Multiple SH3 domain interactions regulate NADPH oxidase assembly in whole cells.

Src homology 3 (SH3) domains mediate specific protein-protein interactions crucial for signal transduction and protein subcellular localization. Upon phagocyte stimulation, two SH3 domain-containing cytosolic components of the NADPH oxidase, p47phox and p67phox, are recruited to the membrane where they interact with flavocytochrome b558 to form an activated microbicidal oxidase. Deletion analysis of p47phox and p67phox in transfected K562 cells demonstrated multiple SH3-mediated interactions between p47phox and the transmembrane flavocytochrome b558 and also between the cytosolic components themselves. The core region of p47phox (residues 151-284), spanning both SH3 domains, was required for flavocytochrome-dependent translocation and oxidase activity in whole cells. Furthermore, translocation of p67phox occurred through interactions of its N-terminal domain (residues 1-246) with p47phox SH3 domains. Both of these interactions were promoted by PMA activation of cells and were influenced by the presence of other domains in both cytosolic factors. Deletion analysis also revealed a third SH3 domain-mediated interaction involving the C-termini of both cytosolic factors, which also promoted p67phox membrane translocation. These data provide evidence for a central role for p47phox in regulation of oxidase assembly through several SH3 domain interactions.     

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.     

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.     

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.     

Mapping of functional domains in the p22(phox) subunit of flavocytochrome b(559) participating in the assembly of the NADPH oxidase complex by "peptide walking".

The superoxide-generating NADPH oxidase complex of phagocytes consists of a membranal heterodimeric flavocytochrome (cytochrome b(559)), composed of gp91(phox) and p22(phox) subunits, and four cytosolic proteins, p47(phox), p67(phox), p40(phox), and the small GTPase Rac (1 or 2). All redox stations involved in electron transport from NADPH to oxygen are located in gp91(phox). NADPH oxidase activation is the consequence of assembly of cytochrome b(559) with cytosolic proteins, a process reproducible in a cell-free system, consisting of phagocyte membranes, and recombinant cytosolic components, activated by an anionic amphiphile. p22(phox) is believed to act as a linker between the cytosolic components and gp91(phox). We applied "peptide walking" to mapping of domains in p22(phox) participating in NADPH oxidase assembly. Ninety one synthetic overlapping pentadecapeptides, spanning the p22(phox) sequence, were tested for the ability to inhibit NADPH oxidase activation in the cell-free system and to bind individual cytosolic NADPH oxidase components. We conclude the following. 1) The p22(phox) subunit of cytochrome b(559) serves as an anchor for both p47(phox) and p67(phox). 2) p47(phox) binds not only to the proline-rich region, located at residues 151-160 in the cytosolic C terminus of p22(phox), but also to a domain (residues 51-63) located on a loop exposed to the cytosol. 3) p67(phox) shares with p47(phox) the ability to bind to the proline-rich region (residues 151-160) and also binds to two additional domains, in the cytosolic loop (residues 81-91) and at the start of the cytosolic tail (residues 111-115). 4) The binding affinity of p67(phox) for p22(phox) peptides is lower than that of p47(phox). 5) Binding of both p47(phox) and p67(phox) to proline-rich p22(phox) peptides occurs in the absence of an anionic amphiphile. A revised membrane topology model of p22(phox) is proposed, the core of which is the presence of a functionally important cytosolic loop (residues 51-91).     

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.     

Activation of the superoxide-producing phagocyte NADPH oxidase requires co-operation between the tandem SH3 domains of p47phox in recognition of a polyproline type II helix and an adjacent alpha-helix of p22phox

Activation of the superoxide-producing phagocyte NADPH oxidase, crucial for host defence, requires an SH3 (Src homology 3)-domain-mediated interaction of the regulatory protein p47phox with p22phox, a subunit of the oxidase catalytic core flavocytochrome b558. Although previous analysis of a crystal structure has demonstrated that the tandem SH3 domains of p47phox sandwich a short PRR (proline-rich region) of p22phox (amino acids 151-160), containing a polyproline II helix, it has remained unknown whether this model is indeed functional in activation of the oxidase. In the present paper we show that the co-operativity between the two SH3 domains of p47phox, as expected from the model, is required for oxidase activation. Deletion of the linker between the p47phox SH3 domains results not only in a defective binding to p22phox but also in a loss of the activity to support superoxide production. The present analysis using alanine-scanning mutagenesis identifies Pro152, Pro156 and Arg158 in the p22phox PRR as residues indispensable for the interaction with p47phox. Pro152 and Pro156 are recognized by the N-terminal SH3 domain, whereas Arg158 contacts with the C-terminal SH3 domain. Amino acid substitution for any of the three residues in the p22phox PRR abrogates the superoxide-producing activity of the oxidase reconstituted in intact cells. The bis-SH3-mediated interaction of p47phox with p22phox thus functions to activate the phagocyte oxidase. Furthermore, we provide evidence that a region C-terminal to the PRR of p22phox (amino acids 161-164), adopting an a-helical conformation, participates in full activation of the phagocyte oxidase by fortifying the association with the p47phox SH3 domains.     

Role of the small GTPase Rac in p22phox-dependent NADPH oxidases

The superoxide-producing phagocyte NADPH oxidase gp91(phox)/Nox2 and the non-phagocytic oxidases Nox1 and Nox3 each form a complex in the membrane with p22(phox), which provides both stabilization and a docking site for organizer proteins. The p22(phox)-complexed Nox2 and Nox1 are dormant on their own, and their activation requires soluble supportive proteins such as a Nox organizer (p47(phox) or Noxo1) and a Nox activator (p67(phox) or Noxa1). The small GTPase Rac directly binds to the activators, and thus plays an essential role in the Nox2-based oxidase containing p47(phox) and p67(phox) or a positive role in Nox1 activity supported by Noxo1 and Noxa1. Although Nox3 complexed with p22(phox) constitutively produce superoxide, the production can be enhanced by supportive proteins. Here we compare the roles of Rac in these p22(phox)-dependent oxidases using the organizer and activator in different combinations. Expression of constitutively active Rac1(Q61L) is essential for activation of the Nox2- or Nox1-based oxidase containing the organizer p47(phox) and either p67(phox) or Noxa1. When these oxidases use Noxo1 as an organizer instead of p47(phox), they produce a small but significant amount of superoxide without expression of Rac1(Q61L), although the production is enhanced by Rac1(Q61L). Thus p47(phox) is likely related to strict dependence on Rac. The Nox3-based oxidase has a similar tendency in the change of the dependence: Rac plays a positive role in Nox3 activation in the presence of p47(phox) and either p67(phox) or Noxa1, whereas Rac fails to upregulate Nox3 activity when p47(phox) is replaced with Noxo1. We also demonstrate that, in the Nox3-based oxidase containing solely p67(phox) as supportive protein, expression of Rac1(Q61L) enhances not only superoxide production but also membrane translocation of p67(phox). Since the enhancements are not observed with a mutant p67(phox) defective in binding to Rac, this GTPase appear to directly recruit p67(phox) to the membrane.     

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.     

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.     

Rac activation induces NADPH oxidase activity in transgenic COSphox cells, and the level of superoxide production is exchange factor-dependent

Transient expression of constitutively active Rac1 derivatives, (G12V) or (Q61L), was sufficient to induce phagocyte NADPH oxidase activity in a COS-7 cell model in which human cDNAs for essential oxidase components, gp91(phox), p22(phox), p47(phox), and p67(phox), were expressed as stable transgenes. Expression of constitutively active Rac1 in "COS(phox)" cells induced translocation of p47(phox) and p67(phox) to the membrane. Furthermore, translocation of p47(phox) was induced in the absence of p67(phox) expression, even though Rac does not directly bind p47(phox). Rac effector domain point substitutions (A27K, G30S, D38A, Y40C), which can selectively eliminate interaction with different effector proteins, impaired Rac1V12-induced superoxide production. Activation of endogenous Rac1 by expression of constitutively active Rac-guanine nucleotide exchange factor (GEF) derivatives was sufficient to induce high level NADPH oxidase activity in COS(phox) cells. The constitutively active form of the hematopoietic-specific GEF, Vav1, was the most effective at activating superoxide production, despite detection of higher levels of Rac1-GTP upon expression of constitutively active Vav2 or Tiam1 derivatives. These data suggest that Rac can play a dual role in NADPH oxidase activation, both by directly participating in the oxidase complex and by activating signaling events leading to oxidase assembly, and that Vav1 may be the physiologically relevant GEF responsible for activating this Rac-regulated complex.     

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.     

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.     

Small-angle X-ray scattering reveals an extended organization for the autoinhibitory resting state of the p47(phox) modular protein

In response to microbial infection, neutrophiles promote the assembly of the NADPH oxidase complex in order to produce superoxide anions. This reaction is activated by the association of cytosolic factors, p47(phox), p67(phox), p40(phox), and a small G protein Rac with the membranous heterodimeric flavocytochrome b(558), composed of gp91(phox) and p22(phox). In the activation process, p47(phox) plays a central role as the target of phosphorylations and as a scaffolding protein conducting the translocation and assembly of cytosolic factors onto the membranous components. The PX and tandem SH3s of p47(phox) have been highlighted as being key determinants for the interaction with membrane lipids and the p22(phox) component, respectively. In the resting state, the two corresponding interfaces are thought to be masked allowing its cytoplasmic localization. However, the resting state modular organization of p47(phox) and its autoinhibition mode are still not fully understood despite available structural information on separate modules. More precisely, it raises the question of the mutual arrangement of the PX domain and the tandem SH3 domains in the resting state. To address this question, we have engaged a study of the entire p47(phox) molecule in solution using small-angle X-ray scattering. Despite internal autoinhibitory interactions, p47(phox) adopts an extended conformation. First insights about the domain arrangement in whole p47(phox) can be derived. Our data allow to discard the usual representation of a globular and compact autoinhibited resting state.     

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.     

Phosphatidylinositol 3-phosphate-dependent and -independent functions of p40phox in activation of the neutrophil NADPH oxidase

In response to bacterial infection, the neutrophil NADPH oxidase assembles on phagolysosomes to catalyze the transfer of electrons from NADPH to oxygen, forming superoxide and downstream reactive oxygen species (ROS). The active oxidase is composed of a membrane-bound cytochrome together with three cytosolic phox proteins, p40(phox), p47(phox), and p67(phox), and the small GTPase Rac2, and is regulated through a process involving protein kinase C, MAPK, and phosphatidylinositol 3-kinase. The role of p40(phox) remains less well defined than those of p47(phox) and p67(phox). We investigated the biological role of p40(phox) in differentiated PLB-985 neutrophils, and we show that depletion of endogenous p40(phox) using lentiviral short hairpin RNA reduces ROS production and impairs bacterial killing under conditions where p67(phox) levels remain constant. Biochemical studies using a cytosol-reconstituted permeabilized human neutrophil cores system that recapitulates intracellular oxidase activation revealed that depletion of p40(phox) reduces both the maximal rate and total amount of ROS produced without altering the K(M) value of the oxidase for NADPH. Using a series of mutants, p47PX-p40(phox) chimeras, and deletion constructs, we found that the p40(phox) PX domain has phosphatidylinositol 3-phosphate (PtdIns(3)P)-dependent and -independent functions. Translocation of p67(phox) requires the PX domain but not 3-phosphoinositide binding. Activation of the oxidase by p40(phox), however, requires both PtdIns(3)P binding and an Src homology 3 (SH3) domain competent to bind to poly-Pro ligands. Mutations that disrupt the closed auto-inhibited form of full-length p40(phox) can increase oxidase activity approximately 2.5-fold above that of wild-type p40(phox) but maintain the requirement for PX and SH3 domain function. We present a model where p40(phox) translocates p67(phox) to the region of the cytochrome and subsequently switches the oxidase to an activated state dependent upon PtdIns(3)P and SH3 domain engagement.     

Characterization of a mutation in the Phox homology domain of the NADPH oxidase component p40phox identifies a mechanism for negative regulation of superoxide production

The phagocyte oxidase (Phox) protein p40(phox) contains a Phox homology (PX) domain which, when expressed alone, interacts with phosphatidylinositol 3-phosphate (PtdIns (3)P). The functions of the PX domain in p40(phox) localization, association with the cytoskeleton, and superoxide production were examined in transgenic COS-7 cells expressing gp91(phox), p22(phox), p67(phox), and p47(phox) (COS(phox) cells). Full-length p40(phox) exhibited a cytoplasmic localization pattern in resting cells. Upon stimulation with phorbol 12-myristate 13-acetate or fMet-Leu-Phe, p40(phox) translocated to plasma membrane in a p67(phox)- and p47(phox)-dependent manner. Heterologous expression of p40(phox) markedly enhanced superoxide production in phorbol 12-myristate 13-acetate - and fMet-Leu-Phe-stimulated COS(phox) cells. Unexpectedly, mutation of Arg-57 in the PX domain to Gln, which abrogated PtdIns (3)P binding, produced a dominant inhibitory effect on agonist-induced superoxide production and membrane translocation of p47(phox) and p67(phox). The mutant p40(phox) (p40R57Q) displayed increased association with actin and moesin and was found enriched in the Triton X-100-insoluble fraction along with p67(phox) and p47(phox). The enhanced cytoskeleton association of p67(phox) and p47(phox) and the dominant inhibitory effect produced by the p40R57Q were alleviated when a second mutation at Asp-289, which eliminated p40(phox) interaction with p67(phox), was introduced. Likewise, cytochalasin B treatment abolished the dominant inhibitory effect of p40R57Q on superoxide production. These findings suggest a dual regulatory mechanism through the PX domain of p40(phox); its interaction with the actin cytoskeleton may stabilize NADPH oxidase in resting cells, and its binding of PtdIns (3)P potentiates superoxide production upon agonist stimulation. Both functions require the association of p40(phox) with p67(phox).     

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).     

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).     

Diverse recognition of non-PxxP peptide ligands by the SH3 domains from p67(phox), Grb2 and Pex13p

The basic function of the Src homology 3 (SH3) domain is considered to be binding to proline-rich sequences containing a PxxP motif. Recently, many SH3 domains, including those from Grb2 and Pex13p, were reported to bind sequences lacking a PxxP motif. We report here that the 22 residue peptide lacking a PxxP motif, derived from p47(phox), binds to the C-terminal SH3 domain from p67(phox). We applied the NMR cross-saturation method to locate the interaction sites for the non-PxxP peptides on their cognate SH3 domains from p67(phox), Grb2 and Pex13p. The binding site of the Grb2 SH3 partially overlapped the conventional PxxP-binding site, whereas those of p67(phox) and Pex13p SH3s are located in different surface regions. The non-PxxP peptide from p47(phox) binds to the p67(phox) SH3 more tightly when it extends to the N-terminus to include a typical PxxP motif, which enabled the structure determination of the complex, to reveal that the non-PxxP peptide segment interacted with the p67(phox) SH3 in a compact helix-turn-helix structure (PDB entry 1K4U).