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

Abstract

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.

FIGURE 1.

H₂O₂ induces EE targeting of p40^phox. A, transfected GFP-p40^phox is localized in the cytoplasm (left); in contrast, GFP-p40^phox(PX) (center; arrow) and p40^phox(F320A) (right; arrow) are localized at dotlike structures, in resting WT HEK293 cells. Bar, 10 μm. B, p40^phox(F320A) co-expressed with p47^phox + p67^phox supports moderate constitutive (32.0 ± 6.2%) and high BIgG-stimulated ROS production in HEK293^{Nox2/FcγRIIa} cells when compared with cells expressing WT p40^phox + p47^phox + p67^phox. Western blotting detects comparable levels of cytoplasmic Phox proteins in both transfection experiments. C, in response to exogenous 1 mm H₂O₂ (120 s after stimulation), GFP-p40^phox expressed in WT HEK293 cells translocates to dotlike structures (arrowheads). D, the dotlike structures are co-stained by an EE marker, EEA1 (arrowheads). E, cytoplasmic GFP-p67^phox co-expressed with mKO-p40^phox in WT HEK293 cells translocates to dotlike structures (arrowheads) after stimulation (110 s) with 1 mm H₂O₂. Similar effects of H₂O₂ are observed in RAW 264.7 cells (supplemental Fig. 3). F, effect of H₂O₂ on the PX-PB1 domain interaction in vitro. The interaction between purified His₆-p40^phox(PX) and GST-p40^phox(PB1) proteins detected by pull-down assays is weakened by the addition of 0.01 mm H₂O₂. Similar results are obtained in three independent experiments. G, effect of H₂O₂ on binding of p40^phox to PI(3)P in vitro. The interaction between purified full-length His₆-p40^phox protein and PI(3)P-containing liposomes detected by pull-down assays is strengthened by the addition of 0.01 mm H₂O₂. Similar results were obtained in three independent experiments. Error bars, S.E.

FIGURE 2.

Direct or indirect membrane targeting of p40^phox induces EE-targeting of p40^phox in WT HEK293 cells. A, transfected GFP-p40^phoxpp is localized at dotlike structures (left; arrowheads) in addition to PM (left; arrow). The dotlike structures are co-stained with EEA1 (right; arrowheads). Bar, 10 μm. B, the dotlike localization of GFP-p40^phoxpp disappears in the case of GFP-p40^phox(ΔPX)pp. Arrow, PM. C, GFP-p40^phoxp is co-stained with EEA1 (arrowheads). A movie of accumulation on phagosome of p40^phoxp during ingestion of BIgG in HEK293^{Nox2/FcγRIIa} cells is available (supplemental Video 1). D, the dotlike localization of GFP-p40^phoxp disappears in the case of GFP-p40^phox(R105K)p. E, PM-targeting mutant of cytoplasmic GFP-p67^phox, GFP-p67^phoxpp, shows PM localization (arrows) in addition to nuclear localization. F, when cytoplasmic mKO-p40^phox is co-expressed with GFP-p67^phoxpp, both proteins are co-localized at EEs stained by EEA1 (using Cy5-conjugated secondary Ab) (arrowheads) in addition to the PM (arrow). G, the dotlike localization of both proteins disappears in the case of mKO-p40^phox(R105K) + GFP-p67^phoxpp. Arrow, PM.

FIGURE 3.

Indirect membrane targeting of p40^phoxby p47^phoxp or Noxo1-p47^phox through the p47^phox-p67^phox-p40^phox interaction induces EE localization of p40^phox in HEK293^p67phox cells. A, both GFP-p47^phox and mKO-p40^phox are localized in the cytoplasm. Bar, 10 μm. B, GFP-p47^phoxp has a reticular, nuclear membrane and Golgi complex localization. C, in the case of co-expression of GFP-p47^phoxp and mKO-p40^phox, dotlike structures with GFP-p47^phoxp and mKO-p40^phox appear (arrows). Co-staining of stably expressed p67^phox at the dotlike structures is shown in supplemental Fig. 5B. D, the dotlike structures disappear in the case of co-expression of GFP-p47^phoxp and mKO-p40^phox(R105K). E, GFP-Noxo1-p47^phox shows PM localization (arrow) in addition to nuclear localization. F, with co-expression of GFP-Noxo1-p47^phox and mKO-p40^phox, mKO-p40^phox is localized at dotlike structures (arrowheads) in addition to PM (arrow) with GFP-Noxo1-p47^phox. In contrast, without co-expression of GFP-Noxo1-p47^phox, mKO-p40^phox is localized in the cytoplasm (asterisks). G, the dotlike localization, but not PM localization, of both proteins disappears in the case of co-expression of GFP-Noxo1-p47^phox and mKO-p40^phox(R105K). H, both the dotlike and PM localizations of both proteins disappear with co-expression of GFP-Noxo1(R40Q)-p47^phox and mKO-p40^phox.

FIGURE 4.

High affinity PI(3)P-binding PX domain is a critical determinant of p40^phox phagosomal accumulation and oxidase function in HEK293^{Nox2/FcγRIIa} cells. A, weak accumulation of GFP-p40^phox on phagosome 105 s after the start of BIgG ingestion is shown (arrow). A movie is available (supplemental Video 2). B, no accumulation of GFP-FYVE-p40^phox on phagosome is observed (arrow). A movie is available (supplemental Video 3). C, accumulation of GFP-PH(TAPP1)-p40^phox on nascent phagosomal cup is observed 65 s after starting BIgG ingestion (arrow). No accumulation of GFP-PH(TAPP1)-p40^phox on mature phagosome is observed (arrowhead). A movie is available (supplemental Video 4). D, FYVE-p40^phoxp is localized at dotlike structures that co-stained with EEA1 (arrowheads). A movie of accumulation on phagosome of FYVE-p40^phoxp during ingestion of BIgG is available (supplemental Video 5). E, BIgG-stimulated ROS production is enhanced 4-fold by p47^phox + p67^phox + PH(TAPP1)-p40^phox and is statistically decreased by p47^phox + p67^phox + FYVE-p40^phox when compared with p47^phox + p67^phox + p40^phox. *, p < 0.05. Error bars, S.E.

FIGURE 5.

Dependence of PI(3)P-binding of p40^phox in ROS production by Nox2 is determined by the phosphorylation status of p47^phox in both HEK293^{Nox2/FcγRIIa} (A–C) and RAW264.7^p40/p47KD cells (D). A, ROS production using p47^phox(ΔAIR) is constitutive, shows small BIgG dependence, and is effectively inhibited by 10 μm diphenylene iodonium. The addition of p40^phox enhances ROS production by p47^phox(ΔAIR) and p67^phox. ROS production by p47^phox(ΔAIR) + p67^phox + p40^phox is independent of the PI(3)P-binding capabilities of p40^phox (p40^phox(R105K)). n.s., not statistically significant. B, ROS production using one- or two-site phosphorylation-mimicking mutants of p47^phox as well as WT p47^phox shows PI(3)P-binding of p40^phox dependence; however, p47^phox(S303D/S304D/S328D) is independent of PI(3)P binding to p40^phox. All mimicking mutants showed higher ROS production than WT p47^phox. 3D, S303D/S304D/S328D. C, PI(3)P dependence in ROS production using WT or one- or two-site phosphorylation-mimicking mutants of p47^phox. Data shown in B are normalized to activities supported by WT p40^phox. All mimicking mutants show less PI(3)P dependence than WT p47^phox. The mimicking mutants having Asp-328 (S328D, S303D/S328D, and S304D/S328D) show constitutive activity without BIgG and less PI(3)P dependence than S303D, S304D, or S303D/S304D. *, p < 0.01. D, in RAW264.7^p40/p47KD cells, ROS production using WT p47^phox shows almost complete dependence on PI(3)P binding to p40^phox. In contrast, reconstituted ROS production by p47^phox(S303D/S304D/S328D) or p47^phox(ΔAIR) shows only moderate PI(3)P dependence. *, p < 0.01. 3D, S303D/S304D/S328D. Error bars, S.E.

FIGURE 6.

Membrane-associating mutants of p40^phox can rescue the function of the PX domain of p47^phox. A, in HEK293^{Nox2/FcγRIIa} cells, ROS production by p47^phox(R90K) + p67^phox + p40^phox is almost absent, whereas p47^phox(R90K)p dramatically rescues reconstituted ROS production. p40^phoxpp, but not p40^phoxp, is able to completely rescue ROS production by p47^phox(R90K) + p67^phox + p40^phox. p40^phox(F320A), which is targeted to EE by disrupted PX-PB1 intermolecular interaction (Fig. 1A), effectively rescues ROS production by p47^phox(R90K) + p67^phox + p40^phox, whereas p40^phox(R105K/F320A) does not. B, in RAW264.7^p40/p47KD cells, WT p40^phox, but not p40^phox(R105K), moderately rescues ROS production with p47^phox(R90K). Membrane-associating mutants, p47^phox(R90K)p, p40^phoxpp, and p40^phox(F320A) almost completely rescue reconstituted ROS production with p47^phox(R90K). Error bars, S.E.