A prenylated p67phox-Rac1 chimera elicits NADPH-dependent superoxide production by phagocyte membranes in the absence of an activator and of p47phox: conversion of a pagan NADPH oxidase to monotheism

Activation of the superoxide-generating NADPH oxidase of phagocytes is the result of the assembly of a membrane-localized flavocytochrome (cytochrome b(559)) with the cytosolic components p47(phox), p67(phox), and the small GTPase Rac. Activation can be reproduced in an in vitro system in which cytochrome b(559)-containing membranes are mixed with cytosolic components in the presence of an anionic amphiphile. We proposed that the essential event in activation is the interaction between p67(phox) and cytochrome b(559) and that Rac and p47(phox) serve as carriers for p67(phox) to the membrane. When prenylated, Rac can fulfill the carrier function by itself, supporting oxidase activation by p67(phox) in the absence of p47(phox) and amphiphile. We now show that a single chimeric protein, consisting of residues 1-212 of p67(phox) and full-length Rac1 (residues 1-192), prenylated in vitro and exchanged to GTP, becomes a potent oxidase activator in the absence of any other component or stimulus. Oxidase activation by prenylated chimera p67(phox) (1-212)-Rac1 (1-192) is accompanied by its spontaneous association with membranes. Prenylated chimeras p67(phox) (1-212)-Rac1 (178-192) and p67(phox) (1-212)-Rac1 (189-192), containing specific C-terminal regions of Rac1, are inactive; the activity of the first but not of the second chimera can be rescued by supplementation with exogenous nonprenylated Rac1-GTP. An analysis of prenylated p67(phox)-Rac1 chimeras suggests that the basic requirements for oxidase activation are: (i) a "two signals" membrane-localizing motif present in Rac, comprising the prenyl group and a C-terminal polybasic sequence and (ii) an intrachimeric or extrachimeric protein-protein interaction between p67(phox) and Rac1, causing a conformational change in the "activation domain" in p67(phox).     

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.     

Role of the phospholipid binding sites,PX of p47phox and PB region of Rac1, in the formation of the phagocyte NADPH oxidase complex NOX2

In phagocytes, superoxide anion (O₂⁻), the precursor of reactive oxygen species, is produced by the NADPH oxidase complex to kill pathogens. Phagocyte NADPH oxidase consists of the transmembrane cytochrome b₅₅₈ (cyt b₅₅₈) and four cytosolic components: p40^phox, p47^phox, p67^phox, and Rac1/2. The phagocyte activation by stimuli leads to activation of signal transduction pathways. This is followed by the translocation of cytosolic components to the membrane and their association with cyt b₅₅₈ to form the active enzyme.To investigate the roles of membrane-interacting domains of the cytosolic proteins in the NADPH oxidase complex assembly and activity, we used giant unilamellar phospholipid vesicles (GUV). We also used the neutrophil-like cell line PLB-985 to investigate these roles under physiological conditions. We confirmed that the isolated proteins must be activated to bind to the membrane. We showed that their membrane binding was strengthened by the presence of the other cytosolic partners, with a key role for p47^phox. We also used a fused chimera consisting of p47^phox(aa 1–286), p67^phox(aa 1–212) and Rac1Q61L, as well as mutated versions in the p47^phox PX domain and the Rac polybasic region (PB). We showed that these two domains have a crucial role in the trimera membrane-binding and in the trimera assembly to cyt b₅₅₈. They also have an impact on O₂.- production in vitro and in cellulo: the PX domain strongly binding to GUV made of a mix of polar lipids; and the PB region strongly binding to the plasma membrane of neutrophils and resting PLB-985 cells.     

Targeting of Rac1 to the phagocyte membrane is sufficient for the induction of NADPH oxidase assembly

The superoxide (O(2))-generating NADPH oxidase complex of phagocytes consists of a membrane-associated flavocytochrome (cytochrome b(559)) and four cytosolic proteins, p47(phox), p67(phox), p40(phox), and the small GTPase Rac (Rac1 or -2). NADPH oxidase activation (O(2) production) is elicited as the consequence of assembly of some or all cytosolic components with cytochrome b(559). This process can be reproduced in an in vitro system consisting of phagocyte membranes, p47(phox), p67(phox), and Rac, activated by an anionic amphiphile. We now show that post-translationally processed (prenylated) Rac1 initiates NADPH oxidase assembly, expressed in O(2) production, in a cell-free system containing phagocyte membrane vesicles and p67(phox), in the absence of an activating amphiphile and of p47(phox). Prenylated Cdc42Hs, a GTPase closely related to Rac, is inactive under the same conditions. Results obtained with phagocyte membrane vesicles can be reproduced fully by replacing these with partially purified cytochrome b(559), incorporated in phosphatidylcholine vesicles. Prenylated, but not nonprenylated, Rac1 binds spontaneously to phagocyte membrane vesicles and also to artificial, protein-free, phosphatidylcholine vesicles, a process counteracted by GDP dissociation inhibitor for Rho. Binding of prenylated Rac1 to membrane vesicles is accompanied by the recruitment of p67(phox) to the same location and the formation of an assembled NADPH oxidase complex, producing O(2) upon the addition of NADPH. Amphiphile and p47(phox)-independent NADPH oxidase activation by prenylated Rac1 is inhibited by Rho GDP dissociation inhibitor and by phosphatidylcholine vesicles, both competing with membrane for prenylated Rac1. We conclude that, in vitro, targeting of Rac to the phagocyte membrane is sufficient for the induction of NADPH oxidase assembly, suggesting that the principal or, possibly, the only role of Rac is to recruit cytosolic p67(phox) to the membrane environment, to be followed by the interaction of p67(phox) with cytochrome b(559).     

p47phox Phox Homology Domain Regulates Plasma Membrane but Not Phagosome Neutrophil NADPH Oxidase Activation

The assembly of cytosolic subunits p47^phox, p67^phox, and p40^phox with flavocytochrome b₅₅₈ at the membrane is required for activating the neutrophil NADPH oxidase that generates superoxide for microbial killing. The p47^phox subunit plays a critical role in oxidase assembly. Recent studies showed that the p47^phox Phox homology (PX) domain mediates phosphoinositide binding in vitro and regulates phorbol ester-induced NADPH oxidase activity in a K562 myeloid cell model. Because the importance of the p47^phox PX domain in neutrophils is unclear, we investigated its role using p47^phox knock-out (KO) mouse neutrophils to express human p47^phox and derivatives harboring R90A mutations in the PX domain that result in loss of phosphoinositide binding. Human p47^phox proteins were expressed at levels similar to endogenous murine p47^phox, with the exception of a chronic granulomatous disease-associated R42Q mutant that was poorly expressed, and wild type human p47^phox rescued p47^phox KO mouse neutrophil NADPH oxidase activity. Plasma membrane NAPDH oxidase activity was reduced in neutrophils expressing p47^phox with Arg⁹⁰ substitutions, with substantial effects on responses to either phorbol ester or formyl-Met-Leu-Phe and more modest effects to particulate stimuli. In contrast, p47^phox Arg⁹⁰ mutants supported normal levels of intracellular NADPH oxidase activity during phagocytosis of a variety of particles and were recruited to phagosome membranes. This study defines a differential and agonist-dependent role of the p47^phox PX domain for neutrophil NADPH oxidase activation.     

A Conserved Region between the TPR and Activation Domains of p67phox Participates in Activation of the Phagocyte NADPH Oxidase

The phagocyte NADPH oxidase, dormant in resting cells, is activated during phagocytosis to produce superoxide, a precursor of microbicidal oxidants. The membrane-integrated protein gp91^phox serves as the catalytic core, because it contains a complete electron-transporting apparatus from NADPH to molecular oxygen for superoxide production. Activation of gp91^phox requires the cytosolic proteins p67^phox, p47^phox, and Rac (a small GTPase). p67^phox, comprising 526 amino acids, moves upon cell stimulation to the membrane together with p47^phox and there interacts with Rac; these processes are prerequisite for gp91^phox activation. Here we show that a region of p67^phox (amino acids 190–200) C-terminal to the Rac-binding domain is evolutionarily well conserved and participates in oxidase activation at a later stage in conjunction with an activation domain. Alanine substitution for Tyr-198, Leu-199, or Val-204 abrogates the ability of p67^phox to support superoxide production by gp91^phox-based oxidase as well as its related oxidases Nox1 and Nox3; the activation also involves other invariant residues such as Leu-193, Asp-197, and Gly-200. Intriguingly, replacement of Gln-192 by alanine or that of Tyr-198 by phenylalanine or tryptophan rather enhances superoxide production by gp91^phox-based oxidase, suggesting a tuning role for these residues. Furthermore, the Y198A/V204A or L199A/V204A substitution leads to not only a complete loss of the activity of the reconstituted oxidase system but also a significant decrease in p67^phox interaction with the gp91^phox NADPH-binding domain, although these mutations affect neither the protein integrity nor the Rac binding activity. Thus the extended activation domain of p67^phox (amino acids 190–210) containing the D(Y/F)LGK motif plays an essential role in oxidase activation probably by interacting with gp91^phox.     

Arachidonic Acid Induces Direct Interaction of the p67phox-Rac Complex with the Phagocyte Oxidase Nox2, Leading to Superoxide Production

The phagocyte NADPH oxidase Nox2, heterodimerized with p22^phox in the membrane, is dormant in resting cells but becomes activated upon cell stimulation to produce superoxide, a precursor of microbicidal oxidants. Nox2 activation requires two switches to be turned on simultaneously: a conformational change of the cytosolic protein p47^phox and GDP/GTP exchange on the small GTPase Rac. These proteins, in an active form, bind to their respective targets, p22^phox and p67^phox, leading to productive oxidase assembly at the membrane. Although arachidonic acid (AA) efficiently activates Nox2 both in vivo and in vitro, the mechanism has not been fully understood, except that AA induces p47^phox conformational change. Here we show that AA elicits GDP-to-GTP exchange on Rac at the cellular level, consistent with its role as a potent Nox2 activator. However, even when constitutively active forms of p47^phox and Rac1 are both expressed in HeLa cells, superoxide production by Nox2 is scarcely induced in the absence of AA. These active proteins also fail to effectively activate Nox2 in a cell-free reconstituted system without AA. Without affecting Rac-GTP binding to p67^phox, AA induces the direct interaction of Rac-GTP-bound p67^phox with the C-terminal cytosolic region of Nox2. p67^phox-Rac-Nox2 assembly and superoxide production are both abrogated by alanine substitution for Tyr-198, Leu-199, and Val-204 in the p67^phox activation domain that localizes the C-terminal to the Rac-binding domain. Thus the “third” switch (AA-inducible interaction of p67^phox·Rac-GTP with Nox2) is required to be turned on at the same time for Nox2 activation.     

Consequences of the constitutive NOX2 activity in living cells: Cytosol acidification,apoptosis, and localized lipid peroxidation

The phagocyte NADPH oxidase (NOX2) is a key enzyme of the innate immune system generating superoxide anions (O₂^?-), precursors of reactive oxygen species. The NOX2 protein complex is composed of six subunits: two membrane proteins (gp91^phox and p22^phox) forming the catalytic core, three cytosolic proteins (p67^phox, p47^phox and p40^phox) and a small GTPase Rac. The sophisticated activation mechanism of the NADPH oxidase relies on the assembly of cytosolic subunits with the membrane-bound components. A chimeric protein, called ‘Trimera’, composed of the essential domains of the cytosolic proteins p47^phox (aa 1–286), p67^phox (aa 1–212) and full-length Rac1Q61L, enables a constitutive and robust NOX2 activity in cells without the need of any stimulus. We employed Trimera as a single activating protein of the phagocyte NADPH oxidase in living cells and examined the consequences on the cell physiology of this continuous and long-term NOX activity. We showed that the sustained high level of NOX activity causes acidification of the intracellular pH, triggers apoptosis and leads to local peroxidation of lipids in the membrane. These local damages to the membrane correlate with the strong tendency of the Trimera to clusterize in the plasma membrane observed by FRET-FLIM microscopy.     

Assembly of phagocyte NADPH oxidase: A concerted binding process?

Background

The phagocyte NADPH-oxidase is a multicomponent enzyme that generates superoxide anions. It comprises a membrane redox component flavocytochrome b₅₅₈ and four cytosolic proteins (p67^phox, p47^phox, p40^phox and Rac) that must assemble to produce an active system. In this work we focused on the spatio-temporal control of the activation process of phagocyte NADPH oxidase.

Methods

A wide range of techniques including fast kinetics with a stopped-flow apparatus and various combinations of the activating factors was used to test the order of assembly and the role of the p47^phox–p67^phox complex.

Results

The data presented here are consistent with the absence of a catalytic role of the p47^phox–p67^phox interacting state and support the idea of independent binding sites for the cytosolic proteins on the flavocytochrome b₅₅₈ allowing random binding order. However, the formation of the active complex appears to involve a synergistic process of binding of the activated cytosolic subunits to cytochrome b₅₅₈. All partners should be in the vicinity for optimal assembly, a delay or the absence of one of the partners in this process seems to lead to a decrease in the efficiency of the catalytic core.

Conclusion and general significance

The activation and assembly of the NADPH oxidase components have to be achieved simultaneously for the formation of an efficient and optimal enzyme complex. This mechanism appears to be incompatible with continuous fast exchanges of the cytosolic proteins during the production of superoxide ion in the phagosome.     

Rac1-NADPH oxidase-regulated generation of reactive oxygen species mediates glutamate-induced apoptosis in SH-SY5Y human neuroblastoma cells

Reactive oxygen species (ROS) are known to play an important role in glutamate-induced neuronal cell death. In the present study, we examined whether NADPH oxidase serves as a source of ROS production and plays a role in glutamate-induced cell death in SH-SY5Y human neuroblastoma cells. Stimulation of the cells with glutamate (100 mM) induced apoptotic cell death and increase in the level of ROS, and these effects of glutamate were significantly suppressed by the inhibitors of the NADPH oxidase, diphenylene iodonium, apocynin, and neopterine. In addition, RT-PCR revealed that SH-SY5Y cells expressed mRNA of gp91^phox, p22^phox and cytosolic p47^phox, p67^phox and p40^phox, the components of the plasma membrane NADPH oxidase. Treatment with glutamate also resulted in activation and translocation of Rac1 to the plasma membrane. Moreover, the expression of Rac1N17, a dominant negative mutant of Rac1, significantly blocked the glutamate-induced ROS generation and cell death. Collectively, these results suggest that the plasma membrane-bound NADPH oxidase complex may play an essential role in the glutamate-induced apoptotic cell death through increased production of ROS.     

Characterization of P-Rex1 for its role in fMet-Leu-Phe-induced superoxide production in reconstituted COSphox cells

P-Rex1 (phosphatidylinositol 3,4,5-trisphosphate-dependent Rac exchanger 1) is a Rac-specific guanine nucleotide exchange factor activated by Gβγ subunits and by PtdIns_(3,4,5)P₃. Recent studies indicate that P-Rex1 plays an important role in signaling downstream of neutrophil chemoattractant receptors. Here we report that heterologous expression of P-Rex1, but not Vav1, reconstitutes formyl peptide receptor 1 (FPR1)-mediated NADPH oxidase activation in the transgenic COS^phox cells expressing gp91^phox, p22^phox, p67^phox and p47^phox. A successful reconstitution requires the expression of a full-length P-Rex1 with intact DH and PH domains, and is accompanied by P-Rex1 membrane localization as well as Rac1 activation. P-Rex1-dependent superoxide generation in the reconstituted COS^phox cells was further enhanced by expression of the novel PKC isoform PKCδ and by overexpression of Akt. Heterologous expression of P-Rex1 in COS^phox cells potentiated fMet-Leu-Phe-induced Akt phosphorylation, whereas expression of a constitutively active form of Akt enhanced Rac1 activation. In contrast, a dominant negative Akt mutant reduced the fMet-Leu-Phe stimulated superoxide generation as well as Rac1 activation. These results demonstrate that in COS^phox cells, P-Rex1 is a critical component for FPR1-mediated signaling leading to NADPH oxidase activation, and there is a crosstalk between the P-Rex1-Rac pathway and Akt in superoxide generation.     

Dual role of Rac in the assembly of NADPH oxidase, tethering to the membrane and activation of p67phox: a study based on mutagenesis of p67phox-Rac1 chimeras

NADPH oxidase activation involves the assembly of membrane-localized cytochrome b559 with the cytosolic components p47phox, p67phox, and the small GTPase Rac. Assembly is mimicked by a cell-free system consisting of membranes and cytosolic components, activated by an anionic amphiphile. We reported that a chimeric construct, consisting of residues 1-212 of p67phox and full-length Rac1, activates the oxidase in vitro in an amphiphile-dependent manner, and when prenylated, in the absence of amphiphile and p47phox. We subjected chimera p67phox-(1-212)-Rac1 to mutational analysis and found that: 1) replacement of a single basic residue at the C terminus of the Rac1 moiety by glutamine is sufficient for loss of activity by the non-prenylated chimera; replacement of all six basic residues by glutamines is required for loss of activity by the prenylated chimera. 2) A V204A mutation in the activation domain of the p67phox moiety leads to a reduction in activity. 3) Mutating residues, known to participate in the interaction between free p67phox and Rac1, in the p67phox-(R102E) or Rac1 (A27K, G30S) moieties of the chimera, leads to a marked decrease in activity, indicating a requirement for intrachimeric bonds, in addition to the engineered fusion. 4) Chimeras, inactive because of mutations A27K or G30S in the Rac1 moiety, are reactivated by supplementation with exogenous Rac1-GTP but not with exogenous p67phox. This demonstrates that Rac has a dual role in the assembly of NADPH oxidase. One is to tether p67phox to the membrane; the other is to induce an "activating" conformational change in p67phox.     

A Fluorescently Tagged C-Terminal Fragment of p47phox Detects NADPH Oxidase Dynamics during Phagocytosis

The assembly of cytosolic p47^phox and p67^phox with flavocytochrome b₅₅₈ at the membrane is crucial for activating the leukocyte NADPH oxidase that generates superoxide for microbial killing. p47^phox and p67^phox are linked via a high-affinity, tail-to-tail interaction involving a proline-rich region (PRR) and a C-terminal SH3 domain (SH3b), respectively, in their C-termini. This interaction mediates p67^phox translocation in neutrophils, but is not required for oxidase activity in model systems. Here we examined phagocytosis-induced NADPH oxidase assembly, showing the sequential recruitment of YFP-tagged p67^phox to the phagosomal cup, and, after phagosome internalization, a probe for PI(3)P followed by a YFP-tagged fragment derived from the p47^phox PRR. This fragment was recruited in a flavocytochrome b₅₅₈-dependent, p67^phox-specific, and PI(3)P-independent manner. These findings indicate that p47PRR fragment probes the status of the p67^phox SH3b domain and suggest that the p47^phox/p67^phox tail-to-tail interaction is disrupted after oxidase assembly such that the p67^phox-SH3b domain becomes accessible. Superoxide generation was sustained within phagosomes, indicating that this change does not correlate with loss of enzyme activity. This study defines a sequence of events during phagocytosis-induced NADPH oxidase assembly and provides experimental evidence that intermolecular interactions within this complex are dynamic and modulated after assembly on phagosomes.     

NADPH oxidase activator p67phox behaves in solution as a multidomain protein with semi-flexible linkers

The NADPH oxidase complex is involved in the destruction of phagocytosed pathogens through the production of reactive oxygen species. This activatable complex consists of a membranous heterodimeric flavocytochrome b, a small G-protein Rac1/Rac2 and cytosolic factors, p47^phox, p67^phox and p40^phox. p67^phox, due to its modular structure, is the NADPH oxidase component for which global structure information is most scarce despite its mandatory role in activation and its central position in the whole complex organization. Indeed, p67^phox is the only factor establishing interaction with all others. In this study, we report the SAXS analysis of p67^phox. Our data reveals that p67^phox behaves as a multidomain protein with semi-flexible linkers. On the one hand, it appears to be a very elongated molecule with its various domains organized as beads on a string. Linkers are predicted to be partially or mainly unstructured and features of our experimental data do point towards inter-domain flexibility. On the other hand, our work also suggests that the protein is not as extended as unstructured linkers could allow, thereby implying the existence of intra-molecular interactions within p67^phox. We suggest that the dual character of p67^phox conformation in solution is central to ensure the numerous interactions to be accommodated.     

Conformational changes in truncated p47phox proteins monitored by fluorescent labeling

The leukocyte NADPH oxidase of neutrophils is a membrane-bound enzyme that catalyzes the reduction of oxygen to O^– ₂ at the expense of NADPH. During activation, the cytosolic oxidase components p47^phox and p67^phox, each containing two Src homology 3 (SH3) domains, migrate to the plasma membrane. p47^phox and p67^phox associate with cytochrome b₅₅₈, 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, as an activating agent. Activators of the oxidase in vitro cause exposure of the SH3 domains of p47^phox, which has probably been masked by the C-terminal region of this protein in a resting state. We show here that the fluorescence exhibited by the covalently labeled N,N-di-methyl-N(iodoacetyl)-N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) ethyleneamine (IANBD) was increased when N-terminal-truncated p47^phox-(SH3)₂-C was treated with anionic amphiphiles. This finding was similar to the results obtained with the full-length p47^phox. However, the fluorescence of C-terminal-truncated p47^phox-N-(SH3)₂ and that of both C-terminal and N-terminal truncated p47^phox-(SH3)₂ 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.     

Activation of the superoxide-generating NADPH oxidase by chimeric proteins consisting of segments of the cytosolic component p67(phox) and the small GTPase Rac1.

Activation of the superoxide (O2(-))-generating NADPH oxidase of phagocytes is the consequence of the assembly of a membrane-associated flavocytochrome b(559) with the cytosolic proteins p47(phox) and p67(phox) and the small GTPase Rac (1 or 2). We proposed that Rac1 serves as a membrane-targeting molecule for p67(phox). This hypothesis was tested by constructing recombinant chimeric proteins, joining various functional domains of p67(phox) and Rac1, and expressing these in Escherichia coli. Chimeras were assayed for the ability to support O2(-) production by phagocyte membranes in an amphiphile-activated cell-free system in the presence or absence of p47(phox). A chimera consisting of p67(phox) truncated at residue 212 and fused to a full-length Rac1 [p67(phox)(1-212)-Rac1(1-192)] was a potent NADPH oxidase activator. A p67(phox)(1-212)-Rac1(178-192) chimera, to which Rac1 contributed only the C-terminal polybasic domain, was a weaker but consistent activator. Chimeras comprising the full length of Rac1 bound GTP/GDP, like bona fide GTPases. The activity of p67(phox)-Rac1 chimeras was dependent on the presence of the tetratricopeptide repeat and activation domains, in the p67(phox) segment, and on an intact polybasic region, at the C terminus of the Rac1 segment, but not on the insert region of Rac1. Partial activation by chimeras, in the GTP-bound form, was also possible in the absence of p47(phox). Evidence is offered in support of the proposal that the GTP- and GDP-bound forms of chimera p67(phox)(1-212)-Rac1(1-192) have distinct conformations, corresponding to the presence and absence of intrachimeric bonds, respectively.     

Assembly of the phagocyte NADPH oxidase

Stimulated phagocytes undergo a burst in respiration whereby molecular oxygen is converted to superoxide anion through the action of an NADPH-dependent oxidase. The multicomponent phagocyte oxidase is unassembled and inactive in resting cells but assembles at the plasma or phagosomal membrane upon phagocyte activation. Oxidase components include flavocytochrome b₅₅₈, an integral membrane heterodimer comprised of gp91phox and p22phox, and three cytosolic proteins, p47phox, p67phox, and Rac1 or Rac2, depending on the species and phagocytic cell. In a sense, the phagocyte oxidase is spatially regulated, with critical elements segregated in the membrane and cytosol but ready to undergo nearly immediate assembly and activation in response to stimulation. To achieve such spatial regulation, the individual components in the resting phagocyte adopt conformations that mask potentially interactive structural domains that might mediate productive intermolecular associations and oxidase assembly. In response to stimulation, post-translational modifications of the oxidase components release these constraints and thereby render potential interfaces accessible and interactive, resulting in translocation of the cytosolic elements to the membrane where the functional oxidase is assembled and active. This review summarizes data on the structural features of the phagocyte oxidase components and on the agonist-dependent conformational rearrangements that contribute to oxidase assembly and activation.     

Phospholipase D-mediated Activation of IQGAP1 through Rac1 Regulates Hyperoxia-induced p47phox Translocation and Reactive Oxygen Species Generation in Lung Endothelial Cells

Phosphatidic acid generated by the activation of phospholipase D (PLD) functions as a second messenger and plays a vital role in cell signaling. Here we demonstrate that PLD-dependent generation of phosphatidic acid is critical for Rac1/IQGAP1 signal transduction, translocation of p47^phox to cell periphery, and ROS production. Exposure of [³²P]orthophosphate-labeled human pulmonary artery endothelial cells (HPAECs) to hyperoxia (95% O₂ and 5% CO₂) in the presence of 0.05% 1-butanol, but not tertiary-butanol, stimulated PLD as evidenced by accumulation of [³²P]phosphatidylbutanol. Infection of HPAECs with adenoviral constructs of PLD1 and PLD2 wild-type potentiated hyperoxia-induced PLD activation and accumulation of /reactive oxygen species (ROS). Conversely, overexpression of catalytically inactive mutants of PLD (hPLD1-K898R or mPLD2-K758R) or down-regulation of expression of PLD with PLD1 or PLD2 siRNA did not augment hyperoxia-induced [³²P]phosphatidylbutanol accumulation and ROS generation. Hyperoxia caused rapid activation and redistribution of Rac1, and IQGAP1 to cell periphery, and down-regulation of Rac1, and IQGAP1 attenuated hyperoxia-induced tyrosine phosphorylation of Src and cortactin and ROS generation. Further, hyperoxia-mediated redistribution of Rac1, and IQGAP1 to membrane ruffles, was attenuated by PLD1 or PLD2 small interference RNA, suggesting that PLD is upstream of the Rac1/IQGAP1 signaling cascade. Finally, small interference RNA for PLD1 or PLD2 attenuated hyperoxia-induced cortactin tyrosine phosphorylation and abolished Src, cortactin, and p47^phox redistribution to cell periphery. These results demonstrate a role of PLD in hyperoxia-mediated IQGAP1 activation through Rac1 in tyrosine phosphorylation of Src and cortactin, as well as in p47^phox translocation and ROS formation in human lung endothelial cells.Phagocytic cells of the immune system (neutrophils, eosinophils, monocytes, and macrophages) generate superoxide ()² instrumental in the killing of invading pathogens solely by NADPH oxidase (1-3). Deficiency of results in the genetically inherited disorder chronic granulomatous disease, a condition in which the affected individuals are susceptible to infection (4). Phagocytic NADPH oxidase is activated when cytosolic p47^phox, p67^phox, and Rac2 translocate to the phagosomes and plasma membrane and form a complex with integral membrane cytochrome b₅₅₈, which, in turn, is a Nox2 (gp91^phox)/p22^phox heterodimer (5, 6). Assembly of phagocytic NADPH oxidase is initiated by two signals. The first is the phosphorylation of multiple serine and tyrosine residues in the p47^phox domain, which leads to unmasking of p47^phox SH3 domains that bind to a proline-rich target in the C terminus of p22^phox (7-10). The interaction between p47^phox and p22^phox seems to be an essential requirement for the translocation of other cytosolic components of the oxidase. The second signal is the binding of GTP to Rac2, which leads to the dissociation of Rac from Rho-GDI and binding to p67^phox, followed by translocation of p67^phox/GTP-Rac2 to the membrane (11). Nonphagocytic cells express predominantly Rac1, Tiam1 (a GEF involved in Rac1 activation), Nox1-5, and most of the other cytosolic phagocytic oxidase components (12); however, the oxidative output of non-phagocytes is much smaller compared with the phagocytes. A recent study indicates that IQGAP1, an effector of Rac1, may link Nox2 to actin, thereby enhancing ROS production and contributing to cell motility in ECs (13). The one or more mechanisms responsible for differences in the oxidative burst between the phagocytic and non-phagocytic cells are yet to be defined.We have demonstrated previously that hyperoxia activates lung endothelial NADPH oxidase, which in part is mediated by ERK, p38 MAPK (14, 15), and Src (16), and hyperoxia-induced p47^phox tyrosine phosphorylation and translocation to cell periphery is dependent on Src (16). Further, tyrosine phosphorylation of cortactin mediated by Src is essential for hyperoxia-induced p47^phox translocation and /ROS generation in HPAECs (17). In addition to Src, phosphatidic acid (PA) or diacylglycerol also stimulated phosphorylation of p47^phox and p22^phox in neutrophils both in vivo and in vitro (18-20). PA is generated in mammalian cells via de novo biosynthesis or hydrolysis of membrane phospholipids catalyzed by phospholipase D (PLD) (21-25). Activation of polymorphonuclear leukocytes with formyl-Met-Leu-Phe enhanced the oxidative burst that correlated with PA accumulation, and inclusion of short-chain primary alcohols attenuated the NADPH oxidase mediated /ROS generation, suggesting a potential role for PLD in the regulation of NADPH oxidase (12, 26, 27). However, the downstream targets of PLD that signal NADPH oxidase activation have not been fully characterized.Here, we identify for the first time that activation of IQGAP1 by Rac1 is downstream of PLD in hyperoxia-induced ROS generation. In addition, we show that activation of Rac1/IQGAP1 by PLD also regulates Src-dependent tyrosine phosphorylation of cortactin and p47^phox translocation to cell periphery. Thus, our results define a novel molecular mechanism for hyperoxia-induced NADPH oxidase activation by PLD/PA-mediated p47^phox membrane translocation via Rac1/IQGAP1/Src/cortactin signaling cascade.     

Role for the first SH3 domain of p67 in activation of superoxide-producing NADPH oxidases

The membrane-bound NADPH oxidase in phagocytes, gp91^phox (a.k.a. Nox2), produces superoxide, a precursor of microbicidal oxidants, thereby playing a crucial role in host defense. Activation of gp91^phox/Nox2 requires assembly with the cytosolic proteins p67^phox and p47^phox, each containing two SH3 domains. Although the C-terminal SH3 domain of p67^phox is responsible for binding to p47^phox, little is known about the role for the first (N-terminal) SH3 domain [SH3(N)]. Here we show that truncation of p67^phox-SH3(N), but not substitution of arginine for the invariant residue Trp-277 in SH3(N), results in an impaired activation of gp91^phox/Nox2. The impairment is overcome by higher expression of an SH3(N)-defective p67^phox in cells, suggesting that SH3(N) primarily increases the affinity of p67^phox for the oxidase complex. On the other hand, p67^phox-SH3(N) is not involved in activation of Nox1 and Nox3, closely-related homologues of gp91^phox/Nox2. Thus p67^phox-SH3(N) specifically functions in gp91^phox/Nox2 activation probably via facilitating oxidase assembly.