A Regulated Adaptor Function of p40^phox: Distinct p67^phox Membrane Targeting by p40^phox and by p47^phox

Abstract

In the phagocytic cell, NADPH oxidase (Nox2) system, cytoplasmic regulators (p47^phox, p67^phox, p40^phox, and Rac) translocate and associate with the membrane-spanning flavocytochrome b₅₅₈, leading to activation of superoxide production. We examined membrane targeting of phox proteins and explored conformational changes in p40^phox that regulate its translocation to membranes upon stimulation. GFP-p40^phox translocates to early endosomes, whereas GFP-p47^phox translocates to the plasma membrane in response to arachidonic acid. In contrast, GFP-p67^phox does not translocate to membranes when expressed alone, but it is dependent on p40^phox and p47^phox for its translocation to early endosomes or the plasma membrane, respectively. Translocation of GFP-p40^phox or GFP-p47^phox to their respective membrane-targeting sites is abolished by mutations in their phox (PX) domains that disrupt their interactions with their cognate phospholipid ligands. Furthermore, GFP-p67^phox translocation to either membrane is abolished by mutations that disrupt its interaction with p40^phox or p47^phox. Finally, we detected a head-to-tail (PX–Phox and Bem1 [PB1] domain) intramolecular interaction within p40^phox in its resting state by deletion mutagenesis, cell localization, and binding experiments, suggesting that its PX domain is inaccessible to interact with phosphatidylinositol 3-phosphate without cell stimulation. Thus, both p40^phox and p47^phox function as diverse p67^phox “carrier proteins” regulated by the unmasking of membrane-targeting domains in distinct mechanisms.

INTRODUCTION

In phagocytic cells, reactive oxygen species (ROS) are produced by NADPH oxidase (Nox2 system). The enzyme is a multiprotein complex assembled from a membrane-spanning flavocytochrome b₅₅₈ (composed of gp91^phox [Nox2] and p22^phox) and four cytoplasmic components (p47^phox, p67^phox, p40^phox, and Rac) (Leto, 1999; Nauseef, 2004; Quinn and Gauss, 2004). In unstimulated phagocytes, the oxidase is dissociated and inactive: the flavocytochrome b₅₅₈ is stored on the membranes of intracellular granules (Jesaitis et al., 1990), Rac is maintained in a GDP-bound cytoplasmic complex dimerized with Rho-guanine nucleotide dissociation inhibitor (GDI) (Bokoch et al., 1994), and the other phox proteins associate in a separate ternary cytoplasmic complex (p47^phox-p67^phox-p40^phox) in a dephosphorylated state (Bolscher et al., 1989; Rotrosen and Leto, 1990; Kuribayashi et al., 2002; Lapouge et al., 2002). During phagocyte activation, intracellular granules containing flavocytochrome b₅₅₈ fuse with phagosomes; p47^phox is phosphorylated, thereby inducing conformational changes in p47^phox that promote the interaction of the cytoplasmic complex with the flavocytochrome b₅₅₈; and Rac dissociates from Rho-GDI and translocates independently to the membrane after exchange of GDP for GTP (Heyworth et al., 1994; Zhao et al., 2003), resulting in generation of superoxide anion by the transfer of electrons from cytoplasmic NADPH to molecular oxygen.

Chronic granulomatous disease (CGD), characterized by defective microbial killing by phagocytic cells, is caused by defects or deficiencies in any one of four oxidase components: Nox2, p22^phox, p47^phox, or p67^phox (Leto, 1999). An essential role for Rac1 or Rac2 in Nox2 activation was also identified in cell-free reconstitution studies (Abo et al., 1991; Knaus et al., 1991). This role was later confirmed in an oxidase-deficient patient who expressed mutant Rac2 (Ambruso et al., 2000; Williams et al., 2000) and in mice rendered genetically deficient in Rac2 (Roberts et al., 1999; Gu et al., 2003). Rac and p67^phox together have a direct roles in regulating electron flow through the flavocytochrome b₅₅₈ through GTP-dependent interactions; hence, p67^phox is called an “activator” component (Bokoch and Diebold, 2002). Alternatively, p47^phox is called an “adaptor” or “organizer” component because it binds to membrane lipids [phosphatidylinositol-(3,4)-bisphosphate [PI(3,4)P₂] and phosphatidic acid (PA)] through its phox (PX) domain (Kanai et al., 2001; Karathanassis et al., 2002), is tethered to the flavocytochrome b₅₅₈ through direct interactions between p22^phox and its Src homology (SH) 3 domain, and is linked to other cytoplasmic phox proteins to this complex (Leto et al., 1994; Sumimoto et al., 1994). CGD patients who lack p47^phox show impaired translocation of p67^phox to the particulate or membrane fraction, whereas CGD patients who lack p67^phox show normal translocation of p47^phox to the particulate fraction, indicating the adaptor function of p47^phox in recruitment of p67^phox to the membrane (Heyworth et al., 1991; Dusi et al., 1996; Allen et al., 1999). However, Nox2 activity can be reconstituted in vitro in the absence of p47^phox, when p67^phox and Rac1 are provided in excess (Freeman and Lambeth, 1996; Koshkin et al., 1996) or when p67^phox is adapted with the membrane-binding sequences from Rac1, although GTP-bound Rac is still required for oxidase activation (Gorzalczany et al., 2000; Alloul et al., 2001), indicating p67^phox and Rac1 are minimum essential cytoplasmic components in the Nox2 system. p40^phox also has a PX domain that specifically binds to phosphatidylinositol 3-phosphate [PI(3)P] (Bravo et al., 2001; Kanai et al., 2001), a phospholipid enriched in the early endosome (Gillooly et al., 2000) and produced in the phagosomal membrane during phagocytosis (Ellson et al., 2001a; Gillooly et al., 2001). Thus, p40^phox is also thought to serve as an adaptor component that recruits p67^phox to phagosomal membranes (Kuribayashi et al., 2002). There are, however, no reports of p40^phox defects or deficiencies resulting in CGD. There is some controversy on the precise function of p40^phox, because both Nox2-inhibitory (Sathyamoorthy et al., 1997; Vergnaud et al., 2000; Lopes et al., 2004) and Nox2-supporting effects of p40^phox (Tsunawaki et al., 1996; Cross, 2000; Ellson et al., 2001b; Kuribayashi et al., 2002; He et al., 2004) have been reported. However, recent studies in p40^phox-deficient mice or in FcγIIA receptor-reconstituted cells indicate p40^phox is an essential component of the Nox2 system (Ellson et al., 2006; Suh et al., 2006).

Arachidonic acid (AA) has been used frequently as a activator of Nox2 in cell-free assay systems (Bromberg and Pick, 1984; Curnutte, 1985). Although relatively high concentrations of AA (50–200 μM) are required for activation of Nox2 both in vitro and in vivo, functional roles for cytosolic phospholipase A₂ (cPLA₂), which produces AA, in Nox2 activation have been demonstrated at the cellular level (Dana et al., 1998; Zhao et al., 2002; Shmelzer et al., 2003). Furthermore, recent studies have shown that the orchestration of low concentrations of AA produced by PLA₂ together with protein kinase C (PKC)-dependent phosphorylation promotes translocation of p47^phox and enhances ROS production by Nox2 (Shiose and Sumimoto, 2000; Peng et al., 2003).

In recent work, we described mechanisms for translocation of p47^phox (Ueyama et al., 2004) and isoform-specific translocation of Rac to the phagosome (Ueyama et al., 2005). To understand and clarify targeting of p67^phox, an essential cytoplasmic Nox2 activator that acts through Rac, we used AA as a stimulant and explored the adaptor functions of p40^phox and p47^phox in recruitment of p67^phox to membranes. In the present study, we show that p67^phox is not targeted to the membrane by itself but that it relies on diverse targeting and adaptor functions of p40^phox and p47^phox for its translocation to membranes. Furthermore, we examine the mechanism by which p40^phox acquires its function as a p67^phox “adaptor protein.” We propose here that an intramolecular interaction between the PX and the PB1 domains of p40^phox in the resting state renders p40^phox inaccessible to bind PI(3)P and that upon stimulation the intramolecular interaction can be disrupted, enabling p40^phox to bind to PI(3)P-enriched membranes.

MATERIALS AND METHODS

Materials

Goat polyclonal antibody (Ab) against human p47^phox or p67^phox and rabbit polyclonal Ab against human p40^phox were described previously (Leto et al., 1991; Sathyamoorthy et al., 1997). Mouse monoclonal Ab against human p47^phox or p67^phox was from BD Biosciences (San Jose, CA). Rabbit polyclonal Ab against green fluorescent protein (GFP) or early endosome antigen-1 (EEA1) was from Clontech (Mountain View, CA) or from ABR-Affinity BioReagents (Golden, CO), respectively. Rabbit polyclonal Ab against glutathione S-transferase (GST) and mouse monoclonal Ab against (His)₆ were from Santa Cruz Biotechnology (Santa Cruz, CA) and GE Healthcare (Little Chalfont, Buckinghamshire, United Kingdom), respectively. AA was purchased from Doosan Serdary Research Laboratories (Kyungki-Do, Korea).

Cell Culture

RAW 264.7 macrophages (Ueyama et al., 2005) and COS-7 cells (American Type Culture Collection, Manassas, VA) were maintained in DMEM (Invitrogen, Carlsbad, CA) supplemented with 10% heat-inactivated fetal bovine serum (Invitrogen) and antibiotics (100 U/ml penicillin and 100 μg/ml streptomycin) at 37°C in 5% CO₂.

Construction of Plasmids

The pEGFP-C1 plasmids (Clontech) containing human p47^phox, p47^phox (W193R), p40^phox, p40^phox(PX; aa 1-167), p40^phox(PX:R105K), and p47^phox(R90K) were described previously (Ago et al., 2001, 2003; Ueyama et al., 2004). cDNA encoding p67^phox and p67^phox(ΔSH3, aa 1-457) were described previously (de Mendez et al., 1994; Ueyama et al., 2004), transferred into pEGFP-C1, and designated GFP-p67^phox and GFP-p67^phox(ΔSH3), respectively. The fragment encoding p67^phox(ΔAD, deletion of aa 199-212) was made by polymerase chain reaction (PCR) by using appropriate primers, transferred into pEGFP-C1, and designated GFP-p67^phox(ΔAD). The fragments encoding p47^phox, p47^phox(ΔPR; aa 1-359), and p40^phox were amplified by PCR, cloned into pIRES2-DsRed2, and designated p47^phox-IRES2-DsRed2, p47^phox(ΔPR)-IRES2-DsRed2, and p40^phox-IRES2-DsRed2, respectively. The fragments encoding p47^phox and GFP-p67^phox(ΔAD) were amplified by PCR, cloned into BglII and EcoRI sites of multiple cloning site of pIRES2-DsRed2 and BstXI and XbaI sites of pIRES2-DsRed2 in place of DsRed2, respectively, and designated p47^phox-IRES2-GFP-p67^phox(ΔAD). GFP-p40^phox(R105K), GFP-p67^phox(ΔAD:K355A), and p40^phox (D289A)-IRES2-DsRed2 were made using the QuikChange II XL site-directed mutagenesis kit protocol (Stratagene, La Jolla, CA). We confirmed that GFP-p47^phox and GFP-p67^phox support Nox2 activity at levels comparable with unfused, wild-type p47^phox, and p67^phox (data not shown).

To study the intramolecular binding of p40^phox, the DNA fragments encoding p40^phox(aa 1-229), p40^phox(aa 1-261), p40^phox(aa 1-284), p40^phox(aa 1-306), p40^phox(aa 1-317), and p40^phox(aa 1-328) were amplified by PCR, cloned into pEGFP-C1, and designated GFP-p40^phox(1-229), GFP-p40^phox(1-261), GFP-p40^phox(1-284), GFP-p40^phox(1-306), GFP-p40^phox(1-317), and GFP-p40^phox(1-328), respectively (Figure 9A). The DNA fragment encoding p40^phox(Δ318-328; deletion of aa 318-328) was amplified by PCR by using GFP-p40^phox(1-328) as a template with appropriate primers, cloned into pEGFP-C1, and designated GFP-p40^phox(Δ318-328). p40^phox(318-328:11A) in pEGFP-C1, in which residues 318-328 are replaced by 11 alanine residues, was made using GFP-p40^phox as a template by the QuikChange II XL site-directed mutagenesis kit. All modified expression vectors were sequenced to confirm their identities.

Figure 9.

Inhibitory effects of the p40^phox PB1 domain (residues 318-328) in PX domain-mediated targeting of p40^phox to vesicular structures in RAW 264.7 cells. (A) Structure of GFP-tagged p40^phox constructs used in the present study. (B) GFP-p40^phox(PX), GFP-p40^phox(1-229), GFP-p40^phox(1-261), GFP-p40^phox(1-284), GFP-p40^phox(1-306), and GFP-p40^phox(1-317) are localized on vesicular structures. In contrast, full-length GFP-p40^phox(1–339) and GFP-p40^phox(1-328) remain localized in the cytoplasm. Bar, 10 μm. Representative of n ≥ 6. (C) GFP-p40^phox(Δ318-328) is localized on vesicular structures. Bar, 10 μm. Representative of n ≥ 6. (D) GFP-p40^phox(318-328:11A) is localized on vesicular structures. Bar, 10 μm. Representative of n ≥ 6. (E) Exogenously expressed wild-type p40^phox is primarily detected by immunofluorescence in the cytoplasm in COS-7 cells (left). p40^phox expressing cell is detected by DsRed2 fluorescence by using p40^phox-IRES2-DsRed2 plasmid (right). Bar, 10 μm. Representative of n ≥ 6.

Immunoprecipitation and Immunoblotting

COS-7 cells were transfected using electroporation methods (Shirai et al., 1998). Forty-eight hours after the transfection, cells were lysed in homogenizing buffer in the presence of protease inhibitors (Ueyama et al., 2001) by sonication. The total cell lysates were centrifuged at 20,000 × g for 30 min at 4°C, the supernatants were incubated with the Ab (α-p47^phox, α-p67^phox or α-p40^phox, α-GFP) or control IgG (goat or rabbit; Santa Cruz Biotechnology, Santa Cruz, CA) for 2 h at 4°C and then with protein G-Sepharose 4B (GE Healthcare) for an additional 12 h at 4°C. The precipitates were washed three times, and the aliquots of precipitates were subjected to SDS-PAGE and followed by immunoblotting using primary Ab for α-p47^phox, α-p67^phox, α-p40^phox, or α-GFP (1/2000, room temperature [RT] for 2 h). Bound antibodies were detected with secondary antibody-horseradish peroxidase (HRP) conjugates (Jackson ImmunoResearch Laboratories, West Grove, PA) by using the ECL detection system (GE Healthcare).

Cell Imaging Studies

Fluorescently labeled (Alexa-568) and IgG-opsonized phagocytosis targets (BIgG) were prepared using 2-μm glass beads (Duke Scientific, Fremont, CA), as described previously (Larsen et al., 2002). In total, 1.0 × 10⁵ RAW 264.7 cells were seeded on 35-mm glass bottom dishes (MatTek chambers; MatTek, Ashland, MA) and transfected using Superfect (QIAGEN, Valencia, CA). Twenty-four to 32 h after the transfection, the culture medium was replaced with Hanks' balanced salt solution (HBSS)⁺⁺ (Larsen et al., 2002). As a stimulant, HBSS⁺⁺ containing BIgG (five targets per cell) or HBSS⁺⁺ containing AA (indicated final concentrations) was added to each plate. Images were collected using an LSM 510 invert (Carl Zeiss, Thornwood, NY) confocal laser scanning fluorescence microscope with a heated stage and objective (40× oil or 63× oil) as described previously (Ueyama et al., 2004). The images were collected at 5 s intervals for 5 min.

For immunocytochemical studies, cells (transfected or untransfected) were fixed with 4% paraformaldehyde in 0.1 M phosphate buffer (Ueyama et al., 2001). After permeabilization, cells were stained using primary Ab (p40^phox, diluted 1/500; EEA-1, diluted 1/250) for 2 h at RT. Primary Abs were visualized by confocal microscopy using Alexa-488 or -594–conjugated anti-rabbit IgG (1/2000, 0.5 h at RT; Invitrogen).

All time-lapsed imaging experiments were performed in triplicate and were repeated in at least three independent transfection experiments (n ≥ 9). All imaging experiments were very reproducible (≥80%), except for the experiment of GFP-p40^phox stimulated with BIgG (≤10%; Figure 2A and Supplemental Video 1).

Figure 2.

Accumulation of GFP-p40^phox(PX) and full-length GFP-p40^phox on membranes during FcγR-mediated phagocytosis in RAW 264.7 cells. (A) During FcγR-mediated phagocytosis, transient vesicular accumulation of GFP-p40^phox (arrows) is observed occasionally, which fuses with newly forming phagosomes. Time-lapsed photography of this process is available in Supplemental Video 1. BIgG, Alexa-568 labeled– and IgG-opsonized 2-μm glass beads. Addition of BIgG occurs at time 0. Representative of n ≥ 30. (B) GFP-p40^phox(PX) is localized prominently on vesicular structures in the cytoplasm, which accumulates on phagosomes (arrows and arrowheads) during FcγR-mediated phagocytosis after phagosome sealing. Time-lapsed photography is available in Supplemental Video 2. Representative of n ≥ 9. (C) Vesicular structures containing GFP-p40^phox(PX) in the cytoplasm are reactive with antibodies against EEA-1, a marker of early endosomes in RAW 264.7 cells. Bar, 10 μm. Representative of n ≥ 6.

In Vitro Binding Assay

The cDNA for the PCR-amplified PX domain of p40^phox(aa 1-167) was cloned into BamHI and EcoRI sites of pProEx-Htb (Invitrogen) and designated (His)₆-p40^phox(PX). Forward and reverse oligonucleotides for p40^phox(aa 307-317), p40^phox(aa 318-328), and p40^phox(aa 329-339) were annealed and cloned into the BamHI and EcoRI sites of pGEX-6P-1 and designated GST-p40^phox(307-317), GST-p40^phox(318-328), and GST-p40^phox(329-339), respectively. PCR-amplified fragments of p40^phox(SH3 domain; aa 170-229) and p40^phox(PB1 domain; aa 237-339) were cloned into the BamHI and EcoRI sites of pGEX-6P-1 and designated GST-p40^phox(SH3) and GST-p40^phox(237-339), respectively. All constructs were sequenced to confirm their identities. Tagged proteins were expressed in Escherichia coli strain BL21-CodonPlus (DE3)-RIL (Stratagene). When the bacteria reached an OD₆₀₀ of ∼0.2, protein expression was induced by 0.1 mM isopropyl β-d-thiogalactoside at 21°C for 16 h and purified using nickel-Agarose [Invitrogen; in the case of (His)₆-p40^phox(PX), the tagged protein was solubilized in 50 mM 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate] or glutathione-Sepharose 4B (GE Healthcare). The purified (His)₆-p40^phox(PX) (500 nM) was mixed with each purified GST-tagged fragment of p40^phox (500 nM) in 500 μl of buffer (25 mM HEPES, 150 mM NaCl, and 2% fatty acid-free bovine serum albumin, pH 7.5). Glutathione-Sepharose 4B beads were added to the solution, rotated for 30 min at 4°C, and then washed three times with the same buffer. The material absorbed to beads was eluted with 10 mM glutathione, and the elutants were separated on a 15% SDS-polyacrylamide gel. Western blotting was performed using primary (His)₆ Ab and secondary Ab-HRP conjugates, and detected by the ECL detection system.

Online Supplemental Material

The supplemental time-lapsed photography shows the “kiss and run” fusion-like accumulation of GFP-p40^phox on phagosomes (Video 1; total time 330 s) and the transient accumulation of GFP-p40^phox(PX) on phagosomes by fusion of early endosomes and from a cytoplasmic pool of GFP-p40^phox(PX) (Video 2; total 310 s) during Fcγ receptor (FcγR)-mediated phagocytosis in RAW 264.7 cells. The supplemental time-lapsed photography shows translocation of GFP-p67^phox(ΔAD) both to early endosomes and to the plasma membrane (Video 3; total time 150 s) after AA stimulation in RAW 264.7 cells coexpressing p40^phox and p47^phox.

RESULTS

Accumulation of p40^phox(PX) at the Phagosome during FcγR-mediated Phagocytosis

Although a recent study reported that p40^phox and p67^phox exist as a heterodimer in the resting state and that the p47^phox-containing ternary complex forms only after cell activation (Brown et al., 2003), most other studies to date indicate that the phox proteins exist as a stable 1:1:1 ternary cytoplasmic complex (p47^phox-p67^phox-p40^phox) in their dephosphorylated state even without cell stimulation (Figure 1A; Bolscher et al., 1989; Rotrosen and Leto, 1990; Lapouge et al., 2002; Kuribayashi et al., 2002). We examined ternary complex formation by using GFP-p67^phox coexpressed with p47^phox and p40^phox in COS-7 cells and confirmed that all three proteins exist at least partially in a complex that could be detected by immunoprecipitation with antibodies against any of these components (Figure 1B). Furthermore, we examined ROS production using GFP-p67^phox or p67^phox plus p47^phox and Nox2 in human embryonic kidney 293 and COS-7 cell models, and confirmed that GFP-p67^phox supports Nox2 activity at levels comparable with the unfused, wild-type p67^phox (data not shown). These results indicate that GFP-p67^phox behaves like wild-type p67^phox, at least with regard to its interactions and assembly with other phox proteins and its ability to support ROS production in the Nox2 system. Therefore, we used GFP-p67^phox to monitor its interactions with other oxidase components in intact cells.

Figure 1.

Phox proteins exist in a complex in resting COS-7 cells. (A) Schematic representation of structural domains and their interactions among p47^phox, p67^phox, and p40^phox. Additional interactions between Rac and p67^phox, p22^phox and p47^phox, PI(3,4)P₂, PA and the PX domain of p47^phox, and PI(3)P and the PX domain of p40^phox are also shown. AIR, autoinhibitory region; TPR, tetratricopeptide repeat; AD, activation domain; PB1, Phox and Bem1; OPCA, OPR-PC-AID. Mutations that disrupt these interactions are shown beside arrows; those used in the current study are in bold. (B) Supernatants from COS-7 cells expressing p47^phox, GFP-p67^phox, and p40^phox were immunoprecipitated by rabbit polyclonal antibodies against GFP or rabbit control IgG, goat polyclonal antibodies against p47^phox or goat control IgG, or rabbit polyclonal antibodies against p40^phox or rabbit control IgG and then immunoblotted with antibodies against p47^phox (monoclonal), p67^phox (monoclonal), and p40^phox (polyclonal). p47^phox and p40^phox are coimmunoprecipitated with GFP-p67^phox (left), GFP-p67^phox and p40^phox are coimmunoprecipitated with p47^phox (middle), and p47^phox and GFP-p67^phox are coimmunoprecipitated with p40^phox (right). Similar results were obtained in two independent experiments

Because we described the mechanism of GFP-p47^phox accumulation on phagosomal cups and phagosomes during FcγR-mediated phagocytosis (et al., 2004), the present study focused on p40^phox. GFP-p40^phox is detected diffusely throughout the cytoplasm of resting RAW 264.7 cells (Figure 2A). During FcγR-mediated phagocytosis, the accumulation of GFP-p40^phox on newly forming phagosomes occurs as a kiss and run phenomenon, in which a transient fusion of GFP-p40^phox is observed between phagosomes and endosomes without showing much evidence of complete intermixing of their membranes (Duclos et al., 2000). This phenomenon is not observed constantly (≤10%), but it occurred only occasionally when followed by time-lapsed photography (Figure 2A and Supplemental Video 1). We then used GFP-p40^phox(PX) for further studies, because it was reported to bind specifically to PI(3)P. Consistent with a previous report (Ellson et al., 2001a), GFP-p40^phox(PX) is localized faintly in the cytoplasm and predominantly on vesicular structures (Figure 2B), which could be colabeled with antibodies against early endosomal antigen (EEA)-1 (Figure 2C), a marker of the early endosome that is enriched in PI(3)P (Gillooly et al., 2000) (Figure 2B). The vesicular structures and free-form of GFP-p40^phox(PX) seems to fuse readily with phagosomes in later stages of FcγR-mediated phagocytosis, after the phagosome is sealed (Figure 2B and Supplemental Video 2). The fusion of early endosomes, as detected by GFP fluorescence, resembles the same kiss and run pattern (transient interaction); however, the GFP-p40^phox(PX) fluorescence seems to reflect significant amounts membrane material fusing with the phagosomes (Supplemental Video 2). This accumulation of GFP-p40^phox(PX) on the phagosome persists longer on phagosomes (>100 s) than observed with GFP-p47^phox, which starts accumulating before phagosome sealing (on phagosomal cups) and is observed on phagosomal cups and retained on phagosomes for 67 ± 7 s (Ueyama et al., 2004). Accumulation of GFP-p67^phox on phagosomes was not evident in RAW 264.7 during FcγR-mediated phagocytosis, perhaps reflecting low levels of expression of the endogenous phox proteins (data not shown). However, GFP-p67^phox accumulated at phagosomal cups and phagosomes after coexpression with p47^phox (data not shown). The differences in localization patterns between GFP fusion proteins of full-length p40^phox and its isolated PX domain suggest that the PX domain of p40^phox is masked or is otherwise maintained in a state that is relatively inaccessible to early endosomes or PI(3)P-enriched phagosomes. The differences in localization between p40^phox and p47^phox fusions proteins suggest that the two proteins are targeted to phagosomes by distinctly different signals.

Translocation of p40^phox and p47^phox, but Not p67^phox, to Membranes after AA Stimulation

To examine the mechanism by which p40^phox acquires the capacity to bind PI(3)P, we explored the effects of cell stimulation by phorbol 12-myristate 13-acetate (PMA), N-formyl-l-methionyl-l-leucyl-l-phenylalanine (fMLP), or AA. Among these stimulants, only AA can induce any detectable translocation of GFP-p40^phox in our real-time confocal microscopy system, which shows that GFP-p40^phox translocates to vesicular structures in the cytoplasm (Figure 3A, left) that are colabeled with antibodies against EEA-1 (Figure 3B). The same translocation pattern of AA-induced GFP-p40^phox translocation is observed using a C-terminally tagged GFP fusion with p40^phox, p40^phox-GFP (data not shown). In contrast, GFP-p47^phox translocates to the plasma membrane after cell stimulation by AA (Figure 3A, middle), whereas GFP-p67^phox shows no translocation to any membranes after treatments by any of these three stimulants (Figure 3A, right). These data suggest that AA induces conformational changes in p40^phox that render the PX domain accessible to bind to early endosomes, as occurs in AA-treated p47^phox to expose its SH3 domains to bind to p22^phox (Shiose and Sumimoto, 2000; Zhao et al., 2002). It is intriguing that GFP-p40^phox and GFP-p47^phox, but not GFP-p67^phox, have the ability to bind to the membrane only after AA stimulation, which likely induces conformational change in both proteins.

Figure 3.

AA-stimulated translocation of GFP-p40^phox and GFP-p47^phox, but not GFP-p67^phox, to the membrane in RAW 264.7 cells. (A) Left, AA triggers GFP-p40^phox translocation to vesicular structures in the cytoplasm. (A) Middle, AA stimulates GFP-p47^phox translocation to the plasma membrane. (A) Right, GFP-p67^phox shows no significant translocation to membranes. AA was added at time 0. Bar, 10 μm. Representative of n ≥ 9. (B) Vesicular structures in the cytoplasm containing GFP-p40^phox are labeled with antibodies against EEA-1, a marker of early endosomes. Bar, 10 μm. Representative of n ≥ 6.

There are reports that phosphorylation occurs within p40^phox at Thr154 and Ser315 (Bouin et al., 1998) and that phosphorylation of Thr154, but not Ser315, inhibits Nox2 activation (Lopes et al., 2004). Therefore, we made mutants of GFP-p40^phox that could not be phosphorylated [GFP-p40^phox(T154A) and GFP-p40^phox(S315A)] or mimic the phosphorylated states [GFP-p40^phox(T154D) and GFP-p40^phox (S315D)]. However, these mutants are localized throughout the cytoplasm and seem to translocate normally to early endosomes after AA stimulation, like GFP-p40^phox (data not shown).

Function of PX Domains of p40^phox and p47^phox for Their Translocation

We then explored the translocation mechanisms of p40^phox and p47^phox by examining the effects of mutating their PX domains. GFP-p40^phox(PX:R105K), which loses its capacity to bind PI(3)P (Ago et al., 2001), shows no vesicular localization pattern, nor does it translocate to early endosomes after AA stimulation (Figure 4A). GFP-p40^phox(R105K) also shows no translocation to the early endosome after AA stimulation (Figure 4B). These observations indicate that translocation of p40^phox to early endosomes requires binding of the PX domain to PI(3)P.

Figure 4.

Requirement of phosphatidylinositol phosphate-specific binding of PX domains for translocation of p40^phox and p47^phox in AA-stimulated RAW 264.7 cells. (A) GFP-p40^phox(PX:R105K), which loses the capacity for binding PI(3)P, shows no vesicular localization pattern nor translocation to early endosomes. Representative of n ≥ 9. (B) GFP-p40^phox(R105K) shows no translocation to early endosomes. Representative of n ≥ 9. (C) GFP-p47^phox(R90K), which loses the capacity for binding PI(3,4)P₂, shows no translocation to the plasma membrane. Representative of n ≥ 9. (D) GFP-p47^phox(W193R), which cannot bind to p22^phox, shows no translocation to the plasma membrane. Bar, 10 μm. Representative of n ≥ 9.

In the case of p47^phox, GFP-p47^phox(R90K), which loses its capacity to bind PI(3,4)P₂ (Ago et al., 2003), shows no translocation to the plasma membrane by AA (Figure 4C). GFP-p47^phox(W193R), which cannot translocate to the phagosome to bind to proline-rich (PR) motif of p22^phox (Ueyama et al., 2004) nor produce ROS (Sumimoto et al., 1996; de Mendez et al., 1997), also shows no translocation to the plasma membrane after AA stimulation (Figure 4D). Together, these data indicate that translocation of p47^phox to the plasma membrane after AA stimulation requires both the binding of the PX domain to PI(3,4)P₂ and the SH3 domain interaction with p22^phox.

Targeting of p67^phox to Early Endosomes or to the Plasma Membrane by Coexpressed Carrier Proteins, p40^phox or p47^phox

To clarify the function of p40^phox as a potential membrane “adaptor protein” for p67^phox, we expressed GFP-p67^phox and p40^phox-IRES2-DsRed2, which identifies transfected cells expressing wild-type p40^phox by DsRed2 fluorescence. When GFP-p67^phox and p40^phox-IRES2-DsRed2 are cotransfected into RAW 264.7 cells, we cannot easily detect cells coexpressing these products. We speculate that overexpression of p67^phox and p40^phox may be too toxic to cells due to the enhanced production of ROS. Therefore, further studies were performed using p67^phox(ΔAD), which cannot support ROS production (Han et al., 1998). In RAW 264.7 cells coexpressing GFP-p67^phox(ΔAD) and p40^phox-IRES2-DsRed2, GFP-p67^phox(ΔAD) translocates to vesicular structures (Figure 5A, top). However, in cells coexpressing p40^phox-IRES2-DsRed2 and GFP-p67^phox(ΔAD:K355A), which does not bind p40^phox (Kuribayashi et al., 2002), GFP-p67^phox(ΔAD:K355A) does not translocate to the vesicular structures (Figure 5A, bottom). This result is further confirmed by complementary experiments using GFP-p67^phox(ΔAD) and p40^phox(D289A)-IRES2-DsRed2, which does not interact with p67^phox (Kuribayashi et al., 2002). In cells coexpressing GFP-p67^phox (ΔAD) and p40^phox(D289A)-IRES2-DsRed2, GFP-p67^phox(ΔAD) shows no translocation to the vesicular structure (data not shown). The interaction between p40^phox and GFP-p67^phox(ΔAD), but not GFP-p67^phox(ΔAD:K355A), is confirmed by immunoprecipitation experiments (Figure 5B).

Figure 5.

Targeting of GFP-p67^phox(ΔAD) to early endosomes by coexpression of p40^phox in RAW 264.7 cells. (A) Top, coexpression of GFP-p67^phox(ΔAD) and p40^phox-IRES2-DsRed2 enables GFP-p67^phox(ΔAD) translocation to vesicular structures. DsRed2 fluorescence (left) identifies cells expressing p40^phox. Bar, 10 μm. Representative of n ≥ 9. (A) Bottom, GFP-p67^phox(ΔAD:K355A) shows no translocation to vesicular structures when coexpressed with p40^phox-IRES2-DsRed2. DsRed2 fluorescence (left) identifies cells expressing p40^phox. Bar, 10 μm. Representative of n ≥ 9. (B) Supernatants from COS-7 cells expressing p40^phox and GFP-p67^phox(ΔAD) or GFP-p67^phox(ΔAD:K355A) were immunoprecipitated by goat polyclonal antibodies against p67^phox or goat control IgG and immunoblotted with rabbit polyclonal antibodies against p40^phox and GFP. p40^phox is coimmunoprecipitated with GFP-p67^phox(ΔAD) but not GFP-p67^phox(ΔAD:K355A). Similar results were obtained in two independent experiments.

In the case where p47^phox is used as a membrane adaptor protein for p67^phox, GFP-p67^phox translocates to the plasma membrane in RAW 264.7 cells (Figure 6A, top). However, in cells coexpressing p47^phox-IRES2-DsRed2 and GFP-p67^phox(ΔSH3), which disrupts the interaction with p47^phox (de Mendez et al., 1994), no GFP-p67^phox(ΔSH3) translocation to the plasma membrane is seen (Figure 6A, bottom). This result was further confirmed by complementary experiment using GFP-p67^phox and p47^phox(ΔPR)-IRES2-DsRed2, which also disrupts the interaction with p67^phox (Kami et al., 2002; Massenet et al., 2005). In the cells coexpressing GFP-p67^phox and p47^phox(ΔPR)-IRES2-DsRed2, GFP-p67^phox shows no the translocation to the plasma membrane (data not shown). The interaction between p47^phox and GFP-p67^phox, but not GFP-p47^phox(ΔSH3), is confirmed in immunoprecipitation experiments (Figure 6B). These data indicate that both p40^phox and p47^phox can function as adaptors of p67^phox to translocate p67^phox to the early endosome and to the plasma membrane, respectively.

Figure 6.

Targeting of GFP-p67^phox to the plasma membrane by coexpression of p47^phox in RAW 264.7 cells. (A) Top, coexpression of GFP-p67^phox and p47^phox-IRES2-DsRed2 enables GFP-p67^phox translocation to the plasma membrane. DsRed2 fluorescence (left) shows cells expressing p47^phox. Bar, 10 μm. Representative of n ≥ 9. (A) Bottom, GFP-p67^phox(ΔSH3) shows no translocation to the plasma membrane when coexpressed with p47^phox-IRES2-DsRed2. DsRed2 fluorescence (left) shows cells expressing p47^phox. Bar, 10 μm. Representative of n ≥ 9. (B) Supernatants from COS-7 cells expressing p47^phox and GFP-p67^phox or GFP-p67^phox(ΔSH3) were immunoprecipitated by rabbit polyclonal antibodies against GFP or control rabbit IgG and then immunoblotted with goat polyclonal antibodies against p47^phox and p67^phox. p47^phox is coimmunoprecipitaed with GFP-p67^phox, but not GFP-p67^phox(ΔSH3). Similar results were obtained in two independent experiments.

Because there are a few studies reporting weak interactions between p47^phox and p40^phox detected in vitro and in the yeast two-hybrid system (Sathyamoorthy et al., 1997; Grizot et al., 2001; Lapouge et al., 2002; Massenet et al., 2005), we examined the effect of this interaction on membrane translocation of p47^phox and p40^phox. Coexpression of p40^phox-IRES2-DsRed2 does not influence the translocation of GFP-p47^phox to the plasma membrane in RAW 264.7 cells (Figure 7A, top). Furthermore, coexpression of p47^phox-IRES2-DsRed2 does not influence the translocation of GFP-p40^phox to the vesicular structures (Figure 7A, bottom). Finally, no apparent interaction is detected between p40^phox and p47^phox (Figure 7B) by immunoprecipitation experiments. These data indicate that the interaction between p40^phox and p47^phox is below detectable levels and does not seem to be a factor in membrane targeting of these proteins in this system.

Figure 7.

Independent membrane targeting of p40^phox and p47^phox in RAW 264.7 cells. (A) Top, p40^phox does not influence GFP-p47^phox targeting to the plasma membrane after AA stimulation. DsRed2 fluorescence (left) shows cells expressing p40^phox. Bar, 10 μm. Representative of n ≥ 9. (A) Bottom, p47^phox does not influence the GFP-p40^phox targeting to vesicular structures after AA stimulation. DsRed2 fluorescence (left) shows cells expressing p47^phox. Bar, 10 μm. Representative of n ≥ 9. (C) Supernatants from COS-7 cells expressing p40^phox and p47^phox were immunoprecipitated by goat polyclonal antibodies against p47^phox or goat control IgG and were immunoblotted with rabbit polyclonal antibodies against p40^phox and goat polyclonal antibodies against p47^phox. p40^phox is not coimmunoprecipitated with p47^phox. Similar results were obtained in two independent experiments.

Finally, we examined the case where all of three phox proteins are overexpressed in RAW 264.7 cells using p47^phox-IRES2-GFP-p67^phox(ΔAD) and p40^phox-IRES2-DsRed2, which identifies transfected cells expressing wild-type p47^phox and GFP-p67^phox(ΔAD) by GFP fluorescence and wild-type p40^phox by DsRed2 fluorescence. In the RAW 264.7 cells coexpressing GFP-p67^phox(ΔAD), p47^phox, and p40^phox, GFP-p67^phox(ΔAD) translocates both to the plasma membrane and to vesicular structures after AA stimulation (Figure 8A and Supplemental Video 3). Expression of all three phox proteins by the internal ribosome entry site (IRES) plasmids is confirmed by Western blotting by using COS-7 cells (Figure 8B).

Figure 8.

Targeting of GFP-p67^phox(ΔAD) both to early endosomes and to the plasma membrane by coexpression of p40^phox and p47^phox in RAW 264.7 cells. (A) Cotransfection of p47^phox-IRES2-GFP-p67^phox(ΔAD) and p40^phox-IRES2-DsRed2 enables GFP-p67^phox(ΔAD) translocation both to vesicular structures and to the plasma membrane (arrows). DsRed2 fluorescence (left) shows cells expressing p40^phox, and GFP fluorescence shows cells expressing p47 ^phox and GFP-p67^phox(ΔAD) (middle). Time-lapsed photography is available in supplemental Video 3. Bar, 10 μm. Representative of n ≥ 9. (B) Supernatants from COS-7 cells expressing p47^phox and GFP-p67^phox(ΔAD) by p47^phox-IRES2-GFP-p67^phox(ΔAD) and p40^phox by p40^phox-IRES2-DeRed2 were immunoblotted with antibodies against p47^phox, p67^phox, and p40^phox. Expression of all three phox proteins by IRES plasmids is confirmed. Similar results were obtained in two independent experiments.

PB1 Domain of p40^phox Masks the PX Domain of p40^phox

It has been suggested that p47^phox exists in the cytoplasm in the resting state because of two intramolecular interactions masking both the N- and C-terminal SH3 domains and the PX domain (Hiroaki et al., 2001; Karathanassis et al., 2002; Groemping et al., 2003; Durand et al., 2006). When phagocytes are activated after cell stimulation, the autoinhibitory region (AIR) (aa 286-340) is phosphorylated, thereby disrupting the intramolecular interactions and rendering both SH3 domains and the PX domain accessible to bind p22^phox and membrane lipids, respectively (Karathanassis et al., 2002; Ago et al., 2003; Groemping et al., 2003)_. It was reported that the interaction between the PR motif in the PX domain and the C-terminal SH3 domain of p47^phox (Hiroaki et al., 2001). p40^phox also has a PR motif in the PX domain and one SH3 domain, which have the possibility of interacting each other. Therefore, we examined this possibility using purified (His)₆-p40^phox(PX) and GST-p40^phox(SH3) in in vitro pull-down binding assays. However, we did not detect binding between these two proteins (data not shown). This result is consistent with crystallographic data on p40^phox(PX) showing that the PR motif in the PX domain of p40^phox is buried within the PX domain, although this study was performed using the lipid-bound form of p40^phox(PX) (Bravo et al., 2001).

Then, we examined other regions within p40^phox that might interact with its own PX domain, by using serially deleted mutants of GFP-p40^phox expressed in RAW 264.7 cells. Among the various C-terminal deletions of GFP-p40^phox, GFP-p40^phox(1-328) loses the punctate localization on early endosomes, as seen with full-length GFP-p40^phox (Figure 9B). GFP-p40^phox(Δ318-328) and GFP-p40^phox(318-328:11A) are localized at vesicular structures in the cytoplasm (Figure 9, C and D). The cytoplasmic localization of wild-type p40^phox is confirmed by indirect immunofluorescence of COS-7 cells transfected with wild-type p40^phox (Figure 9E). To examine further, we used in vitro pull-down assays between purified (His)₆-p40^phox(PX) and various forms of purified GST-tagged proteins. We could not detect interactions between (His)₆-p40^phox(PX) and GST-p40^phox (307-317), GST-p40^phox(318-328), GST-p40^phox(329-339), or GST alone; however, (His)₆-p40^phox(PX) interacts strongly with GST-p40^phox(PB1:237-339) (Figure 10).These results suggested that the PB1 domain of p40^phox masks the PX domain of p40^phox, making this domain inaccessible to bind to early endosomes in the absence of AA stimulation (Figure 11).

Figure 10.

Interaction of PX domain and PB1 domain of p40^phox. (A) Structure of (His)₆-tagged PX domain (1–167) of p40^phox and GST-tagged PBI domain (237–339) of p40^phox constructs used in pull-down assays. (B) Top blot, purified GST-p40^phox(PBI: 237–339), but not GST, interacts with (His)₆-p40^phox(PX) in GST-based pull-down assays. Middle and bottom blots, comparable amounts of input proteins are confirmed by Ponsceau S staining (GST proteins) and by (His)₆ blotting [(His)₆-p40^phox(PX)], respectively. Representative of three independent experiments.

Figure 11.

Model of activation and assembly of Nox2 system during phagocytosis. In the resting state, the ternary complex of p47^phox, p67^phox, and p40^phox is in the cytoplasm. p47^phox is folded and inactive, due to inhibitory intramolecular interactions (Hiroaki et al., 2001; Groemping et al., 2003; Durand et al., 2006). p40^phox is also folded and inactive due to inhibitory intramolecular head-to-tail (PX–PB1) interactions. In the early stages of activation of Nox2, the ternary complex translocates to the phagosome after conformational changes of p47^phox that render the PX domain and SH3 domains of p47^phox accessible to phospholipids [PI(3,4)P₂, and PA; produced during this stage] and the PR motif of p22^phox, respectively. In later stages of activation of Nox2, the ternary complex is retained on the phagosome or may exchange with a cytoplasmic pool after conformational changes in p40^phox that render the PX domain of p40^phox accessible to bind PI(3)P. In both stages, PKC may stay on the phagosome and phosphorylate p47^phox, whereas AA may be a key modulator of conformational changes in p40^phox and p47^phox. Rac independently translocates to the phagosome.

DISCUSSION

In this study, we propose that both p40^phox and p47^phox function as regulated carrier or adaptor proteins of p67^phox through distinct membrane-targeting mechanisms during NADPH oxidase assembly. We reported that p47^phox accumulates transiently on phagocytic cups and mature phagosomes during FcγR-mediated phagocytosis (Ueyama et al., 2004), consistent with earlier studies showing p47^phox and p67^phox associate with both nascent and mature phagosomes in neutrophils and neutrophil-like PLB-985 cells (Allen et al., 1999; van Bruggen et al., 2004). The present study shows that GFP-p40^phox also transiently associates on phagosomes, although this targeting is initiated at later stages after phagosome sealing. This accumulation is even more apparent with GFP-p40^phox(PX), suggesting the PX domain of p40^phox is masked or otherwise inhibited from interacting with membranes in the context of the full-length protein, because the isolated PX domain binds readily to PI(3)P-enriched endosome-like vesicles even before phagocytic stimulation. Finally, we show that AA has dramatic affects in unmasking the PX domain, thereby enabling p40^phox to bind these vesicles along with p67^phox. We suggest two mechanisms for the targeting p40^phox to phagosomes during FcγR-mediated phagocytosis: 1) p40^phox can bind initially to PI(3)P-enriched early endosomes, followed by their fusion to phagosomes, and/or 2) p40^phox can target directly to phagosomes, where PI(3)P is transiently produced during phagosytosis (Ellson et al., 2001a; Gillooly et al., 2001). Because the cytosolic phox proteins exist as a ternary complex (Kuribayashi et al., 2002; Lapouge et al., 2002), we propose a model for Nox2 complex assembly involving these spatiotemporal factors during the FcγR-mediated respiratory burst (Figure 11): 1) in early stages, before phagosome sealing, p47^phox functions as the predominant carrier, mediating translocation of the ternary complex to newly forming phagosomes, 2) in later stages after phagosome sealing, the carrier or adaptor function of p47^phox is replaced by p40^phox, whereby the ternary complex interacts with sealed phagosome through PI(3)P specific-binding of the PX domain of p40^phox. In this series of interactions, both PKC and AA derived from PLA₂ likely orchestrate conformational changes in p40^phox and p47^phox that promote their assembly with Nox2 complexes. This model is consistent with recent studies showing that p40^phox contributes significantly to phagosomal superoxide production in mouse neutrophils or in FcγIIA receptor and phox-reconstituted COS-7 cells (Ellson et al., 2006; Suh et al., 2006) and that this requires interactions with the PX, SH3, and PB1 domains of p40^phox (Suh et al., 2006).

Several lines of evidence suggest AA has important roles in activation of the Nox2 system, although the mechanism of its action is still debated. Many reports emphasize the effects of AA on p47^phox. Early studies showed AA or other anionic amphiphiles activate ROS production in cell-free assays of Nox2 activity (Bromberg and Pick, 1984; Curnutte, 1985). It is thought that AA induces conformational changes in p47^phox that may mimic the effects of phosphorylation, resulting in exposure of its binding PX and SH3 domains and translocation of p47^phox and p67^phox (Shiose and Sumimoto, 2000). It was reported that PLA₂ is required for translocation of phox proteins without their phosphorylation (Uhlinger et al., 1993; Zhao et al., 2002). Others suggested both AA produced by PLA₂- and PKC-dependent phosphorylation together promote translocation of p47^phox and enhance ROS production by Nox2 (Sellmayer et al., 1996; Shiose and Sumimoto, 2000; Peng et al., 2003). We confirmed AA can trigger membrane translocation of p47^phox, through interactions involving its PX domain and SH3 domain and that this enables cotranslocation of p67^phox (Figures 4 and 6). Other reports suggest exogenous AA has indirect effects on Nox2 activation through downstream AA-derived mediators (Liu et al., 2003; Kerkhoff et al., 2005; Kim and Dinauer, 2006). Direct involvement of cPLA₂ in human Nox2 activation (neutrophils, PLB-985 cells, or monocytes) has been demonstrated using inhibitors or antisense molecules targeted to cPLA₂ (Dana et al., 1994; Li and Cathcart, 1997; Dana et al., 1998; Zhao et al., 2002). However, macrophages or neutrophils from cPLA₂-deficient mice exhibited normal stimulated ROS release (Gijon et al., 2000; Rubin et al., 2005), suggesting the murine systems are less dependent on AA. Surprisingly, human cPLA₂-deficient cells show normal translocation of phox proteins (Dana et al., 1994; Dana et al., 1998; Shmelzer et al., 2003), suggesting cPLA₂ serves other roles in oxidase activation during Nox2 assembly. Interestingly, both cPLA₂ and secretory PLA₂ accumulate on newly formed phagosomes (Shmelzer et al., 2003; Girotti et al., 2004; Balestrieri et al., 2006). Furthermore, cells lacking Nox2 show no translocation of cPLA₂ to membranes, and it seems that human cPLA₂ associates directly with the cytoplasmic phox proteins and the membrane-bound Nox2 complex after stimulation by several agonists that activate the oxidase (Shmelzer et al., 2003).

We have now shown that AA triggers the movement of p40^phox to PI(3)P-enriched membranes independently of other cytoplasmic phox proteins and that this can enable cotranslocation of p67^phox. Although high concentrations of AA (200 μM) were used to observe this phenomenon, 50–100 μM AA also induced translocation (data not shown). Based on our experience, high concentrations of agonists (including AA) are usually required to visualize translocation of GFP-tagged molecules (Shirai et al., 1998). It is difficult to estimate the concentrations of AA achieved in whole cells, although high local concentrations of AA may be reached on phagosomes when PLA₂ is recruited during phagocytosis. AA-dependent translocation of p40^phox to early endosomes occurs with either N- or C-terminally GFP-tagged p40^phox, suggesting the fusions do not influence this process. We propose that this translocation involves AA-induced conformational changes in p40^phox that disrupt intramolecular interactions and expose its PX domain to bind membrane lipids, as was suggested in p47^phox (Shiose and Sumimoto, 2000). However, in this case we mapped the intramolecular contacts of the p40^phox PX domain to its C-terminal PB1 domain.

It is known that p40^phox and p67^phox associate through homotypic interactions between their PB1 domains (Ito et al., 2001; Kuribayashi et al., 2002; Wilson et al., 2003). The region of p40^phox involved was reported as the PC motif (residues 287-302) (Nakamura et al., 1998; Ito et al., 2001), now referred as the OPR-PC-AID (OPCA) motif (Ponting et al., 2002). Later, Wilson et al. (2003) showed that Thr337 and Pro339 in the region C-terminal region also participate in the heterodimerization. The importance of this interaction with p67^phox was demonstrated with the p40^phox(D289A) mutant, which, unlike wild-type p40^phox, is unable to stimulate Nox2 activity in PMA or muscarinic receptor-stimulated cells (Kuribayashi et al., 2002). This mutation not only interferes with p40^phox membrane translocation but also leads to decreased p67^phox and p47^phox translocation stimulated by PMA. In contrast, in our AA-stimulated model, wild-type or mutant (D289A) GFP-p40^phox translocates to early endosomes, even in the absence of p67^phox (Figure 3; data not shown). However, we show that a mutation of the PB1 domain in p67^phox(K355A) inhibits its cotranslocation with p40^phox in AA-stimulated cells (Figure 5). This is the first report proposing an autoinhibitory intramolecular interaction within p40^phox between its PX domain and its PB1 domain, because deleting residues 318-328 or replacing this sequence with alanine residues resulted in localization of GFP-p40^phox on early endosomes without AA stimulation (Figure 9). Furthermore, we observed the interaction between (His)₆-p40^phox(PX) and the GST-p40^phox(PB1:237-339) (Figure 10B). Crystallographic studies indicate that residues 318-328, encompassing the β-5 strand, do not participate in heterodimerization of p40^phox and p67^phox (Wilson et al., 2003). However, we could not determine which part of the PB1 domain participates in the PX–PB1 interaction of p40^phox, because β-5 is part of a β-sheet core structure and deletion or changes of residues 318-328 to alanines could affect the global folding of the PB1 domain of p40^phox. It is interesting that sites of phosphorylation in p40^phox (Thr154 and Ser315) lie close to the PX domain and residues 318-328 (Bouin et al., 1998). Phosphorylation of Thr154, but not Ser315, was proposed as a negative regulator of ROS production by Nox2 (Lopes et al., 2004). However, we could not observed significant differences in the subcellular localization between GFP-p40^phox and its phosphorylation site mutants [GFP-p40^phox(T154A), GFP-p40^phox(T154D), GFP-p40^phox(S315A), or GFP-p40^phox(S315D)], suggesting phosphorylation does not disrupt intramolecular interactions within GFP-p40^phox, as seen with AA stimulation. Further studies are needed to clarify whether phosphorylation of p40^phox can affect interactions with its PX or PB1 domain or the dynamics of its assembly with the oxidase on phagosomes.

Abbreviations used:

AA

arachidonic acid

FcγR

Fcγ receptor

PA

phosphatidic acid

ROS

reactive oxygen species.