Characterization of a mutation in the Phox homology domain of the NADPH oxidase component p40^phox identifies a mechanism for negative regulation of superoxide production

The phagocyte oxidase (Phox) protein p40^phox contains a Phox homology (PX) domain which, when expressed alone, interacts with phosphatidylinositol 3-phosphate (PtdIns (3)P). The functions of the PX domain in p40^phox localization, association with the cytoskeleton, and superoxide production were examined in transgenic COS-7 cells expressing gp91^phox, p22^phox, p67^phox, and p47 ^phox (COS^phox cells). Full-length p40^phox exhibited a cytoplasmic localization pattern in resting cells. Upon stimulation with phorbol 12-myristate 13-acetate or fMet-Leu-Phe, p40^phox translocated to plasma membrane in a p67^phox- and p47 ^phox-dependent manner. Heterologous expression of p40^phox markedly enhanced superoxide production in phorbol 12-myristate 13-acetate - and fMet-Leu-Phe-stimulated COS^phox cells. Unexpectedly, mutation of Arg-57 in the PX domain to Gln, which abrogated PtdIns (3)P binding, produced a dominant inhibitory effect on agonist-induced superoxide production and membrane translocation of p47^phox and p67^phox. The mutant p40 ^phox (p40R57Q) displayed increased association with actin and moesin and was found enriched in the Triton X-100-insoluble fraction along with p67^phox and p47^phox. The enhanced cytoskeleton association of p67^phox and p47^phox and the dominant inhibitory effect produced by the p40R57Q were alleviated when a second mutation at Asp-289, which eliminated p40^phox interaction with p67^phox, was introduced. Likewise, cytochalasin B treatment abolished the dominant inhibitory effect of p40R57Q on superoxide production. These findings suggest a dual regulatory mechanism through the PX domain of p40^phox; its interaction with the actin cytoskeleton may stabilize NADPH oxidase in resting cells, and its binding of PtdIns (3)P potentiates superoxide production upon agonist stimulation. Both functions require the association of p40 ^phox with p67^phox.