Mechanism for Phosphorylation-induced Activation of the Phagocyte NADPH Oxidase Protein p47 phox

Activation of the superoxide-producing phagocyte NADPH oxidase requires interaction between p47 phox and p22 phox , which is mediated via the SH3 domains of the former protein. This interaction is considered to be induced by exposure of the domains that are normally masked by an intramolecular interaction with the C-terminal region of p47 phox . Here we locate the intramolecular SH3-binding site at the region of amino acid residues 286–340, where Ser-303, Ser-304, and Ser-328 that are among several serines known to become phosphorylated upon cell stimulation exist. Simultaneous replacement of the three serines in p47 phox with aspartates or glutamates, each mimicking phosphorylated residues, is sufficient for disruption of the intramolecular interaction and resultant access to p22 phox . The triply mutated proteins are also capable of activating the NADPH oxidase without in vitro activators such as arachidonate under cell-free conditions. In a whole-cell system where expression of the wild-type p47 phox reconstitutes the stimulus-dependent oxidase activity, substitution of the kinase-insensitive residue alanine for Ser-328 as well as for Ser-303/Ser-304 leads to a defective production of superoxide. These findings suggest that phosphorylation of the three serines in p47 phox induces a conformational change to a state accessible to p22 phox , thereby activating the NADPH oxidase.

Protein-protein interactions form the basis of a variety of cellular processes. The interactions often depend on modular domains that serve as specific protein-binding structures (reviewed in Refs. [1][2][3][4]. Among them, SH3 1 domains, found in a wide array of proteins involved in intracellular signal transduction and cytoskeletons, interact with proline-rich ligands via direct binding to the PX⌽P motif (where P denotes proline residue; X denotes any amino acid residue; and ⌽ denotes a hydrophobic residue) (1)(2)(3)(4). SH3-mediated interactions were initially considered to be constitutive, e.g. the adaptor protein Grb2 associates with the Ras activator Sos in a preformed heterodimeric complex, which is mediated via binding of the Grb2 SH3 domains to the C-terminal proline-rich tail of Sos. There exist, however, currently increasing examples in which SH3-mediated interactions are regulated (5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16), although molecular mechanisms underlying their regulation remain largely unknown.
The first example to be described as a regulatory SH3-mediated interaction is the one involved in the signaling system for activation of the superoxide-producing NADPH oxidase in phagocytes as well as B lymphocytes (5,6). During phagocytosis or with appropriate stimuli, the phagocyte NADPH oxidase, dormant in resting cells, becomes activated to produce superoxide, a precursor of microbicidal oxidants (reviewed in Refs. [17][18][19][20][21][22]. The significance of the enzyme in host defense is exemplified by recurrent and life-threatening infections that occur in patients with chronic granulomatous disease, whose phagocytes are deficient in the superoxide-producing activity. The catalytic core of the oxidase is membrane-bound flavocytochrome b 558 , comprising the two subunits gp91 phox and p22 phox , that transfers electrons upon activation from NADPH to oxygen molecule. When cells are stimulated, the three cytosolic proteins p47 phox , p67 phox , and the small GTPase Rac, each indispensable for the oxidase activation, translocate to the membrane where they assemble with the cytochrome. p47 phox harbors two SH3 domains, which specifically interact with the C-terminal cytoplasmic PRR of p22 phox upon activation (5,6). This induced interaction plays a crucial role in activation of the NADPH oxidase; both the interaction and superoxide production are completely abrogated by replacement of the conserved  in the N-terminal SH3 domain with Arg or by substitution of Gln for Pro-156 in the PRR of p22 phox , a mutation that occurs in a patient with chronic granulomatous disease (5,6,(23)(24)(25). Since the full-length wild-type p47 phox in resting phagocytes or the one expressed in Escherichia coli or budding yeast is incapable of binding to p22 phox , a resting form of p47 phox is likely in a closed inactive conformation in which the SH3 domain is masked (5,26). We and Leto et al. (5,6) have previously proposed a model that the Cterminal region (residues 286 -390) of p47 phox intramolecularly interacts with the SH3 domains to render this protein in the closed state, and, upon activation, the SH3 domains are unmasked to bind to the target p22 phox . Anionic amphiphiles such as arachidonate and SDS, activators of the oxidase in vitro (27), cause a conformational change of p47 phox to expose the SH3 domains, as suggested by analyses using an anti-SH3 monoclonal antibody (5) and tryptophan fluorescence spectroscopy (28). This "unmasking-masking" model for SH3-mediated regulatory interactions has been supported by a recent observation that a C-terminally truncated p47 phox (p47-⌬C; amino acid residues 1-286), in which the intramolecular interaction does not occur because of a lack of the SH3 target, is capable of both binding to p22 phox and activating the oxidase in the absence of the amphiphile activators (26).
It is well established that, upon cell stimulation, p47 phox becomes extensively phosphorylated (29 -31). An intensive study by Babior's group (32) has revealed that 9 to 10 serine residues within the C-terminal region of p47 phox (Ser-303, Ser-304, Ser-315, Ser-320, Ser-328, Ser-345, Ser-348, Ser-359, Ser-370, and Ser-379) are phosphorylated when human neutrophils were stimulated with the protein kinase C activator PMA or the chemotactic formyl peptide fMLP. The phosphorylation is likely involved in activation of the phagocyte NADPH oxidase, because the mutant p47 phox carrying the double substitution S303A/S304A only marginally corrects the defect in superoxide production in EBV-transformed p47 phox -deficient B cells (33). However, the molecular link between the phosphorylation event and the oxidase activation remains to be elucidated.
To address this question, we focused on relationship between phosphorylation of p47 phox and a conformational change that leads to the oxidase activation. As an initial step of the analyses, we replaced the serines of p47 phox with aspartates or glutamates, each mimicking phosphorylated residues in various proteins (14, 34 -36), and we tested the effects of replacements on the SH3-mediated intramolecular and intermolecular interactions. The experiments reveal that simultaneous replacement of Ser-303, Ser-304, and Ser-328 is sufficient for disruption of the intramolecular interaction and resultant access of the SH3 domains to p22 phox . Furthermore, the triply mutated p47 phox can activate the oxidase in vitro without the amphiphiles. Substitution of alanine for Ser-328 as well as for Ser-303/Ser-304 results in a defective production of superoxide in vivo. Thus phosphorylation of the three serines of p47 phox induces a conformational change to a state accessible to p22 phox , thereby activating the NADPH oxidase.
Two-hybrid Experiments-Various combinations between pGBT9 and pACT2 plasmids were co-transformed into competent yeast Y190 cells containing HIS3 and lacZ reporter genes using a lithium-acetate method (37). Following the selection for Trp ϩ and Leu ϩ phenotype, the transformants were tested for their ability to grow on plates lacking histidine supplemented with 25 mM 3-aminotriazole to suppress the background growth. Activation of lacZ reporter was examined by the ␤-galactosidase filter assay according to the manufacturer's recommendation (CLONTECH).
An in Vitro Binding Assay Using Purified Proteins-Proteins fused to GST or to MBP were expressed in E. coli strain BL21 and purified by glutathione-Sepharose -4B (Amersham Pharmacia Biotech) or amylose resin (New England Biolabs), respectively, according to the manufac-turers' protocols. For in vitro pull-down binding assays, a pair of a GST fusion and an MBP-tagged protein were mixed in 500 l of phosphatebuffered saline (137 mM NaCl, 2.68 mM KCl, 8.1 mM Na 2 HPO 4 , and 1.47 mM KH 2 PO 4 ) containing 1% Triton X-100 and incubated for 30 min at 4°C. A slurry of glutathione-Sepharose 4B or amylose resin was subsequently added, followed by further incubation for 30 min at 4°C. After washing three times with phosphate-buffered saline, proteins were eluted from glutathione-Sepharose 4B or amylose resin, with 5 mM glutathione in 50 mM Tris-HCl (pH 8.0) or with 10 mM amylose in 50 mM Tris-HCl (pH 8.0), respectively. The eluates were subjected to SDS-PAGE and stained with Coomassie Brilliant Blue.
Cell-free Activation of the Phagocyte NADPH Oxidase-The membrane fraction of human neutrophils was prepared as described previously (5,24,26). The membranes (10 g of protein/ml) were mixed with the indicated concentrations of the wild-type or mutant p47 phox fused to GST, an N-terminal fragment of p67 phox (p67-N; amino acids 1-242) as a GST fusion protein (10 g/ml), and His-tagged Rac2 (10 g/ml) preloaded with 100 M GTP␥S, followed by incubation with or without SDS (100 M) for 2.5 min at room temperature in 100 mM potassium phosphate, pH 7.0, containing 75 M cytochrome c, 10 M FAD, 1.0 mM EGTA, 1.0 mM MgCl 2 , and 1.0 mM NaN 3 . The reaction was initiated by addition of NADPH (250 M) to the reaction mixture. The NADPH-dependent superoxide-producing activity was measured by determining the rate of superoxide dismutase-inhibitable ferricytochrome c reduction at 550 to 540 nm with a dual-wavelength spectrophotometer (Hitachi 557) (5,24,26).
Activation of the NADPH Oxidase in the Whole-cell System-We used a retroviral vector system, pSXLC/pHa, that utilizes an internal ribosome entry site fragment of encephalomyocarditis virus (38) to transduce the gp91 phox gene into the leukemia cell line K562 that expresses p22 phox but not gp91 phox (39). Cells highly expressing gp91 phox were selected using FACS scan with the monoclonal antibody 7D5 to detect functional cytochrome b 558 comprising the two subunits gp91 phox and p22 phox (40). A bicistronic retrovirus vector encoding a human multidrug resistance gene (MDR1) and the p67 phox gene (pHa-MDR-IRES-p67) (41) was further transduced to the stably transduced gp91 phoxexpressing K562 cells. The doubly transduced cells were selected with 4 ng/ml vincristine, expanded in a drug-free medium, and used for the following experiments.
Complementary DNAs encoding the full-length of the wild-type and mutant p47 phox carrying the S328A or S303A/S304A substitution were subcloned into pREP4 (Invitrogen), which were transfected by electroporation to the K562 cells that stably express both gp91 phox and p67 phox . The K562 cells (2 ϫ 10 7 cells/ml) were electroporated in the presence of 10 g of the wild-type or mutant form of p47 phox plasmid DNA at 170 V, 960 microfarads using Gene Pulser (Bio-Rad). To obtain stable transformants, cells were selected for over 30 days with 100 mg/ml hygromycin B. For detection of p47 phox and p67 phox , K562 cells (1 ϫ 10 5 cells) were lysed by sonication, and the sonicates were applied to 10% SDS-PAGE. Proteins were transferred to a polyvinylidene difluoride membrane (Millipore) and probed with anti-p47 phox and anti-p67 phox monoclonal antibodies (both from Transduction Laboratories). The blots were developed using ECL-plus (Amersham Pharmacia Biotech) to visualize the antibodies.
Superoxide production by K562 cells (1 ϫ 10 5 cells) expressing the wild-type or mutant p47 phox was determined as superoxide dismutaseinhibitable chemiluminescence detected with an enhancer-containing luminol-based detection system (DIOGENES; National Diagnostics) as described by de Mendez et al. (39). After the addition of the enhanced luminol-based substrate, the cells were stimulated with 200 ng/ml PMA. The chemiluminescence was assayed using luminometer (Auto Lumat LB953; EG & G Berthold).

RESULTS
C-terminally Truncated p47 phox Is Capable of Binding to p22 phox -The two SH3 domains of p47 phox are tandemly arranged in the central portion in the primary sequence: the N-terminal one of amino acid residues 154 -219 (SH3(N)) and the C-terminal one of residues 223-286 (SH3(C)) ( Fig. 1). We have previously shown that the C-terminal region of p47 phox (amino acids 286 -390) interacts with the SH3 domains in an intramolecular fashion, which likely prevents the domains from binding to p22 phox (5,26). To map precisely the region responsible for the prevention, we expressed and purified the full-length p47 phox (p47-F; amino acids 1-390) and a series of C-terminally truncated p47 phox (p47-(1-340), p47-(1-327), p47-(1-314), p47-(1-302), and p47-⌬C-(1-286)) as GST fusion proteins ( Fig. 2A), and we tested their ability to bind to the Cterminal cytoplasmic tail of p22 phox (amino acids 132-195), p22-C. As shown in Fig. 2B, GST-p47-⌬C-(1-286) was fully precipitated with amylose resin coupled to MBP-p22-C, whereas GST alone was hardly recovered. The interaction seems to be mediated via the SH3 domains of p47 phox , since p47-⌬C was not recovered when resins were coupled to MBP alone or a mutant p22-C carrying the P156Q substitution (data not shown). p47-(1-302) also bound to MBP-p22-C but to a lesser extent (Fig. 2B). On the other hand, neither p47-(1-314) nor p47 phox proteins with a shorter deletion could interact with p22-C. Although there were substantial small protein fragments in the sample of p47-(1-314) ( Fig. 2A), the possibility that they act as an inhibitor in binding can be excluded, since the sample of p47-(1-314) did not affect the interaction of p22-C with p47-⌬C or with p47-(1-302) (data not shown). These results were confirmed by an in vivo binding assay using the yeast two-hybrid system; both histidine-independent growth and ␤-galactosidase activity were observed, only when the yeast Y190 cells were co-transformed with both the DNA-binding domain fusion vector pGBT9 encoding p22-C (pGBT9-p22-C) and the transactivation domain fusion vector pACT2 encoding p47-⌬C or p47-(1-302) (data not shown).
The Tandem SH3 Domains Synergistically Interact with the Region of Amino Acids 286 -340 in p47 phox -To clarify the role for each of the two SH3 domains of p47 phox in the intramolecular interaction, we compared the ability to bind to the target region of amino acids 286 -340 among p47-(SH3)2, p47-SH3(N), and p47-SH3(C). As shown in Fig. 4A, a negligible binding activity was observed when p47-SH3(N) or p47-SH3(C) was used instead of p47-(SH3)2. Thus the tandem SH3 domains appear to interact synergistically with the intramolecular target site to keep p47 phox in a closed inactive conformation.
The Stretch PPRR of Amino Acids 299 -302 Plays an Essential Role in the Intramolecular Interaction of p47 phox -Structural analyses of several SH3-ligand complexes have revealed that SH3 domains bind to a proline-rich region in the polyproline II (PPII) helix conformation with extremely high preference to the sequence RX⌽PX⌽P (type I ligand) and/or ⌽PX⌽PXR (type II ligand), and thus PX⌽P is generally accepted as the target motif of SH3 domains accordingly (43)(44)(45)(46). Such a PX⌽P motif, however, is absent in the intramolecular SH3 target region of p47 phox (amino acids 286 -340); there exist only two proline residues at positions 299 and 300 (Fig. 1). Instead, the sequence of 299 -302, PPRR, that is present in the region minimally required for the intramolecular interaction (amino acids 286 -314), appears to be a remnant of the type II SH3 ligand and is thereby expected to play a role in the interaction with the SH3 domains (Fig. 1). To test this possibility, we substituted glutamines for Pro-299 and Pro-300 (P299Q/ P300Q) or glutamates for Arg-301 and Arg-302 (R301E/ R302E). A weak binding of p47-(286 -314) to p47-(SH3)2 was abrogated by these substitutions (Fig. 3C). The P299Q/P300Q substitution also resulted in a loss of a stronger interaction between p47-(286 -340) and the SH3 domains (Fig. 3C). Thus the PPRR stretch of 299 -302 likely conforms the core-binding site for the SH3 domains in the intramolecular interaction.
A proline residue in an SH3-targeted peptide directly contacts a conserved tryptophan residue in the binding surface of the SH3 domain (43)(44)(45)(46). To know which SH3 domain of p47 phox acts as the partner for the PPRR stretch, we introduced a substitution of arginine for either of the conserved tryptophans in the two SH3 domains as follows: p47-(SH3)2 (W193R) with the substitution in the N-terminal SH3 domain and p47-(SH3)2 (W263R) with that in the C-terminal one. The W193R substitution abolished the interaction with p47-(286 -340), whereas the replacement of Trp-263 led to only a partial defect (Fig. 4B). The finding indicates that the intramolecular interaction requires, probably as the partner for the PPRR stretch, the Trp-193-centered binding surface of SH3(N), which also participates in the intermolecular interaction with p22 phox (24).
Mutations in the PPRR Stretch Render p47 phox in a Conformation Capable of Binding to p22 phox -The dual role for the N-terminal SH3 domain of p47 phox , i.e. its involvement in both intra-and intermolecular interactions via the same binding surface, suggests that disruption of the intramolecular interaction in p47 phox directly promotes the accessibility of the SH3 domain to p22 phox . To investigate this possibility, we expressed the full-length mutant protein of p47 phox carrying the P299Q/ P300Q or R301E/R302E substitution, in each of which the intramolecular interaction is expected to be disrupted. As shown in Fig. 5A, proteins with these substitutions were capable of interacting with p22 phox . The results were consistent with those by the pull-down binding assay using purified proteins (Fig. 5B); MBP-p22-C directly interacted with the mutated proteins but not with the wild-type one (Fig. 5C). Taken together, we concluded that p47 phox becomes accessible to p22 phox solely by disrupting the intramolecular interaction.
Triple Replacement of Ser-303, Ser-304, and Ser-328 with Aspartates or Glutamates Is Sufficient for p47 phox to Interact with p22 phox -It is known that, upon cell stimulation, p47 phox becomes extensively phosphorylated at more than nine serine residues of the C-terminal quarter (32). They include five serines present in the region required for the stable intramolecular interaction with the p47 phox SH3 domains (residues 286 -340) as follows: Ser-303, Ser-304, Ser-315, Ser-320, and Ser-328. Among these serine residues, we initially focused on Ser-303 and Ser-304. Since these residues are intensively phosphorylated in stimulated cells (32) and exist just adjacent to the SH3-binding PPRR sequence of 299 -302, phosphorylation of these serines may disrupt the SH3-mediated intramolecular interaction, thereby activating p47 phox . To test this possibility, we replaced the serines by aspartates or glutamates, which are known to mimic phosphorylated residues in various proteins (14, 34 -36). Unexpectedly, a full-length p47 phox carrying the double substitution of aspartates for Ser-303 and Ser-304, designated p47-F (S303D/S304D), could not interact with p22-C in the two-hybrid experiment (Fig. 6A), raising the possibility that additional modification may be required for conversion of p47 phox to a state accessible to p22 phox .
All the three serines (Ser-303, Ser-304, and Ser-328) to be replaced are likely required for p47 phox to interact with p22 phox , since neither p47-F (S303D/S328D) nor p47-F (S304D/S328D) was capable of interacting with p22-C (Table I). In addition, replacement of Ser-315, Ser-320, or both, instead of Ser-328, in p47-F (S303/304D) did not lead to interaction with p22-C (Table I). Furthermore, any interactions could not be promoted by substitutions of aspartates for serines that lie outside of the SH3-targeted region: double substitution for Ser-345 and Ser-348 (Table I), the two serines that can be phosphorylated by the MAP kinases ERK and p38 (47,48), or triple substitution for the three C-terminal serines at positions 359, 370, and 379 (Table I). Thus the simultaneous substitution for Ser-303, Ser-304, and Ser-328 appears to be sufficient for promoting the interaction with p22 phox .
Triple Replacement of Ser-303, Ser-304, and Ser-328 Results in Disruption of the SH3-mediated Intramolecular Interaction in p47 phox -It seems likely that the binding of the triply mutated p47 phox (S303D/S304D/S328D) to p22 phox results from a defect of the SH3-mediated intramolecular interaction. To confirm this, we prepared the SH3-targeted fragment (amino acids 286 -340) with the substitution S303D/S304D and/or S328D, and we tested their ability to interact with p47-(SH3)2. The fragment carrying the S303D/S304D or S328D substitution bound to p47-(SH3)2 more weakly than the wild-type one did (Fig. 7). The triple substitution for Ser-303, Ser-304, and Ser-328 resulted in a completely defective interaction (Fig. 7), which is consistent with the finding that the only triply mutated p47 phox gains access to p22 phox (Figs. 5C and 6A). Taken together, simultaneous phosphorylation of Ser-303, Ser-304, and Ser-328 of p47 phox appears to primarily disrupt the SH3mediated intramolecular interaction, thereby leading to the interaction of the unmasked SH3 domain with p22 phox . Mutant p47 phox Proteins That Are Accessible to p22 phox Can Support Superoxide Production in an Anionic Amphiphile-independent Manner under Cell-free Activation Conditions of the NADPH Oxidase-As described above, a mutant p47 phox carrying the triple replacement of Ser-303, Ser-304, and Ser-328 with aspartates, mimicking a phosphorylated form, is in a conformation capable of binding to p22 phox . We next tested how this mutant protein serves in activation of the phagocyte NADPH oxidase.
The NADPH oxidase can be activated by anionic amphiphiles such as arachidonate and SDS in a cell-free system reconstituted with human neutrophil membranes that contain a high amount of the catalytic core cytochrome b 558 and three cytosolic proteins: p47 phox , p67 phox , and the small GTPase Rac1/2 in the GTP-bound state (24,26). We have recently shown that, even without the amphiphiles, the oxidase can be activated in vitro by p47 phox and p67 phox , both in C-terminally truncated forms, in the presence of the GTP-bound Rac (26). When the full-length p47 phox (p47-F) is used instead of the truncated p47 phox , p47-⌬C (residues 1-286), the activation absolutely requires the amphiphiles (Ref. 26 and Fig. 8). The finding implies that p47 phox is a target of the amphiphiles and that p47-⌬C serves as an active form of p47 phox . By using this system, we tested whether mutant p47-F proteins mimicking a phosphorylated form can replace p47-⌬C to activate the oxidase in vitro without the amphiphiles. As shown in Fig. 8A, p47-F (S303D/S304D/S328D) was capable of supporting superoxide production in the anionic amphiphile-independent system for the oxidase activation, although higher concentrations were required for fully activating the oxidase, TABLE I Interaction between full-length p47 phox carrying replacement of serines and p22 phox in the yeast two-hybrid system Histidine-independent growth was tested using yeast Y190 cells that were co-transformed by pairs of pGBT9 encoding p22 phox and pACT encoding full-length p47 phox carrying replacement of serines as described under "Experimental Procedures." p47 phox Histidine-independent growth compared with p47-⌬C. Both p47-F (S303E/S304E/S328E) and p47-F (S(303-379)D) were as active as p47-F (S303D/S304D/ S328D) in the oxidase activation (data not shown). On the other hand, p47-F (S303D/S304D) or p47-F (S328D), each lacking the p22 phox binding activity (Fig. 5C), was incapable of activating the oxidase without amphiphiles (Fig. 8A), although these mutant proteins are as active as the wild-type p47 phox in the presence of the amphiphile activator SDS (Fig. 8B). Thus triple replacement of Ser-303, Ser-304, and Ser-328 renders p47 phox in a conformation capable of not only binding to p22 phox but also activating the NADPH oxidase.
These experiments also show that the ability of p47 phox to activate the NADPH oxidase in the amphiphile-independent system appears to be parallel with that to bind to p22 phox . We next tested the ability of full-length mutant proteins carrying substitutions in the core intramolecular binding site for the SH3 domains of p47 phox , p47-F (P299Q/P300Q) and p47-F (R301E/R302E), both of which were capable of interacting with p22 phox (Fig. 5). As shown in Fig. 8A, these proteins could activate the oxidase in an amphiphile-independent manner.
The S328A or S303A/S304A Substitution in p47 phox Results in Defective Activation of the NADPH Oxidase in a Whole-cell System-The present observations suggest that phosphorylation of Ser-328 as well as Ser-303 and Ser-304 of p47 phox causes a conformational change to a state accessible to p22 phox , thereby activating the phagocyte NADPH oxidase. To investigate the role of the phosphorylation at a cell level, we have developed a whole-cell system of the K562 leukemic cell line. The cells are known to express Rac1/2 and a low level of endogenous p22 phox and to require expression of the other three oxidase factors (gp91 phox , p47 phox , and p67 phox ) to exhibit superoxide production in response to PMA (39). To explore the function of p47 phox , we transduced K562 cells for stable expression of gp91 phox and p67 phox using retroviral vectors encoding the proteins. The transduced cells expressed functional cytochrome b 558 comprising the two subunits gp91 phox and p22 phox (data not shown; see "Experimental Procedures") and p67 phox (Fig. 9A).
The K562 cells expressing both cytochrome b 558 and p67 phox were subsequently transfected with the episomal vector pREP4 that contained cDNA encoding the full-length wild-type p47 phox (p47-F) or full-length mutant proteins carrying replacement of serines by alanine, a residue that does not become phosphorylated, namely p47-F (S328A) and p47-F (S303A/S304A). The wild-type p47 phox -expressing cells fully produced superoxide when stimulated with PMA (Fig. 9, B and C). On the other hand, in the cells transfected with the p47-F (S328A) cDNA, the stimulant induced superoxide production but to a much lesser extent (Fig. 9, B and C), although the p47 phox protein was expressed at a similar level to the wild-type one in the control cells (Fig. 9A). Only a marginal production of superoxide was also detected upon stimulation in the cells expressing p47-F (S303A/S304A), consistent with the previous report showing that this mutant protein is essentially inactive when expressed in EBV-transformed p47 phox -deficient B cells (33). These substitutions unlikely lead to a loss of the ability to activate the oxidase, since bacterially expressed p47 phox with the S303A/ S304A and S328A substitutions were both capable of supporting superoxide production in the amphiphile-dependent cellfree activation system in the same dose-dependent manner as the wild-type p47 phox (data not shown). Thus the S328A as well as S303A/S304A substitution of p47 phox resulted in a drastically decreased activation of the NADPH oxidase under the whole-cell conditions, indicating that phosphorylation of the three serines plays a crucial role.

DISCUSSION
The SH3-mediated Intramolecular Interaction as the Major Determinant for Active and Inactive Conformations of p47 phox -We have previously shown that activation of the phagocyte NADPH oxidase absolutely requires interaction between p47 phox and p22 phox , which is mediated via the SH3 domains of the former protein (5,24). This interaction is considered to be induced by exposure of the domains that are normally masked by an intramolecular interaction with the C-terminal region of p47 phox (5,24,26). However, properties of the intramolecular interaction have remained largely unknown as follows: which region acts as the SH3 target; which SH3 domain is involved; and, most importantly, how the interaction functions, and how it is regulated.
In this study, we locate the intramolecular SH3-binding site at the region of amino acids 286 -340. The fragment of amino acids 286 -314 is essential for a minimal interaction, whereas its C-terminal one (amino acids 315-340) is further required for a stable association (Fig. 3B). The SH3-binding site, somewhat to our surprise, lacks the canonical SH3 target motif PX⌽P; there exist only two proline residues at positions 299 and 300 in the region of amino acids 286 -340 (Fig. 1). The present results show that the PPRR stretch (amino acids 299 -302), a remnant of the type II SH3 ligand ⌽PX⌽PXR, likely conforms the binding core, since the interaction is completely abolished by the P299Q/P300Q substitution in both the minimal and full-length SH3 target (amino acids 286 -314 and 286 -340, respectively) (Fig. 3C). In addition, full-length mutant proteins of p47 phox , P299Q/P300Q and R301E/R302E, are capable of not only binding to the intermolecular SH3 target p22 phox (Fig. 5, A and C) but also activating the NADPH oxidase under cell-free conditions without the amphiphile activators (Fig. 8A). These observations establish that the SH3-mediated intramolecular interaction is the major determinant for keeping p47 phox in a closed inactive conformation.
The interaction of p47 phox with p22 phox appears to be a ratelimiting step in the oxidase activation, as indicated by the observation that the p22 phox binding activity of various mutant p47 phox is completely parallel with their ability to activate the oxidase (Fig. 8A). Thus a conformation capable of engaging p22 phox , resulting from disruption of the SH3-mediated intramolecular interaction, represents an active state of p47 phox .
Reason for the Two SH3 Domains to Be Tandemly Arrayed in p47 phox : For the Refined Regulation of the Conformational Change?-The intramolecular interaction that determines active and inactive conformation of p47 phox requires both SH3 domains. The N-terminal SH3 domain of p47 phox (amino acids 154 -219) appears to contact directly with the PPRR stretch (amino acids 299 -302), because the intramolecular interaction is abrogated by the substitution of Arg for Trp-193, the conserved residue among all SH3 domains that is expected to interact directly with a proline of the target (43)(44)(45)(46), not by the corresponding mutation in the C-terminal SH3 domain (SH3(C)) (Fig. 4B). In addition to the core stretch, its C-terminal flanking region of about 40 residues is required for a stable SH3-mediated intramolecular interaction in p47 phox . The allosteric effect of the region outside the binding core may suggest a role for SH3(C) as a binding partner for the extra core region, since SH3(C) also participates in the interaction, i.e. the two SH3 domains synergistically bind to the fragment of amino acids 286 -340 (Fig. 4A). A similar mechanism underlies regulation of the Src family of protein kinases, in which the tandem SH3 and SH2 domains synergistically keep the enzyme in a closed inactive conformation via intramolecular interactions; the SH3 domain interacts with the linker between the SH2 and catalytic domains, whereas the SH2 domain binds to the phosphotyrosine-containing C-terminal tail of the kinase in a resting state (49,50). Interestingly, the intramolecular SH3 target in Src lacks the PX⌽P motif as well (49). Such a synergism may occur in p47 phox , where the tandemly arrayed SH3 domains likely contribute to a refined regulation of the intramolecular interaction-dependent conformational change. This may explain why the tandem SH3 domains are present in p47 phox .
Triple Replacement of Ser-303, Ser-304, and Ser-328 in p47 phox with Aspartates, a Mutation That Is Sufficient for Disruption of the SH3-mediated Intramolecular Interaction, Induction of Binding to p22 phox , and Activation of the NADPH Oxidase-It is well established that stimulation of human neutrophils leads to extensive phosphorylation of p47 phox in parallel with superoxide production (29 -31). However, it has remained unknown about a molecular link between the phosphorylation event and activation of the phagocyte NADPH oxidase.
Here we demonstrate that simultaneous replacement of Ser-303, Ser-304, and Ser-328 in p47 phox with aspartates or glutamates, each mimicking phosphorylated residues (14, 44 -46), is sufficient for disruption of the SH3-mediated intramolecular interaction and resultant access of the unmasked SH3 domains to p22 phox . The three serine residues, all being present in the intramolecular SH3 target site (amino acids 286 -340), are known to become intensively phosphorylated when human FIG. 9. Effect of the S328A or S303A/S304A substitution in p47 phox on the NADPH oxidase activation in a whole-cell system. A, expression of p47 phox in gp91 phox and p67 phox -transduced K562 cells. The doubly transduced K562 cells were transfected with the pREP4 vector alone or pREP4 encoding the wild-type or mutant p47 phox carrying the substitution S303A/S304A or S328A. In the upper panel, cell lysates were resolved by SDS-PAGE, transferred to a polyvinylidene difluoride membrane, and immunoblotted with an anti-p47 phox monoclonal antibody. In the lower panel, cell lysates were immunoblotted with an anti-p67 phox monoclonal antibody. B, PMA-induced chemiluminescence by gp91 phox and p67 phox -transduced K562 cells transfected with pREP4 encoding the wild-type or mutant p47 phox carrying the substitution S303A/S304A or S328A. The K562 cells expressing the indicated form of p47 phox (1 ϫ 10 5 cells) were stimulated with PMA (200 ng/ml), and chemiluminescence change was continuously monitored with an enhanced luminol-based substrate, DIOGENES. Superoxide dismutase (SOD) (50 g/ml) was added where indicated. C, relative superoxide-producing activities in PMA-stimulated K562 cells transfected with the wild-type or mutant of p47 phox . Superoxide production is expressed as the percent activity relative to control cells transfected with the wild-type p47 phox . Each graph represents the mean of data from three independent transfections, with bars representing the standard deviation of percent activity. neutrophils are stimulated with PMA or fMLP (32). The triply mutated p47 phox is considered to be in an active conformation, since it activates the phagocyte NADPH oxidase under cell-free conditions in a manner independent of the anionic amphiphile activators (Fig. 8A). On the other hand, mutant p47 phox with either S328D or S303D/S304D substitution is inactive in the cell-free system. Thus simultaneous phosphorylation of Ser-303, Ser-304, and Ser-328 in p47 phox likely functions as a switch from a closed inactive conformation to a state capable of both binding to p22 phox and activating the oxidase. The requirement for phosphorylation of the three serines is supported by the observation that substitution of the kinase-insensitive residue alanine for Ser-328 as well as for both Ser-303 and Ser-304 results in defective production of superoxide in PMA-stimulated cells (Fig. 9). Thus phosphorylation of Ser-303, Ser-304, and Ser-328 appears to primarily disrupt the intermolecular interaction to activate p47 phox .
Role for Phosphorylation of Ser-328 as Well as Ser-303 and Ser-304 in p47 phox -A recent study has suggested the importance of phosphorylation at serines 303 and 304 in the oxidase activation; the double mutant p47 phox S303A/S304A is much less active than the wild-type one when expressed in EBVtransformed p47 phox -deficient B cells (33). It has remained, however, unknown what is induced in p47 phox carrying these phosphorylated residues. The present results show that phosphorylation of Ser-303 and Ser-304 is likely required for disruption of the SH3-mediated intramolecular interaction. In addition to both serines, Ser-328 also appears to be necessarily phosphorylated for activation of p47 phox , since the S328A substitution results in defective activation of the oxidase in vivo (Fig. 9). The strict requirement for the three serines to be phosphorylated is also suggested by the finding that a protein without substitution of aspartate for any of Ser-303, Ser-304, or Ser-328 is incapable of interacting with p22 phox , even when all other serines to be phosphorylated are replaced with aspartates (Fig. 6C).
Role for Phosphorylation of Other Serines in p47 phox -It has been also suggested that a phosphorylated serine at position 359 or 370 participates in the oxidase activation by facilitating phosphorylation of the remaining serines (51). The two serine residues lie outside the intramolecular SH3 binding region of amino acids 286 -340. In addition, a full-length p47 phox carrying the S359D/S370D/S379D substitution fails to interact with p22 phox (Table I), whereas the triple replacement of Ser-303, Ser-304, and Ser-328, all being present in the SH3 target site, with aspartates or glutamates, is sufficient for both disrupting the interaction and binding to p22 phox (Figs. 5 and 6 and Table  I). Serines 359 and 370 are located just N-and C-terminally to the PRR of p47 phox (amino acids 360 -369; KPQPAVPPRP), respectively. The PRR is constitutively occupied by the C-terminal SH3 domain of p67 phox , a binding that occurs in a manner independent of the SH3-mediated intramolecular interaction in p47 phox (26). Thus both Ser-359 and Ser-370 are likely sequestered from the intramolecularly interacting moiety of p47 phox , and phosphorylation of these residues does not appear to be directly involved in disruption of the intramolecular interaction.
A Model for Phosphorylation-dependent Activation of p47 phox -Based on the present findings, here we propose a model that phosphorylation of p47 phox induces a conformational change to a state accessible to p22 phox , thereby activating the NADPH oxidase (Fig. 10). In a resting state, p47 phox is folded in a closed inactive conformation by an intramolecular interaction that is synergistically mediated via the tandem SH3 domains. Phosphorylation of Ser-303, Ser-304, and Ser-328 primarily disrupts the SH3-mediated intramolecular inter-action. The disruption renders p47 phox in an open conformation capable of interacting with p22 phox via the unmasked SH3 domains. The induced interaction between the oxidase factors serves as a rate-limiting step to activate the NADPH oxidase. Since at least one tryptophan residue, Trp-193, plays a critical role in keeping p47 phox in the SH3-mediated closed conformation, this model likely explains an observation by Quinn's group (28) that phosphorylation of p47 phox by protein kinase C in vitro results in quenching in the intrinsic tryptophan fluorescence, which correlates well with NADPH oxidase activity.