Assembly and activation of the phagocyte NADPH oxidase. Specific interaction of the N-terminal Src homology 3 domain of p47phox with p22phox is required for activation of the NADPH oxidase.

The phagocyte NADPH oxidase is activated during phagocytosis to produce superoxide, a precursor of microbicidal oxidants. The activation involves assembly of membrane-integrated cytochrome b558 comprising gp91(phox) and p22(phox), two specialized cytosolic proteins (p47(phox) and p67(phox)), each containing two Src homology 3 (SH3) domains, and the small G protein Rac. In the present study, we show that the N-terminal SH3 domain of p47(phox) binds to the C-terminal cytoplasmic tail of p22(phox) with high affinity (KD = 0.34 microM). The binding is specific to this domain among several SH3 domains including the C-terminal one of p47(phox) and the two of p67(phox) and requires the Pro156-containing proline-rich sequence but not other putative SH3 domain-binding sites of p22(phox). Replacement of Trp193 by Arg in the N-terminal SH3 domain completely abrogates the association with p22(phox). A mutant p47(phox) with this substitution is incapable of supporting superoxide production under cell-free activation conditions. These findings provide direct evidence that the interaction between the N-terminal SH3 domain of p47(phox) and the proline-rich region of p22(phox) is essential for activation of the NADPH oxidase.

During phagocytosis of microorganisms, neutrophils and other phagocytic cells produce superoxide (O 2 . ), a precursor of microbicidal oxidants (for reviews, see Refs. [1][2][3][4][5]. This process involves activation of the phagocyte NADPH oxidase, dormant in resting cells, that catalyzes reduction of molecular oxygen to superoxide in conjunction with oxidation of NADPH. 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), 1 a hereditary disease resulting in defective NADPH oxidase activity (1)(2)(3)(4)(5).
For an active NADPH oxidase, four specialized proteins are required, gp91 phox and p22 phox , tightly associated subunits constituting a phagocyte-specific membrane-integrated b-type cytochrome (cytochrome b 558 ) (6 -11), and the two cytosolic proteins p47 phox and p67 phox that assemble with the cytochrome during activation of the enzyme (12)(13)(14)(15). Genetic lesions affecting any of the four proteins can cause CGD. In addition, as a third cytosolic factor, the small GTP-binding protein Rac (either Rac1 or Rac2) is needed for the activation (16 -18), the protein that also migrates to the membrane upon cell stimulation and interacts with cytochrome b 558 (19,20). Cytochrome b 558 is now considered to be a flavocytochrome comprising a complete apparatus transporting electrons from NADPH via FAD and then heme to molecular oxygen (21)(22)(23)(24)(25). Current models postulate that assembly of the three cytosolic factors with cytochrome b 558 causes a conformational change in the flavocytochrome, which may enhance NADPH binding and/or facilitate electron transfer between NADPH and FAD and/or from FAD to heme (26). Increasing attention thus has been paid to mechanism of their assembly leading to activation of the enzyme. The first indication for the mechanism emerges from experiments using neutrophils from CGD patients (27,28). Neither p47 phox nor p67 phox translocates to the membrane in stimulated neutrophils that lack cytochrome b 558 , indicating that the cytochrome provides a membrane docking site for the cytosolic components. In p47 phox -deficient neutrophils, p67 phox fails to migrate, whereas p47 phox does bind to the membrane in stimulated neutrophils from patients deficient in p67 phox . Similar dependence of p67 phox translocation on the presence of p47 phox is observed using a cell-free system consisting of the neutrophil membrane and recombinant cytosolic proteins (29). The protein p47 phox probably participates in the active oxidase complex by direct interaction with cytochrome b 558 , whereas p67 phox does via binding to p47 phox . Recent efforts have been made to understand the molecular nature of these interactions between oxidase factors (30 -36). Both p47 phox and p67 phox contain two Src homology 3 (SH3) domains (15,37), which are present in many signaling proteins and known to mediate interactions via binding to proline-rich regions in target proteins (38 -40). Finan et al. (31) have shown that the C-terminal SH3 domain of p67 phox can interact with p47 phox via binding to the C-terminal proline-rich region. The binding is not required for the NADPH oxidase activity in the cell-free system (33,34), although the SH3 domain is essential for restoration of the oxidase activity in Epstein-Barr virustransformed B lymphocytes that lack p67 phox (34). Also in interaction between p47 phox and cytochrome b 558 , SH3 domains seem to play a crucial role. We and Leto et al. (30,32) have presented that the region of the tandem SH3 domains of p47 phox expressed as a glutathione S-transferase (GST) fusion protein binds to the C-terminal cytoplasmic tail of p22 phox but fails to interact with a mutant p22 phox carrying the Pro 156 3 Gln substitution in a proline-rich region. This substitution occurs in a CGD patient whose cytochrome b 558 is normal in both appearance and abundance as determined by visible spectroscopy and by immunoblot analyses but is devoid of activity in the cell-free oxidase activation system (41). Membrane translocation of p47 phox is impaired in activated neutrophils from the same patient (42). Thus the interaction via the SH3 domains of p47 phox appears to be involved in both assembly and activation of the phagocyte NADPH oxidase. Several questions, however, remain to be answered. Which SH3 domain of p47 phox directly binds to p22 phox ? What is the nature of the binding? Is the domain essential for the oxidase activation?
Here we show that the N-terminal SH3 domain of p47 phox binds to the C-terminal cytoplasmic tail of p22 phox with high affinity. The binding is highly specific to this SH3 domain and requires the Pro 156 -containing proline-rich region but not other putative SH3 domain-binding sites of p22 phox . Furthermore, a mutant p47 phox carrying the Trp 193 3 Arg substitution in the N-terminal SH3 domain, a mutation leading to defective interaction with p22 phox , is incapable of supporting superoxide production under cell-free activation conditions. This specific interaction, thus, is required for activation of the NADPH oxidase.
Binding of SH3 Domains to the C-terminal Cytoplasmic Tail of p22 phox -The GST fusion proteins containing various regions of p22 phox (10 pmol) purified by glutathione-Sepharose-4B beads were subjected to 10% SDS-PAGE (polyacrylamide gel electrophoresis) and transferred to nitrocellulose membrane. The membrane was blocked by buffer A (500 mM NaCl, 20 mM Tris, pH 7.5) containing 3% non-fat dry milk for 2 h at room temperature. The GST-fusion proteins with various SH3 domains (3 g/ml) were added to buffer A plus 0.25% gelatin, incubated for 1 h at room temperature, and washed four times with buffer A containing 0.1% Tween 20. The filter was probed with an anti-GST monoclonal antibody (43), which was kindly provided by Dr. Yoichi Tachibana (Nippon Zeon Corp.), in buffer A plus 0.25% gelatin for 2 h at room temperature and washed three times with buffer A plus 0.1% Tween 20. Complexes were detected using anti-mouse IgG antibodies. Under the condition used in this study, the anti-GST monoclonal antibody did not recognize GST fusion proteins transferred to nitrocellulose membranes after SDS-PAGE.
All the possible pairs between the pGBT and pGAD plasmids were cotransformed into competent yeast HF7c cells with HIS3 and lacZ reporter genes using a modified lithium-acetate method (44). Following the selection for Leu ϩ and Trp ϩ phenotype, transformants were tested for their ability to grow on plates lacking histidine. These indicator plates were supplemented with 10 mM 3-aminotriazole to suppress the background growth due to leaky expression of HIS3 gene in HF7c cells. The pairs of the plasmids were also cotransformed into competent SFY526 cells with HIS3 and lacZ reporter genes, and activation of lacZ reporter was examined by ␤-galactosidase filter assay according to the manufacturer's recommendation.
Real-time Interaction Analysis of SH3 Domains with p22 phox -The dissociation equilibrium constant (K D ) for the indicated SH3 domains with immobilized GST-p22-(132-195) or GST-p22-(132-195, P156Q) was calculated from data obtained using the interactive analysis system (IAsys, Fisons Applied Sensor Technology) where a laser biosensor measures real-time optical changes occurring when an analyte binds to its partner (45,46). GST-p22-(132-195), GST-p22-(132-195, P156Q), or GST alone was coupled to the reaction cuvette at concentrations of 1-2 g/mm 2 and kinetic analysis was completed at various concentrations of the indicated SH3 domains. For a pseudo-first-order reaction, the rate is described by Equation 1: where k a ϭ association rate constant, k d ϭ dissociation rate constant, R max ϭ maximal binding, R ϭ amount bound (biosensor response), and C ϭ concentration of SH3 domain. In this study, measured on-rate constant (k on ) can be obtained from analysis of association curve, and then k a and k d were determined by plotting k on (equal to negative of the slope of dR/dt versus R) for several concentrations of the SH3 domains against their respective concentrations. Each will result in a plot with slope equal to k a with intercept on the y axis equal to k d according to Equation 2: K D was calculated from Equation 3: Isolation and Fractionation of Human Neutrophils-Human neutrophils were isolated from healthy volunteers by dextran sedimentation, hypotonic lysis, and the Conray-Ficoll centrifugation (47). Membrane and cytosolic fractions of the neutrophils were prepared by sequential centrifugations as described previously (48).
Preparation of Rac2-enriched Cytosol Fraction-Rac2-enriched fraction was prepared by the method as described previously (47). Briefly, the neutrophil cytosolic fraction was applied to a 2Ј,5Ј-ADP-Sepharose CL-6B column to which both p47 phox and p67 phox bound. The flowthrough fraction was dialyzed and then applied onto a DEAE-Sepharose CL-6B column. After washing, the Rac fraction was eluted with 0.2 M NaCl. The fraction contained Rac2 but was free of p47 phox and p67 phox as confirmed by immunodetection.
Cell-free Activation of the NADPH Oxidase-The NADPH oxidase activity in a cell-free system was assayed using human neutrophil membrane, the Rac2-enriched fraction, and recombinant p47 phox and p67 phox , each expressed as a GST-fusion protein, as described by Arg mutant (GST-p47-F(W193R)), followed by incubation with an optimal concentration of SDS (100 M) for 2 min at room temperature. The reaction was initiated by addition of NADPH (1.0 mM) to the reaction mixture, and NADPH-dependent superoxide-producing activity was measured by determining the rate of superoxide dismutase-inhibitable ferricytochrome c reduction using a dual-wavelength spectrophotometer (Hitachi 557). Under the assay condition used, a negligible activity was detected in the absence of p67 phox or Rac2.

RESULTS
The N-terminal SH3 Domain of p47 phox Specifically Binds to the C-terminal Cytoplasmic Tail of p22 phox -We and others (30,32) have previously shown that the region of the tandem SH3 domains of p47 phox expressed as a GST fusion protein (GST-p47-(SH3) 2 ) binds to the C-terminal cytoplasmic tail of p22 phox but fails to interact with a mutant p22 phox carrying the Pro 156 3 Gln substitution in a proline-rich region. It remained unsolved which one of the two SH3 domains directly interacts with p22 phox . To address this question, we expressed and purified each SH3 domain as a GST fusion protein (GST-p47-SH3(N) and GST-p47-SH3(C)) and tested their ability to bind to p22 phox . In the present study, the anti-GST monoclonal antibody (43) was used for detection of an SH3 domain-target complex, instead of using biotinylated GST-SH3 domain-fusion proteins as in the previous studies (30,32). Biotinylation may affect binding activity of the proteins, depending on their amino acid sequences. The present method thus enabled us to compare binding activities of various SH3 domains under the same experimental condition. As shown in Fig. 1, GST-p47-SH3(N) strongly bound to the C-terminal cytoplasmic tail of p22 phox (lane 1). On the other hand, no binding could be observed when GST-p47-SH3(C) was used (lane 3). The binding of GST-p47-SH3(N) was completely abolished by a single amino acid substitution of Gln for Pro 156 in a proline-rich region of p22 phox (lane 2), indicating that this region is involved in the interaction with the SH3 domain. To study further the nature of the interaction, we introduced a mutation to the N-terminal SH3 domain of p47 phox (p47-SH3(N)), resulting in replacement of Trp 193 by Arg. This tryptophan residue is the most conserved one in SH3 domains (49), and it directly interacts with a proline of target peptides (50,51). Equivalent mutations (substitution of Arg or Leu for Trp) in Src and CRK result in loss of function (43,52). The mutant p47-SH3(N) carrying the Trp 193 3 Arg substitution failed to interact with p22 phox (lane 5), suggesting that p47-SH3(N) binds to p22 phox in a manner common to SH3 domains. This raised a question whether the binding to p22 phox is specific to p47-SH3(N) among SH3 domains. We tested several SH3 domains from various proteins. Neither N-terminal nor C-terminal SH3 domain of p67 phox could interact with p22 phox (lanes 6 and 7). No binding was observed with SH3 domains of other signaling proteins, v-Src, CRK, Abl, or phospholipase C-␥1 (lanes 8 -11). Thus, p47-SH3(N) specifically binds to the C-terminal cytoplasmic domain of p22 phox .
The Pro 156 -containing Proline-rich Region of p22 phox Is the Target of p47-SH3(N)-The finding that replacement of Pro 156 by Gln in a proline-rich region of p22 phox abolished the activity to interact with p47-SH3(N) (Fig. 1, lane 2) suggests that this region is at least one of the targets of the SH3 domain. The search for SH3 domain-binding sites using peptide libraries and structural analysis of SH3 domains complexed with proline-rich peptides have revealed that a Pro-Xaa-Xaa-Pro motif is the minimal requisite for SH3 domain binding in several signaling proteins (50,51). In p22 phox , the motif exists in three regions of the C-terminal cytoplasmic domain: Pro 133 -Ile-Glu-Pro, Pro 152 -Ser-Asn-Pro-Pro-Pro-Arg-Pro-Pro, and Pro 180 -Gly-Gly-Pro. To determine the target site(s) of p47-SH3(N), we used GST-fusion proteins with several fragments derived from the C-terminal cytoplasmic domain of p22 phox (Fig. 2A). Strong bindings were observed with the fusion proteins that contained the region of amino acids 132-195, 132-170, 145-195, and 145-170 (Fig. 2B, lanes 1, 3, 4, and 6, respectively), all of which possessed the stretch Pro 152 -Ser-Asn-Pro-Pro-Pro-Arg-Pro-Pro. In addition, p47-SH3(N) bound to the Pro 156 -containing proline-rich peptide of p22 phox (Pro 151 -Pro-Ser-Asn-Pro-Pro-Pro-Arg-Pro-Pro) fused to GST but to a lesser extent (lane 8). On the other hand, proteins without this region, GST-p22 (132-150) and GST-p22 (163-195), were not capable of interacting with p47-SH3(N) (lanes 5 and 7, respectively). These findings indicate that the Pro 156 -containing proline-rich region of p22 phox is required for the interaction with p47-SH3(N) and that other regions containing a Pro-Xaa-Xaa-Pro motif are not targets of the SH3 domain.
In Vivo Interaction between p22 phox and p47 phox -We next used a two-hybrid system in the yeast to test whether interaction between p22 phox and p47 phox also occurs under in vivo conditions. The C-terminal cytoplasmic domain of p22 phox and its Pro 156 3 Gln mutant were fused to the DNA-binding domain of GAL4 on the pGBT9g vector to construct pGBT::p22-(132-195) and pGBT::p22- (132-195, P156Q), respectively. We also prepared a construct comprising the SH3 domains of p47 phox (amino acids 154 -286), as well as one carrying the Trp 193 3 Arg substitution, and fused them to the activation domain of GAL4 on the vector pGAD424g to obtain pGAD::p47-(SH3) 2 and pGAD::p47-(SH3) 2 (W193R), respectively. All the possible pairs between the pGBT::p22 and pGAD::p47 plasmids were introduced into competent HF7c cells and tested for activation of HIS3 reporter gene. The various cotransformed cells were able to grow at similar rates in the presence of histidine, indicating no particular toxicity of expressed hybrid proteins (data not shown). The cell bearing both pGBT::p22-(132-195) and pGAD::p47-(SH3) 2 , but not other combinations, grew on the plate lacking histidine (Fig.  3A). We also used another yeast strain SFY526, cotransformed with the pGBT::p22 and pGAD::p47 plasmids, and tested ability to activate lacZ reporter gene. As shown in Fig. 3B, only the transformant expressing both p22-(132-195) and p47-(SH3) 2 showed a significant induction of the reporter gene. The reciprocal combination GAL4 activation domain-p22 phox /GAL4 DNA-binding domain-47 phox had the same effect (data not shown). These findings indicate that p47-SH3(N) interacts with the Pro 156 -containing proline-rich region of p22 phox in vivo as well as in vitro.
Resonance Mirror Studies of Binding of p47-SH3(N) to p22 phox -To estimate precise affinity for binding of SH3 domains to p22 phox , we employed the technique of resonance mirror (45,46) for analysis of this interaction. As shown in Fig.  3A, p47-SH3(N) rapidly bound to the C-terminal cytoplasmic domain of p22 phox (GST-p22(132-195)) immobilized to a biosensor tip. This binding was considered due to specific interaction between these proteins by the following reasons. First, no binding was observed by addition of GST alone (data not shown) or the Trp 193 3 Arg mutant (GST-p47-SH3(N)(W193R)) (Fig. 4A). Second, GST-p47-SH3(N) did not elicit any signal changes, when GST alone or the mutant p22 phox with the Pro 156 3 Gln substitution (GST-p22(132-195, P156Q)) was immobilized to a biosensor tip (data not shown). Finally, the C-terminal cytoplasmic tail of p22 phox did not interact with any GST fused to p47-SH3(C), p67-SH3(N), p67-SH3(C), Src-SH3, or CRK-SH3(N) (data not shown). To determine the dissociation equilibrium constant (K D ) for the interaction between p22 phox and p47 phox , we obtained k on (measured on-rate constant) from analysis of association curves at various concentrations of p47-SH3(N). Fig. 4B shows a plot of k on for five different concentrations of p47-SH3(N) against their respective concentrations. The K D value calculated from this plot was 0.34 M (for calculation, see "Experimental Procedures"), which is one to two orders lower than those reported for interaction between SH3 domains and proline-rich peptides in other systems (50,53). The tandem SH3 domains of p47 phox , 47-(SH3) 2 , also bound to p22 phox with a K D of 0.36 M (data not shown). Again, the Trp 193 3 Arg substitution completely abolished the binding (data not shown). Thus this new technique, the resonance mirror, clearly shows that p47-SH3(N) binds to the C-terminal cytoplasmic tail of p22 phox specifically and with high affinity.
Activation of the NADPH Oxidase in the Cell-free Reconstitution System-To investigate a role of the interaction between p47-SH3(N) and p22 phox in activation of the NADPH oxidase, we expressed and purified the full-length of p47 phox and the one carrying the Trp 193 3 Arg substitution as GST-fusion proteins and tested their activities in a cell-free activation system. The system was reconstituted with human neutrophil membranes, recombinant p67 phox , and the Rac2-enriched fraction. Wild- type p47 phox was fully active in the reconstitution system, whereas the mutant p47 phox carrying the Trp 193 3 Arg substitution in the N-terminal SH3 domain is incapable of supporting superoxide production (Fig. 5). This one amino acid substitution thus completely abrogated both activities to bind to p22 phox and to activate the NADPH oxidase, providing evidence that activation of the NADPH oxidase requires the interaction between p47-SH3(N) and p22 phox .

DISCUSSION
In the present study, we demonstrate that the N-terminal SH3 domain of p47 phox (p47-SH3(N)) binds to the C-terminal cytoplasmic tail of p22 phox . The binding is highly specific to p47-SH3(N) and requires the Pro 156 -containing proline-rich region but not other putative SH3 domain-binding sites of p22 phox . Furthermore, we show that a mutant p47 phox carrying the Trp 193 3 Arg substitution in p47-SH3(N) fails to interact with p22 phox and is unable to support superoxide production in the reconstituted activation system. Based on these findings, we conclude that this specific interaction is required for activation of the NADPH oxidase. The conclusion is also supported by the present observation that p47-SH3(N) can not bind to a mutant p22 phox with the Pro 156 3 Gln substitution, since cytochrome b 558 carrying this substitution is defective in forming a stable complex with p47 phox in stimulated neutrophils (42) and incapable of producing superoxide when activated both in vivo and in vitro (41,42).
Leto et al. (32) have reported, using a filter binding assay, that probes comprising single SH3 domains from p47 phox , in contrast to the one with both SH3 domains, bind less avidly to p22 phox , and binding to p22 phox by those probes was not greatly affected by the CGD-associated mutation (Pro 156 3 Gln). On the other hand, the present filter assay shows and analysis by the biosensor IAsys confirms that the N-terminal SH3 domain of p47 phox is the one that directly binds to p22 phox with high affinity. The Pro 156 3 Gln substitution results in complete loss of the binding, and the calculated K D for interaction between p47-SH3(N) and p22 phox (0.34 M) is essentially the same as that between p47-(SH3) 2 and p22 phox (0.36 M). The reason for the discrepancy between the two studies is presently unknown. One possible explanation may be that it is due to differences in experimental conditions; they used biotinylated proteins as probes whereas we used unmodified ones in in vitro binding assays; in their study sources of p22 phox were crude lysates from E. coli expressing GST fused to p22 phox , whereas purified proteins were used in this study.
There are now many examples of SH3 domain-mediated protein-protein interactions via binding to proline-rich sequences in target proteins (38 -40). Their functional roles, how-FIG. 3. Interaction of p22 phox with p47 phox analyzed by the yeast two-hybrid system. A, the yeast strain HF7c was cotransformed with pairs of recombinant plasmids pGBT9 and pGAD424, the former encoding the C-terminal cytoplasmic tail of p22 phox fused to GAL4 DNA-binding domain (pGBT::p22 (132-195)) or its mutant (pGBT::p22 (132-195, P156Q)), and the latter encoding the SH3 domains of p47 phox fused to GAL4 activation domain (pGAD::p47-(SH3) 2 ) or the Trp 193 3 Arg mutant (pGAD::p47-(SH3) 2 (W193R)). All the possible pGBT9 and pGAD424 plasmid pairs were tested for the His ϩ phenotype. B, the pairs of the plasmids were cotransformed into the yeast strain SFY526, and activation of lacZ reporter was examined by ␤-galactosidase filter assay. ever, are not easy to elucidate. For example, even with a wellknown interaction between SH3 domains of Grb2 and the C-terminal proline-rich tail of the Ras-activating protein Sos, it is still controversial whether this binding is required for downstream signal transduction (54). In the NADPH oxidase system, the C-terminal SH3 domain of p67 phox can bind to p47 phox via the C-terminal proline-rich region (31)(32)(33), a binding that is not required for superoxide-producing activity under cell-free activation conditions; both a mutant p67 phox without the SH3 domain and a mutant p47 phox deleting the binding partner are fully active under these conditions (33,34). In contrast, the interaction between p47 phox and p22 phox via an SH3 domain seems to play an essential role in activation of the oxidase. A mutant p47 phox carrying the Trp 193 3 Arg substitution in the N-terminal SH3 domain fails to interact with p22 phox and is incapable of supporting superoxide production in the reconstituted activation system. These results are consistent with that the Pro 156 3 Gln mutant of p22 phox , devoid of activity binding to p47-SH3(N), is also inactive in the cell-free system (41). At present, however, it is unknown whether the proline-rich region of p22 phox gives a simple docking site for p47 phox or the binding itself is involved in a conformational change of cytochrome b 558 leading to the enzyme activation.
The present findings, combined with our previous results (30), suggest the following mechanism for activation of the phagocyte NADPH oxidase. Normally inaccessible SH3 domains of p47 phox become exposed upon cell stimulation, and the unmasked p47-SH3(N) specifically interacts with the C-terminal cytoplasmic domain of p22 phox to form an active oxidase complex. This interaction is required but is probably not enough for stable binding to cytochrome b 558 . A single mutation in the large subunit of cytochrome b 558 (gp91 phox ) of a CGD patient (replacement of Asp 500 by Gly) results in defective translocation of p47 phox to the membrane upon neutrophil activation, although the cytochrome is normal in spectroscopic appearance and abundance (55). Elucidation of another interaction between p47 phox and cytochrome b 558 will be important to understand the more detailed mechanism of activation of the NADPH oxidase.