Novel Human Homologues of p47phox and p67phox Participate in Activation of Superoxide-producing NADPH Oxidases*

Ryu Takeya {ddagger}, Noriko Ueno {ddagger}, Keiichiro Kami {ddagger}, Masahiko Taura {ddagger}, Motoyuki Kohjima {ddagger}, Tomoko Izaki {ddagger}, Hiroyuki Nunoi § and Hideki Sumimoto {ddagger} 

From the {ddagger}Medical Institute of Bioregulation, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan and the §Department of Pediatrics, Miyazaki Medical College, Miyazaki 889-1692, Japan

Received for publication, December 17, 2002 , and in revised form, April 9, 2003.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
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.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Current attention has been increasingly paid to a gene family encoding superoxide-producing NAD(P)H oxidases, termed Nox1 (1, 2). The mammalian members occur in various types of cells. The founder Nox protein gp91phox (Nox2) is predominantly expressed in professional phagocytes such as neutrophils and plays a crucial role in host defense against microbial infection (39). On the other hand, Nox1 and Nox4 are abundant in epithelial cells in colon and kidney (1014), respectively, but their biological roles are not well understood at present. The Nox oxidases are membrane-integrated proteins containing a complete electron-transferring apparatus (from NADPH to molecular oxygen) with binding sites for heme, FAD, and NADPH (39). In phagocytes, gp91phox is complexed with p22phox to form flavocytochrome b558. The cytochrome is dormant in resting cells, but becomes activated during phagocytosis to generate superoxide, a precursor of microbicidal oxidants. The significance of the phagocyte oxidase in host defense is exemplified by recurrent and life-threatening infections that occur in patients with chronic granulomatous disease due to an impaired superoxide-producing activity of their phagocytes (9). On the other hand, the oxidase activity is strictly regulated, since inappropriate or excessive production of reactive oxygen species results in inflammatory disorders.

The activation of cytochrome b558 requires stimulus-induced membrane translocation of cytosolic proteins including the small GTPase Rac and the two specialized cytosolic proteins p67phox and p47phox, each containing two SH3 domains (39). In this process, p47phox translocates to the membrane by itself, whereas p67phox is recruited via p47phox (15, 16); they constitutively associate via a tail-to-tail interaction (1719). p47phox binds via its SH3 domains to a proline-rich region (PRR) of p22phox, the small subunit of cytochrome b558; interaction plays a crucial role in the oxidase activation (20, 21). The SH3 domains are normally masked via an intramolecular interaction with the autoinhibitory region (AIR) that exists C-terminal to the domains (22). Upon cell stimulation, p47phox becomes phosphorylated at multiple serines (23, 24), which induces a conformational change of this protein to render the domains in a state accessible to p22phox (22, 25, 26). On the other hand, Rac is recruited upon cell simulation to the membrane independently of p47phox or p67phox (27). At the membrane, p67phox directly interacts with GTP-bound Rac via the N-terminal domain that harbors four tetratricopeptide repeat (TPR) motifs (28, 29) and is thus targeted to cytochrome b558 (3033), leading to superoxide production. Hence, the formation of the active phagocyte oxidase in cells requires the five proteins, namely gp91phox, p22phox, p67phox, p47phox, and Rac in the GTP-bound state; chronic granulomatous disease is caused by a defect of any of the genes encoding these proteins except Rac (9). The active oxidase can be formed in a cell-free system reconstituted with the purified proteins described above and anionic amphiphiles such as arachidonic acid (39). In the presence of excess amounts of p67phox and Rac, however, p47phox is not essential for the cell-free activation of the oxidase (3335). It is thus considered that p67phox is a protein that directly activates gp91phox together with Rac, whereas p47phox functions as an organizer in the activation.

In contrast to the phagocyte oxidase system, the activation mechanism for other members of the Nox family has remained largely unknown, except that Nox5 is shown to be activated by elevation of intracellular Ca2+ (36). Although it may be possible that Nox oxidases other than gp91phox/Nox2 can also be regulated by p47phox and p67phox, this possibility has not been intensively tested. On the other hand, solely p47phox or p67phox exists in some types of cells containing one or more Nox oxidases, whereas neither p47phox nor p67phox is present in other types of Nox-expressing cells (3739). In these cells, a heretofore unidentified protein possibly functions instead of p47phox or p67phox in activation of the phagocyte or other oxidases.

Here we describe the primary structure and function of novel human homologues of p47phox and p67phox. The novel proteins exhibit almost the same domain arrangement as the classical ones and can interact with the target proteins thus far identified for p47phox or p67phox, except that the p67phox homologue fails to bind to p40phox, a dispensable but significant regulator of the phagocyte oxidase (40). Consistent with these findings, the novel homologues are capable of supporting the activation of the phagocyte NADPH oxidase (i.e. gp91phox). We also show that Nox1 becomes activated when it is coexpressed with both novel homologues of p47phox and p67phox or with a novel and a classical homologue.


    EXPERIMENTAL PROCEDURES
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Cloning of Human cDNAs Encoding Novel Proteins Homologous to p47phox and p67phoxA search of EST data bases with the C-terminal region of p47phox yielded an EST clone encoding a protein homologous to p47phox of 370 amino acids (GenBankTM accession number BC015917 [GenBank] ). Based on the sequences of these cDNA and genome DNA clones, we performed PCR using human multiple tissue cDNA panels (Clontech) and obtained a clone (GenBankTM accession number AB097667 [GenBank] ) that encodes the p47phox homologue comprising 371 amino acids: Lys-49 of this cDNA clone is missing in the EST clone. The novel protein homologous to p47phox was tentatively designated p41nox, the gene of which is termed Nox organizer 1 (NOXO1; for details, see "Results").

We also performed a search of EST data bases with the C-terminal SH3 domain of human p67phox, which yielded several cDNA clones containing partial sequences similar to those of p67phox. Using the sequences, we further carried out the data base search to find several EST clones that encode N-terminal partial sequences of the protein homologous to p67phox. Based on these partial sequences of these cDNA, we performed PCR using Human Multiple Tissue cDNA panels (Clontech) and obtained a cDNA clone encoding the full-length protein (GenBankTM accession number AB095031 [GenBank] ). The novel homologue of p67phox was tentatively named p51nox, the gene of which is termed Nox activator 1 (NOXA1; for details, see "Results").

Plasmid Construction—Complementary DNA fragments encoding various lengths of p47phox, p67phox, p40phox, p22phox, gp91phox/Nox2, Nox1, Rac1, Rac2, and Cdc42 were prepared as previously described (13, 20, 22, 28). We also amplified the DNA fragments that encode p41nox-F (amino acid residues 1–371), p41nox-(SH3)2 (residues 154–292), p41nox-C (residues 293–371), p51nox-F (residues 1–476), p51nox-N (residues 1–224), and p51nox-SH3 (residues 397–476) by PCR using specific primers from the cloned cDNAs encoding human p41nox or p51nox. The DNA fragments were ligated to the indicated expression vectors. Mutations leading to the indicated amino acid substitutions were introduced by PCR-mediated site-directed mutagenesis, and the mutated fragments were cloned into the indicated vectors. All of the constructs were sequenced to confirm their identities.

Two-hybrid Experiments—Various combinations between pGBT9 (Clontech) and pGADGH (Clontech) plasmids, each encoding an oxidase protein, were cotransformed into competent yeast HF7c cells containing a HIS3 reporter gene, as previously described (28). Following the selection for Trp+ and Leu+ phenotypes, the transformants were tested for their ability to grow on plates lacking histidine, according to the manufacturer's recommendation (Clontech).

An in Vitro Binding Assay Using Purified Proteins—For expression in Escherichia coli, cDNA fragments were ligated to the following vectors: pGEX-2T (Amersham Biosciences) for glutathione S-transferase (GST) fusion protein; pMALc2 (New England Biolabs) for maltose-binding protein (MBP) fusion protein; or pProEX-HTb (Invitrogen) for His-tagged protein. GST-, MBP-, or His-tagged proteins were purified by glutathione-Sepharose-4B (Amersham Biosciences), amylose resin (New England Biolabs), or His-bind resin (Novagen), respectively, according to the manufacturers' protocols. For in vitro pull-down binding assays, a pair of a GST and an MBP fusion protein or a pair of a GST fusion and a His-tagged protein were mixed in 400 µl of phosphate-buffered saline (137 mM NaCl, 2.68 mM KCl, 8.1 mM Na2HPO4, and 1.47 mM KH2PO4) containing 10 mM dithiothreitol and incubated for 30 min at 4 °C. A slurry of glutathione-Sepharose-4B was subsequently added, followed by further incubation for 60 min at 4 °C. After washing four times with phosphate-buffered saline containing 0.5% Triton X-100 and 10 mM dithiothreitol, proteins were eluted from glutathione-Sepharose-4B with 10 mM glutathione in 100 mM Tris-HCl (pH 8.0) and 200 mM NaCl. The eluates were subjected to SDS-PAGE and stained with Coomassie Brilliant Blue or analyzed by immunoblot with an anti-His monoclonal antibody (Qiagen), followed by development using ECL-plus (Amersham Biosciences).

In Vivo Interaction between Oxidase Proteins—For expression of proteins in mammalian cells as HA- or Myc-tagged proteins, cDNAs were ligated to pEF-BOS (41), as previously described (42, 43). COS-7 cells transfected using LipofectAMINE (Invitrogen) with the indicated cDNA were cultured for 36 h and broken with a lysis buffer composed of 1% Nonidet P-40, 142.5 mM NaCl, 2 mM EDTA, 10% glycerol, leupeptin (40 µg/ml), aprotinin (10 µg/ml), 2 mM phenylmethylsulfonyl fluoride, and 20 mM HEPES, pH 7.4. Proteins of the lysate were precipitated with protein G-Sepharose using an anti-Myc monoclonal antibody (9E10; Roche Applied Science). The precipitants were analyzed by immunoblot with anti-HA or anti-Myc polyclonal antibodies (both from Santa Cruz Biotechnology.). The blots were developed using ECL-plus (Amersham Biosciences) to visualize the antibodies.

Activation of gp91phox in K562 Cells—We transduced the gp91phox gene into the leukemia cell line K562 and prepared cells that stably express a high amount of functional cytochrome b558 (K562-gp91phox cells) as previously described (28). We also obtained a stable transformant expressing both p67phox and gp91phox (K562-gp91phox/p67phox cells) by further transducing the p67phox gene to K562-gp91phox cells, as previously described (22). The cDNA encoding the full-length of p41nox, p51nox, p47phox, or p67phox ligated to pEF-BOS was transfected by electroporation to K562-gp91phox or K562-gp91phox/p67phox cells. The K562 cells (2 x 107 cells/ml) were electroporated in the presence of 20 µg of the plasmids at 170 V, 1070 microfarads using a Gene Pulser (Bio-Rad). Cells were cultured for 36 h and tested for superoxide-producing activity.

Superoxide production by the indicated cells was determined by superoxide dismutase (SOD)-inhibitable chemiluminescence with an enhancer-containing luminol-based detection system (DIOGENES; National Diagnostics), as previously described (22, 28). After the addition of the enhanced luminol-based substrate, the cells were stimulated with 200 ng/ml phorbol 12-myristate 13-acetate (PMA). The chemiluminescence was assayed using a luminometer (Auto Lumat LB953; EG&G Berthold).

Activation of gp91phox and Nox1 in COS-7, HEK293, or CHO Cells— The cDNAs for gp91phox/Nox2 and Nox1 (13) were ligated to the expression vector pcDNA3.0 (Invitrogen). The monkey kidney COS-7 cells or human embryonic kidney HEK293 cells were transfected using LipofectAMINE (Invitrogen) with the following combinations of cDNAs: pcDNA3.0-gp91phox/Nox2 or pcDNA3.0-Nox1; pEF-BOS-p47phox or pEF-BOS-p41nox; and pEF-BOS-p67phox or pEF-BOS-p51nox. Although HEK293 cells lack Nox1 and gp91phox, they express a small amount of Nox4 (13). However, Nox4 in HEK293 cells does not seem to affect the assay system; a weak superoxide-producing activity could be detected in a cell-free system composed of the membrane fraction of the cells and NAD(P)H, whereas no activity was observed at the cellular level, indicating that Nox4 in HEK293 cells produces superoxide within the cells but not outside of the cells (13). In some cases, the Chinese hamster ovary CHO cells were transfected with pEF-BOS-p41nox, pEF-BOS-p51nox, pEF-BOS-p22phox, and pcDNA3.0-gp91phox/Nox2 or pcDNA3.0-Nox1. After being cultured for 30 h, adherent cells were harvested by incubating with trypsin/EDTA for 1 min at 37 °C and washed with Hepes-buffered saline (120 mM NaCl, 5 mM KCl, 5 mM glucose, 1 mM MgCl2, 0.5 mM CaCl2, and 17 mM Hepes, pH 7.4). Superoxide production by these cells was determined by SOD-inhibitable chemiluminescence, as described above.

Expression of the p51nox and p41nox Gene in Various Human Tissues—Real time PCR analyses were performed using the ABI PRISM® 7000 Sequence Detection System (Applied Biosystems) according to the manufacturer's instruction. The reaction mixture (25 µl) contained SYBR® Green PCR Master Mix (Applied Biosystems), 0.3 µM each primer, and 2.5 µl of the first strand cDNA from different human tissues (Human MTCTM panels I and II; Clontech) as a template. To quantitate the transcript levels of p41nox, p51nox, Nox1, and glyceraldehyde-3-phosphate dehydrogenase, we used the relative standard curve method as described by Johnson et al. (44). The primers used are as follows: 5'-TTCTCTGTGCGCTGGTCAGA-3' (forward primer) and 5'-TCTTGAGCTGCCTGAATTCGT-3' (reverse primer) for the p41nox cDNA; 5'-TGGGAGGTGCTACACAATGTG-3' (forward primer) and 5'-TTGGACATGGCCTCCCTTAG-3' (reverse primer) for the p51nox cDNA; 5'-TGTGGCCCTCGGACTTTG-3' (forward primer) and 5'-CCAGACTGGAATATCGGTGACA-3' (reverse primer) for the Nox1 cDNA; and 5'-GAAATCCCATCACCATCTTCCA-3' (forward primer) and 5'-CCTTCTCCATGGTGGTGAAGAC-3' (reverse primer) for the glyceraldehyde-3-phosphate dehydrogenase cDNA.

For Northern blot analysis, human multiple tissue Northern (MTNTM) blots (Clontech) were hybridized with a 32P-labeled cDNA fragment, encoding the region that corresponds to amino acids 1–153 of p51nox or amino acids 307–476 of p41nox, under high stringency conditions using ExpressHybTM (Clontech).


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Primary Structure and Domain Architecture of p41nox, a Novel Homologue of p47phoxWe have obtained a novel cDNA clone that encodes a protein similar to p47phox (GenBankTM accession number AB097667 [GenBank] ; for details, see "Experimental Procedures"). Since p47phox is considered to act as an organizer rather than a direct activator of the phagocyte oxidase (see Introduction), the novel gene encoding the p47phox homologue is named NADPH oxidase organizer 1 (NOXO1) after discussion with colleagues working in the field and consultation with Dr. Ruth Lovering (the Human Genome Organization Nomenclature Committee). The NOXO1 cDNA contains an open reading frame of 1,113 nucleotides with the first methionine codon surrounded by a sequence similar to the Kozak consensus (ACAGCCATGG) and an in-frame preceding termination codon. The encoded protein comprises 371 amino acids with a calculated molecular mass of about 41 kDa (Fig. 1), and thus the novel protein was designated as p41nox. When the protein was expressed in COS-7 cells, an apparent molecular mass was estimated at 43–44 kDa by SDS-PAGE analysis (data not shown). Searching the sequence data base revealed that a human genomic contig (GenBankTM accession number NT-010552) that is located on chromosome 16p13 contains an entire region of the p41nox cDNA. The p41nox protein described here is encoded by at least eight exons with exon-intron boundaries, covering a minimum of about 3 kb.



View larger version (40K):
[in this window]
[in a new window]
 
FIG. 1.
Structure of human p41nox, a novel homologue of p47phox. A, deduced amino acid sequence of human p41nox in comparison with human p47phox. Identical amino acid residues are indicated by asterisks. Shaded are the PX domain, the N-terminal and C-terminal SH3 domains (SH3(N) and SH3(C), respectively), the AIR, and the PRR. Boxed are the PRR and its C-terminal flanking region. B, a schematic representation of the domain arrangement of human p41nox and p47phox.

 

Although the overall amino acid sequence of human p41nox shows only about 23% identity with human p47phox (Fig. 1A), the domain arrangements of both proteins are basically conserved; p41nox contains the N-terminal PX domain (45, 46), the two SH3 domains tandemly arranged in the middle, and the C-terminal PRR (Fig. 1B). In addition to the PRR of p41nox, its C-terminally flanking region is also similar to that of p47phox, which plays an important role in binding to p67phox together with the PRR (19). On the other hand, p41nox lacks a region that is expected to mask its SH3 domains; the corresponding region of p47phox (amino acids 286–340), the AIR, intramolecularly binds to the SH3 domains, and thereby prevents the domains from binding to the target p22phox (22). The stretch Pro299-Pro300-Arg301-Arg303 in the AIR of p47phox directly interacts with the N-terminal SH3 domain (22), and phosphorylation of this region, particularly at Ser-303, Ser-304, and Ser-328, culminates in induction of a conformational change, which renders the SH3 domains in a state accessible to p22phox (22, 26). These amino acids are all missing in p41nox, suggesting that accessibility of the SH3 domains of p41nox is not regulated, and thus the novel protein may act as a constitutively active organizer of the oxidase accordingly.

Primary Structure and Domain Architecture of p51nox, a Novel Homologue of p67phoxWe have also cloned a novel human cDNA encoding a protein homologous to p67phox (GenBankTM accession number AB095031 [GenBank] ; for details, see "Experimental Procedures"). After discussion with colleagues working in the field and Dr. Lovering, the gene has been termed NADPH oxidase activator 1 (NOXA1), because p67phox is likely to function as a direct activator of the phagocyte NADPH oxidase (see Introduction). The cDNA clone contains an open reading frame of 1,428 nucleotides with the first methionine codon, which is surrounded by a sequence that completely agrees with the Kozak consensus (GCCGCCATGG). The predicted protein product of the NOXA1 gene consists of 476 amino acids (Fig. 2A). Because of a calculated molecular mass of about 51 kDa, this protein is tentatively designated as p51nox; a slightly higher molecular mass (53–54 kDa) was estimated by SDS-PAGE when expressed in COS-7 cells (data not shown).



View larger version (41K):
[in this window]
[in a new window]
 
FIG. 2.
Structure of human p51nox, a novel homologue of p67phox. A, deduced amino acid sequence of human p51nox in comparison with human p67phox. Identical amino acid residues are indicated by asterisks. Shaded are the TPR motifs 1–4, a so-called activation domain, the N-terminal SH3 domain (SH3(N)), the PB1 domain, and the C-terminal SH3 domain (SH3(C)). B, a schematic representation of the domain arrangement of human p51nox and p67phox.

 

Although the overall amino acid sequence of human p51nox exhibits only about 28% identity with human p67phox, the domain architectures of both proteins are very similar, except that the N-terminal SH3 domain is absent in p51nox (Fig. 2, A and B). The most conserved region is the C-terminal SH3 domain, with 52% identity, a domain of p67phox known to directly bind to the C-terminal PRR of p47phox (1719). It is also known that p67phox interacts with p40phox via the Phox and Bem 1 (PB1) domain between the SH3 domains (47). There also exists the PB1-like region in p51nox, but without the conserved lysine, corresponding to Lys-355 of p67phox, that plays an essential role in the interaction with p40phox (40, 47).

The small GTPase Rac directly binds to the N-terminal region of p67phox containing ~200 amino acid residues, an interaction that is essential for activation of the phagocyte NADPH oxidase (28). There exist four TPR motifs in the Rac-binding domain (28, 29), and Arg-102 on the third motif plays a critical role (28); the R102E substitution results in a loss of both the interaction with Rac and the oxidase activation (28). The N-terminal region of p51nox is well conserved, with 39% amino acid identity to that of p67phox; it contains four TPR motifs and keeps the corresponding arginine residue (Arg-103) in the third TPR, suggesting that p51nox may interact with Rac. In addition, an about 10-amino acid stretch, C-terminal to the Rac-binding domain, is highly conserved between p51nox and p67phox. The stretch of p67phox, a so-called activation domain, is required for the phagocyte oxidase activation (48, 49); deletion of the stretch or the V204A substitution leads to an impaired activation of the oxidase (4850). Thus, p51nox is expected to function as an activator of the phagocyte oxidase gp91phox/Nox2.

p41nox Interacts with p22phox and p67phoxAs an initial step to investigate the function of p41nox, we tested the ability of p41nox to bind to other oxidase factors. It is established that p47phox binds via the SH3 domains to the C-terminal tail of p22phox, which plays an essential role in activation of the phagocyte NADPH oxidase. Similarly, the SH3 domains of p41nox bound to the C terminus of p22phox, as estimated in the yeast two-hybrid system (Fig. 3A) and by an in vitro binding assay using purified proteins (Fig. 3B). However, the domains could not interact with a p22phox carrying the P156Q substitution, a mutation that occurs in a patient with chronic granulomatous disease (51, 52). Full-length p47phox (p47phox-F) was incapable of binding to p22phox, since the SH3 domains of p47phox are normally masked via an intramolecular interaction with the AIR (Fig. 3, A and B; see Refs. 20 and 22). Interestingly, full-length p41nox (p41nox-F) interacted with p22phox (Fig. 3A), which appears to be in agreement with the fact that the AIR is absent in p41nox (Fig. 1).



View larger version (29K):
[in this window]
[in a new window]
 
FIG. 3.
Interaction of human p41nox with other oxidase factors. Interaction of p41nox with p22phox was estimated by the yeast two-hybrid system (A) and by an in vitro pull-down assay using purified proteins (B). A, the yeast HF7c cells were cotransformed with a pair of pGBT9 encoding the wild-type p22phox-C (amino acids 132–195) or a p22phox-C carrying the P156Q substitution and pGADGH encoding the isolated tandem SH3 domains or full-length protein of p41nox and p47phox: p41nox-(SH3)2, p47phox-(SH3)2, p41nox-F, or p47phox-F. Following the selection for Trp+ and Leu+ phenotype, its histidine-dependent (right) and -independent (left) growth was tested as described under "Experimental Procedures." B, GST alone, GST-p22phox-C, or GST-p22phox-C(P156Q) was incubated with MBP-p41nox-(SH3)2 and pulled down with glutathione-Sepharose. The precipitated proteins were subjected to SDS-PAGE, followed by staining with Coomassie Brilliant Blue. Positions for marker proteins are indicated in kDa. Interaction of p41nox or p47phox with p51nox or p67phox was estimated by the yeast two-hybrid system (C) and by an immunoprecipitation assay using cells expressing the proteins (D). C, the yeast HF7c cells were cotransformed with a pair of pGBT9 encoding the C terminus of p41nox (p41nox-C) or p47phox (p47phox-C) and pGADGH encoding p51nox-SH3 or p67phox-SH3(C). Following the selection for Trp+ and Leu+ phenotype, its histidine-dependent (right) and -independent (left) growth was tested. D, COS-7 cells were transfected with a pair of the expression constructs as indicated above each lane; the constructs were pEF-BOS-Myc-p41nox, pEF-BOS-Myc-p47phox, pEF-BOS-HA-p51nox, and pEF-BOS-HA-p67phox. Lysates of the transfected cells were analyzed by immunoprecipitation (IP) with the anti-Myc monoclonal antibody, followed by immunoblot (Blot) with the anti-Myc (upper panels) or anti-HA (lower panels) polyclonal antibodies. For details, see "Experimental Procedures." These experiments have been repeated more than three times with similar results.

 

It is also known that p47phox associates with p67phox via the C-terminal region containing the PRR (1719). As shown in Fig. 3 (C and D), p41nox was capable of interacting with p67phox as well. The findings suggest the possibility that p41nox may activate the phagocyte oxidase instead of p47phox by interacting with the oxidase factors.

p51nox Interacts with Rac but Fails to Interact with p40phoxIn the yeast two-hybrid system, p51nox interacted with Rac1 (Q61L), a constitutively active form, but not with a dominant negative form of Rac (T17N), as p67phox did (Fig. 4A). The same interaction was observed when Rac2, but not Cdc42, was used instead of Rac1 (Fig. 4A). In addition, p51nox bound to GTP-bound Rac1 in an in vitro binding assay using purified proteins, whereas it failed to interact with GDP-bound Rac1 (Fig. 4B). Thus, p51nox appears to directly bind to GTP-bound Rac. The substitution of Glu for the conserved arginine Arg-103 in p51nox led to a complete loss of the interaction with Rac (Fig. 4A). Thus, p51nox probably recognizes Rac in a similar manner as does p67phox, suggesting that the novel homologue also acts as an oxidase activator.



View larger version (27K):
[in this window]
[in a new window]
 
FIG. 4.
Interaction of human p51nox with other oxidase factors. Interaction of p51nox with Rac (A and B) or p40phox (C and D) was estimated by the yeast two-hybrid system (A) and by an in vitro pull-down assay using purified proteins (B). A, the yeast HF7c cells were cotransformed with recombinant plasmids pGBT9 encoding Rac1, Rac2, or Cdc42 carrying a mutation and pGADGH encoding the N terminus of the wild-type or a mutant p51nox or p67phox: Q61L (a constitutively active form of the GTPases) and T17N (a dominant negative form of the GTPases). Following the selection for Trp+ and Leu+ phenotype, its histidine-dependent (right) and independent (left) growth was tested as described under "Experimental Procedures." B, GST alone or GST-p51nox-N (amino acids 1–224) was incubated with His-Rac1 (Q61L) containing GTP{gamma}S or wild-type Rac1 containing GDP and pulled down with glutathione-Sepharose. The precipitated proteins were subjected to SDS-PAGE, followed by immunoblot analysis with an anti-His anti-body. Positions for marker proteins are indicated in kDa. Interaction of p51nox with p40phox was estimated by the yeast two-hybrid system (C) and by an immunoprecipitation assay using cells expressing the proteins (D). C, the yeast HF7c cells were cotransformed with a pair of pGBT9 encoding the full length of p51nox (p51nox-F) or p67phox (p67phox-F) and pGADGH encoding the full length (amino acids 1–339) or the C terminus (residues 234–339) of p40phox (p40phox-F and p40phox-C, respectively). Following the selection for Trp+ and Leu+ phenotype, its histidine-dependent (right) and -independent (left) growth was tested. D, COS-7 cells were cotransfected with either pEF-BOS-HA-p51nox or pEF-BOS-HA-p67phox and pEF-BOS-Myc-p40phox, as indicated above each lane. Lysates of the transfected cells were analyzed by immunoprecipitation (IP) with the anti-Myc monoclonal anti-body, followed by immunoblot (Blot) with the anti-Myc (upper panels) or anti-HA (lower panels) polyclonal antibodies. Proteins in cell lysates were also analyzed directly by immunoblot. For details, see "Experimental Procedures." These experiments have been repeated more than three times with similar results.

 

Furthermore, p51nox was also capable of binding to p47phox and p41nox (Fig. 3D). The binding is considered to be mediated via the SH3 domain; the substitution of Arg for Trp-436, the invariant residue among SH3 domains, resulted in a loss of the interaction (Fig. 3C). Since p51nox retains the PB1 domain, albeit with a weak similarity (Fig. 2), it seemed possible that p51nox also can bind to p40phox. However, we could not detect interaction between p51nox and p40phox in the yeast two-hybrid system (Fig. 4C) or in an immunoprecipitation assay using cells expressing both proteins (Fig. 4D), whereas p67phox fully bound to p40phox under the same conditions. These findings appear to be consistent with the fact that the PB1-like region of p51nox lacks the conserved lysine residue corresponding to Lys-355 of p67phox (Fig. 2).

p41nox and p51nox Are Capable of Supporting Activation of the Phagocyte NADPH Oxidase in K562 Cells—To explore the function of p41nox, we transfected the K562-gp91phox/p67phox cells with the mammalian expression vector pEF-BOS encoding cDNA for p41nox or p47phox. The K562-gp91phox/p67phox cells stably express functional cytochrome b558 comprising the two subunits gp91phox and p22phox as well as p67phox, but they do not contain p47phox (22). As shown in Fig. 5A, the p41nox-expressing cells fully produced superoxide in response to PMA, a potent stimulant of the phagocyte NADPH oxidase in vivo, indicating that p41nox is a functional homologue of p47phox. The activity of p41nox appeared to be higher than that of p47phox (Fig. 5A); the difference is not due to their expression level, since immunoblot analysis revealed that essentially the same amounts of these proteins were expressed in the reconstitution system (data not shown). The cooperation of p41nox with p67phox in the oxidase activation agrees with the present finding that p41nox, as well as p47phox, binds to p67phox (Fig. 3, C and D).



View larger version (17K):
[in this window]
[in a new window]
 
FIG. 5.
Effects of p41nox and p51nox on activation of the phagocyte NADPH oxidase in K562 cells. A, the K562-gp91phox/p67phox cells were transfected with the mammalian expression vector pEF-BOS encoding cDNA for either p41nox or p47phox or pEF-BOS alone. The transfected cells (1 x 105 cells) were stimulated with PMA (200 ng/ml), and chemiluminescence change was continuously monitored with an enhanced luminol-based substrate, DIOGENES. SOD (50 µg/ml) was added where indicated. B, the K562-gp91phox cells were transfected with a pair of pEF-BOS-p41nox and pEF-BOS-p51nox or a pair of pEF-BOS-p47phox and pEF-BOS-p67phox. The doubly transfected cells (1 x 105 cells) were stimulated with PMA (200 ng/ml). Chemiluminescence change was continuously monitored with DIOGENES, and SOD (50 µg/ml) was added where indicated. For details, see "Experimental Procedures." These experiments have been repeated more than three times with similar results.

 

Since p41nox can interact not only with p67phox but also with p51nox (Fig. 3, C and D), it seems likely that p41nox also functions together with p51nox. To test this possibility, we expressed both p41nox and p51nox in K562-gp91phox cells that express functional cytochrome b558 but lack p47phox and p67phox (28). In response to PMA, the K562-gp91phox cells cotransfected with p41nox and p51nox produced superoxide at a level comparable with the cells transfected with p47phox and p67phox (Fig. 5B) and the cells transfected with p41nox and p67phox (data not shown). In contrast, the cells expressing both p47phox and p51nox only marginally produced superoxide, when simulated with PMA (data not shown). Thus, p41nox likely functions in combination with p51nox as well as with p67phox in activation of the phagocyte oxidase gp91phox.

Activation of gp91phox/Nox2 by p41nox and p51nox in COS-7 Cells—It has recently been reported that the monkey kidney COS-7 cells are useful for functional reconstitution of the phagocyte NADPH oxidase system at the cellular level (50). As expected, superoxide was produced in response to PMA by COS-7 cells transfected simultaneously with pcDNA3.0-gp91phox, pEF-BOS-p47phox, and pEF-BOS-p67phox (Fig. 6). The superoxide production was not observed when cells were transfected with the gp91phox cDNA alone or with solely the p47phox and p67phox cDNA (data not shown). These findings indicate that the phagocyte oxidase is successfully reconstituted in COS-7 cells. We next coexpressed gp91phox with a pair of p41nox and p51nox in COS-7 cells, instead of a pair of p47phox and p67phox. Interestingly, the cells expressing the three proteins produced superoxide in the absence of any stimulants. The superoxide production was increased in response to PMA (Fig. 6). Furthermore, cells containing gp91phox, p41nox, and p67phox also generated superoxide spontaneously, which was also enhanced by the addition of PMA (Fig. 6). On the other hand, gp91phox was marginally activated by a pair of p47phox and p51nox, even when cells were stimulated with PMA (Fig. 6).



View larger version (27K):
[in this window]
[in a new window]
 
FIG. 6.
Effects of p41nox and p51nox on activation of gp91phox in COS-7 cells. COS-7 cells were transfected simultaneously with pcDNA3.0-gp91phox and the following pair of pEF-BOS vectors encoding the oxidase organizer and activator homologues: p41nox and p51nox; p41nox and p67phox; p47phox and p51nox;orp47phox and p67phox. The transfected cells (1 x 105 cells) were preincubated for 5 min at 37 °C and then stimulated with PMA (200 ng/ml). Chemiluminescence change was continuously monitored with DIOGENES, and SOD (50 µg/ml) was added where indicated. For details, see "Experimental Procedures." The experiments have been repeated more than three times with similar results.

 

Expression of mRNAs for p41nox and p51noxWe next studied expression of the NOXO1 and NOXA1 genes encoding p41nox and p51nox, respectively. To quantitatively compare the expression of the two genes among various human tissues, we performed real time PCR analyses. As shown in Fig. 7A, the mRNA for p41nox was abundant in colon and testis and also present in tissues such as pancreas, liver, thymus, and small intestine, but to a lesser extent. The p51nox mRNA abundantly existed in pancreas; it was less but significantly expressed in liver, kidney, spleen, prostate, small intestine, and colon. Intriguingly, both mRNAs occurred together in several tissues, including colon, small intestine, liver, and pancreas (Fig. 7B). On the other hand, the NOX1 mRNA was most predominantly expressed in colon among adult tissues tested (Fig. 7C), which is in agreement with previous findings by other groups (10, 11, 14).



View larger version (18K):
[in this window]
[in a new window]
 
FIG. 7.
Expression of p41nox, p51nox, and Nox1 in human tissues. The expression levels of mRNAs for the NOXO1, NOXA1, and NOX1 genes encoding p41nox (A), p51nox (B), and Nox1 (C), respectively, were analyzed by real time PCR using Human Multiple Tissue cDNA panels (Clontech). sk. muscle, skeletal muscle; small intest., small intestine. Each graph represents the mean of the content of the indicated transcript normalized to glyceraldehyde-3-phosphate dehydrogenase content in triplicate samples, with bars representing the S.D. For details, see "Experimental Procedures."

 

The NOXO1 and NOXA1 genes appear to be expressed solely in restricted cells of the tissues or at a low level in most cells, since detection of the PCR products for the two genes in any tissues required much more PCR cycles than that for the NOX1 gene (data not shown). Essentially the same patterns of tissue expression of the p51nox and p41nox genes were observed by Northern blot analysis (data not shown), although only weak signals could be obtained, which is consistent with the results of the PCR detection as mentioned above.

Activation of Nox1 by p41nox and p51nox in HEK293 and COS-7 Cells—Expression of both p41nox and p51nox in human colon suggests that they may be involved in activation of the Nox1 oxidase, which is abundantly present in this organ (Fig. 7). To test this possibility, we transfected the human embryonic kidney HEK293 cells with pcDNA3.0-Nox1, pEF-BOS-p41nox, and pEF-BOS-p51nox. As shown in Fig. 8A, cells cotransfected with all the three plasmids produced superoxide without any stimulants added. On the other hand, the production was not observed in cells that were individually transfected with Nox1, p41nox, or p51nox (data not shown) or in cells expressing both p41nox and p51nox but not Nox1 (Fig. 8A). Since p41nox and p51nox are adaptor proteins without any known catalytic domains, as shown here, it is conceivable that superoxide is directly produced by Nox1. This proposal is supported by the observation that superoxide production is almost completely blocked by treatment of the cells with 5 µM diphenylene iodonium, an inhibitor of the Nox family oxidases (data not shown). The superoxide production by Nox1 was enhanced by the addition of PMA (Fig. 8A).



View larger version (13K):
[in this window]
[in a new window]
 
FIG. 8.
Effects of p41nox and p51nox on activation of Nox1 in HEK293 and COS-7 cells. A, HEK293 cells were transfected simultaneously with pcDNA3.0-Nox1, pEF-BOS-p41nox, and pEF-BOS-p51nox or with pcDNA3.0 alone, pEF-BOS-p41nox, and pEF-BOS-p51nox. The transfected cells (1 x 105 cells) were incubated for 5 min at 37 °C and then stimulated with PMA (200 ng/ml). Chemiluminescence change was continuously monitored with DIOGENES, and SOD (50 µg/ml) was added where indicated. For details, see "Experimental Procedures." The experiments have been repeated more than three times with similar results. B, COS-7 cells were transfected simultaneously with pcDNA3.0-Nox1 and the following pair of pEF-BOS vectors encoding the oxidase organizer and activator homologues: p41nox and p51nox; p41nox and p67phox; p47phox and p51nox;orp47phox and p67phox. The transfected cells (1 x 105 cells) were incubated for 5 min at 37 °C and then stimulated with PMA (200 ng/ml). Chemiluminescence change was continuously monitored with DIOGENES. Superoxide production is expressed as the percentage of activity relative to control cells transfected with pcDNA3.0-Nox1, pEF-BOS-p41nox, and pEF-BOS-p51nox. Each graph represents the mean of data from three independent transfections.

 

To confirm that Nox1 can be activated by a pair of p41nox and p51nox, we next used COS-7 cells. Coexpression of Nox1 with both p41nox and p51nox in these cells also resulted in a constitutive production of superoxide (Fig. 8B). The addition of PMA to these cells increased the superoxide production, consistent with the results of the experiments using HEK293 cells (Fig. 8A). These findings indicate that p41nox and p51nox support activation of Nox1; activation appears to be partially constitutive.

We further tested the effect of other pairs of the p47phox and p67phox homologues in activation of Nox1. In the COS-7 cells where Nox1 was coexpressed with a pair of p41nox and p67phox, production was produced without stimuli, and the production was facilitated upon stimulation with PMA (Fig. 8B). Although Nox1 was normally inactive in cells that expressed both p47phox and p51nox, the oxidase became active in response to PMA. Thus, stimulus-independent production of superoxide occurred only when p41nox was present instead of p47phox. On the other hand, Nox1 could not be activated either with or without stimuli by a pair of the classical homologues p47phox and p67phox under the present conditions.

Effect of p22phox on Nox1-dependent Superoxide Production—It is well established that gp91phox tightly associates with p22phox in phagocytes, whereas it has remained unclear whether Nox1 is also capable of forming a complex with p22phox. To address this question, we tested the effect of expression of p22phox on Nox1-dependent superoxide production. For this purpose, we used CHO cells, since it has been reported that CHO cells scarcely express the mRNA of p22phox (53). The p22phox mRNA is known to be present in a wide variety of cells (14); COS-7 and HEK293 cells used in the present study indeed expressed a significant amount of the p22phox mRNA, which is consistent with the observation that transfection of these cells with the p22phox cDNA was not required for the gp91phox activity (Fig. 6; data not shown). As expected, gp91phox was almost dormant in CHO cells cotransfected with the p41nox and p51nox cDNAs but not with the p22phox cDNA, even when cells were stimulated with PMA (Fig. 9A). Simultaneous transfection with gp91phox and p22phox led to superoxide production in CHO cells expressing both p41nox and p51nox, a production that was facilitated upon cell stimulation with PMA (Fig. 9A). When Nox1 was expressed in CHO cells instead of gp91phox under these conditions, a small amount of superoxide was produced without cotransfection with the p22phox cDNA (Fig. 9B). Treatment of cells with PMA slightly increased the Nox1-dependent superoxide production. Intriguingly, coexpression of p22phox culminated in a great enhancement of superoxide production by Nox1, either with or without PMA stimulation (Fig. 9B). These findings suggest that Nox1 can be complexed with p22phox.



View larger version (14K):
[in this window]
[in a new window]
 
FIG. 9.
Effect of p22phox on gp91phox- and Nox1-dependent superoxide production in CHO cells expressing both p41nox and p51nox. A, CHO cells were transfected simultaneously with pcDNA3.0-gp91phox, pEF-BOS-p41nox, and pEF-BOS-p51nox or with pcDNA3.0-gp91phox, pEF-BOS-p41nox, pEF-BOS-p51nox, and pEF-BOS-p22phox. The transfected cells (1 x 105 cells) were incubated for 5 min at 37 °C and then stimulated with PMA (200 ng/ml). Chemiluminescence change was continuously monitored with DIOGENES. Superoxide production is expressed as the percentage of activity relative to control cells transfected with pcDNA3.0-gp91phox, pEF-BOS-p41nox, pEF-BOS-p51nox, and pEF-BOS-p22phox. Each graph represents the mean of data from three independent transfections. B, CHO cells were transfected simultaneously with pcDNA3.0-Nox1, pEF-BOS-p41nox, and pEF-BOS-p51nox or with pcDNA3.0-Nox1, pEF-BOS-p41nox, pEF-BOS-p51nox, and pEF-BOS-p22phox. The transfected cells (1 x 105 cells) were incubated for 5 min at 37 °C and then stimulated with PMA (200 ng/ml). Chemiluminescence change was continuously monitored with DIOGENES. Superoxide production is expressed as the percentage of activity relative to control cells transfected with pcDNA3.0-Nox1, pEF-BOS-p41nox, pEF-BOS-p51nox, and pEF-BOS-p22phox. Each graph represents the mean of data from three independent transfections.

 


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
In this study, we have identified novel functional homologues of the phagocyte oxidase proteins p47phox and p67phox, tentatively designated p41nox (the NOXO1 gene product) and p51nox (the NOXA1 gene product), respectively. The homologues retain most of the functional modules for protein-protein interactions: the SH3 domains of p41nox interact with p22phox; p41nox, as well as p47phox, binds to p51nox and p67phox via a tail-to-tail interaction; and the N-terminal TPR domain of p51nox interacts with Rac in a GTP-bound state. Consistent with the conservation of the crucial interactions between oxidase factors, p41nox and p51nox function together or in combination with a classical one to support superoxide production by the phagocyte NADPH oxidase. We also show that Nox1 also can be activated by the novel homologues, a finding that will help our understanding of the regulation of Nox1. During the completion of the present study, Bánfi et al. (54) have reported the cloning of mouse NOXO1 and NOXA1 and shown that they are capable of activating Nox1.

Activation of the phagocyte NADPH oxidase gp91phox is known to require, as a switch, at least two events elicited during intracellular signal transduction in stimulated cells. One of the events is a conformational change of p47phox. The SH3 domains of p47phox are normally masked via an intramolecular interaction with the AIR; upon cell stimulation, p47phox is phosphorylated, which leads to its conformational change that allows the domains to become accessible to p22phox. The resultant interaction between p47phox and p22phox plays an essential role in the oxidase activation (2022). The other crucial event is conversion of Rac to the active state; only the GTP-bound Rac, but not the GDP-bound one, binds to p67phox, thereby activating the phagocyte oxidase. In resting phagocytes, both switches are kept "off," preventing the oxidase from producing superoxide. In contrast, the present findings have demonstrated that novel homologues of p47phox and p67phox can activate gp91phox and Nox1 without cell stimulation under certain conditions (Figs. 6 and 8). In COS-7 cells expressing a pair of p41nox and p51nox or a pair of p41nox and p67phox, gp91phox seems to be constitutively active, albeit not in a state fully activated (Fig. 6). On the other hand, PMA is absolutely required for activation of gp91phox in cells that contain both p47phox and p67phox (Fig. 6). Similarly, Nox1 spontaneously generated superoxide when it was cotransfected in COS-7 cells with both cDNAs for p41nox and p51nox or with those for p41nox and p67phox (Fig. 8). In the presence of p47phox and p51nox, however, activation of this oxidase completely depends on the addition of PMA as a stimulant (Fig. 8).

Thus stimulus-independent activation of Nox1 and gp91phox seems to require p41nox. On the other hand, Nox1 and gp91phox are both dormant in cells expressing p47phox instead of p41nox, but become activated in response to PMA. In this context, it should be noted that p41nox does not harbor a region homologous to the AIR that prevents the SH3 domains of p47phox from interacting with p22phox (Fig. 1). Indeed, the SH3 domains of p41nox are considered to be in a state accessible to its target, since full-length p41nox binds to p22phox (Fig. 3). On the other hand, full-length p47phox is incapable of interacting with p22phox under the conditions where its isolated SH3 domains fully associate with the target (Fig. 3). It is thus likely that p41nox occurs in a constitutively active form, whereas p47phox is normally inactive but becomes accessible to p22phox via the stimulus-induced conformational change. The difference may explain the reason why p41nox is more active than p47phox even in activation of gp91phox (Figs. 5A and 6) and Nox1 (Fig. 8B), since it seems very unlikely that all of the p47phox molecules become activated at the same time in cells; it is well known that only a part (about 10%) of p47phox translocates to the membrane when neutrophils are stimulated with PMA, one of the most potent activators of the phagocyte oxidase in vivo (39).

The present study suggests that Nox1 also forms a heterodimer with p22phox as gp91phox/Nox2 does (Fig. 9). In CHO cells containing p41nox and p51nox, stimulus-independent superoxide production by Nox1 is greatly enhanced by ectopic expression of p22phox (Fig. 9). This finding supports the above mentioned hypothesis that p41nox functions, via binding to p22phox without stimulation, in constitutive activation of Nox1. Whereas gp91phox is almost inactive in CHO cells that are not transfected with the cDNA of p22phox, Nox1 can produce a small amount of superoxide without the transfection (Fig. 9). The reason for the difference is currently unknown. It may be possible that Nox1 interacts with p22phox with a much higher affinity than does gp91phox if there is a trace, undetectable amount of p22phox in CHO cells. It seems also possible that Nox1 might be complexed with a heretofore unidentified homologue of p22phox in CHO cells. This question should be addressed in the future studies.

In addition to the difference between p41nox and p47phox, the regulation of p51nox also does not seem to be the same as that of its homologue p67phox. We have recently shown that p40phox enhances the phagocyte oxidase activation by facilitating the membrane translocation of p67phox and p47phox and that this effect is totally dependent on the interaction of p40phox with p67phox (40). Since p51nox is incapable of interacting with p40phox (Fig. 4), the regulation mediated via p40phox cannot be expected in an activation system involving p51nox. Although the target protein for the N-terminal SH3 domain of p67phox, the most conserved module of this protein (55), still remains unidentified, the absence of this domain in p51nox may suggest a difference in oxidase activation between the homologues.

Activation of gp91phox by p41nox and p51nox in K562 cells is entirely dependent on stimulation with PMA (Fig. 5), whereas, even without the addition of any stimulants, gp91phox can produce superoxide in COS-7 cells containing p41nox and p51nox (Fig. 6). The reason for this discrepancy is presently unknown. One possible explanation is that the state of Rac, a switch of the oxidase activation, may be different between types of cells; for instance, the amount of GTP-bound Rac might be higher in COS-7 cells than K562 cells. In the presence of GTP-bound Rac, a rate-limiting step will be the conformational change of p47phox, another switch of the oxidase activation; thus, PMA is still required in COS-7 cells with p47phox but not in ones with the constitutively active homologue p41nox (Fig. 6). Even in the latter case, the amount of GTP-bound Rac may be insufficient, because superoxide production is enhanced by the addition of PMA (Fig. 6). This stimulant is known to facilitate the formation of GTP-bound Rac at the cellular level (56). It is also possible that, in K562 cells, p41nox is kept in an inactive state by an unknown mechanism.

In summary, the present study demonstrates that distinct pairs of the novel and classical homologues of p47phox and p67phox are capable of supporting activation of gp91phox/Nox2 and Nox1, suggesting the existence of a common mechanism underlying the activation of Nox family oxidases. Future studies should clarify whether other Nox oxidases such as Nox4 are similarly regulated or not. In addition to the common features of the homologues of p47phox and p67phox, there are also differences between them in regulation of Nox oxidases. First, gp91phox likely prefers p67phox to p51nox as its activator (Fig. 6). In contrast, p51nox appears to activate Nox1 more efficiently than p67phox (Fig. 8). Second, distinct types of regulation for Nox oxidases are considered to be achieved by different combinations of the oxidase organizers p47phox and p41nox with the oxidase activators p67phox and p51nox; a phosphorylation-induced conformational change of p47phox upon cell stimulation serves as a switch to activate the oxidase, whereas p41nox seems to be constitutively in an active state; p67phox, but not p51nox, can regulate the oxidase activation in cooperation with p40phox. Thus, a certain Nox oxidase can be regulated in a manner dependent on the oxidase factors expressed in cells. For example, since p41nox and p51nox, as well as Nox1, are expressed in the colon, Nox1 is mainly controlled by the two novel homologues in this organ. The present findings suggest that the active oxidase complex in the colon comprises Nox1, p22phox, p41nox, p51nox, and Rac: Nox1 is likely complexed with p22phox at the membrane (Fig. 9); p41nox constitutively associates with the Nox1-p22phox complex via direct binding to p22phox (Fig. 3); and p51nox directly interacts with Rac and p41nox via the N-terminal TPR domain (Fig. 4) and the SH3 domain at the C terminus (Fig. 3), respectively. On the other hand, it is known that vascular smooth muscle cells express Nox1 (57) and produce superoxide in a stimulus-dependent manner (37). As expected from the dependence, p47phox exists in the cells and plays a crucial role in superoxide production (58), whereas the cells do not contain p67phox (37), raising the possibility that p51nox may be present instead.


    FOOTNOTES
 
The nucleotide sequence(s) reported in this paper has been submitted to the DDBJ/GenBankTM/EBI Data Bank with accession number(s) AB097667 [GenBank] and AB095031 [GenBank] .

* This work was supported in part by grants-in-aid for Scientific Research and National Project on Protein Structural and Functional Analyses from the Ministry of Education, Culture, Sports, Science, and Technology of Japan, ONO Medical Research Foundation, and the BIRD project of JST Corp. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

To whom correspondence should be addressed. Tel.: 81-92-642-6806; Fax: 81-92-642-6807; E-mail: hsumi{at}bioreg.kyushu-u.ac.jp.

1 The abbreviations used are: Nox, NAD(P)H oxidase; NOXO1, Nox organizer 1; NOXA1, Nox activator 1; PRR, proline-rich region; AIR, autoinhibitory region; PB1, Phox and Bem 1; GST, glutathione S-transferase; MBP, maltose-binding protein; PMA, phorbol 12-myristate 13-acetate; SOD, superoxide dismutase; SH3, Src homology 3; EST, expressed sequence tag; CHO, Chinese hamster ovary; TPR, tetratricopeptide repeat; GTP{gamma}S, guanosine 5'-3-O-(thio)triphosphate. Back


    ACKNOWLEDGMENTS
 
We are grateful to Yohko Kage (Kyushu University) for technical assistance.



    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Lambeth, J. D., Cheng, G., Arnold, R. S., and Edens, W. E. (2000) Trends Biochem. Sci. 25, 459–461[CrossRef][Medline] [Order article via Infotrieve]
  2. Lambeth, J. D. (2002) Curr. Opin. Hematol. 9, 11–17[CrossRef][Medline] [Order article via Infotrieve]
  3. DeLeo, F. R., and Quinn, M. T. (1996) J. Leukocyte Biol. 60, 677–691[Abstract]
  4. Babior, B. M. (1999) Blood 93, 1464–1476[Free Full Text]
  5. Nauseef, W. M. (1999) Proc. Assoc. Am. Physicians 111, 373–382[Medline] [Order article via Infotrieve]
  6. Clark, R. A. (1999) J. Infect. Dis. 179, Suppl. 2, S309–S317[Medline] [Order article via Infotrieve]
  7. Bokoch, G. M., and Diebold, B. A. (2002) Blood 100, 2692–2696[Abstract/Free Full Text]
  8. Sumimoto, H., Ito, T., Hata, K., Mizuki, K., Nakamura, R., Kage, Y., Sakaki, Y., Nakamura, M., and Takeshige, K. (1997) in Membrane Proteins: Structure, Function, and Expression Control (Hamasaki, N., and Mihara, K., eds) pp. 235–245, Kyushu University Press, Fukuoka, Japan
  9. Roos, D., de Boer, M., Kuribayashi, F., Meischl, C., Weening, R. S., Segal, A. W., Åhlin, A., Nemet, K., Hossle, J. P., Bernatowska-Matuszkiewicz, E., and Middleton-Price, H. (1996) Blood 87, 1663–1681[Free Full Text]
  10. Suh, Y. A., Arnold, R. S., Lassegue, B., Shi, J., Xu, X., Sorescu, D., Chung, A. B., Griendling, K. K., Lambeth, J. D. (1999) Nature 401, 79–82[CrossRef][Medline] [Order article via Infotrieve]
  11. Bánfi, B., Maturana, A., Jaconi, S., Arnaudeau, S., Laforge, T., Sinha, B., Ligeti, E., Demaurex, N., Krause, K.-H. (2000) Science 287, 138–142[Abstract/Free Full Text]
  12. Geiszt, M., Kopp, J. B., Várnai, P., and Leto, T. L. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 8010–8014[Abstract/Free Full Text]
  13. Shiose, A., Kuroda, J., Tsuruya, K., Hirai, M., Hirakata, H., Naito, S., Hattori, M., Sakaki, Y., and Sumimoto, H. (2001) J. Biol. Chem. 276, 1417–1423[Abstract/Free Full Text]
  14. Cheng, G., Cao, Z., Xu, X., van Meir, E. G., and Lambeth, J. D. (2001) Gene (Amst.) 269, 131–140[CrossRef][Medline] [Order article via Infotrieve]
  15. Heyworth, P. G., Curnutte, J. T., Nauseef, W. M., Volpp, B. D., Pearson, D. W., Rosen, H., and Clark, R. A. (1991) J. Clin. Invest. 87, 352–356[Medline] [Order article via Infotrieve]
  16. Dusi, S., Donini, M., and Rossi, F. (1996) Biochem. J. 314, 409–412[Medline] [Order article via Infotrieve]
  17. Finan, P., Shimizu, Y., Gout, I., Hsuan, J., Truong, O., Butcher, C., Bennett, P., Waterfield, M. D., and Kellie, S. (1994) J. Biol. Chem. 269, 13752–13755[Abstract/Free Full Text]
  18. Leusen, J. H., Fluiter, K., Hilarius, P. M., Roos, D., Verhoeven, A. J., and Bolscher, B. G. (1995) J. Biol. Chem. 270, 11216–11221[Abstract/Free Full Text]
  19. Kami, K., Takeya, R., Sumimoto, H., and Kohda, D. (2002) EMBO J. 21, 4268–4276[Abstract/Free Full Text]
  20. Sumimoto, H., Kage, Y., Nunoi, H., Sasaki, H., Nose, T., Fukumaki, Y., Ohno, M., Minakami, S., and Takeshige, K. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 5345–5349[Abstract]
  21. Leto, T. L., Adams, A. G., and de Mendez, I. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 10650–10654[Abstract/Free Full Text]
  22. Ago, T., Nunoi, H., Ito, T., and Sumimoto, H. (1999) J. Biol. Chem. 274, 33644–33653[Abstract/Free Full Text]
  23. Rotrosen, D., and Leto, T. L. (1990) J. Biol. Chem. 265, 19910–19915[Abstract/Free Full Text]
  24. El Benna, J., Faust, L. P., and Babior, B. M. (1994) J. Biol. Chem. 269, 23431–23436[Abstract/Free Full Text]
  25. Huang, J., and Kleinberg, M. E. (1999) J. Biol. Chem. 274, 19731–19737[Abstract/Free Full Text]
  26. Shiose, A., and Sumimoto, H. (2000) J. Biol. Chem. 275, 13793–13801[Abstract/Free Full Text]
  27. Heyworth, P. G., Bohl, B. P., Bokoch, G. M., and Curnutte, J. T. (1994) J. Biol. Chem. 269, 30749–30752[Abstract/Free Full Text]
  28. Koga, H., Terasawa, H., Nunoi, H., Takeshige, K., Inagaki, F., and Sumimoto, H. (1999) J. Biol. Chem. 274, 25051–25060[Abstract/Free Full Text]
  29. Lapouge, K., Smith, S. J. M., Walker, P. A., Gamblin, S. J., Smerdon, S. J., and Rittinger, K. (2000) Mol. Cell 6, 899–907[Medline] [Order article via Infotrieve]
  30. Nisimoto, Y., Motalebi, S., Han, C.-H., and Lambeth, J. D. (1999) J. Biol. Chem. 274, 22999–23005[Abstract/Free Full Text]
  31. Diebold, B. A., and Bokoch, G. M. (2001) Nat. Immunol. 2, 211–215[CrossRef][Medline] [Order article via Infotrieve]
  32. Dang, P. M., Cross, A. R., and Babior, B. M. (2001) Proc. Natl. Acad. Sci. U. S. A. 98, 3001–3005[Abstract/Free Full Text]
  33. Gorzalczany, Y., Alloul, N., Sigal, N., Weinbaum, C., and Pick, E. (2002) J. Biol. Chem. 277, 18605–18610[Abstract/Free Full Text]
  34. Freeman, J. L., and Lambeth, J. D. (1996) J. Biol. Chem. 271, 22578–22582[Abstract/Free Full Text]
  35. Koshkin, V., Lotan, O., and Pick, E. (1996) J. Biol. Chem. 271, 30326–30329[Abstract/Free Full Text]
  36. Bánfi, B., Molnár, G., Maturana, A., Steger, K., Hegedûs, B., Demaurex, N., Krause, K.-H. (2001) J. Biol. Chem. 276, 37594–37601[Abstract/Free Full Text]
  37. Patterson, C., Ruef, J., Madamanchi, N. R., Barry-Lane, P, Hu, Z., Horaist, C., Ballinger, C. A., Brasier, A. R., Bode, C., and Runge, M. S. (1999) J. Biol. Chem. 274, 19814–19822[Abstract/Free Full Text]
  38. Griendling, K. K., and Ushio-Fukai, M. (1997) Trends Cardiovasc. Med. 7, 301–307[CrossRef]
  39. Brar, S. S., Kennedy, T. P., Sturrock, A. B., Huecksteadt, T. P., Quinn, M. T., Whorton, A. R., and Hoidal, J. R. (2002) Am. J. Physiol. 282, C1212–C1224
  40. Kuribayashi, F., Nunoi, N., Wakamatsu, K., Tsunawaki, S., Sato, K., Ito, T., and Sumimoto, H. (2002) EMBO J. 21, 6312–6320[Abstract/Free Full Text]
  41. Mizushima, S., and Nagata, S. (1990) Nucleic Acids Res. 18, 5322[Medline] [Order article via Infotrieve]
  42. Noda, Y., Takeya, R., Ohno, S., Naito, S., Ito, T., and Sumimoto, H. (2001) Genes Cells 6, 107–119[Abstract/Free Full Text]
  43. Kohjima, M., Noda, Y., Takeya, R., Saito, N., Takeuchi, K., and Sumimoto, H. (2002) Biochem. Biophys. Res. Commun. 299, 641–646[CrossRef][Medline] [Order article via Infotrieve]
  44. Johnson, M. R., Wang, K., Smith, J. B., Heslin, M. J., and Diasio, R. B. (2000) Anal. Biochem. 278, 175–184[CrossRef][Medline] [Order article via Infotrieve]
  45. Hiroaki, H., Ago, T., Ito, T., Sumimoto, H., and Kohda, D. (2001) Nat. Struct. Biol. 6, 526–530
  46. Ago, T., Kuribayashi, F., Hiroaki, H., Takeya, R., Ito, T., Kohda, D., and Sumimoto, H. (2003) Proc. Natl. Acad. Sci. U. S. A. 100, 4474–4479[Abstract/Free Full Text]
  47. Ito, T., Matsui, Y., Ago, T., Ota, K., and Sumimoto, H. (2001) EMBO J. 20, 3938–3946[Abstract/Free Full Text]
  48. Hata, K., Takeshige, K., and Sumimoto, H. (1997) Biochem. Biophys. Res. Commun. 241, 226–231[CrossRef][Medline] [Order article via Infotrieve]
  49. Han, C.-H., Freeman, J. L. R., Lee, T., Motalebi, S. A., and Lambeth, J. D. (1998) J. Biol. Chem. 273, 16663–16668[Abstract/Free Full Text]
  50. Price, M. O., McPhail, L. C., Lambeth, J. D., Han, C.-H., Knaus, U. G., and Dinauer, M. C. (2002) Blood 99, 2653–2661[Abstract/Free Full Text]
  51. Dinauer, M. C., Pierce, E. A., Erickson, R. W., Muhlebach, T. J., Messner, H., Orkin, S. H., Seger, R. A., and Curnutte, J. T. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 11231–11235[Abstract]
  52. Leusen, J. H. W., Bolscher, B. G. J. M., Hilarius, P. M., Weening, R. S., Kaulfersch, W., Segar, R. A., Roos, D., and Verhoeven, A. J. (1994) J. Exp. Med. 180, 2329–2334[Abstract]
  53. Biberstine-Kinkade, K. J., Yu, L., Stull, N., LeRoy, B., Bennett, S., Cross, A., and Dinauer, M. C. (2002) J. Biol. Chem. 277, 30368–30374[Abstract/Free Full Text]
  54. Bánfi, B., Clark, R. A., Steger, K., and Krause, K.-H. (2003) J. Biol. Chem. 278, 3510–3513[Abstract/Free Full Text]
  55. Mizuki, K., Kadomatsu, K., Hata, K., Ito, T., Fan, Q.-W., Kage, Y., Fukumaki, Y., Sakaki, Y., Takeshige, K., and Sumimoto, H. (1998) Eur. J. Biochem. 251, 573–582[Abstract]
  56. Akasaki, T., Koga, H., and Sumimoto, H. (1999) J. Biol. Chem. 274, 18055–18059[Abstract/Free Full Text]
  57. Lassègue, B., Sorescu, D., Szöcs, K., Yin, Q., Akers, M., Zhang, Y., Grant, S. L., Lambeth, J. D., and Griendling, K. K. (2001) Circ. Res. 88, 888–894[Abstract/Free Full Text]
  58. Lavigne, M. C., Malech, H. L., Holland, S. M., and Leto, T. L. (2001) Circulation 104, 79–84[Abstract/Free Full Text]