Activation of the phagocyte NADPH oxidase protein p47(phox). Phosphorylation controls SH3 domain-dependent binding to p22(phox).

Activation of phagocyte NADPH oxidase requires interaction between p47(phox) and p22(phox). p47(phox) in resting phagocytes does not bind p22(phox). Phosphorylation of serines in the p47(phox) C terminus enables binding to the p22(phox) C terminus by inducing a conformational change in p47(phox) that unmasks the SH3A domain. We report that an arginine/lysine-rich region in the p47(phox) C terminus binds the p47(phox) SH3 domains expressed in tandem (SH3AB) but does not bind the individual N-terminal SH3A and C-terminal SH3B domains. Peptides matching amino acids 301-320 and 314-335 of the p47(phox) arginine/lysine-rich region block the p47(phox) SH3AB/p22(phox) C-terminal and p47(phox) SH3AB/p47(phox) C-terminal binding and inhibit NADPH oxidase activity in vitro. Peptides with phosphoserines substituted for serines 310 and 328 do not block binding and are poor inhibitors of oxidase activity. Mutated full-length p47(phox) with aspartic acid substitutions to mimic the effects of phosphorylations at serines 310 and 328 bind the p22(phox) proline-rich region in contrast to wild-type p47(phox). We conclude that the p47(phox) SH3A domain-binding site is blocked by an interaction between the p47(phox) SH3AB domains and the C-terminal arginine/lysine-rich region. Phosphorylation of serines in the p47(phox) C terminus disrupts this interaction leading to exposure of the SH3A domain, binding to p22(phox), and activation of the NADPH oxidase.     

A region C-terminal to the proline-rich core of p47phox regulates activation of the phagocyte NADPH oxidase by interacting with the C-terminal SH3 domain of p67phox

Activation of the phagocyte NADPH oxidase requires the regulatory proteins p47(phox) and p67(phox), each harboring two SH3 domains. p67(phox) interacts with p47(phox) via simultaneous binding of the p67(phox) C-terminal SH3 domain to both the proline-rich region (PRR) of amino acid residues 360-369 and its C-terminally flanking region of p47(phox); the role of the interaction in oxidase regulation has not been fully understood. Here we show that the p47(phox)-p67(phox) interaction is disrupted not only by deletion of the PRR but also by substitution for basic residues in the extra-PRR (K383E/K385E). The substitution impaired oxidase activation partially in vitro and much more profoundly in vivo, indicating the significance of the p47(phox) extra-PRR. Replacement of Ser-379 in the extra-PRR, a residue known to undergo phosphorylation in stimulated cells, by aspartate attenuates the interaction and thus results in a defective superoxide production, suggesting that phosphorylation of Ser-379 is involved in oxidase regulation.     

Arachidonic acid and phosphorylation synergistically induce a conformational change of p47phox to activate the phagocyte NADPH oxidase

The superoxide-producing phagocyte NADPH oxidase can be activated by arachidonic acid (AA) or by phosphorylation of p47(phox) under cell-free conditions. The molecular mechanism underlying the activation, however, has remained largely unknown. Here we demonstrate that AA, at high concentrations (50-100 micrometer), induces direct interaction between the oxidase factors p47(phox) and p22(phox) in parallel with superoxide production. The interaction, being required for the oxidase activation, is mediated via the Src homology 3 (SH3) domains of p47(phox) (p47-(SH3)(2)), which are intramolecularly masked in a resting state. We also show that AA disrupts complexation of p47-(SH3)(2) with its intramolecular target fragment (amino acids 286-340) without affecting association of p47-(SH3)(2) with p22(phox), indicating that the disruption plays a crucial role in the induced interaction with p22(phox). Phosphorylation of p47(phox) by protein kinase C partially replaces the effects of AA; treatment of the SH3 target fragment with PKC in vitro results in a completely impaired interaction with p47-(SH3)(2), and the same treatment of the full-length p47(phox) leads to both interaction with p22(phox) and oxidase activation without AA, but to a lesser extent. Furthermore, phosphorylated p47(phox) effectively binds to p22(phox) and activates the oxidase in the presence of AA at low concentrations (1-5 micrometer), where an unphosphorylated protein only slightly supports superoxide production. Thus AA, at high concentrations, fully induces the interaction of p47(phox) with p22(phox) by itself, whereas, at low concentrations, AA synergizes with phosphorylation of p47(phox) to facilitate the interaction, thereby activating the NADPH oxidase.     

Mechanism for phosphorylation-induced activation of the phagocyte NADPH oxidase protein p47(phox). Triple replacement of serines 303, 304, and 328 with aspartates disrupts the SH3 domain-mediated intramolecular interaction in p47(phox), thereby activating the oxidase

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.     

NMR solution structure of the tandem Src homology 3 domains of p47phox complexed with a p22phox-derived proline-rich peptide

The phagocyte NADPH oxidase plays a crucial role in host defense against microbial infections by generating reactive oxygen species. It is a multisubunit enzyme composed of membrane-bound flavocytochrome b558 as well as cytosolic components, including p47phox, which is essential for assembly of the complex. When phagocytes are activated, the cytosolic components of the NADPH oxidase translocate to flavocytochrome b558 due to binding of the tandem Src homology 3 (SH3) domains of p47phox to a proline-rich region in p22phox, a subunit of flavocytochrome b558. Using NMR titration, we first identified the proline-rich region of p22phox that is essential for binding to the tandem SH3 domains of p47phox. We subsequently determined the solution structure of the p47phox tandem SH3 domains complexed with the proline-rich peptide of p22phox using NMR spectroscopy. In contrast to the intertwined dimer reported for the crystal state, the solution structure is a monomer. The central region of the p22phox peptide forms a polyproline type II helix that is sandwiched by the N- and C-terminal SH3 domains, as was observed in the crystal structure, whereas the C-terminal region of the peptide takes on a short alpha-helical conformation that provides an additional binding site with the N-terminal SH3 domain. Thus, the C-terminal alpha-helical region of the p22phox peptide increases the binding affinity for the tandem SH3 domains of p47phox more than 10-fold.     

Characterization of surface structure and p47phox SH3 domain-mediated conformational changes for human neutrophil flavocytochrome b

The heterodimeric, integral membrane protein flavocytochrome b (Cyt b) is the catalytic core of the phagocyte NADPH oxidase and generates superoxide which plays a critical role in host defense. To better define the activation of superoxide production by this multisubunit enzyme complex, Cyt b-specific monoclonal antibodies (mAbs) and the p47phox SH3 domains (p47SH3AB) were used in the present study as probes to map surface structure and conformational dynamics in human neutrophil Cyt b. In pull-down and co-immunoprecipitation studies with detergent-solubilized Cyt b, the oxidase-inhibitory mAb CS9 was shown to share an overlapping binding site with p47SH3AB on the C-terminal region of the p22phox subunit. Similar studies demonstrated a surprising lack of overlap between the mAb 44.1 and CS9/p47SH3AB binding sites, and they indicated that the oxidase-inhibitory mAb NL7 binds a region physically separated from the p22phox C-terminal domain. Resonance energy transfer and size exclusion chromatography confirmed the above results for functionally reconstituted Cyt b and provided evidence that binding of both mAb CS9 and p47SH3AB altered the conformation of Cyt b. Further support that binding of the p47phox SH3 domains modulates the structure of Cyt b was obtained using a cell-free assay system where p47SH3AB enhanced superoxide production in the presence of a p67phox (1-212)-Rac1(Q61L) fusion protein. Taken together, this study further characterizes the structure of human neutrophil Cyt b in both detergent micelles and reconstituted membrane bilayers, and it provides evidence that the cytosolic regulatory subunit p47phox modulates the conformation of Cyt b (in addition to serving as an adapter protein) during oxidase activation.     

The adaptor protein p40(phox) as a positive regulator of the superoxide-producing phagocyte oxidase

Activation of the superoxide-producing phagocyte NADPH oxidase, crucial in host defense, requires the cytosolic proteins p67(phox) and p47(phox). They translocate to the membrane upon cell stimulation and activate flavocytochrome b(558), the membrane-integrated catalytic core of this enzyme system. The activators p67(phox) and p47(phox) form a ternary complex together with p40(phox), an adaptor protein with unknown function, comprising the PX/PB2, SH3 and PC motif- containing domains: p40(phox) associates with p67(phox) via binding of the p40(phox) PC motif to the p67(phox) PB1 domain, while p47(phox) directly interacts with p67(phox) but not with p40(phox). Here we show that p40(phox) enhances membrane translocation of p67(phox) and p47(phox) in stimulated cells, which leads to facilitated production of superoxide. The enhancement cannot be elicited by a mutant p40(phox) carrying the D289A substitution in PC or a p67(phox) with the K355A substitution in PB1, each being defective in binding to its respective partner. Thus p40(phox) participates in activation of the phagocyte oxidase by regulating membrane recruitment of p67(phox) and p47(phox) via the PB1-PC interaction with p67(phox).     

p40phox as an alternative organizer to p47phox in Nox2 activation: a new mechanism involving an interaction with p22phox

p40(phox) activated phagocyte NADPH oxidase without p47(phox) in a cell-free system consisting of p67(phox), Rac and cytochrome b(558) relipidated with phosphatidylinositol 3-phosphate. The activation reached to 70% of that by p47(phox). Addition of p47(phox) slightly increased the activation, but not additively. p40(phox) improved the efficiency of p67(phox) in the activation. The C-terminus-truncated p67(phox), p40(phox)(D289A), p40(phox)(R58A), or p40(phox)(W207R) showed an impaired activation. A peptide corresponding to the p22(phox) Pro-rich region suppressed the activation, and far-western blotting revealed its interaction with p40(phox) SH3 domain. Thus, p40(phox) can substitute for p47(phox) in the activation, interacting with p22(phox) and p67(phox) through their specific regions.     

Novel human homologues of p47phox and p67phox participate in activation of superoxide-producing NADPH oxidases

The catalytic core of a superoxide-producing NADPH oxidase (Nox) in phagocytes is gp91phox/Nox2, a membrane-integrated protein that forms a heterodimer with p22phox to constitute flavocytochrome b558. The cytochrome becomes activated by interacting with the adaptor proteins p47phox and p67phox as well as the small GTPase Rac. Here we describe the cloning of human cDNAs for novel proteins homologous to p47phox and p67phox, designated p41nox and p51nox, respectively; the former is encoded by NOXO1 (Nox organizer 1), and the latter is encoded by NOXA1 (Nox activator 1). The novel homologue p41nox interacts with p22phox via the two tandem SH3 domains, as does p47phox. The protein p51nox as well as p67phox can form a complex with p47phox and with p41nox via the C-terminal SH3 domain and binds to GTP-bound Rac via the N-terminal domain containing four tetratricopeptide repeat motifs. These bindings seem to play important roles, since p47phox and p67phox activate the phagocyte oxidase via the same interactions. Indeed, p41nox and p51nox are capable of replacing the corresponding classical homologue in activation of gp91phox. Nox1, a homologue of gp91phox, also can be activated in cells, when it is coexpressed with p41nox and p51nox, with p41nox and p67phox, or with p47phox and p51nox; in the former two cases, Nox1 is partially activated without any stimulants added, suggesting that p41nox is normally in an active state. Thus, the novel homologues p41nox and p51nox likely function together or in combination with a classical one, thereby activating the two Nox family oxidases.     

Activation state-dependent interaction between Galphai and p67phox

The phagocyte NADPH oxidase consists of multiple protein subunits that interact with each other to form a functional superoxide-generating complex. Although the essential components for superoxide production have been well characterized, other proteins potentially involved in the regulation of NADPH oxidase activation remain to be identified. We report here that the Galphai subunit of heterotrimeric G proteins is a novel binding partner for p67phox in transfected HEK293T cells and peripheral blood polymorphonuclear leukocytes. p67phox preferably interacted with inactive Galphai. Expression of p67phox caused a dose-dependent decrease in intracellular cyclic AMP concentration, suggesting altered function of Galphai. We identified a fragment of p67phox, consisting of the PB1 domain and the C-terminal SH3 domain, to be critical for the interaction with Galphai. Because these domains are involved in the interaction with p47phox and p40phox, the relationship between the respective binding events was investigated. Wild-type Galphai, but not its QL mutant, could promote the interaction between p67phox and p47phox. However, the interaction between p67phox and p40phox was not affected by either Galphai form. These results provide the first evidence for an interaction between p67phox and an alpha subunit of heterotrimeric G proteins, suggesting a potential role for Galphai in the regulation or activation of NADPH oxidase.     

Specificity of p47phox SH3 domain interactions in NADPH oxidase assembly and activation.

The delineation of molecular structures that dictate Src homology 3 (SH3) domain recognition of specific proline-rich ligands is key to understanding unique functions of diverse SH3 domain-containing signalling molecules. We recently established that assembly of the phagocyte NADPH oxidase involves multiple SH3 domain interactions between several oxidase components (p47phox, p67phox, and p22phox). p47phox was shown to play a central role in oxidase activation in whole cells by mediating interactions with both the transmembrane component p22phox and cytosolic p67phox. To understand the specific roles of each SH3 domain of p47phox in oxidase assembly and activation, we mutated critical consensus residues (Tyr167 or Tyr237-->Leu [Y167L or Y237L], W193R or W263R, and P206L or P276L) on each of their binding surfaces. The differential effects of these mutations indicated that the first SH3 domain is responsible for the p47phox-p22phox interaction and plays a predominant role in oxidase activity and p47phox membrane assembly, while the second p47phox SH3 domain interacts with the NH2-terminal domain of p67phox. Binding experiments using the isolated first SH3 domain also demonstrated its involvement in intramolecular interactions within p47phox and showed a requirement for five residues (residues 151 to 155) on its N-terminal boundary for binding to p22phox. The differential effects of nonconserved-site mutations (W204A or Y274A and E174Q or E244Q) on whole-cell oxidase activity suggested that unique contact residues within the third binding pocket of each SH3 domain influence their ligand-binding specificities.     

Effects of p47phox C terminus phosphorylations on binding interactions with p40phox and p67phox. Structural and functional comparison of p40phox and p67phox SH3 domains

The neutrophil NADPH oxidase produces superoxide anions in response to infection. This reaction is activated by association of cytosolic factors, p47phox and p67phox, and a small G protein Rac with the membranous flavocytochrome b558. Another cytosolic factor, p40phox, is associated to the complex and is reported to play regulatory roles. Initiation of the NADPH oxidase activation cascade has been reported as consecutive to phosphorylation on serines 359/370 and 379 of the p47phox C terminus. These serines surround a polyproline motif that can interact with the Src homology 3 (SH3) module of p40phox (SH3p40) or the C-terminal SH3 of p67phox (C-SH3p67). The latter one presents a higher affinity in the resting state for p47phox. A change in SH3 binding preference following phosphorylation has been postulated earlier. Here we report the crystal structures of SH3p40 alone or in complex with a 12-residue proline-rich region of p47phox at 1.46 angstrom resolution. Using intrinsic tryptophan fluorescence measurements, we compared the affinity of the strict polyproline motif and the whole C terminus peptide with both SH3p40 and C-SH3p67. These data reveal that SH3p40 can interact with a consensus polyproline motif but also with a noncanonical motif of the p47phox C terminus. The electrostatic surfaces of both SH3 are very different, and therefore the binding preference for C-SH3p67 can be attributed to the polyproline motif recognition and particularly to the Arg-368p47 binding mode. The noncanonical motif contributes equally to interaction with both SH3. The influence of serine phosphorylation on residues 359/370 and 379 on the affinity for both SH3 domains has been checked. We conclude that contrarily to previous suggestions, phosphorylation of Ser-359/370 does not modify the SH3 binding affinity for both SH3, whereas phosphorylation of Ser-379 has a destabilizing effect on both interactions. Other mechanisms than a phosphorylation induced switch between the two SH3 must therefore take place for NADPH oxidase activation cascade to start.     

Tripartite chimeras comprising functional domains derived from the cytosolic NADPH oxidase components p47phox, p67phox, and Rac1 elicit activator-independent superoxide production by phagocyte membranes: an essential role for anionic membrane phospholipids

The superoxide-generating NADPH oxidase is converted to an active state by the assembly of a membrane-localized cytochrome b(559) with three cytosolic components: p47(phox), p67(phox), and GTPase Rac1 or Rac2. Assembly involves two sets of protein-protein interactions: among cytosolic components and among cytosolic components and cytochrome b(559) within its lipid habitat. We circumvented the need for interactions among cytosolic components by constructing a recombinant tripartite chimera (trimera) consisting of the Phox homology (PX) and Src homology 3 (SH3) domains of p47(phox), the tetratricopeptide repeat and activation domains of p67(phox), and full-length Rac1. Upon addition to phagocyte membrane, the trimera was capable of oxidase activation in vitro in the presence of an anionic amphiphile. The trimera had a higher affinity (lower EC(50)) for and formed a more stable complex (longer half-life) with cytochrome b(559) compared with the combined individual components, full-length or truncated. Supplementation of membrane with anionic but not neutral phospholipids made activation by the trimera amphiphile-independent. Mutagenesis, truncations, and domain replacements revealed that oxidase activation by the trimera was dependent on the following interactions: 1) interaction with anionic membrane phospholipids via the poly-basic stretch at the C terminus of the Rac1 segment; 2) interaction with p22(phox) via Trp(193) in the N-terminal SH3 domain of the p47(phox) segment, supplementing the electrostatic attraction; and 3) an intrachimeric bond among the p67(phox) and Rac1 segments complementary to their physical fusion. The PX domain of the p47(phox) segment and the insert domain of the Rac1 segment made only minor contributions to oxidase assembly.     

Interaction between the SH3 domains and C-terminal proline-rich region in NADPH oxidase organizer 1 (Noxo1)

NADPH oxidase organizer 1 (Noxo1), harboring a PX domain, two SH3 domains, and a proline-rich region (PRR), participates in activation of superoxide-producing Nox-family NADPH oxidases. Here, we show that Noxo1 supports superoxide production in a cell-free system for gp91(phox)/Nox2 activation by arachidonic acid. This lipid enhances an SH3-mediated binding of Noxo1 to p22(phox), a protein complexed with Nox oxidases; the binding is known to be required for Nox activation. We also demonstrate that the bis-SH3 domain directly interacts with the Noxo1 PRR. The interaction appears to prevent the bis-SH3 domain and PRR from binding to their target proteins; disruption of the intramolecular interaction facilitates Noxo1 binding to p22(phox) and also allows the PRR to associate with the Nox activator Noxa1, which association is crucial for Nox activation as well. These findings suggest that Nox activation involves a conformational change leading to disruption of the bis-SH3-PRR interaction in Noxo1.     

Solution structure of the tandem Src homology 3 domains of p47phox in an autoinhibited form

The phagocyte NADPH oxidase is a multisubunit enzyme responsible for the generation of superoxide anions (O(2).) that kill invading microorganisms. p47(phox) is a cytosolic subunit of the phagocyte NADPH oxidase, which plays a crucial role in the assembly of the activated NADPH oxidase complex. The molecular shapes of the p47(phox) tandem SH3 domains either with or without a polybasic/autoinhibitory region (PBR/AIR) at the C terminus were studied using small angle x-ray scattering. The tandem SH3 domains with PBR/AIR formed a compact globular structure, whereas the tandem SH3 domains lacking the PBR/AIR formed an elongated structure. Alignment anisotropy analysis by NMR based on the residual dipolar couplings revealed that the tandem SH3 domains with PBR/AIR were in good agreement with a globular module corresponding to the split half of the intertwisted dimer in crystalline state. The structure of the globular module was elucidated to represent a solution structure of the tandem SH3 domain in the autoinhibited form, where the PBR/AIR bundled the tandem SH3 domains and the linker forming a closed structure. Once PBR/AIR is released by phosphorylation, rearrangements of the SH3 domains may occur, forming an open structure that binds to the cytoplasmic proline-rich region of membrane-bound p22(phox).     

Point mutations in the proline-rich region of p22phox are dominant inhibitors of Nox1- and Nox2-dependent reactive oxygen generation

The integral membrane protein p22phox is an indispensable component of the superoxide-generating phagocyte NADPH oxidase, whose catalytic core is the membrane-associated gp91phox (also known as Nox2). p22phox associates with gp91phox and, through its proline-rich C terminus, provides a binding site for the tandem Src homology 3 domains of the activating subunit p47phox. Whereas p22phox is expressed ubiquitously, its participation in regulating the activity of other Nox enzymes is less clear. This study investigates the requirement of p22phox for Nox enzyme activity and explores the role of its proline-rich region (PRR) for regulating activity. Coexpression of specific Nox catalytic subunits (Nox1, Nox2, Nox3, Nox4, or Nox5) along with their corresponding regulatory subunits (NOXO1/NOXA1 for Nox1; p47phox/p67phox/Rac for Nox2; NOXO1 for Nox3; no subunits for Nox4 or Nox5) resulted in marked production of reactive oxygen. Small interfering RNAs decreased endogenous p22phox expression and inhibited reactive oxygen generation from Nox1, Nox2, Nox3, and Nox4 but not Nox5. Truncated forms of p22phox that disrupted the PRR-inhibited reactive oxygen generation from Nox1, Nox2, and Nox3 but not from Nox4 and Nox5. Similarly, p22phox (P156Q), a mutation that disrupts Src homology 3 binding by the PRR, potently inhibited reactive oxygen production from Nox1 and Nox2 but not from Nox4 and Nox5. Expression of p22phox (P156Q) inhibited NOXO1-stimulated Nox3 activity, but co-expression of NOXA1 overcame the inhibitory effect. The P157Q and P160Q mutations of p22phox showed selective inhibition of Nox2/p47phox/p67phox, and selectivity was specific for the organizing subunit (p47phox or NOXO1) rather than the Nox catalytic subunit. These studies stress the importance of p22phox for the function of Nox1, Nox2, Nox3, and Nox4, and emphasize the key role of the PRR for regulating Nox proteins whose activity is dependent upon p47phox or NOXO1.     

Small angle neutron scattering and gel filtration analyses of neutrophil NADPH oxidase cytosolic factors highlight the role of the C-terminal end of p47phox in the association with p40phox

The NADPH oxidase of phagocytic cells is regulated by the cytosolic factors p47(phox), p67(phox), and p40(phox) as well as by the Rac1-Rho-GDI heterodimer. The regulation is a consequence of protein-protein interactions involving a variety of protein domains that are well characterized in signal transduction. We have studied the behavior of the NADPH oxidase cytosolic factors in solution using small angle neutron scattering and gel filtration. p47(phox), two truncated forms of p47(phox), namely, p47(phox) without its C-terminal end (residues 1-358) and p47(phox) without its N-terminal end (residues 147-390), and p40(phox) were found to be monomeric in solution. The dimeric form of p67(phox) previously observed by gel filtration experiments was confirmed. Our small angle neutron scattering experiments show that p40(phox) binds to the full-length p47(phox) in solution in the absence of phosphorylation. We demonstrated that the C-terminal end of p47(phox) is essential in this interaction. From the comparison of the presence or absence of interaction with various truncated forms of the proteins, we confirmed that the SH3 domain of p40(phox) interacts with the C-terminal proline rich region of p47(phox). The radii of gyration observed for p47(phox) and the truncated forms of p47(phox) (without the C-terminal end or without the N-terminal end) show that all these molecules are elongated and that the N-terminal end of p47(phox) is globular. These results suggest that the role of amphiphiles such as SDS or arachidonic acid or of p47(phox) phosphorylation in the elicitation of NADPH oxidase activation could be to disrupt the p40(phox)-p47(phox) complex rather than to break an intramolecular interaction in p47(phox).     

Cloning of rat p47phox and comparison with human p47phox.

A cDNA encoding rat p47phox was cloned from rat spleen cDNA library, utilizing rapid amplification of cDNA ends. The open reading frame corresponded to 389 amino acids: It contained the phagocyte oxidase homology domain, two Src homology 3 domains and a proline rich region, all of which are conserved in mammalian p47phox sequences. Rat p47phox displayed the highest degree of identity to mouse p47phox (94%). We expressed and purified rat p47phox as a glutathione S-transferase fusion protein, and found that the rat protein could replace human p47phox in a cell-free activation system for human NADPH oxidase, giving about half activity. Although rat 12-lipoxygenase interacted with human p47phox in a yeast two-hybrid system, this was not the case for rat p47phox.     

C-terminal region of the cytosolic subunit p47(phox) is a primary target of conformational change during the activation of leukocyte NADPH oxidase

The leukocyte NADPH oxidase of neutrophils is a membrane-bound enzyme that catalyzes the production of O(2(-)) from oxygen using NADPH as the electron donor. During activation, the cytosolic oxidase components p47(phox) and p67(phox), each containing two Src homology 3 (SH3) domains, migrate to the plasma membrane, where they associate with cytochrome b(558), a membrane-integrated flavohemoprotein, to assemble the active oxidase. Oxidase activation can be mimicked in a cell-free system using an anionic amphiphile, such as sodium dodecyl sulfate or arachidonic acid and the phosphorylation of p47(phox )with protein kinase C. Activators of the oxidase in vitro cause exposure of p47(phox)-SH3, which has probably been masked by the C-terminal region of this protein in a resting state. We show here that the total protein steady-state intrinsic fluorescence exhibited by the tryptophan residues of p47(phox) substantially decreased when N-terminal truncated p47(phox)-SH3-C was treated with anionic amphiphiles or phosphorylated with protein kinase C. This finding was similar to the results obtained with full-length p47(phox). However, the fluorescence of C-terminal truncated p47(phox)-N-SH3 and both C-terminal and N-terminal truncated p47(phox)-SH3 were not altered by the activators. These results indicate that the C-terminal region of p47(phox) is a primary target of the conformational change during the activation of NADPH oxidase.