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.     

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.     

Involvement of Rac1 in activation of multicomponent Nox1- and Nox3-based NADPH oxidases

Several Nox family NADPH oxidases function as multicomponent enzyme systems. We explored determinants of assembly of the multicomponent oxidases Nox1 and Nox3 and examined the involvement of Rac1 in their regulation. Both enzymes are supported by p47phox and p67phox or homologous regulators called Noxo1 and Noxa1, although Nox3 is less dependent on these cofactors for activity. Plasma membrane targeting of Noxa1 depends on Noxo1, through tail-to-tail interactions between these proteins. Noxa1 can support Nox1 without Noxo1, when targeted to the plasma membrane by fusing membrane-binding sequences from Rac1 (amino acids 183 to 192) to the C terminus of Noxa1. However, membrane targeting of Noxa1 is not sufficient for activation of Nox1. Both the Noxo1-independent and -dependent Nox1 systems involve Rac1, since they are affected by Rac1 mutants or Noxa1 mutants defective in Rac binding or short interfering RNA-mediated Rac1 silencing. Nox1 or Nox3 expression promotes p22phox transport to the plasma membrane, and both oxidases are inhibited by mutations in the p22phox binding sites (SH3 domains) of the Nox organizers (p47phox or Noxo1). Regulation of Nox3 by Rac1 was also evident from the effects of mutant Rac1 or mutant Nox3 activators (p67phox or Noxa1) or Rac1 silencing. In the absence of Nox organizers, the Nox activators (p67phox or Noxa1) colocalize with Rac1 within ruffling membranes, independently of their ability to bind Rac1. Thus, Rac1 regulates both oxidases through the Nox activators, although it does not appear to direct the subcellular localization of these activators.     

Molecular composition and regulation of the Nox family NAD(P)H oxidases

Reactive oxygen species (ROS) are conventionally regarded as inevitable deleterious by-products in aerobic metabolism with a few exceptions such as their significant role in host defense. The phagocyte NADPH oxidase, dormant in resting cells, becomes activated during phagocytosis to deliberately produce superoxide, a precursor of other microbicidal ROS, thereby playing a crucial role in killing pathogens. The catalytic center of this oxidase is the membrane-integrated protein gp91(phox), tightly complexed with p22(phox), and its activation requires the association with p47(phox), p67(phox), and the small GTPase Rac, which normally reside in the cytoplasm. Since recent discovery of non-phagocytic gp91(phox)-related enzymes of the NAD(P)H oxidase (Nox) family--seven homologues identified in humans--deliberate ROS production has been increasingly recognized as important components of various cellular events. Here, we describe a current view on the molecular composition and post-translational regulation of Nox-family oxidases in animals.     

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.     

Expression and function of Noxo1gamma, an alternative splicing form of the NADPH oxidase organizer 1

Activation of the superoxide-producing NADPH oxidase Nox1 requires both the organizer protein Noxo1 and the activator protein Noxa1. Here we describe an alternative splicing form of Noxo1, Noxo1gamma, which is expressed in the testis and fetal brain. The Noxo1gamma protein contains an additional five amino acids in the N-terminal PX domain, a phosphoinositide-binding module; the domain plays an essential role in supporting superoxide production by NADPH oxidase (Nox) family oxidases including Nox1, gp91(phox)/Nox2, and Nox3, as shown in this study. The PX domain isolated from Noxo1gamma shows a lower affinity for phosphoinositides than that from the classical splicing form Noxo1beta. Consistent with this, in resting cells, Noxo1gamma is poorly localized to the membrane, and thus less effective in activating Nox1 than Noxo1beta, which is constitutively present at the membrane. On the other hand, cell stimulation with phorbol 12-myristate 13-acetate (PMA), an activator of Nox1-3, facilitates membrane translocation of Noxo1gamma; as a result, Noxo1gamma is equivalent to Noxo1beta in Nox1 activation in PMA-stimulated cells. The effect of the five-amino-acid insertion in the Noxo1 PX domain appears to depend on the type of Nox; in activation of gp91(phox)/Nox2, Noxo1gamma is less active than Noxo1beta even in the presence of PMA, whereas Noxo1gamma and Noxo1beta support the superoxide-producing activity of Nox3 to the same extent in a manner independent of cell stimulation.     

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).     

The NADPH oxidase Nox3 constitutively produces superoxide in a p22phox-dependent manner: its regulation by oxidase organizers and activators

Nox3, a member of the superoxide-producing NADPH oxidase (Nox) family, participates in otoconia formation in mouse inner ears, which is required for perception of balance and gravity. The activity of other Nox enzymes such as gp91(phox)/Nox2 and Nox1 is known to absolutely require both an organizer protein (p47(phox) or Noxo1) andanactivatorprotein (p67(phox) or Noxa1); for the p47(phox)-dependent activation of these oxidases, treatment of cells with stimulants such as phorbol 12-myristate 13-acetate is also indispensable. Here we show that ectopic expression of Nox3 in various types of cells leads to phorbol 12-myristate 13-acetate-independent constitutive production of a substantial amount of superoxide under the conditions where gp91(phox) and Nox1 fail to generate superoxide, i.e. in the absence of the oxidase organizers and activators. Nox3 likely forms a functional complex with p22(phox); Nox3 physically interacts with and stabilizes p22(phox), and the Nox3-dependent superoxide production is totally dependent on p22(phox). The organizers p47(phox) and Noxo1 are capable of enhancing the superoxide production by Nox3 in the absence of the activators, and the enhancement requires the interaction of the organizers with p22(phox), further indicating a link between Nox3 and p22(phox). The p47(phox)-enhanced Nox3 activity is further facilitated by p67(phox) or Noxa1, whereas the activators cancel the Noxo1-induced enhancement. On the other hand, the small GTPase Rac, essential for the gp91(phox) activity, is likely dispensable to the Nox3 system. Thus Nox3 functions together with p22(phox) as an enzyme constitutively producing superoxide, which can be distinctly regulated by combinatorial use of the organizers and activators.     

Direct involvement of the small GTPase Rac in activation of the superoxide-producing NADPH oxidase Nox1

Activation of the non-phagocytic superoxide-producing NADPH oxidase Nox1, complexed with p22(phox) at the membrane, requires its regulatory soluble proteins Noxo1 and Noxa1. However, the role of the small GTPase Rac remained to be clarified. Here we show that Rac directly participates in Nox1 activation via interacting with Noxa1. Electropermeabilized HeLa cells, ectopically expressing Nox1, Noxo1, and Noxa1, produce superoxide in a GTP-dependent manner, which is abrogated by expression of a mutant Noxa1(R103E), defective in Rac binding. Superoxide production in Nox1-expressing HeLa and Caco-2 cells is decreased by depletion or sequestration of Rac; on the other hand, it is enhanced by expression of the constitutively active Rac1(Q61L), but not by that of a mutant Rac1 with the A27K substitution, deficient in binding to Noxa1. We also demonstrate that Nox1 activation requires membrane recruitment of Noxa1, which is normally mediated via Noxa1 binding to Noxo1, a protein tethered to the Nox1 partner p22(phox): the Noxa1-Noxo1 and Noxo1-p22(phox) interactions are both essential for Nox1 activity. Rac likely facilitates the membrane localization of Noxa1: although Noxa1(W436R), defective in Noxo1 binding, neither associates with the membrane nor activates Nox1, the effects of the W436R substitution are restored by expression of Rac1(Q61L). The Rac-Noxa1 interaction also serves at a step different from the Noxa1 localization, because the binding-defective Noxa1(R103E), albeit targeted to the membrane, does not support superoxide production by Nox1. Furthermore, a mutant Noxa1 carrying the substitution of Ala for Val-205 in the activation domain, which is expected to undergo a conformational change upon Rac binding, fully localizes to the membrane but fails to activate Nox1.     

Cooperation of p40(phox) with p47(phox) for Nox2-based NADPH oxidase activation during Fcγ receptor (FcγR)-mediated phagocytosis: mechanism for acquisition of p40(phox) phosphatidylinositol 3-phosphate (PI(3)P) binding

During activation of the phagocyte (Nox2-based) NADPH oxidase, the cytoplasmic Phox complex (p47(phox)-p67(phox)-p40(phox)) translocates and associates with the membrane-spanning flavocytochrome b(558). It is unclear where (in cytoplasm or on membranes), when (before or after assembly), and how p40(phox) acquires its PI(3)P-binding capabilities. We demonstrated that in addition to conformational changes induced by H(2)O(2) in the cytoplasm, p40(phox) acquires PI(3)P-binding through direct or indirect membrane targeting. We also found that p40(phox) is essential when p47(phox) is partially phosphorylated during FcγR-mediated oxidase activation; however, p40(phox) is less critical when p47(phox) is adequately phosphorylated, using phosphorylation-mimicking mutants in HEK293(Nox2/FcγRIIa) and RAW264.7(p40/p47KD) cells. Moreover, PI binding to p47(phox) is less important when the autoinhibitory PX-PB1 domain interaction in p40(phox) is disrupted or when p40(phox) is targeted to membranes. Furthermore, we suggest that high affinity PI(3)P binding of the p40(phox) PX domain is critical during its accumulation on phagosomes, even when masked by the PB1 domain in the resting state. Thus, in addition to mechanisms for directly acquiring PI(3)P binding in the cytoplasm by H(2)O(2), p40(phox) can acquire PI(3)P binding on targeted membranes in a p47(phox)-dependent manner and functions both as a "carrier" of the cytoplasmic Phox complex to phagosomes and an "adaptor" of oxidase assembly on phagosomes in cooperation with p47(phox), using positive feedback mechanisms.     

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.     

Direct interaction of the novel Nox proteins with p22phox is required for the formation of a functionally active NADPH oxidase

Nox1 and Nox4, homologues of the leukocyte NADPH oxidase subunit Nox2 (gp91phox) mediate superoxide anion formation in various cell types. However, their interactions with other components of the NADPH oxidase are poorly defined. We determined whether a direct interaction of Nox1 and Nox4 with the p22phox subunit of the NADPH oxidase occurs. Using confocal microscopy, co-localization of p22phox with Nox1, Nox2, and Nox4 was observed in transiently transfected vascular smooth muscle cells (VSMC) and HEK293 cells. Plasmids coding for fluorescent fusion proteins of p22phox and the Nox proteins with cyan- and yellow-fluorescent protein (cfp and yfp, respectively) were constructed and expressed in VSMC and HEK293 cells. The cfp-tagged p22phox expression level increased upon cotransfection with Nox1 or Nox4. Protein-protein interaction between the fluorescent fusion proteins of p22phox and the Nox partners was observed using the fluorescence resonance energy transfer technique. Immunoprecipitation of native Nox1 from human VSMC revealed co-precipitation of p22phox. Immunoprecipitation from transfected HEK293 cells revealed co-precipitation of native p22phox with yfp-tagged Nox1, Nox2, and Nox4. Following mutation of a histidine (corresponding to the position 115 in human Nox2) to leucine, this interaction was abolished. Transfection of rat p22phox (but not Noxo1 and Noxa1) increased the radical generation in cells expressing Nox4. We provide evidence that p22phox directly interacts with Nox1 and Nox4, to form an superoxide-generating NADPH oxidase and demonstrate that mutation of the potential heme binding site in the Nox proteins disrupts the complex formation of Nox1 and Nox4 with p22phox.     

Structure, regulation and evolution of Nox-family NADPH oxidases that produce reactive oxygen species

NADPH oxidases of the Nox family exist in various supergroups of eukaryotes but not in prokaryotes, and play crucial roles in a variety of biological processes, such as host defense, signal transduction, and hormone synthesis. In conjunction with NADPH oxidation, Nox enzymes reduce molecular oxygen to superoxide as a primary product, and this is further converted to various reactive oxygen species. The electron-transferring system in Nox is composed of the C-terminal cytoplasmic region homologous to the prokaryotic (and organelle) enzyme ferredoxin reductase and the N-terminal six transmembrane segments containing two hemes, a structure similar to that of cytochrome b of the mitochondrial bc(1) complex. During the course of eukaryote evolution, Nox enzymes have developed regulatory mechanisms, depending on their functions, by inserting a regulatory domain (or motif) into their own sequences or by obtaining a tightly associated protein as a regulatory subunit. For example, one to four Ca(2+)-binding EF-hand motifs are present at the N-termini in several subfamilies, such as the respiratory burst oxidase homolog (Rboh) subfamily in land plants (the supergroup Plantae), the NoxC subfamily in social amoebae (the Amoebozoa), and the Nox5 and dual oxidase (Duox) subfamilies in animals (the Opisthokonta), whereas an SH3 domain is inserted into the ferredoxin-NADP(+) reductase region of two Nox enzymes in Naegleria gruberi, a unicellular organism that belongs to the supergroup Excavata. Members of the Nox1-4 subfamily in animals form a stable heterodimer with the membrane protein p22(phox), which functions as a docking site for the SH3 domain-containing regulatory proteins p47(phox), p67(phox), and p40(phox); the small GTPase Rac binds to p67(phox) (or its homologous protein), which serves as a switch for Nox activation. Similarly, Rac activates the fungal NoxA via binding to the p67(phox)-like protein Nox regulator (NoxR). In plants, on the other hand, this GTPase directly interacts with the N-terminus of Rboh, leading to superoxide production. Here I describe the regulation of Nox-family oxidases on the basis of three-dimensional structures and evolutionary conservation.     

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.     

Noxa1 as a moderate activator of Nox2-based NADPH oxidase

Noxa1 was discovered as an activating factor for Nox1, an O(2)(-)-generating enzyme. Subsequent studies have shown that Noxa1 is colocalized with Nox2 in several cell types, including vascular cells. Nox2 activation by Noxa1 has been examined in reconstituted model cells. However, little is known about the kinetic properties of Noxa1 in Nox2 activation. In the present study, we used purified cyt.b(558) (Nox2 plus p22(phox)), Rac(Q61L), and Noxo1 to examine the ability of Noxa1 to activate Nox2. In the pure reconstitution system, Noxa1 activated Nox2 with lower efficiency than p67(phox), a canonical activator of Nox2. The EC(50) value of Noxa1 was considerably higher than that of p67(phox). The V(max) value with Noxa1 and Noxo1 was one-third of that with p67(phox) and p47(phox). The EC(50) value of Noxo1 or Rac(Q61L) was also higher when Noxa1 was used. The affinity of FAD for the oxidase and the stability of the active complex were remarkably low when Noxa1 and Noxo1 were used compared with p67(phox) and p47(phox). The stability was not improved by fusion of Noxa1 with Rac(Q61L). These findings show that Noxa1 has quite different kinetic properties from p67(phox) and suggest that Noxa1 may function as a moderate activator of Nox2.     

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.     

Subcellular localization and function of alternatively spliced Noxo1 isoforms

Nox organizer 1 (Noxo1), a p47(phox) homolog, is produced as four isoforms with unique N-terminal PX domains derived by alternative mRNA splicing. We compared the subcellular distribution of these isoforms or their isolated PX domains produced as GFP fusion proteins, as well as their ability to support Nox1 activity in several transfected models. Noxo1alpha, beta, gamma, and delta show different subcellular localization patterns, determined by their PX domains. In HEK293 cells, Noxo1beta exhibits prominent plasma membrane binding, Noxo1gamma shows plasma membrane and nuclear associations, and Noxo1alpha and delta localize primarily on intracellular vesicles or cytoplasmic aggregates, but not the plasma membrane. Nox1 activity correlates with Noxo1 plasma membrane binding in HEK293 cells, since Noxo1beta supports the highest activity and Noxo1gamma and Noxo1alpha support moderate or low activities, respectively. In COS-7 cells, where Noxo1alpha localizes on the plasma membrane, the activities supported by the three isoforms (alpha, beta, and gamma) do not differ significantly. The PX domains of beta and gamma bind the same phospholipids, including phosphatidic acid. These results indicate that the variant PX domains are unique determinants of Noxo1 localization and Nox1 function. Finally, the overexpressed Noxo1 isoforms do not affect p22(phox) localization, although Nox1 is needed to transport p22(phox) to the plasma membrane.     

Expression of isoforms of NADPH oxidase components in rat pancreatic islets

Increased oxidative stress plays a role in the pathogenesis of beta-cell dysfunction and death. We studied isoforms of NADPH oxidase components in islets of Langerhans isolated from rat pancreas and tumoral rat beta-cell line RINm5F cells by RT-PCR and sequencing of its products. RT-PCR revealed that isolated islets constitutively expressed mRNA of NADPH oxidase components, Nox1, Nox2, Nox4 and p22(phox) as membrane-associated components and p47(phox), Noxo1 (homologue of p47(phox)), Noxa1 (homologue of p67(phox)), and p40(phox) as cytosolic components. RINm5F cells showed a similar pattern of expression but Nox2 mRNA was not detected. Expression of Nox1, Nox4, Noxo1 and Noxa1 was confirmed by sequencing the PCR products. Immunohistochemistry revealed the expression of NADPH oxidase component in beta-cells of rat pancreatic islets. Glucose-stimulated insulin secretion from isolated islets was suppressed by diphenyleneiodonium, a flavocytochrome inhibitor, but not by apocynin, an inhibitor of p47(phox) translocation to membranes. Our results suggest that the functional significance of NADPH oxidase in insulin secretion may merit further investigation.     

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.