The adaptor protein p40phox as a positive regulator of the superoxide‐producing phagocyte oxidase (original) (raw)

Introduction

Professional phagocytes, including neutrophils and macrophages, play a crucial role in host defense against microbial infection. During phagocytosis of invading microorganisms or upon cell stimulation with soluble agents, phagocytes produce superoxide (O2−), a precursor of microbicidal oxidants, by catalysis of an activated NADPH oxidase (Sumimoto et al., 1997; Babior, 1999; Clark, 1999; Nauseef, 1999). The soluble stimulants include phorbol myristate acetate (PMA) and chemoattractants, the latter of which bind to their own receptors to activate the oxidase via the trimeric GTPase Gi (Bokoch, 1995). The significance of the oxidase in host defense is exemplified by recurrent and life‐threatening infections that occur in patients with chronic granulomatous disease (CGD), whose phagocytes are defective in the superoxide‐producing activity (Roos et al., 1996).

The redox core of the phagocyte NADPH oxidase is a membrane‐spanning flavocytochrome b_558, comprising the two subunits gp91_phox and p22_phox_. The cytochrome is catalytically inactive in resting cells but acquires the capacity to transfer electrons from NADPH to molecular oxygen upon cell stimulation, leading to superoxide production (Roos et al., 1996; Babior, 1999). The activation of cytochrome b_558 requires stimulus‐induced membrane translocation of cytosolic proteins, namely the small GTPases Rac and two SH3 domain‐harboring oxidase factors p47_phox and p67_phox_ (Sumimoto et al., 1997; Babior, 1999; Clark, 1999; Nauseef, 1999). In CGD neutrophils lacking cytochrome b_558, neither p47_phox nor p67_phox_ can be recruited to the membrane upon cell stimulation (Heyworth et al., 1991). In p47_phox_‐deficient phagocytes, membrane targeting of p67_phox_ does not occur, whereas p47_phox_ is independently targeted in p67_phox_‐defective cells (Heyworth et al., 1991; Dusi et al., 1996). Thus p47_phox_ appears to directly interact with cytochrome b_558 and recruits p67_phox to the membrane. Indeed the SH3 domains of p47_phox_ bind to a proline‐rich region (PRR) of p22_phox_, the small subunit of cytochrome b_558 (Leto et al., 1994; Sumimoto et al., 1994). Although the SH3 domains are normally masked via an intramolecular interaction, the conformation of p47_phox becomes changed upon cell stimulation to render the domains in a state accessible to p22_phox_, which leads to the activation of the NADPH oxidase (Sumimoto et al., 1994; Ago et al., 1999). The P156Q substitution in the p22_phox_ PRR, a mutation that has occurred in a case of CGD (Dinauer et al., 1991), results in not only impaired interaction between p22_phox_ and p47_phox_ in vitro (Sumimoto et al., 1994), but also defective translocation of p47_phox_ to the membrane in vivo (Leusen et al., 1994). It is also known that p67_phox_ constitutively associates with p47_phox_ via a tail‐to‐tail interaction: the C‐terminal SH3 domain of p67_phox_ directly binds to a PRR in the C‐terminal region of p47_phox_ (Leusen et al., 1994; Ito et al., 1996). On the other hand, membrane translocation of Rac is independent of the presence of cytochrome b_558, p47_phox and p67_phox_: even in CGD neutrophils lacking one of these proteins, the GTPase is normally recruited to the membrane (Heyworth et al., 1994).

Another SH3 domain‐containing oxidase factor, p40_phox_, has been identified as a protein that constitutively associates with p67_phox_ in the cytosol of resting phagocytes (Someya et al., 1993; Wientjes et al., 1993; Tsunawaki et al., 1994). It comprises three portions: a PX (Phox) domain [also known as PB2 (Phox and Bem 2) domain] at the N‐terminus, an SH3 domain in the middle and the C‐terminal region containing a PC (Phox and Cdc) motif (Ponting, 1996; Sumimoto et al., 1997; Mizuki et al., 1998; Nakamura et al., 1998; Hiroaki et al., 2001) (Figure 1). The PC motif is present in a variety of signaling proteins such as the yeast polarity protein Cdc24p, the protein kinase MEK5 and Zip, a protein that interacts with the ζ isoform of protein kinase C, and serves as a target of PB1 (Phox and Bem 1) domain, thereby mediating modular protein–protein interactions (Ito et al., 2001; Terasawa et al., 2001). The protein p67_phox_ contains a PB1 domain between the two SH3 domains, which recognizes the PC motif of p40_phox_ and plays an essential role in interaction between p67_phox_ and p40_phox_ (Ito et al., 2001). Thus, in a ternary oxidase complex of resting phagocytes, p67_phox_ is thought to tether p47_phox_ to p40_phox_: the C‐terminal SH3 domain binds to the PRR of p47_phox_ and the PB1 domain directly interacts with the PC motif of p40_phox_ (Figure 1).

Figure 1

Protein–protein interactions in a complex of the phagocyte NADPH oxidase factors p67_phox_, p47_phox_ and p40_phox_. The NADPH oxidase proteins p67_phox_, p47_phox_ and p40_phox_ form a complex in resting phagocytes. The three proteins harbor multiple modular domains: p67_phox_ comprises a domain containing four units of TPR (tetratricopeptide repeat), the first SH3 domain, PB1 domain and the second SH3 domain; p47_phox_ contains PX or PB2 domain, two SH3 domains and the C‐terminal PRR; and p40_phox_ harbors PX/PB2 and SH3 domains, followed by PC motif. In the oxidase complex, p67_phox_ directly binds to p47_phox_ via a tail‐to‐tail interaction between the C‐terminal SH3 of p67_phox_ and the PRR of p47_phox_; and p67_phox_ also directly binds to p40_phox_ via a novel modular interaction between the PB1 domain of p67_phox_ and the PC motif‐containing region of p40_phox_. Thus p67_phox_ tethers p47_phox_ to p40_phox_.

The role of p40_phox_ in the phagocyte NADPH oxidase activation has remained largely elusive, although it probably resides in a complex of the oxidase as described above. The oxidase can be activated in a cell‐free system reconstituted with cytochrome b_558, cytosolic proteins (p47_phox, p67_phox_ and Rac in the GTP‐bound form) and anionic amphiphiles such as arachidonate and sodium dodecyl sulfate (Bromberg and Pick, 1985; Hata et al., 1998). It is well known that the cell‐free activation does not require p40_phox_. The effect of the addition of p40_phox_ to the cell‐free system, however, seems controversial: Cross (2000) has shown that p40_phox_ only slightly elevates the oxidase activity under cell‐free conditions, while other investigators have reported that p40_phox_ represses the oxidase activation at high concentrations (Sathyamoorthy et al., 1997). Although the cell‐free system has greatly contributed to understanding of the molecular mechanism for activation of the NADPH oxidase, results obtained using this system sometimes seem inconsistent with those using intact cells. For instance, p67_phox_ lacking the C‐terminal SH3 domain is incapable of activating the NADPH oxidase when it is expressed in cells, while the mutant p67_phox_ almost fully activates the oxidase under cell‐free conditions (de Mendez et al., 1994).

Here we describe the role of p40_phox_ in activation of the phagocyte NADPH oxidase at the cellular level. This protein enhances membrane translocation of the cytosolic activators p67_phox_ and p47_phox_, and subsequent activation of the NADPH oxidase by 2‐ to 3‐fold, when cells are stimulated with PMA. The enhancements are totally dependent on the binding of p40_phox_ to p67_phox_, which is mediated via the PB1–PC interaction. Intriguingly, in cells treated with a Gi‐activating peptide instead of PMA, p40_phox_ serves as a much more efficient activator of the oxidase, thereby underscoring its importance in a physiological context that cannot be reproduced under cell‐free conditions.

Results

Expression of p40 phox leads to enhanced production of superoxide in PMA‐stimulated cells

To investigate the role of p40_phox_ in activation of the phagocyte NADPH oxidase, we transfected the plasmid vector pREP4 encoding the full‐length cDNA of p40_phox_ or vector alone to K562 cells that express functional cytochrome b_558, p47_phox and p67_phox_ (Ago et al., 1999; Koga et al., 1999), and cloned seven independent transformants, each expressing p40_phox_ at the same level (for details see Materials and methods). The transformants stably express p40_phox_ as estimated by flow cytometric analysis (Figure 2A). The p40_phox_‐expressing cells harbored the same amounts of functional gp91_phox_ as the p40_phox_‐deficient cells, containing the vector pREP4 that did not encode this protein (Figure 2B). In addition, analyses by immunoblot (Figure 2C) and flow cytometry (data not shown) revealed that no difference existed between these cells in expression of the proteins p22_phox_, p47_phox_, p67_phox_ and the small GTPase Rac.

Figure 2

Stable expression of p40_phox_ in K562 cells transfected with pREP4 encoding the full‐length of this protein. (A) Expression of p40_phox_ in the K562 cells. Parent K562 cells stably expressing gp91_phox_, p67_phox_ and p47_phox_ were transfected with pREP4 encoding the full‐length p40_phox_ (p40_phox_) or vector alone (vector). The cells were stained with anti‐p40_phox_ serum (filled histogram) or pre‐immune serum (open histogram) and analyzed by flow cytometry. For details see Materials and methods. (B) Expression of functional gp91_phox_. The cells were stained with the monoclonal antibody 7D5 to detect gp91_phox_ in functional cytochrome b_558 (filled histogram) or control IgG (open histogram) and analyzed by flow cytometry. (C) Expression of p47_phox, p67_phox_, p40_phox_ and p22_phox_. The lysates of the K562 cells were analyzed by immunoblot with an anti‐p47_phox_, an anti‐p67_phox_, an anti‐p40_phox_ or an anti‐p22_phox_ antibody, as described in Materials and methods.

When the K562 cells expressing p40_phox_ were stimulated with PMA, they produced a large amount of superoxide, which was estimated by superoxide dismutase (SOD)‐inhibitable cytochrome c reduction (Figure 3A). We also tested four other clones (data not shown) and found that the cells exhibited essentially the same superoxide‐producing activity of 4.2 ± 0.14 nmol/min per 106 cells (n = 5). On the other hand, the activity of the cells without p40_phox_ was 1.8 ± 0.12 nmol/min per 106 cells (n = 5 independent clones) (Figure 3A; data not shown). Using another method, SOD‐inhibitable chemiluminescence, we also tested the effect of p40_phox_ in activation of the phagocyte NADPH oxidase (Figure 3B). In response to PMA, seven independent clones of the p40_phox_‐expressing K562 cells all produced 2‐ to 3‐fold more superoxide than the cells lacking p40_phox_ (Figure 3B). Thus expression of p40_phox_ leads to enhanced production of superoxide in PMA‐stimulated cells, raising the possibility that p40_phox_ facilitates the assembly of the phagocyte oxidase factors.

Figure 3

p40_phox_‐enhanced superoxide production and membrane translocation of p47_phox_ and p67_phox_ upon cell stimulation with PMA. (A) Superoxide production by the K562 cells with stable expression of p40_phox_ or without p40_phox_ was measured as reduction of cytochrome c. The cells (5.0 × 105 cells/ml) were stimulated with PMA (200 ng/ml) (arrow), and the superoxide‐producing activity was measured by determining the rate of ferricytochrome c reduction at 550 to 540 nm. The reduction was stopped by addition of superoxide dismutase (SOD) (50 μg/ml) (arrowhead). (B) Superoxide production by the K562 cells with p40_phox_ or without p40_phox_ was measured as change of chemiluminescence. The K562 cells (5.0 × 103 cells) were stimulated with PMA (200 ng/ml), and the chemiluminescence change was continuously monitored with DIOGENES. SOD (50 μg/ml) was added where indicated (arrowhead). For details see Materials and methods. (C) Membrane translocation of p47_phox_, p67_phox_, p40_phox_ and Rac upon PMA stimulation. The K562 cells with or without p40_phox_ (1.0 × 107 cells) were incubated with PMA (200 ng/ml) for the indicated time. After the incubation, the cells were sonicated and ultracentrifuged as described in Materials and methods. The membrane fractions were analyzed by immunoblot with the antibody against p47_phox_, p67_phox_, p40_phox_, Rac or p22_phox_. The data are representative of results from five independent experiments.

p40 phox facilitates membrane translocation of p47 phox and p67 phox but not of Rac upon cell stimulation

It is well established that stimulus‐elicited translocation of p47_phox_ and p67_phox_ from the cytosol to the membrane is required for activation of the phagocyte NADPH oxidase (Roos et al., 1996; Clark, 1999; Nauseef, 1999). Since p40_phox_ is constitutively associated with p67_phox_ and enhances the oxidase activation as shown above (Figure 3A and B), it seems likely that p40_phox_ affects the membrane recruitment of p47_phox_ and p67_phox_. To test this possibility, we fractionated the membrane of the K562 cells with or without p40_phox_ and estimated the amounts of p47_phox_ and p67_phox_. When the p40_phox_‐lacking cells were stimulated with PMA, both p47_phox_ and p67_phox_ translocated to the membrane in a time‐dependent manner (Figure 3C). In the cells expressing p40_phox_, this protein as well as p47_phox_ and p67_phox_ was targeted upon cell stimulation to the membrane (Figure 3C). The amounts of p47_phox_ and p67_phox_ at the membrane were increased by 2‐ to 3‐fold, compared with those of the p40_phox_‐deficient cells (Figure 3C). On the other hand, p40_phox_ did not affect PMA‐elicited membrane targeting of Rac (Figure 3C). Thus the enhancement of the oxidase activation by p40_phox_ is likely to be due to the p40_phox_‐induced facilitation of membrane translocation of p47_phox_ and p67_phox_.

p40 phox carrying the D289A substitution is incapable of binding to p67 phox and fails to enhance superoxide production and membrane translocation of p47 phox and p67 phox

We next tested whether the interaction of p40_phox_ with p67_phox_ participates in the p40_phox_‐enhanced translocation of p47_phox_ and p67_phox_. As we have previously shown, this interaction is mediated via a direct binding of the p40_phox_ PC motif to the p67_phox_ PB1 domain (Nakamura et al., 1998; Ito et al., 2001). Consistent with previous observations, the maltose‐binding protein (MBP)‐tagged full‐length protein of p40_phox_, MBP–p40_phox_ (WT), directly interacted with the p67_phox_ PB1 domain as a glutathione S_‐transferase (GST) fusion (GST–p67‐PB1) as analyzed by a pull‐down assay (Figure 4A). To confirm the role for the PC motif of p40_phox, we purified a mutant p40_phox_ carrying the amino acid substitution of Ala for Asp289, an invariant residue among PC motifs (Nakamura et al., 1998); the corresponding residue in the PC motif of the yeast polarity protein Cdc24p plays an essential role in its binding to the PB1 domain of Bem1p (Ito et al., 2001). As shown in Figure 4A, the D289A substitution resulted in a complete loss of the interaction of p40_phox_ with p67_phox_.

Figure 4

Effect of the D289A substitution in the PC motif of p40_phox_ on its interaction with p67_phox_ both in vivo and in vitro. (A) Direct interaction of p40_phox_ with p67_phox_. GST‐tagged PB1 domain of p67_phox_ (GST–p67‐PB1) or GST alone was incubated with MBP–p40_phox_ (WT) or MBP–p40_phox_ (D289A) and proteins were pulled down with glutathione–Sepharose‐4B. The precipitated proteins were subjected to SDS–PAGE, followed by staining with CBB, as described, see Materials and methods. Positions for marker proteins are indicated in kDa. (B) Expression of p40_phox_ (D289A) in K562 cells. K562 cells stably expressing gp91_phox_, p67_phox_ and p47_phox_ were transfected with pREP4 encoding p40_phox_ with the D289A substitution, p40_phox_ (D289A). The K562 cells were stained with anti‐p40_phox_ serum (filled histogram) or pre‐immune serum (open histogram) and analyzed by flow cytometry. (C) Interaction of p40_phox_ with p67_phox_ in the K562 cells. The cell lysates of the K562 cells were analyzed by immunoprecipitation with an anti‐p67_phox_ or control IgG (cont.) (left panel), or an anti‐p47_phox_ antibody or control IgG (cont.) (right panel) followed by immunoblot with the indicated antibody, as described under Materials and methods.

To examine the effect of the D289A substitution in the PC motif at the cellular level, we prepared seven clones of the K562 cells stably expressing p40_phox_ (D289A). The amount of p40_phox_ as well as those of other oxidase proteins including gp91_phox_, p22_phox_, p47_phox_ and p67_phox_ in the p40_phox_ (D289A)‐expressing cells was identical to that in the cells expressing the wild‐type p40_phox_ (WT) (Figure 4B and C; data not shown). An immunoprecipitation assay demonstrated that p67_phox_ binds to p40_phox_ (WT) but not to p40_phox_ (D289A) in vivo (Figure 4C), indicating that the binding of p40_phox_ to p67_phox_ is mediated solely via the PC motif even at the cellular level. Although p47_phox_ was involved in a complex with p40_phox_ and p67_phox_ (Figure 4C), it should be noted that the interaction between p47_phox_ and p67_phox_ occurs in a manner independent of the presence of p40_phox_ (Figure 4C).

When cells were stimulated with PMA, p40_phox_ (D289A) was incapable of enhancing the activation of the NADPH oxidase: the amount of superoxide produced by the cells expressing p40_phox_ (D289A) was similar to that by the cells without p40_phox_ (Figure 5A). The amounts of p67_phox_ and p47_phox_ in the membrane of the p40_phox_ (D289A)‐expressing cells were half‐to‐one‐third of those of the p40_phox_ (WT)‐expressing ones. In addition, p40_phox_ (D289A) did not translocate to the membrane in response to PMA (Figure 5B), suggesting that its localization is totally dependent on the interaction with p67_phox_. The present observations thus indicate that membrane translocation of p47_phox_ and p67_phox_ is facilitated by the binding of p40_phox_ to p67_phox_, which leads to enhanced activation of the NADPH oxidase.

Figure 5

Effect of the D289A substitution in the PC motif of p40_phox_ on superoxide production and membrane translocation of p47_phox_ and p67_phox_ upon PMA stimulation. (A) Superoxide production by the K562 cells with p40_phox_ (WT) or p40_phox_ (D289A) was measured as change of chemiluminescence. The K562 cells (5.0 × 103 cells) were stimulated with PMA (200 ng/ml) and the chemiluminescence change was measured. Each histogram indicates the average from five independent experiments, with bars representing SD. (B) Membrane translocation of p47_phox_, p67_phox_ and p40_phox_ upon PMA stimulation. The K562 cells stably expressing p40_phox_ (WT) or p40_phox_ (D289A) (1.0 × 107 cells) were stimulated with PMA (200 ng/ml) for the indicated time and the amounts of p47_phox_, p67_phox_ and p40_phox_ in the membrane fractions were analyzed by immunoblot as described under Materials and methods.

p67 phox carrying the K355A substitution is incapable of interacting with p40 phox , and fails to enhance superoxide production and membrane translocation of p47 phox and p67 phox

To further study the importance of the interaction between p40_phox_ and p67_phox_, we used a mutant p67_phox_ with the substitution of Ala for Lys355 (Figure 6A), a conserved residue among PB1 domains (Ito et al., 2001; Terasawa et al., 2001). In an in vitro pull‐down assay, GST–p67‐PB1 (K355A) was incapable of interacting with MBP–p40_phox_, while the wild‐type PB1 domain fully bound to p40_phox_ (data not shown). Similarly, p67_phox_ (K355A) could not associate with p40_phox_ in vivo (Figure 6B). On the other hand, both p67_phox_ (WT) and p67_phox_ (K355A) were co‐immunoprecipitated with p47_phox_ (Figure 6B), indicating that p40_phox_ binds to p47_phox_ indirectly but via p67_phox_ (see Figure 1).

Figure 6

Effect of the K355A substitution in the PB1 domain of p67_phox_ on its interaction with p40_phox_. (A) Expression of p67_phox_ in K562 cells. K562 cells stably expressing gp91_phox_, p47_phox_ and p40_phox_ were transfected with pREP4 encoding p67_phox_ (WT) or p67_phox_ (K355A). The K562 cells were stained with anti‐p67_phox_ serum (filled histogram) or pre‐immune serum (open histogram), and analyzed by flow cytometry. (B) In vivo interaction of p67_phox_ with p40_phox_ in the K562 cells. The cell lysates of the K562 cells were analyzed by immunoprecipitation with the anti‐p67_phox_ or control IgG (cont.) (left panel), or the anti‐p47_phox_ antibody or control IgG (cont.) (right panel) followed by immunoblot (Blot) with the indicated antibody.

We next investigated the effect of the K355A substitution in the p67_phox_ PB1 domain on superoxide production and membrane translocation of p47_phox_ and p67_phox_. As shown in Figure 7A, the amounts of p47_phox_ and p67_phox_ in the membranes of the p67_phox_ (K355A)‐expressing cells were half‐to‐one‐third of those of the p67_phox_ (WT)‐expressing cells. In the cells expressing p67_phox_ (K355A), p40_phox_ failed to translocate to the membranes. As shown in Figure 7B, the amount of superoxide produced by the cells expressing p67_phox_ (K355A) was half‐to‐one‐third of that by those expressing p67_phox_ (WT). In response to PMA, the cells with p67_phox_ (K355A) produced the same low amount of superoxide irrespective of the presence of p40_phox_ (Figure 7B). These observations further supported the idea that the p40_phox_–p67_phox_ interaction enhances the membrane translocation of p47_phox_ and p67_phox_, thereby facilitating the assembly of the active oxidase complex.

Figure 7

Effect of the K355A substitution in the PB1 domain on membrane translocation of p47_phox_ and p67_phox_ and superoxide production upon PMA stimulation. (A) Membrane translocation of p47_phox_, p67_phox_ and p40_phox_ upon PMA stimulation. The K562 cells stably expressing p67_phox_ (WT) or p67_phox_ (K355A) (1.0 × 107 cells) were stimulated with PMA (200 ng/ml) for the indicated time and the amounts of p47_phox_, p67_phox_ and p40_phox_ in the membrane fractions were analyzed by immunoblot. (B) Superoxide production by the K562 cells with p67_phox_ (WT) or p67_phox_ (K355A) was measured as change of chemiluminescence. The K562 cells (5.0 × 103 cells) were stimulated with PMA (200 ng/ml) and the chemiluminescence change was measured. The chemiluminescence change by K562 cells without the expression of p40_phox_ and with p67_phox_ (WT) or p67_phox_ (K355A) was also measured. Each histogram indicates the average from five independent experiments, with bars representing SD.

p40 phox greatly enhances superoxide production in cells stimulated with the muscarinic receptor peptide m4I3C(14), an agent that can activate the trimeric GTPase G i

It is well documented that, in neutrophils, chemoattractants such as N_‐formyl peptides induce superoxide production via the trimeric GTPase Gi (Bokoch, 1995). The chemotactic receptor, however, is not expressed in K562 cells. To further clarify the role of p40_phox in the oxidase activation, we used the muscarinic receptor peptide m4I3C(14) as a stimulant.

In response to m4I3C(14), K562 cells expressing p40_phox_ produced superoxide (Figure 8A). The superoxide production, albeit 10‐fold less than that by PMA‐stimulated cells, is likely to be catalyzed by the phagocyte NADPH oxidase, since the peptide did not act on parental K562 cells that lack oxidase proteins (data not shown) or cells expressing all oxidase factors except p67_phox_ (see later). Furthermore, we did detect, although qualitatively, the peptide‐induced membrane translocation of the cytosolic oxidase proteins (data not shown). The treatment of the K562 cells with pertussis toxin (PTX) resulted in a loss of the m4I3C(14)‐induced superoxide production, while the toxin did not affect that induced by PMA (Figure 8B). The peptide thus activates the oxidase via Gi.

Figure 8

p40_phox_‐enhanced superoxide production upon cell stimulation with the muscarinic receptor peptide m4I3C(14). (A) Superoxide production by the K562 cells with stable expression of p40_phox_ or without p40_phox_. The K562 cells (5.0 × 103 cells) were stimulated with the muscarinic receptor peptide m4I3C(14) (m) (200 μM) and the chemiluminescence change was continuously monitored with DIOGENES, and SOD (50 μg/ml) was added where indicated (arrowhead). For details see Materials and methods. (B) Effect of pertussis toxin (PTX) on superoxide production upon stimulation with m4I3C(14) or PMA. The K562 cells (5.0 × 103 cells) were pretreated with PTX (18 μg/ml) and stimulated with m4I3C(14) (200 μM) or PMA (200 ng/ml) and superoxide production was measured as change of chemiluminescence. Each histogram indicates the average from five independent experiments, with bars representing SD. (C) Effect of the D289A substitution in the PC motif of p40_phox_ on superoxide production upon stimulation with m4I3C(14). The K562 cells with p40_phox_ (WT) or p40_phox_ (D289A) or without p40_phox_ (5.0 × 103 cells) were stimulated with 200 μM m4I3C(14) and superoxide production was measured as change of chemiluminescence. Each histogram represents the average from five independent experiments, with bars representing SD. (D) Effect of the K355A substitution in the PB1 domain of p67_phox_ on superoxide production upon stimulation with m4I3C(14). The K562 cells with p67_phox_ (WT) or p67_phox_ (K355A) or without p67_phox_ (5.0 × 103 cells) were stimulated with 200 μM m4I3C(14), and superoxide production was measured as change of chemiluminescence. Each histogram indicates the average from five independent experiments, with bars representing SD.

Intriguingly, in response to m4I3C(14), K562 cells expressing p40_phox_ produced superoxide ∼20‐fold as much compared with the cells lacking this adaptor protein (Figure 8C). In addition, the peptide served as a weak agonist when cells expressed p40_phox_ (D289A) but not the wild‐type p40_phox_ (Figure 8C). Consistent with this, only a small amount of superoxide was generated in cells expressing p67_phox_ (K355A) instead of the wild‐type p67_phox_, while no superoxide production was detected in cells lacking p67_phox_ (Figure 8D). Thus, in cells stimulated with the Gi activator m4I3C(14), p40_phox_ greatly enhances activation of the phagocyte NADPH oxidase, which is dependent on its interaction with p67_phox_.

Discussion

Although p40_phox_ is known as a protein that constitutively associates with the phagocyte oxidase activator p67_phox_, the role of p40_phox_ in the oxidase activation has remained elusive. In the present study, we show that p40_phox_ facilitates the activation of the phagocyte NADPH oxidase at the cellular level (Figures 3 and 8). The facilitation is thought to be caused by enhancing the stimulus‐induced recruitment of p67_phox_ and p47_phox_ to the membrane, an essential step for the oxidase activation, without affecting the membrane translocation of the small GTPase Rac (Figure 3). We also demonstrate that both enhanced recruitment of the oxidase activators and facilitated activation of the oxidase are totally dependent on the binding of p40_phox_ to p67_phox_, which is mediated via a novel modular interaction between the p40_phox_ PC motif and the p67_phox_ PB1 domain (Figures 5, 7 and 8).

One of the reasons why the role of p40_phox_ has been obscure for a long time is due to the fact that p40_phox_ is dispensable for NADPH oxidase activation under both cell‐free and whole‐cell conditions (de Mendez et al., 1994; Sathyamoorthy et al., 1997). In addition, it has been reported that the presence of p40_phox_ marginally affects the oxidase activity in a cell‐free activation system: this protein only slightly enhances the activation (Cross, 2000). Although studies under cell‐free conditions have shed light on the understanding of the molecular mechanism for the oxidase activation, results obtained in such studies sometimes seem discrepant with those observed in a whole‐cell system. For instance, p67_phox_ binds to p47_phox_ via the C‐terminal SH3 domain of p67_phox_, a domain which is required for the oxidase activation in a whole‐cell system (de Mendez et al., 1994) but not for the cell‐free activation (de Mendez et al., 1994; Leusen et al., 1995; Hata et al., 1998). Here we show, using cells stably expressing p40_phox_, that p40_phox_ enhances activation of the NADPH oxidase. When cells are stimulated with PMA, a potent activator of protein kinase C, expression of p40_phox_ leads to a 2‐ to 3‐fold enhancement (Figure 3). On the other hand, the oxidase activation is much more drastically (∼20‐fold) facilitated by p40_phox_ in response to the muscarinic receptor peptide m4I3C(14), that acts as an activator of Gi (Figure 8). The peptide probably functions as a more physiological stimulant, since PTX blocks the peptide‐induced superoxide production but not the PMA‐elicited one (Figure 8). It seems probable that, in in vivo activation of the oxidase, p40_phox_ plays a role that is much more important than expected from the results of experiments with PMA. Alternatively, the significance of p40_phox_ could be dependent on the types of stimulants or signaling pathways for the oxidase activation. In either case, p40_phox_ is deeply involved in activation of the phagocyte NADPH oxidase.

Although no case has been thus far reported of CGD with the defect of p40_phox_, it is conceivable that its defect causes less severe forms of compromised host defense and hence has escaped the clinical screening for CGD. Alternatively, since the phagocyte oxidase is known to participate not only in host defense but also inflammation, an aberrance in inflammatory response, rather than vulnerability to infection, may be manifested in the cases with defect of p40_phox_. It is thus intriguing to search single nucleotide polymorphisms or mutations in the p40_phox_ gene NCF4 showing significant association with those displaying these symptoms. However, one should note that extra‐phagocytic roles for p40_phox_ and hence its involvement in other diseases are plausible, because mouse Ncf4 gene is also expressed in T cells and neurons, with no expression of p67_phox_ (Mizuki et al., 1998). In any case, pathophysiological roles for p40_phox_ remain largely elusive.

In contrast with the present results, Sathyamoorthy et al. (1997) have reported that transient expression of p40_phox_ leads to a repressed activation of the NADPH oxidase when cells are stimulated with PMA. The reason for this discrepancy is presently unknown. They have also shown that expression of the SH3 domain of p40_phox_ by itself represses the oxidase activation more efficiently than the full‐length p40_phox_ does (Sathyamoorthy et al., 1997). Since the SH3 domain of p40_phox_ is capable of binding to p47_phox_ but to a much lesser extent than the C‐terminal SH3 domain of p67_phox_ (Ito et al., 1996), overexpression of the p40_phox_ SH3 domain may compete with the SH3 domain of p67_phox_ to prevent p67_phox_ from interacting with p47_phox_, thereby inhibiting the oxidase activation.

The adaptor protein p40_phox_ is present in a complex containing not only p67_phox_ but also p47_phox_. In this complex, p40_phox_ directly binds to p67_phox_ via the PB1–PC interaction, while p40_phox_ interacts with p47_phox_ in an indirect manner: p67_phox_ tethers p40_phox_ to p47_phox_ (Figure 1). If the association between p40_phox_ and p67_phox_ is absent, p40_phox_ and p47_phox_ cannot reside in the same complex (Figures 4 and 6). Thus membrane translocation of p47_phox_ is not enhanced by p40_phox_ under conditions where the association between p40_phox_ and p67_phox_ is specifically disrupted (Figures 5 and 7). On the other hand, the membrane translocation of p40_phox_ requires p67_phox_. When a mutant p67_phox_ (K355A), that cannot interact with p40_phox_, is expressed instead of the wild‐type p67_phox_, p40_phox_ is incapable of translocating to the membrane upon cell stimulation (Figure 7). This is consistent with a previous observation that, in neutrophils of a p67_phox_‐deficient CGD patient, p40_phox_ is not targeted to the membrane in response to cell stimuli (Dusi et al., 1996). Even in these cells, stimulus‐induced translocation of p47_phox_ can still be observed (Dusi et al., 1996). Taken together, p47_phox_ plays an essential role in membrane recruitment of the p67_phox_–p47_phox_–p40_phox_ complex, an event which is strongly enhanced by p40_phox_.

This study clearly demonstrates that p40_phox_ facilitates membrane targeting of the essential oxidase factors p67_phox_ and p47_phox_, which requires the interaction of p40_phox_ with p67_phox_. The molecular mechanism whereby p40_phox_ functions, however, is not fully understood at present. In the N‐terminal region, p40_phox_ harbors the PX/PB2 domain (Figure 1), a module that interacts with phosphoinositides (Ago et al., 2001; Kanai et al., 2001; Ellson et al., 2001b). The PX domain of p40_phox_ specifically binds to phosphatidylinositol‐3‐phosphate [PtdIns(3)P] in vitro, and the domain expressed as a fusion to green fluorescent protein localizes to early endosomes where this phosphoinositide is enriched (Ago et al., 2001; Kanai et al., 2001; Ellson et al., 2001b). It is possible that the phospholipid‐binding activity of p40_phox_ is involved in its membrane localization. In this context, it is intriguing to note a current report showing that PtdIns(3)P is enriched in phagosomes (Ellson et al., 2001a), where the NADPH oxidase is activated. It has been also reported that p40_phox_ can interact with coronin, an actin‐binding protein (Grogan et al., 1997). Interaction of p40_phox_ with cytoskeletal elements may participate in a process of membrane translocation of this protein. In addition, stimulus‐induced phosphorylation of p40_phox_ (Bouin et al., 1998) possibly plays a role, an event which has escaped analysis in a cell‐free system. These possibilities should be tested in future studies.

The PB1 domain and PC motif prevail in a variety of proteins and mediate protein–protein interactions: the PB1 domain of Bem1p, scd2, atypical protein kinase Cζ or p67_phox_ recognizes and binds to the PC motif of Cdc24p, scd1, ZIP or p40_phox_, respectively (Ito et al., 2001). In the budding yeast Saccharomyces cerevisiae, the interaction between Bem1p and Cdc24p is essential for polarity establishment of cells (Ito et al., 2001; Butty et al., 2002). The present study provides another example of functional interactions mediated via these modules, i.e. the binding of p40_phox_ to p67_phox_ plays a significant role in activation of the phagocyte NADPH oxidase, an enzyme involved in host defense. Further studies are required to clarify the importance of other PB1–PC interactions.

Materials and methods

Plasmid constructions

cDNA fragments encoding p40_phox_, p47_phox_ and p67_phox_ were prepared as described previously (Hata et al., 1998; Nakamura et al., 1998; Ago et al., 1999) and cloned into the vectors pREP (Invitrogen), pGEX‐4T1 (Amersham Pharmacia Biotech) and pMALc2 (New England Biolabs). Mutations leading to the indicated amino acid substitutions were introduced by PCR‐mediated site‐directed mutagenesis. All the constructs were sequenced to confirm their identities.

Expression of p40 phox in K562 cells containing cytochrome b 558 , p47 phox and p67 phox

The doubly transduced K562 cells that stably express both cytochrome b_558 and p67_phox were prepared using the monoclonal antibody 7D5 to detect gp91_phox_ in functional cytochrome b_558 (Yamauchi et al., 2001) and anti‐p67_phox polyclonal antibodies raised to a C‐terminal peptide of this protein (Imajoh‐Ohmi et al., 1992), as previously described (Ago et al., 1999). The cells were electroporated in the presence of the full‐length cDNA of p47_phox_ in pREP9. After incubation with G418 (1.0 mg/ml), the cells were cloned by limiting dilution and checked by flow cytometry with an anti‐p47_phox_ monoclonal antibody (Transduction Laboratories). The stable transformants were further transfected with pREP4 encoding the wild‐type p40_phox_ or a mutant one carrying the D289A substitution. The transfected cells were cloned by limiting dilution in the presence of hygromycin B (100 μg/ml) and expression of p40_phox_ was confirmed by flow cytometry with anti‐p40_phox_ polyclonal antibodies raised to an N‐terminal peptide of this protein (Tsunawaki et al., 1994).

Expression of p67 phox in K562 cells containing cytochrome b 558 , p47 phox and p40 phox

The doubly transduced K562 cells that stably express both functional cytochrome b_558 and p47_phox were prepared as previously described (Koga et al., 1999). The cells were electroporated in the presence of pREP9 (Invitrogen) encoding the full‐length cDNA of p40_phox_ or vector alone. After incubation with G418 (1.0 mg/ml), the K562 cells were cloned by limiting dilution. The stable transformants were further transfected with pREP10 encoding the wild‐type p67_phox_ or a mutant one carrying the K355A substitution. The transfected cells were cloned by limiting dilution in the presence of hygromycin B (100 μg/ml), and expression of p67_phox_ was confirmed by flow cytometry with the anti‐p67_phox_ antibodies.

Flow cytometric analysis

Cells were washed with phosphate‐buffered saline (PBS; 137 mM NaCl, 2.7 mM KCl, 8.1 mM Na2HPO4 and 1.5 mM KH2PO4 pH 7.4), and fixed with 2.0% paraformaldehyde for 1 h at 25°C. For detection of cytosolic proteins, the fixed cells were permeabilized with PBS including 0.1% Triton X‐100 and 2.0% bovine serum albumin (BSA). The cells were incubated with the indicated antibody, washed twice with 2.0% BSA in PBS, and indirectly labeled with fluorescein isothiocyanate‐conjugated goat‐anti mouse or rabbit IgG (BioSource International). Cells were washed twice with PBS and analyzed by FACS scan.

Activation of the phagocyte NADPH oxidase

Superoxide production was determined as SOD‐inhibitable chemiluminescence, as previously described (Ago et al., 1999; Koga et al., 1999). Cells in HEPES‐buffered saline (120 mM NaCl, 5 mM KCl, 5 mM glucose, 1 mM MgCl2, 0.5 mM CaCl2 and 17 mM HEPES pH 7.4) were stimulated at 37°C with PMA (200 ng/ml), and the reaction was terminated by the addition of SOD (50 μg/ml). The chemiluminescence was counted with an enhancer‐containing luminol‐based detection system (DIOGENES; National Diagnostics) using a luminometer (Auto Lumat LB953; EG&G Berthold).

Alternatively, the superoxide‐producing activity was measured by determining the rate of SOD‐inhibitable cytochrome c reduction at 550–540 nm using a dual‐wavelength spectrophotometer (Hitachi 557) (Sumimoto et al., 1994).

Translocation of p47 phox , p67 phox and p40 phox to the membrane in stimulated K562 cells

Membrane translocation of cytosolic oxidase factors were determined by the method of Leusen et al. (1994) with minor modifications. Briefly, cells were stimulated for the indicated time at 37°C with PMA (200 ng/ml). The incubation was terminated by the addition of ice cold PBS. The cells were resuspended in 3 ml of ice cold buffer A (75 mM NaCl, 170 mM sucrose, 1 mM MgCl2, 0.5 mM EGTA, 10 μM ATP, 2 mM NaN3, 5 μM GTPγS, 100 μg/ml of _p_‐amidinophenyl methanesulfonyl fluoride hydrochloride and 20 mM HEPES pH 7.0) and lyzed by sonication. The sonicate was layered on a discontinuous sucrose gradient consisting of 4 ml of 40% (w/v) sucrose and 3 ml of 15% (w/v) sucrose with 1 mM MgCl2, 40 mM NaCl, 0.5 mM EGTA and 5 μM 5′‐3‐_O_‐ (thio)‐triphosphate (GTPγS). After ultracentrifugation for 45 min at 100 000 g, membrane‐associated proteins (1.0 μg) were analyzed by immunoblot with the indicated antibody, which were developed using ECL‐plus (Amersham Pharmacia Biotech).

An in vitro binding assay using purified recombinant proteins

The PB1 domain of p67_phox_ (amino acids 335–427) and the protein with the K355A substitution were expressed as GST fusion proteins in Escherichia coli and purified by glutathione–Sepharose‐4B (Amersham Pharmacia Biotech) (Ago et al., 1999; Koga et al., 1999). MBP‐tagged full‐length p40_phox_ and the protein with the D289A substitution were also expressed and purified by amylose resin (New England BioLab). For in vitro pull‐down binding assays, a pair of a GST fusion (250 μg) and an MBP‐tagged protein (500 μg) were incubated for 30 min in 1 ml PBS containing 0.5% Triton X‐100. Proteins were precipitated with glutathione–Sepharose‐4B and eluted with 5 mM glutathione. The eluates were subjected to SDS–PAGE and stained with Coomassie Brilliant Blue (CBB).

An in vivo binding assay

Cells were lyzed with 1 ml of a lysis buffer (1% Triton X‐100, 100 mM NaCl, 5 mM MgCl2, 1 mM EDTA and 40 mM HEPES pH 7.4). The lysate was precipitated with the anti‐p47_phox_ or anti‐p67_phox_ antibody in the presence of protein G–Sepharose (Amersham Pharmacia Biotech). After washing with the lysis buffer, precipitated proteins were analyzed by immunoblotting.

Preparation of the muscarinic receptor peptide m4I3C(14)

m4I3C(14) is a 14‐residue peptide fragment corresponding to the C‐terminal portion of the third intracellular loop and the N‐terminal portion of the sixth transmembrane helix of human m4 muscarinic acetylcholine receptor (Bonner et al., 1987) (residues 393–406: RERKVTRTIFAILL). This peptide, like mastoparan (Ross and Higashijima, 1994), activates trimeric Gi reconstituted in phospholipid vesicles in a similar manner as the parental m4 muscarinic acetylcholine receptor (K.Wakamatsu, in preparation) yet does not show any cell‐damaging activity as observed for mastoparan (Nakajima et al., 2000). The peptide was synthesized by a standard fluoren‐9‐ylmethoxycarbonyl‐based solid‐phase method. The purity and the identity of the synthesized peptide were confirmed by analytical HPLC and time‐of‐flight mass spectroscopy.

References

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