Cooperation of p40phox with p47phox for Nox2-based NADPH Oxidase Activation during Fcγ Receptor (FcγR)-mediated Phagocytosis

Background: p40phox acquires PI(3)P-binding capabilities through arachidonic acid-induced and H2O2-induced conformational changes in phagocytes. Results: In addition to conformational changes induced by H2O2 in the cytoplasm, p40phox can acquire PI(3)P binding following membrane targeting. Conclusion: p40phox has novel mechanisms inducing its conformation changes, apart from p47phox. Significance: This study demonstrates both p40phox and p47phox synchronously function as “carriers” and “adaptors” of Nox2-based NADPH oxidase assembly through their conformation changes. During activation of the phagocyte (Nox2-based) NADPH oxidase, the cytoplasmic Phox complex (p47phox-p67phox-p40phox) translocates and associates with the membrane-spanning flavocytochrome b558. It is unclear where (in cytoplasm or on membranes), when (before or after assembly), and how p40phox acquires its PI(3)P-binding capabilities. We demonstrated that in addition to conformational changes induced by H2O2 in the cytoplasm, p40phox acquires PI(3)P-binding through direct or indirect membrane targeting. We also found that p40phox is essential when p47phox is partially phosphorylated during FcγR-mediated oxidase activation; however, p40phox is less critical when p47phox is adequately phosphorylated, using phosphorylation-mimicking mutants in HEK293Nox2/FcγRIIa and RAW264.7p40/p47KD cells. Moreover, PI binding to p47phox is less important when the autoinhibitory PX-PB1 domain interaction in p40phox is disrupted or when p40phox is targeted to membranes. Furthermore, we suggest that high affinity PI(3)P binding of the p40phox 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 H2O2, p40phox can acquire PI(3)P binding on targeted membranes in a p47phox-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 p47phox, using positive feedback mechanisms.

Chronic granulomatous disease (CGD), characterized by defective microbial killing by phagocytic cells, is caused by defects or deficiencies in any one of five oxidase components: Nox2, p22 phox , p47 phox , p67 phox , or p40 phox . An essential role for Rac in NADPH oxidase activation was also demonstrated in an oxidase-deficient patient who expressed mutated Rac2 (1) and in mice rendered genetically deficient in Rac2 or in Rac1 plus Rac2 (17). p47 phox is called a "carrier," "adaptor," or "organizer" component because it binds to membrane lipids (PI(3,4)P 2 , phosphatidic acid, and phosphatidylserine) through its PX domain (18), is tethered to the flavocytochrome b 558 through direct interactions between p22 phox and its tandem SH3 domains, and is linked to other cytoplasmic Phox proteins in this complex (19,20). CGD patients who lack p47 phox show impaired translocation of p67 phox to the particulate fraction or phagosomes in response to PMA (21,22), fMLP (22), or opsonized zymosan (23), whereas CGD patients who lack p67 phox show normal translocation of p47 phox to the particulate fraction (21,22). p40 phox was shown to act as an essential positive regulator of Nox2 in studies in p40 phox -deficient mice (24), in p40 phoxR58A/Ϫ knock-in mice (25), or in Fc␥IIa receptor-reconstituted cells (26). In recent work, we described a model in which p47 phox functions as an early stage carrier and adaptor protein of the cytoplasmic ternary complex, whereas p40 phox functions as a late stage carrier or adaptor protein that links the cytoplasmic ternary complex to closed phagosomes and prolongs retention of the complex on phagosomes using PI(3)P binding during Fc␥R-mediated oxidative burst (12,27). Although mounting evidence suggested that p40 phox functions as an essential positive regulator of the Nox2-based NADPH oxidase, only recently was p40 phox deficiency described in a CGD patient, who has compound heterozygosity for a missense mutation predicting a R105Q substitution in the PX domain and a frameshift mutation at codon 52 (K52R) with a premature stop at codon 79 and exhibited a severe defect in Fc␥R-mediated oxidative burst but not in PMA-or fMLP-stimulated extracellular ROS release (28). Contrary to views on the role of p40 phox serving as a carrier of the cytoplasmic Phox complex (12, 27, 29 -31), a recent report suggested that p40 phox primarily functions in sustaining Nox2 activity on phagosomes rather than in translocation of the cytoplasmic Phox complex to phagosomes (32). Another report suggested that although p40 phox acts as a carrier of the Phox complex, this function is PX domain-dependent but PI(3)P-independent in PMA-stimulated permeabilized PLB-985 neutrophil cores (31). Thus, where (in the cytoplasm or on membranes), when (before or after assembly), and how p40 phox acquires its PI(3)P-binding capabilities is unsolved, and how p40 phox cooperates with p47 phox during oxidase assembly or activation is also unclear. To address these questions, we used membrane-targeted mutants of p40 phox and p47 phox to delineate contributions of various intra-and intermolecular domain interactions affecting their targeting to phagosomes and oxidase activation. Here we show that in addition to acquiring PI(3)P-binding capabilities following exposure to H 2 O 2 in the cytoplasm, p40 phox can acquire PI(3)P binding following membrane targeting, either directly by itself or indirectly in a p47 phox -dependent manner through interactions in the p47 phox -p67 phox -p40 phox complex. We found that the dependence on p40 phox PI(3)P binding for Nox2 activity is determined by the phosphorylation status of p47 phox . p40 phox is essential during Fc␥R-mediated oxidase activation; however, p40 phox is less critical under conditions when p47 phox is adequately phosphorylated, using phosphorylation/activation-mimicking p47 phox mutants. Moreover, PI binding of 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. Taken together, these results indicate that p40 phox and p47 phox cooperate in executing the carrier function directing the cytoplasmic ternary Phox complex to phagosomes and the adaptor function for assembly of the Nox2 complex during the Fc␥R-mediated oxidative burst.

EXPERIMENTAL PROCEDURES
Materials-Goat polyclonal antibody (pAb) against p47 phox or p67 phox and rabbit pAb against p40 phox were described previously (33,34). Rabbit pAb against mouse p40 phox and mouse monoclonal Ab (mAb) against p67 phox were from Millipore and BD Biosciences, respectively. Mouse mAb against the C terminus of p47 phox (196 -390 aa) and rabbit mAb against the C-terminal end of p40 phox were from Santa Cruz Biosciences and Abcam, respectively. Mouse mAb against gp91 phox or p22 phox was a kind gift from Drs. Roos and Verhoeven (35). Goat pAb against Fc␥RIIa and mouse mAb against early endosome antigen-1 (EEA1) were from R&D Systems and BD Biosciences, respectively. H 2 O 2 was from Wako Pure Chemical Industries.
The purified full-length His 6 -p40 phox protein (300 nM) was mixed with biotin-coupled PI(3)P-containing polymerized liposomes (100 M) (PI(3)P PolyPIPsomes TM : Y-P003, Echelon) in 50 l of the binding buffer (12). After 10 min of agitation at 4°C, 0.01 mM H 2 O 2 was added in the solution and incubated for 10 min at 4°C. Then streptavidin-coupled magnetic beads (Dynabeads M-280 Streptavidin, Invitrogen) were added to the solution and agitated for 30 min at 4°C. The precipitates were washed three times using a magnetic rack with the buffer, the material absorbed to beads was eluted in Laemmli sample buffer, and the magnetic beads were removed using a magnetic rack. The aliquots of eluants were subjected to SDS-PAGE and followed by immunoblotting using mouse mAb against His 6 (9C11)-peroxidase-conjugated (Wako; 1:1000 at room temperature for 2 h).
Confocal Fluorescence Imaging Studies Using Fixed Cells or Live Cells-A total of 2.5 ϫ 10 5 cells (HEK293, HEK293 Nox2/Fc␥RIIa , or HEK293 p67phox ) were seeded on 35-mm glass bottom dishes (MatTek chambers) 48 h prior to transfection and transfected using FuGENE 6. 25-30 h after the transfection, cells were fixed using 4% paraformaldehyde in HEPES buffer solution, permeabilized as described previously (37), and stained using primary Abs at room temperature for 2 h. Primary Abs were visualized by a confocal laser-scanning fluorescence microscope (LSM510 or LSM700, Zeiss) using Alexa-conjugated anti-IgG (Invitrogen; 1:2000, 0.5 h at room temperature). BIgG was prepared using 5-m glass beads (Duke Scientific Corp.) at 10 mg/500 l of HBSS ϩϩ (Invitrogen), as described previously (42). 25-30 h after the transfection, the culture medium was replaced with HBSS ϩϩ . After HBSS ϩϩ containing BIgG (five targets per cell) or H 2 O 2 was added to each plate (12), images were collected at 5-s intervals for 15 min using a confocal laser-scanning fluorescence microscope with a heated stage and objective (38). The point of stimulant addition or the starting point of ingestion of added BIgG was chosen as time 0. All imaging experiments were performed in triplicate and were repeated in at least three independent transfection experiments (n Ն 9).
ROS Production Assay-HEK293 Nox2/Fc␥RIIa and RAW246.7 cells were seeded on 6-well dishes at 2.5 ϫ 10 5 cells/well and 1.5 ϫ 10 5 cells/well, respectively, 48 h prior to transfection. HEK293 and RAW246.7 cells were transfected using FuGENE 6 and FuGENE HD in complexes with various combinations of plasmids, respectively. The transfection to RAW246.7 cells was most efficient by FuGENE HD among several reagents tested, and the efficacy was about 70 -80% based on the imaging experiments using various GFP-based plasmids, such as GFP-p40 phox . The cells were fed 5 h post-transfection with complete medium and were used for assay 25-30 h after transfection. ROS release with or without stimulation (3 l of BIgG or 200 ng/ml PMA; Sigma-Aldrich) from 2.0 ϫ 10 5 trypsinized cells was measured by luminol-enhanced chemiluminescence methods in the presence of exogenous 10 units/ml HRP (Sigma-Aldrich) and 200 M luminol (Sigma-Aldrich) for 20 min using a luminometer (Mithras LB940, Berthold). The ROS detected in the present study was the sum of extracellular ROS and intracellular ROS (probably including intraphagosomal ROS detected by luminol ϩ exogenous HRP) but predominantly extracellular ROS (supplemental Fig. 1C). The assay (luminol-HRP without SOD ϩ catalase; also luminol-HRP with SOD ϩ catalase, luminol without HRP, and isoluminol-HRP) clearly shows p40 phox dependence in response to BIgG (supplemental Fig. 1C). NADPH oxidase activity was inhibited by 10 min of prior incubation with 10 M diphenylene iodonium (Sigma-Aldrich). Comparable expression of Phox proteins was adjusted and confirmed by immunoblotting using the total lysates from the same number of cells.
Statistical Analysis-Mean oxidase activities (ROS production) were calculated from at least three independent transfection experiments and were presented as percentages (mean Ϯ S.E.). Significant differences were calculated by Student's t test, and results with p Ͻ 0.05 were considered significant.
These results suggest that H 2 O 2 induces conformational changes within p40 phox in the cytoplasm, enabling it to function as a carrier protein that directs the cytoplasmic Phox complex to PI(3)P-enriched membranes. To support this speculation, we performed a binding (pull-down) experiment using purified His 6 -p40 phox (PX) and GST-p40 phox (PB1) proteins. H 2 O 2 (0.01 mM) weakened the interaction between His 6 -p40 phox (PX) and GST-p40 phox (PB1), further suggesting that H 2 O 2 induces some conformational changes in the PX and/or the PB1 domain of p40 phox (Fig. 1F). Furthermore, in an in vitro binding assay using purified full-length His 6 -p40 phox protein and PI(3)P-containing liposomes, H 2 O 2 (0.01 mM) strengthened the interaction between His 6 -p40 phox and PI(3)P, suggesting that H 2 O 2 induces disruption of the PX-PB1 domain interaction within p40 phox (Fig. 1G).
To further investigate the possibility that indirect membrane targeting of p40 phox also induces conformational changes in p40 phox promoting its binding to PI(3)P, we used a PM-targeted mutant of p67 phox , GFP-p67 phox pp. GFP-p67 phox is a cytoplasmic protein in resting cells (12); however, GFP-p67 phox pp showed PM localization in WT HEK293 cells (Fig. 2E). When cytoplasmic mKO-p40 phox was co-expressed with GFP-p67 phox pp, mKO-p40 phox and GFP-p67 phox pp co-localized at EEs in addition to the PM in WT HEK 293 cells (Fig. 2F). This EE but not PM localization of mKO-p40 phox was abolished in the case of mKO-p40 phox (R105K), which does not bind PI(3)P (39) (Fig. 2G).
These results suggest that PM or endomembrane targeting of p40 phox , whether through direct or indirect means, caused recruitment of p40 phox to PI(3)P-enriched EEs through subcel-lular membrane cycling (PM to EEs in the endocytic pathway (47,48) and endomembranes to EEs in the retrograde-transport and anterograde-transport pathways (49)); finally, p40 phox bound to PI(3)P and accumulated on the membranes of EE. In agreement with our data, a recent study reported that the membrane-spanning Phox protein (heterodimer of Nox2 and p22 phox ) was localized on the recycling endosomes as well as EEs and PM in CHO model cells and macrophages (43). Thus, p40 phox probably develops PI(3)P-binding capabilities also through direct or indirect membrane targeting and may even promote these membrane cycling and trafficking pathways through PI(3)P binding.
These data show that indirect targeting of p40 phox to membranes through other Phox protein interactions enables p40 phox to bind to PI(3)P, thereby redirecting the cytoplasmic ternary Phox complex to PI(3)P-enriched membranes.
These results indicate that the high affinity of the PX domain of p40 phox for PI(3)P, even when masked by the PB1 domain in the resting state, initiates or dictates its accumulation on phagosomes. Despite the limitations of FYVE-p40 phox detection by imaging these fluorescent-tagged proteins, even the basal level affinity for PI(3)P binding of the PX domain of p40 phox or FYVE domain appears to influence ROS production.
(i.e. p47 phox (⌬AIR) and p47 phox (S303D/S304D/S328D)) ( Fig. 5 and supplemental Figs. 6 and 8). In the present study, we showed the dependence on p40 phox PI(3)P binding for Nox2 activity (both for extracellular and intracellular ROS) during Fc␥R-mediated oxidative burst, based on the following methods and observations: 1) in assays using luminol with HRP (measuring total ROS but predominantly extracellular ROS), in assays using luminol with HRP ϩ (SOD ϩ catalase) (measuring intracellular ROS), in assays using luminol without HRP (measuring intracellular ROS), or in assays using isoluminol with HRP (measuring extracellular ROS) in HEK293 Nox2/Fc␥RIIa cells (Fig. 5, A and B, and supplemental Figs. 1 and 6); 2) in assays using RAW264.7 p40/p47KD cells ( Fig. 5D and supplemental Fig.  8); and 3) based on a report demonstrating p40 phox and PI(3)P binding dependence during Fc␥R-mediated oxidative burst (measured using luminol with HRP) in COS7 cells stably expressing Phox proteins and Fc␥RIIa (32). A series of stepwise phosphorylation events at eight distinct phosphorylated sites within p47 phox were reported (8,9), of which only four are prominently phosphorylated in membrane-bound p47 phox fractions in early phases of stimulation in normal neutrophils but not in flavocytochrome b 558 -deficient CGD neutrophils (8). We demonstrated that PI binding (i.e. membrane targeting) (12,18,56), by p47 phox is less critical (Fig. 6) when the autoinhibitory (PX-PB1) interaction within p40 phox is released and allows binding to PI(3)P or when p40 phox is targeted to membranes by other means, as seen with p40 phox (F320A) and p40 phox pp. These observations strongly support the proposed carrier function of p40 phox in delivering the ternary Phox complex to phagosomes in cooperation with p47 phox under conditions when p47 phox is only partially phosphorylated (e.g. in the initial stages of translocation of the Phox complex to phagosomes) or when PI levels are insufficient to bind p47 phox (e.g. in late stages of phagocytosis) (12). Furthermore, although the function of p40 phox as a carrier and adaptor seems to be less prominent than p47 phox (Fig. 6A), we propose that both p40 phox and p47 phox are required for orchestrating optimal phagosometargeting of the cytoplasmic Phox complex and also for stable assembly and retention of the Nox2 complex on phagosomes during the Fc␥R-mediated oxidative burst. Several reports support this concept of a functional partnership of both proteins: p47 phox is required for phagosome-targeting of p67 phox and ROS production in p47 phox -deficient neutrophils (23); p40 phoxdeficient neutrophils exhibit a severe defect in Fc␥R-mediated oxidative burst but not in PMA-or fMLP-stimulated ROS production (28); PI(3)P-binding capabilities of p40 phox are required for prolonged retention of p40 phox (28) and the p40 phox -p67 phox -p47 phox complex (27) on phagosomes; autosomal recessive CGD patients, including a p40 phox -deficient patient, suffer less severe clinical phenotypes than X-linked CGD patients (62); phosphorylation of p40 phox on Thr-154 is required for phagosome targeting of p47 phox and ROS production in reconstituting p40 phox -deficient (or knockdown) neutrophils (63); and both p40 phox and p47 phox are required for ROS production in microvascular endothelial cells (64).
It was reported that translocation of p67 phox , involving the carrier function of p40 phox , is dependent on the PX domain of p40 phox but is PI(3)P-independent and that activation of Nox2 is PI(3)P-dependent in PMA-stimulated, permeabilized PLB-985 neutrophil cores (31). These authors speculated that moesin (65), a cytoskeletal protein, instead of PI(3)P may be a predominant target of the p40 phox PX domain in the PMAstimulated oxidative burst of permeabilized cores (31). In the present study using HEK293 Nox2/Fc␥RIIa cells, we found that the PX domain of p40 phox is much less critical in responses to PMA than the PX domain of p47 phox (supplemental Fig. 9), indicating that PMA, an analog of diacylglycerol, triggers predominantly p47 phox (PX)-dependent but p40 phox -independent oxidase activation, consistent with studies in p40 phox -deficient COS phox Fc␥R cells and neutrophils (26,28). Thus, the p40 phox and PI(3)P dependence in Nox2 activation is determined by stimulus (e.g. BIgG versus PMA).
Cho and Stahelin (58) described a general mechanism of membrane-protein interactions in which membrane adsorption of PX domain-containing proteins such as p47 phox and p40 phox (18,56) is initially driven by nonspecific electrostatic interactions (between anionic lipids in membranes and cationic surfaces of proteins) and by diffusion, which is then followed by specific interaction with PIs and interfacial penetration (66) of hydrophobic and aromatic residues located near its respective binding site of PIs. Crystallographic studies on p40 phox , revealed that intramolecular PX-PB1 domain interactions are sterically inhibiting access of the PX domain with membraneembedded PI(3)P, rather than completely masking the PI(3)Pbinding site (13); in other words, the PX domain is able to access PI(3)P in certain conditions because three-dimensional positioning of membrane-embedded PI(3)P changes during phagosome formation. This speculation is supported by reports that full-length p40 phox binds to soluble PI(3)P to the same extent as the PX domain (39) and that full-length p40 phox binds to PI(3)P in surface plasmon resonance and lipid monolayer assays in which PI(3)P is flexible in the lipid monolayer (56). Considering our finding that p40 phox possessing the high affinity binding PX domain for PI(3)P accumulated on phagosomes (Fig. 4A), even if sterically inhibited, whereas FYVE-p40 phox possessing low affinity PI(3)P binding did not accumulate on phagosomes (Fig.  4B), the "semimasked" high affinity binding PX domain of p40 phox probably fulfills important missions during its translocation, including initiation of translocation.
Enhanced protein tyrosine phosphorylation by H 2 O 2 -induced inhibition of phosphotyrosine phosphatases has been reported (67,68). In addition, it was reported that H 2 O 2 directly induces conformational changes in proteins (69). In the present study, p40 phox tyrosine phosphorylation was not observed in response to H 2 O 2 using anti-phosphotyrosine Ab (4G10) (data not shown), consistent with previous work (70), and conformational changes within p40 phox induced by H 2 O 2 were observed by in vitro binding assays (Fig. 1, F and G). The other adaptor protein, p47 phox , showed no translocation to any membrane site in response to H 2 O 2 . Interestingly, PKCs, which are well known to phosphorylate p47 phox (10,38,71) and accumulate on phagosomes (38), are reported to be activated by H 2 O 2 both on membranes and in the cytoplasm (72,73). Thus, H 2 O 2 may induce several positive feedback effects on both p40 phox and p47 phox , both on membranes and in the cytoplasm during the Fc␥R-mediated oxidative burst. In addition to this positive feedback mechanism of p40 phox directly acquiring PI(3)P binding capabilities by exposure to H 2 O 2 (demonstrated in the present study) or by arachidonic acid (12), p40 phox can also indirectly acquire PI(3)P binding (Figs. 2F and 3, C and F) on targeted membranes in a p47 phox -dependent manner through its associations within the p47 phox -p67 phox -p40 phox complex.