) is disrupted or when p40(phox) is targeted to membranes. Furthermore, we suggest that high affinity PI(3)P binding of the p40(phox) PX domain is critical during its accumulation on phagosomes, even when masked by the PB1 domain in the resting state. Thus, in addition to mechanisms for directly acquiring PI(3)P binding in the cytoplasm by H(2)O(2), p40(phox) can acquire PI(3)P binding on targeted membranes in a p47(phox)-dependent manner and functions both as a "carrier" of the cytoplasmic Phox complex to phagosomes and an "adaptor" of oxidase assembly on phagosomes in cooperation with p47(phox), using positive feedback mechanisms.