Expression and function of Noxo1c, an alternative splicing form of the NADPH oxidase organizer Ryu Takeya1,2, Masahiko Taura1, Tomoko Yamasaki1, Seiji Naito3 and Hideki Sumimoto1,2 Medical Institute of Bioregulation, Kyushu University, Fukuoka, Japan CREST, Japan Science and Technology Agency, Saitama, Japan Department of Urology, Kyushu University Graduate School of Medical Sciences, Fukuoka, Japan Keywords NADPH oxidase (Nox1); Nox organiser (Noxo1); PX domain; phosphoinositide Correspondence H Sumimoto, Medical Institute of Bioregulation, Kyushu University, Fukuoka 812-8582, Japan Fax: +81 92 642 6807 Tel: +81 92 642 6806 E-mail: hsumi@bioreg.kyushu-u.ac.jp (Received 31 May 2006, accepted 12 June 2006) doi:10.1111/j.1742-4658.2006.05371.x 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, Noxo1c, which is expressed in the testis and fetal brain The Noxo1c 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, gp91phox ⁄ Nox2, and Nox3, as shown in this study The PX domain isolated from Noxo1c shows a lower affinity for phosphoinositides than that from the classical splicing form Noxo1b Consistent with this, in resting cells, Noxo1c is poorly localized to the membrane, and thus less effective in activating Nox1 than Noxo1b, 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 Noxo1c; as a result, Noxo1c is equivalent to Noxo1b 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 gp91phox ⁄ Nox2, Noxo1c is less active than Noxo1b even in the presence of PMA, whereas Noxo1c and Noxo1b support the superoxideproducing activity of Nox3 to the same extent in a manner independent of cell stimulation Members of the NADPH oxidase (Nox) family produce superoxide from molecular oxygen in conjunction with oxidation of NADPH [1–10] Superoxide generated serves as a precursor of other reactive oxygen species, which are currently considered to be involved in various physiological processes The founder Nox enzyme gp91phox, also termed Nox2, is predominantly expressed in professional phagocytes, and plays a crucial role in host defense; superoxide generation by gp91phox leads to subsequent formation of microbicidal reactive oxygen species such as hydroxyl radical and hypochlorous acid Nox1, the second member of the Nox family, is abundant in the colon and vascular tissues [11,12] and considered to participate in host defense at the colon and signaling to cell proliferation [11,13,14] Recent studies have revealed that Nox1, expressed heterologously, associates with the membrane-integrated protein p22phox to form a functional heterodimer [15,16] Activation of gp91phox, also complexed with p22phox, absolutely requires the two cytosolic proteins p47phox and p67phox Nox organizer (Noxo1) and Nox Abbreviations CHO, Chinese hamster ovary; GST, glutathione S-transferase; HA, hemaglutinin; Nox, NADPH oxidase; Noxo1, Nox organizer 1; Noxa1, Nox activator 1; PMA, phorbol 12-myristate 13-acetate; PtdIns(3)P, phosphatidylinositol 3-phosphate; PtdIns(4)P, phosphatidylinositol 4-phosphate; PtdIns(3,5)P2, phosphatidylinositol 3,5-bisphosphate; PX, phox homology FEBS Journal 273 (2006) 3663–3677 ª 2006 The Authors Journal compilation ª 2006 FEBS 3663 Expression and function of Noxo1c R Takeya et al activator (Noxa1), novel respective homologs of p47phox and p67phox, have been identified by several groups including ours [15,17,18] Noxo1 and Noxa1 are both required for activation of Nox1 [15,17–19] The organizers p47phox and Noxo1 each contain two SH3 domains, arranged tandemly In p47phox, the SH3 domains normally interact intramolecularly with the autoinhibitory region, which prevents the domains from binding to the target p22phox A conformational change in p47phox, which can be induced by its phosphorylation by protein kinase C, enables the protein to access p22phox, leading to superoxide production On the other hand, Noxo1 lacks the autoinhibitory region [15,17,18]; its SH3 domains are capable of binding to p22phox even in a resting state [15] This seems to explain why cell stimulation with phorbol 12-myristate 13-acetate (PMA), a potent activator of protein kinase C, is not required for Noxo1dependent superoxide-producing activity of Nox1 [15] In addition to the SH3 domains, Noxo1 and p47phox harbor a phagocyte oxidase (phox) homology (PX) domain in the N-terminus PX domains occur in a variety of proteins involved in cell signaling, membrane trafficking, and polarity establishment, and function as phosphoinositide-binding modules in the assembly of proteins at membrane surfaces [20–22] Through the interaction with phosphoinositides, the PX domain of p47phox plays a crucial role in membrane recruitment of the protein and subsequent activation of the phagocyte oxidase [23] The phosphoinositide-binding activity of the Noxo1 PX domain seems also to be involved in activation of Nox1 [19] The third oxidase, Nox3, is involved in otoconia formation in mouse inner ears [24], and appears to be constitutively active even in the absence of an oxidase organizer (p47phox or Noxo1) or an oxidase activator (p67phox or Noxa1) [25] Nox3, like gp91phox and Nox1, forms a functional complex with p22phox in transfected cells; and the organizers p47phox and Noxo1 are capable of enhancing the superoxide production by Nox3 via the interaction of their SH3 domains with p22phox [25– 27] Although the SH3 domain of Noxo1 participates in regulation of Nox3, the role of the PX domain in Nox3 activity remains unknown In the process of cloning of human Noxo1, some spliced transcripts of the NOXO1 gene have been identified [15,17–19] They seem to be formed by alternative splicing at two distinct sites, which results in insertion of one amino acid at one site and ⁄ or five amino acids at another site in the PX domain However, little is known about the expression pattern of the splicing variants, as they could not be distinguished in assays used in previous studies of 3664 expression of the Noxo1 mRNA [15,17,18] In addition, it remained to be elucidated whether each transcript possesses the activity to support activation of the Nox enzymes In this study, we show the expression of alternatively spliced transcripts of the NOXO1 gene, by PCR using variant-specific primers, and the roles of the protein products in activation of Nox oxidases Results and Discussion Alternative splice forms of human Noxo1 We have previously identified a transcript (AB097667) of human NOXO1 gene encoding 371 amino acids ´ [15], which is identical with that reported by Banfi et al (AF539796) and Cheng & Lambeth (AF532984) [17,19] (Fig 1A) On the other hand, Geiszt et al reported the alternative transcript (AY255768) [18], which encodes a protein lacking Lys50 To investigate the relative abundance of spliced variants of Noxo1, we performed PCR experiments using cDNA panels as template, and sequenced the PCR products (for details, see Experimental procedures) The sequencing analysis revealed that the transcript that we have previously reported (AB097667, AF532984, and AF539796), currently referred to as Noxo1b [28], is the major mRNA form in various human tissues including the colon Another alternative transcript, Noxo1c (AF532985), is abundantly expressed in the testis This transcript is generated by the use of the alternative splice donor site of the ends of exon (Fig 1A) and thus contains five additional amino acids in the PX domain (Fig 1B) The five-amino-acid insertion is not expected to alter the overall PX structure of Noxo1, as the insertion is located in a loop between the polyproline II helix and a3 helix of the PX domain [29], where a considerable sequence divergence occurs among various PX domains (Fig 1C) On the other hand, the insertion in the loop may affect the affinity for phosphoinositides This loop is expected to play an important role, because the corresponding loop of p40phox and p47phox is directly involved in the interaction with phosphoinositides [30,31] The loop region in the PX domain of p40phox faces the phosphoinositide-binding pocket as shown by the crystal structure of the p40phox PX domain bound to phosphatidylinositol 3-phosphate [PtdIns(3)P]; Lys92 in the loop is critical for binding to phosphoinositides [30] Lys79 in the loop of p47phox also seems to contribute to phosphoinositide binding [31] The five-amino-acid insertion in the loop of Noxo1 might alter the configuration of the phosphoinositide-binding region, affecting the affinity for FEBS Journal 273 (2006) 3663–3677 ª 2006 The Authors Journal compilation ª 2006 FEBS R Takeya et al Expression and function of Noxo1c A B C Fig Alternative splice forms of human Noxo1 (A) The genomic organization of human NOXO1 gene Translated sequences are shown as black boxes, and untranslated sequences as open boxes In the lower panel, sequence around splice sites of the 3rd exon are shown Intron sequences are shown in lower case, and exon sequences in upper case A five-amino-acid insertion of Noxo1c is underlined (B) Schematic presentation of domain structures of Noxo1 and the location of the five-amino-acid insertion in the PX domain SH3, Src-homology domain; PRR, proline-rich region (C) Sequence alignments of the PX domains of Noxo1, p47phox, p40phox, SNX3, and Vam7p The alignments take the secondary structure of the p47phox PX domain into account [29] A consensus sequence is shown on the top, where # indicates hydrophobic residues A five-amino-acid insertion of Noxo1c is highlighted Lys92 in p40phox and Lys79 in p47phox, mentioned in the text, are underlined phosphoinositides The other two variants with deletion of Lys50, Noxo1a (AY255768 and AF532983) and Noxo1d (AY191359), have been deposited in the GenBank database: the deletion in these variants is generated by alternative splicing involved in a different splice acceptor site of exon In the present PCR experiments, Noxo1a was expressed in skeletal muscle and Noxo1d in the brain; these two variants were expressed to a much lesser extent than Noxo1b and Noxo1c (data not shown) FEBS Journal 273 (2006) 3663–3677 ª 2006 The Authors Journal compilation ª 2006 FEBS 3665 Expression and function of Noxo1c R Takeya et al Expression of Noxo1b and Noxo1c in various human tissues To compare the distribution pattern of Noxo1b and Noxo1c, we designed variant-specific primers as shown in Fig 2A: the primers ‘a’ and ‘b’, which are specific for Noxo1b and Noxo1c, respectively The specificity of each primer was confirmed by PCR using control plasmids (Fig 2B) With these specific primers, we studied expression of the messengers of Noxo1b and Noxo1c by PCR using the cDNA panel of various human tissues As shown in Fig 2C, the mRNA for Noxo1c was expressed substantially in the testis but only slightly in the colon On the other hand, the Noxo1b mRNA was relatively abundant in the colon and also present in the testis, liver, thymus, and kidney but to a lesser extent (Fig 2C) Among fetal organs tested, the message of Noxo1c was most abundantly expressed in the brain (Fig 2D) Similarly, the Noxo1b mRNA was most abundant in the brain among the fetal organs, although it was also present in the thymus, liver, and kidney To compare the amounts of the two variants expressed in the testis, we performed the PCR using the primers ‘d’ and ‘c’, where the cDNAs for both Noxo1b and Noxo1c are amplified as the insert region is located between the pair of primers (Fig 2A) The lengths of the PCR products of Noxo1b and Noxo1c were 225 and 240 bp, respectively To delineate the difference in the two products, we subjected the PCR fragments to PAGE; the two fragments were clearly separated As shown in Fig 2E, Noxo1c and Noxo1b were almost equally expressed in the testis, whereas Noxo1b was the major form in the colon To investigate the physiological relevance of Noxo1c expression in the testis, we examined expression of Nox1 and Noxa1 by PCR analysis and found a small but significant amount of the Nox1 and Noxa1 mRNAs in the testis (data not shown), which is consistent with the previous observation by Cheng et al [32] It has also been shown that Nox1 is present in the androgen-independent prostate cancer LNCaP cells [33] RT-PCR analysis revealed that LNCaP cells abundantly expressed the mRNA for Noxo1c (Fig 2F) The Noxo1c mRNA also existed in several Nox1-expressing human cancer cell lines: the androgen-independent prostate cancer PC3 and DU145 cells and the testicular germ cell tumor NEC8 cells (Fig 2F) Noxa1, a protein that activates Nox1 in cooperation with Noxo1 [15,17–19], was also expressed (Fig 2F) in LNCaP, PC3, and DU145 cells, suggesting that Nox1 is regulated by Noxo1c and Noxa1 in these cancer cells The role of Nox1 in prostate tumors has been suggested: Nox1 seems to increase tumorigenicity of DU145 prostate cancer cells [34]; and increased expression of endogenous Nox1 is observed in parallel with increasing tumor and metastatic potential in a series of cell lines developed from LNCaP cells [35] As the mRNA for Noxo1c was detected in fetal brain (Fig 2D), we also investigated its expression by PCR using the cDNA panel of the human fetal neural system As shown in Fig 2G, the Noxo1c mRNA as well as the Noxo1b mRNA was expressed in the occipital Fig Expression of Noxo1b and Noxo1c in human tissues (A) Location of primers for PCR analyses The cDNA primers ‘a’ and ‘b’ are designed as specific primers for Noxo1b and Noxo1c, respectively The reverse primer ‘c’ is used in combination with sense primers ‘a’ and ‘b’ in PCR analyses using human adult tissues (C) and fetal tissues (D) The primers ‘d’ and ‘c’ are used in the PCR analyses (E) where both Noxo1b and Noxo1c are simultaneously amplified (B) The specificity of the variant-specific primers With the indicated combination of primers, PCR was performed using cDNAs for Noxo1b or Noxo1c as a template, and the PCR products were subjected to 2% agarose-gel electrophoresis, and stained with ethidium bromide (C) Expression of Noxo1b and Noxo1c in human adult tissues The expression levels of Noxo1b and Noxo1c were analyzed by PCR using Human Multiple Tissue cDNA panels (Clontech): sk muscle, skeletal muscle; small intest., small intestine The PCR products were subjected to 2% agarose-gel electrophoresis, and stained with ethidium bromide The experiments have been repeated more than three times with similar results (D) Expression of Noxo1b and Noxo1c in human fetal tissues The expression levels of Noxo1b and Noxo1c were analyzed by PCR using Human Fetal Multiple Tissue cDNA panels (Clontech) The PCR products were subjected to 2% agarose-gel electrophoresis, and stained with ethidium bromide The experiments have been repeated more than three times with similar results (E) The DNA fragments amplified by PCR using primers ‘d’ and ‘c’ were subjected to 10% polyacrylamide gel electrophoresis For details, see Experimental procedures The experiments have been repeated more than three times with similar results (F) Expression of Noxo1b, Noxo1c, Nox1, and Noxa1 in various human cell lines: androgen-independent prostate cancer cells (LNCaP), androgen-independent prostate cancer cells (PC3 and DU145), and testicular germ cell tumor cells (NEC8) The expression levels were analyzed by RT-PCR using total RNA extracted from each cell line as a template The DNA fragments for Noxo1b and Noxo1c were subjected to 10% polyacrylamide gel electrophoresis (upper panel) as in (E), and the fragments for Nox1 and Noxa1 were subjected to 2% agarose-gel electrophoresis (middle and lower panels) The experiments have been repeated more than three times with similar results (G) Expression of Noxo1b and Noxo1c in human fetal neural tissues The expression levels of Noxo1b and Noxo1c were analyzed by PCR using Human Fetal Neural Tissue cDNA panels (Biochain Institute) The PCR products were subjected to 10% polyacrylamide gel electrophoresis, and stained with ethidium bromide as in (E) The experiments have been repeated more than three times with similar results 3666 FEBS Journal 273 (2006) 3663–3677 ª 2006 The Authors Journal compilation ª 2006 FEBS R Takeya et al Expression and function of Noxo1c A B C D F E G FEBS Journal 273 (2006) 3663–3677 ª 2006 The Authors Journal compilation ª 2006 FEBS 3667 Expression and function of Noxo1c R Takeya et al lobe, parietal lobe, pons, cerebellum, and spinal cord, suggestive of the role in neurons Activation of Nox1 by Noxo1b and Noxo1c in Chinese hamster ovary (CHO) cells It is well known that the classical splicing form Noxo1b is essential for activation of Nox1 [15,17–19] To know the activity of Noxo1c to activate Nox1, we A B C D 3668 transfected Chinese hamster ovary (CHO) cells with pEF-BOS– pcDNA3.0–Nox1, pEF-BOS–p22phox, Noxa1, and pEF-BOS–Noxo1b or pEF-BOS–Noxo1c As shown in Fig 3A, Noxo1b and Noxo1c equally supported Nox1 activation on stimulation with PMA Without stimulants added, Noxo1c also activated Nox1 but to a lesser extent than Noxo1b (Fig 3A,B) On the other hand, Noxo1a, a minor spliced transcript, was much less active even in the presence of PMA (Fig 3B) We further investigated the stimulusindependent activity of Nox1 using CHO cells transfected at various amounts of the Noxo1b or Noxo1c cDNA As shown in Fig 3C, Noxo1c was less active than Noxo1b in resting cells, indicating the difference between Noxo1b and Noxo1c In the above experiments, we used CHO cells detached from culture dishes using trypsin ⁄ EDTA to measure superoxide production It is known that the modulation of cell–cell adhesion can activate certain intracellular signaling pathways including the small GTPase Rac [36]; Rac seems to participate in Nox1 Fig Noxo1c-supported activation of Nox1 (A) CHO cells were cotransfected with pcDNA3.0–Nox1, pEF-BOS–p22phox, pEF-BOS– myc-Noxa1, and simultaneously with pEF-BOS–HA-Noxo1b, pEFBOS–HA-Noxo1c, or pEF-BOS–HA-Noxo1a The transfected cells (1 · 106 cells) were incubated for 10 at 37 °C, and then stimulated with PMA (200 ngỈmL)1) Chemiluminescence change was continuously monitored with Diogenes, and superoxide dismutase (50 lgỈmL)1) was added where indicated (left panel) Expression of variant Noxo1 proteins in the transfected cells was determined by immunoblot analysis with the monoclonal antibody to HA (right panel) (B) CHO cells were cotransfected with pcDNA3.0–Nox1, pEF-BOS–p22phox, pEF-BOS–myc-Noxa1, and simultaneously with pEF-BOS–HA-Noxo1b, pEF-BOS–HA-Noxo1c, or pEF-BOS–HANoxo1a Superoxide production was assayed by chemiluminescence using Diogenes in the presence or absence of PMA (200 ngỈmL)1) Each graph represents the mean ± SD of the peak chemiluminescence values obtained from three independent transfections (C) CHO cells were transfected simultaneously with pcDNA3.0–Nox1 (1 lg), pEF-BOS–p22phox (1 lg), pEF-BOS–mycNoxa1 (1 lg), and the indicated amount of pEF-BOS–HA-Noxo1b (left panel) or pEF-BOS–HA-Noxo1c (right panel) Superoxide production was assayed by chemiluminescence using Diogenes in the presence or absence of PMA (200 ngỈmL)1), and expressed as the percentage activity relative to that of lg pEF-BOS–HA-Noxo1b (left panel) or pEF-BOS–HA-Noxo1c (right panel)-transfected cells in the presence of PMA (D) Superoxide production in adherent CHO cells undetached from culture dishes CHO cells were cotransfected with pcDNA3.0–Nox1, pEF-BOS–p22phox, pEF-BOS–myc-Noxa1, and simultaneously with pEF-BOS–HA-Noxo1b, pEF-BOS–HANoxo1c PMA-independent superoxide production was assayed by chemiluminescence using Diogenes in the presence or absence of superoxide dismutase (50 lgỈmL)1) at 37 °C The graph represents the mean ± SD of chemiluminescence values obtained from three independent transfections FEBS Journal 273 (2006) 3663–3677 ª 2006 The Authors Journal compilation ª 2006 FEBS R Takeya et al Expression and function of Noxo1c A Merge Noxo1γ p22phox Merge Intracellular localization of Noxo1b and Noxo1c Noxo1 appears to exist in a constitutively active form [15], and is reported to be located in the membrane of resting cells [19] The present finding that Noxo1c weakly supports a stimulus-independent activation of Nox1 suggests that Noxo1c is less associated with the membrane in a resting state than Noxo1b To test the possibility, we examined intracellular localization of the Noxo1 proteins ectopically expressed in CHO cells Noxo1b was barely located in the cytoplasm but Noxo1γ PMA: (–) (+) Noxo1γ phox Blot: anti-p22 activation [37] To test the possibility that cell detachment elicits activation of Nox1, we estimated superoxide production by adherent cells Adherent CHO cells, coexpressing Nox1 with both Noxo1b and Noxa1, produced a considerable amount of superoxide (Fig 3D) Under the same conditions, Noxo1c weakly supported superoxide production compared with Noxo1b, confirming that Noxo1c is more effective than Noxo1b in activating Nox1 in unstimulated cells Noxo1β Blot: anti-HA (+) Noxo1β (–) Noxo1γ PMA: Noxo1β C Noxo1γ B Noxo1β Fig Intracellular localization of Noxo1b and Noxo1c in CHO cells (A) Intracellular localization of Noxo1b (upper panels) and Noxo1c (lower panels) in quiescent CHO cells In merged images (right panels), localization of Noxo1b and Noxo1c is shown in green, and p22phox in red Scale bars, 20 lm (B) Membrane translocation of Noxo1c Before or after cell stimulation with PMA (200 ngỈmL)1), the cell lysates were fractionated by centrifugation, and the membrane fractions were analyzed by immunoblot with antibodies to HA or p22phox as a loading control These experiments have been repeated more than three times with similar results (C) The extent of membrane localization of Noxo1b and Noxo1c The intensities of immunoreactive bands for HA-Noxo1b and HA-Noxo1c in (B) were quantified using a LAS-1000plus (Fuji film) image analyzer and expressed as the fold increase relative to that of the band for Noxo1c in the absence of PMA p22phox membrane localization (fold increase) Noxo1β concentrated in punctate intracellular structures (Fig 4A); they resemble fused endosomes on which Noxo1b is reported to be located [19] On the other hand, Noxo1c was located in the cytoplasm and not concentrated in any internal membrane structures within the cytoplasmic compartment Less association of Noxo1c with the membrane-integrated protein p22phox, the partner of Nox1 (Fig 4A), may be consistent with the finding that Noxo1c weakly supports superoxide production by Nox1 in resting CHO cells more weakly than Noxo1c (Fig 3) We also attempted but failed to assess the intracellular localization of Noxo1b after treatment of the cells with PMA, as the cells became rounded without detaching from coverslips To biochemically assess the localization of Noxo1s after treatment with PMA, we prepared the membrane fraction and tested the localization of Noxo1c As shown in Fig 4B, Noxo1c localized to the membrane only partly in resting cells, but was further targeted to the membrane after stimulation with PMA On the other hand, Noxo1b was constitutively FEBS Journal 273 (2006) 3663–3677 ª 2006 The Authors Journal compilation ª 2006 FEBS 3669 Expression and function of Noxo1c A R Takeya et al GST-Noxo1β -PX PI PI3P PI4P PI5P PI(3,5)P2 PI(4,5)P2 PI(3,4)P2 PI(3,4,5)P3 B GST-Noxo1γ -PX PI PI3P PI4P PI5P PI(3,5)P2 PI(4,5)P2 PI(3,4)P2 PI(3,4,5)P3 Fig Phosphoinositide-binding activity of the PX domains of Noxo1b and Noxo1c The GST-fusion proteins of Noxo1b-PX (amino acids 1– 153) (A) and Noxo1c-PX (amino acids 1–158) (B) were tested in an overlay lipid-binding assay using the PIP array, in which serial dilutions of indicated phosphoinositides (100, 50, 25, 12.5, 6.25, 3.13, and 1.56 pmol) were spotted: PI, phosphatidylinositol; PI3P, phosphatidylinositol 3-phosphate; PI4P, phosphatidylinositol 4-phosphate; PI5P, phosphatidylinositol 5-phosphate; PI(3,5)P2, phosphatidylinositol 3,5-bisphosphate; PI(4,5)P2, phosphatidylinositol 4,5-bisphosphate; PI(3,4)P2, phosphatidylinositol 3,4-bisphosphate; PI(3,4,5)P3, phosphatidylinositol 3,4,5-trisphosphate For details, see Experimental procedures associated with the membrane fraction (Fig 4B) As the extent of the membrane localization of Noxo1 correlated well with that of the superoxide-producing activity of Nox1 (Fig 4C), a weaker activity of Noxo1c to support Nox1 activity in unstimulated cells (Fig 3) may be due to the fact that Noxo1c fails to fully localize to the membrane The mechanism for the PMA-dependent membrane recruitment of Noxo1c is at present unknown It is well established that p47phox undergoes phosphorylation in response to PMA, which is essential for membrane translocation of this protein As Noxo1 also has several potential protein kinase C phosphorylation sites, Noxo1 might become phosphorylated in PMAstimulated cells, leading to membrane translocation Phosphoinositide-binding activity of the PX domains of Noxo1b and Noxo1c The membrane localization of Noxo1b is mediated in part by binding of the PX domain to membrane phospholipids [19] Less association of Noxo1c with the membrane (Fig 3) raised the possibility that the phospholipid-binding activity of Noxo1c may be impaired In this context, it should be noted that Noxo1c contains the five-amino-acid insertion in the PX domain (Fig 1) To determine the effect of the insertion, we examined the phosphoinositide-binding activity of the PX domain of Noxo1c by an overlay assay, in which each phosphoinositide was spotted on the membrane and overlaid with glutathione S-transferase (GST)-fused PX domains The PX domain of Noxo1b bound to phosphatidylinositol 3,5-bisphosphate [PtdIns(3,5)P2] with the highest affinity, which is consistent with the recent report of Cheng & Lambeth [19]; it also interacted with 3670 PtdIns(3)P and phosphatidylinositol 4-phosphate [PtdIns(4)P], but to a lesser extent (Fig 5A) On the other hand, the PX domain of Noxo1c showed a weaker binding activity to the phosphoinositides under the same experimental conditions (Fig 5B) Moreover, a liposome-binding assay also showed that the Noxo1c PX domain interacted with phospholipids such as PtdIns(3,5)P2 more weakly than that of Noxo1b (data not shown) Thus the insertion in the PX domain decreases the affinity for phosphoinositides Activation of gp91phox and Nox3 by Noxo1c We next investigated the ability of Noxo1c to activate gp91phox ⁄ Nox2 and Nox3 In CHO cells expressing gp91phox ⁄ Nox2 with p67phox, Noxo1c supported superoxide production to a much lesser extent than Noxo1b (Fig 6A) When coexpressed with Noxa1, the Noxo1csupported superoxide production by gp91phox ⁄ Nox2 was severalfold less than the Noxo1b-supported one (Fig 6B) On the other hand, Noxo1c and Noxo1b showed the same ability to support Nox3 activation in the presence (Fig 6C) or absence (Fig 6D) of Noxa1; the activity was 10-fold higher than those obtained in cells expressing Nox3, with or without Noxa1, but not the Noxo1 proteins (data not shown) Thus the effect of the five-amino-acid insertion in the Noxo1 PX domain depends on the type of Nox Role of the interaction between Noxo1c and p22phox in Nox1-dependent and Nox3-dependent superoxide production It is known that Noxo1 functions via the SH3-mediated interaction with p22phox, which forms a heterodimer FEBS Journal 273 (2006) 3663–3677 ª 2006 The Authors Journal compilation ª 2006 FEBS R Takeya et al PMA(–) PMA(+) Noxo1β Noxo1γ C chemiluminescence ( x 107 cpm) B chemiluminescence ( x 106 cpm) gp91phox + p67phox Nox3 1.2 0.8 0.4 Noxo1β Noxo1γ gp91phox + Noxa1 1.6 1.2 0.8 0.4 Noxo1β Noxo1γ D chemiluminescence ( x 106 cpm) chemiluminescence ( x 106 cpm) A Expression and function of Noxo1c Nox3 + Noxa1 interaction with p22phox To confirm this, we investigated the dependence of Noxo1c-supported Nox activation on p22phox It is known that superoxide production by Nox1 in CHO cells expressing Noxo1b is largely but not completely dependent on the cotransfection with the p22phox cDNA [15], whereas Nox3 activity requires p22phox expression under the same conditions [25] Similarly, Noxo1c-supported superoxide production by Nox1 is partly dependent on p22phox (Fig 7C); on the other hand, the expression of p22phox was a requisite for the Noxo1c-supported Nox3 activity (Fig 7D) Thus Noxo1c probably binds to p22phox in a manner similar to Noxo1b Role of phosphoinositide-binding activity of Noxo1 in Nox3 activation Noxo1β Noxo1γ Fig Noxo1c-supported activation of gp91phox ⁄ Nox2 and Nox3 CHO cells were cotransfected with the following combination of plasmids: pcDNA3.0–gp91phox, pEF-BOS–p22phox, pEF-BOS–mycp67phox, and simultaneously with pEF-BOS–HA-Noxo1b or pEF-BOS–HA-Noxo1c in (A); pcDNA3.0–gp91phox, pEF-BOS– p22phox, pEF-BOS–myc-Noxa1, and simultaneously with pEF-BOS– HA-Noxo1b or pEF-BOS–HA-Noxo1c in (B); pcDNA3.0–Nox3 and pEF-BOS–p22phox, and simultaneously with pEF-BOS–HA-Noxo1b or pEF-BOS–HA-Noxo1c in (C); pcDNA3.0–Nox3 and pEF-BOS– p22phox, pEF-BOS–myc-Noxa1, and simultaneously with pEF-BOS– HA-Noxo1b or pEF-BOS–HA-Noxo1c in (D) Superoxide production was assayed by chemiluminescence using Diogenes in the presence or absence of PMA (200 ngỈmL)1) Each graph represents the mean ± SD of the peak chemiluminescence values obtained from three independent transfections Protein levels of Noxo1b and Noxo1c in the transfected cells were estimated by immunoblot analysis with the monoclonal antibody to HA (lower panels) with Nox1, gp91phox ⁄ Nox2, and Nox3 [25] It may be possible that the interaction with p22phox is blocked by the five-amino-acid insertion in the PX domain, which leads to impaired localization of Noxo1c to the membrane To exclude this possibility, we performed an in vitro binding assay using purified Noxo1b and Noxo1c As shown in Fig 7A, Noxo1c-DC and Noxo1b-DC bound to the C-terminus of p22phox to the same extent; the binding was completely abolished by the P156Q substitution in p22phox, a mutation leading to defective interaction with the SH3 domains of Noxo1 [15] In addition, Noxo1c as well as Noxo1b interacted with p22phox in a similar manner in the yeast two-hybrid system (Fig 7B) Thus the insertion in the PX domain does not seem to affect the SH3-mediated To study the role of the Noxo1 PX domain by itself, we expressed a mutant Noxo1b lacking the PX domain, Noxo1b-DPX, in CHO cells The deletion of the PX domain resulted in complete loss of superoxide production by Nox1 (Fig 8A) and by gp91phox ⁄ Nox1 (Fig 8B) The enhancement of Nox3 activity by Noxo1b [25] was also entirely dependent on the PX domain (Fig 8C) In contrast with the essential role of the PX domain, Noxo1c, containing a PX domain with a weak lipid-binding activity (Fig 5), is capable of fully activating Nox3 (Fig 6) To clarify the role of the lipid-binding activity in Nox3 activation, we examined the effect of substituting Gln for Arg40 in the PX domain, which completely abrogates the phosphoinositide-binding activity [19] As shown in Fig 9A, a mutant Noxo1b carrying the R40Q substitution failed to support the superoxide production by Nox1 Thus the PX-mediated lipid binding is required for Nox1 activation The R40Q substitution in Noxo1c also abolished superoxide production by Nox1 (Fig 9A), supporting the conclusion that Noxo1c retains considerable lipid-binding activity (Fig 5B) On the other hand, in Nox3 activation, the mutant Noxo1 proteins were threefold less active than the wild-type one (Fig 9B), suggesting that PX-mediated binding to phosphoinositides is involved in, but not absolutely required for, Nox3 activity This is in contrast with the observation that the PX domain by itself is essential for Nox3 activation (Fig 8C) The partial dependence on the lipid-binding activity may explain why Noxo1c with a weak but significant lipid-binding activity (Fig 5) is equivalent to Noxo1b in Nox3 activation (Fig 6) The idea may be supported by the observation that a part of Noxo1c as well as Noxo1b was localized to ruffling membranes in the Nox3-transfected CHO cells (data not shown) FEBS Journal 273 (2006) 3663–3677 ª 2006 The Authors Journal compilation ª 2006 FEBS 3671 R Takeya et al B GST–Noxo1γ-ΔC GST–Noxo1β-ΔC GST–Noxo1γ-ΔC A GST–Noxo1β-ΔC Expression and function of Noxo1c p22phox-C (WT) p22phox-C (P156Q) Noxo1β-ΔC Noxo1γ-ΔC MBP–p22phox-C (+) p22phox-C p22phox-C (WT) (P156Q) C His D chemiluminescence ( x 105 cpm) chemiluminescence ( x 106 cpm) Nox1 + Noxa1 + Noxo1γ 2.0 1.6 1.2 0.8 0.4 (–) (–) His Nox3 + Noxo1γ – p22phox + p22phox (+) – p22phox + p22phox Fig Role of the interaction between Noxo1c and p22phox in Nox1-dependent and Nox3-dependent superoxide production (A) Interaction between Noxo1 and p22phox estimated by an in vitro pull-down assay using purified proteins GST–Noxo1b-DC (amino acids 1–292) or GST– Noxo1c-DC (amino acids 1–297) was incubated with MBP–p22phox-C (amino acids 132–195) or MBP–p22phox-C (P156Q) and pulled down with glutathione–Sepahrose 4B The precipitated proteins were subjected to SDS ⁄ PAGE, followed by immunoblot analysis with an antibody to maltose-binding protein (MBP) (B) Interaction between Noxo1 and p22phox estimated by the yeast two-hybrid system The yeast HF7c cells were cotransformed with recombinant plasmids pGBT9g encoding the C-terminus of the wild-type or a mutant p22phox and pGADGH encoding Noxo1b-DC (amino acids 1–292) or Noxo1c-DC (amino acids 1–297) After the selection for Trp+ and Leu+ phenotype, its histidinedependent (right) and independent (left) growth was tested CHO cells were cotransfected with the following combination of plasmids: pcDNA3.0–Nox1, pEF-BOS–myc-Noxa1, pEF-BOS–HA-Noxo1c, and with or without pEF-BOS–p22phox in (C); pcDNA3.0–Nox3, pEF-BOS– myc-Noxa1, pEF-BOS–HA-Noxo1c, and with or without pEF-BOS–p22phox in (D) Superoxide production was assayed by chemiluminescence using Diogenes in the presence of PMA (200 ngỈmL)1) Each graph represents the mean ± SD of the peak chemiluminescence values obtained from three independent transfections Concluding remarks In this study, we show that Noxo1c, a novel alternative splicing form of human Noxo1 containing an additional five amino acids in the PX domain, is expressed in the testis and fetal brain (Fig 2) During the revision of this manuscript, Cheng & Lambeth [28] reported the expression and function of the four splice forms of human Noxo1 The Noxo1c mRNA is also expressed in several Nox1-expressing and Noxa1expressing human cancer cell lines [the androgenindependent prostate cancer LNCaP cells, and the androgen-independent prostate cancer PC3 and DU145 cells (Fig 2)], indicating that Noxo1c regulates Nox1 in co-operation with Noxa1 in a single cell In PMA-stimulated cells, Noxo1c and Noxo1b support Nox1 activation to the same extent (Fig 3) The PX domain of Noxo1c shows a lower affinity for phosphoinositides than that of Noxo1b (Fig 5), which seems to attenuate the membrane localization in resting 3672 cells (Fig 4) Consistent with this, Noxo1c supports the stimulus-independent activity of Nox1 more weakly than Noxo1b (Fig 3) We also demonstrate that Noxo1c fails to fully activate gp91phox even in the presence of PMA, whereas Nox3 activity enhanced by Noxo1c is almost equivalent to that by Noxo1b (Fig 7) The difference may be due to the fact that the significance of the PX-mediated lipid binding depends on the type of Nox, although the PX domain of Noxo1 by itself is indispensable for supporting superoxide production by all the three Nox enzymes Experimental procedures Isolation of cDNA for splice variants of human NOXO1 gene Based on the sequence of mRNA for human NOXO1 (GenBank accession number AB097667), we synthesized the two unique oligonucleotide primers 5¢-GCAGGATCCAT FEBS Journal 273 (2006) 3663–3677 ª 2006 The Authors Journal compilation ª 2006 FEBS R Takeya et al Expression and function of Noxo1c A PMA(–) 1.2 chemiluminescence ( x 106 cpm) chemiluminescence ( x 107 cpm) Nox1 + Noxa1 A Nox1 + Noxa1 1.6 PMA(+) 0.8 0.4 Noxo1β ΔPX vector B 3.0 B 2.0 chemiluminescence ( x 105 cpm) chemiluminescence ( x 105 cpm) 4.0 1.0 C 3.0 chemiluminescence ( x 106 cpm) gp91phox + Noxa1 Noxo1β ΔPX vector Nox3 Nox3 2.0 1.0 Noxo1β ΔPX vector Fig Role of the Noxo1 PX domain in activation of Nox enzymes CHO cells were cotransfected with the following combination of plasmids: pcDNA3.0–Nox1, pEF-BOS–p22phox, pEF-BOS–mycNoxa1, and simultaneously with pEF-BOS–HA-Noxo1b (wild-type), pEF-BOS–HA-Noxo1-DPX, or pEF-BOS vector in (A); pcDNA3.0– gp91phox ⁄ Nox2, pEF-BOS–p22phox, pEF-BOS–myc-Noxa1, and simultaneously with pEF-BOS–HA-Noxo1b (wild-type), pEF-BOS–HANoxo1-DPX, or pEF-BOS vector in (B); pcDNA3.0–Nox3 and pEF-BOS–p22phox, and simultaneously with pEF-BOS–HA-Noxo1b (wild-type), pEF-BOS–HA-Noxo1-DPX, or pEF-BOS vector in (C) Superoxide production was assayed by chemiluminescence using Diogenes in the presence or absence of PMA (200 ngặmL)1) GGCAGGCCCCCGATACCCAG-3Â and 5¢-CGTCTCGA GGAGGCGGCCCGCAGCGCGAGA-3¢; sequences from the mRNA are underlined With the two primers, PCR was performed using Human Multiple Tissue cDNA (MTCTM) panels (Clontech, Mountain View, CA, USA) as a template, and the PCR products were subcloned into pBluescript Noxo1β Noxo1γ Noxo1β Noxo1γ (R40Q) (R40Q) Noxo1β Noxo1γ Noxo1β Noxo1γ (R40Q) (R40Q) Fig Effect of the R40Q substitution in Noxo1c on activation of Nox1 and Nox3 (A) CHO cells were cotransfected with pcDNA3.0– Nox1, pEF-BOS–p22phox, pEF-BOS–myc-Noxa1, and simultaneously with pEF-BOS–HA-Noxo1b, pEF-BOS–HA-Noxo1c, pEF-BOS–HANoxo1b (R40Q), or pEF-BOS–HA-Noxo1c (R40Q) Superoxide production was assayed by chemiluminescence using Diogenes in the presence of PMA (200 ngỈmL)1) Each graph represents the mean ± SD of the peak chemiluminescence values obtained from three independent transfections (B) CHO cells were cotransfected with pcDNA3.0–Nox3, pEF-BOS–p22phox, and simultaneously with pEF-BOS–HA-Noxo1b, pEF-BOS–HA-Noxo1c, pEF-BOS–HA-Noxo1b (R40Q), or pEF-BOS–HA-Noxo1c (R40Q) Superoxide production was assayed by chemiluminescence using Diogenes in the presence of PMA (200 ngỈmL)1) Each graph represents the mean ± SD of the peak chemiluminescence values obtained from three independent transfections Sequencing analysis of the PCR products corroborated four previously reported variants: Noxo1b (AB097667, AF532984, and AF539796), Noxo1c (AF532985), Noxo1a (AY255768 and AF532983), and Noxo1d (AY191359) On the basis of the sequence of splice variants, we synthesized the full-length Noxo1b, Noxo1c, Noxo1a, and Noxo1d by PCR-mediated site-directed mutagenesis, and the DNA fragments were cloned into vectors All the constructs were sequenced to confirm their identities FEBS Journal 273 (2006) 3663–3677 ª 2006 The Authors Journal compilation ª 2006 FEBS 3673 Expression and function of Noxo1c R Takeya et al Expression of Noxo1b and Noxo1c in various human tissues The expression patterns of the Noxo1b and Noxo1c messengers were determined by PCR using Human Multiple Tissue cDNA panels and Human Fetal Neural Tissue cDNA panels (Biochain Institute, Hayward, CA, USA), according to the manufacturer’s protocol Expression of Noxo1c was determined by RT-PCR using total RNA as a template, which was extracted by TRIzol reagent (Invitrogen, Carlsbad, CA, USA) from the following human cell lines; androgen-independent prostate cancer LNCaP cells, androgen-independent prostate cancer PC3 and DU145 cells, and testicular germ cell tumor NEC8 cells [38,39] Splicing-specific PCR was performed using the following primers: ‘a’, 5¢-TCTCCCAAAGCTTCTCGATGC-3¢ (forward primer specific for the Noxo1 cDNA); ‘b’, 5¢-CCCAAAGCTTCTCGGTCAGGC-3¢ (forward primer specific for the Noxo1c cDNA); ‘c’, 5¢-TCTGGGGTGGG CAGGATCACC-3¢ (reverse primer for both the Noxo1b and Noxo1c cDNA) To amplify both the Noxo1b and Noxo1c cDNAs, the following primer, ‘d’, 5¢-CCGCGT TCTCCCAAAGCT-3¢ and primer ‘c’ were used 5¢-GA AATCCCATCACCATCTTCCA-3¢ (forward primer) and 5¢-CCTTCTCCATGGTGGTGAAGAC-3¢ (reverse primer) were used for the glyceraldehyde-3-phosphate dehydrogenase cDNA PCR analyses were performed using ABI PRISMÒ 9700 (Applied Biosystems, Foster City, CA, USA) according to the manufacturer’s instructions The reaction mixture (10 lL) contained KOD-plus DNA polymerase (Toyobo, Osaka, Japan), 0.3 lm each primer, and lL of the first-strand cDNA from different human tissues (Human MTC panels I, II, and Fetal MTC panel; Clontech) as a template, and amplification was carried out for 35 cycles The PCR fragments were subjected to 2% agarose gel electrophoresis, except for those in Fig 2E,F,G, which were subjected to PAGE (10% gel) PCR products were purified with a MERmaid kit (Q-BIOgene, Morgan Irvine, CA, USA) and sequenced to confirm their identities Superoxide-producing activity of CHO cells expressing Nox1, gp91phox, or Nox3 The cDNAs for Nox1, gp91phox, and Nox3 were ligated to the mammalian expression vector pcDNA3.0 (Invitrogen), and cDNAs encoding p22phox, p47phox, p67phox, Noxo1, and Noxa1 were ligated to the mammalian expression vector pEF-BOS [15,25] Noxo1 and p47phox were constructed for expression as a hemaglutinin (HA)tagged protein, Noxa1 and p67phox as a myc-tagged protein, and p22phox as a protein without a tag Transfection of the CHO cells with the cDNAs was performed using FuGENE6 Transfection Reagent (Roche Diagnostics, Mannheim, Germany) After culture for 30 h, adherent cells were harvested by incubating with trypsin ⁄ EDTA 3674 for at 37 °C, and washed with Hepes-buffered saline (120 mm NaCl, mm KCl, mm glucose, mm MgCl2, 0.5 mm CaCl2 and 17 mm Hepes, pH 7.4) Superoxide production by the transfected cells was determined by superoxide dismutase-inhibitable chemiluminescence with an enhancer-containing luminol-based detection system (Diogenes; National Diagnostics, Atlanta, GA, USA), as previously described [15,23,40,41] After the addition of the enhanced luminol-based substrate, the cells were stimulated with 200 ngỈmL)1 PMA The chemiluminescence was assayed using a luminometer (Auto Lumat LB953; Berthold Technologies, Bad Wildbad, Germany) Measurement of superoxide production using adherent cells undetached from culture dishes CHO cells were plated on six-well plates (1 · 105 cells ⁄ well) 18 h before the transfection Cells were transfected with plasmids using FuGENE6 Transfection Reagent, and cultured for 30 h After three washes with Hepes-buffered saline, cells were mixed with Diogenes Chemiluminescence was measured using a multilabel counter Wallac 1420 ARVOsx (PerkinElmer Life Sciences, Turku, Finland) Estimation of expression of cytosolic regulatory proteins Total cell lysates of transfected CHO cells were used to estimate expression of Noxo1b, Noxo1c, and Noxo1a The lysates were subjected to SDS ⁄ PAGE, transferred to a poly(vinylidene difluoride) membrane (Millipore, Billerica, MA, USA), and probed with a monoclonal antibody to HA (Covance Research Products, Berkeley, CA, USA) The blots were developed using ECL-plus (GE Healthcare Biosciences, Piscataway, NJ, USA) for visualization of the antibodies, as previously described [15] Localization of Noxo1c and Noxo1b in CHO cells Localization of HA-tagged Noxo1 proteins was tested using CHO cells as previously described with minor modifications [42] Transfected CHO cells were fixed for 15 at 25 °C in 3.7% formaldehyde The fixed cells were washed four times with phosphate-buffered saline (NaCl ⁄ Pi: 137 mm NaCl, 2.68 mm KCl, 8.1 mm Na2HPO4, and 1.47 mm KH2PO4), and blocked with NaCl ⁄ Pi containing 3% BSA for 60 The sample was subsequently incubated with the monoclonal antibody to HA and probed with Alexa Fluor 488TM-labeled goat anti-mouse IgG (Invitrogen, Carlsbad, CA, USA) as secondary antibodies For detection of p22phox, the sample was incubated with polyclonal antibodies to p22phox, which were raised against the C-terminal 20 amino acids of human p22phox [25] and probed with Alexa Fluor FEBS Journal 273 (2006) 3663–3677 ª 2006 The Authors Journal compilation ª 2006 FEBS R Takeya et al 594TM-labeled goat anti-rabbit IgG (Molecular Probes) as secondary antibodies Images were visualized with a confocal laser-scanning microscope LSM5 PASCAL (Carl Zeiss, Oberkochen, Germany) Membrane translocation of Noxo1 Membrane translocation of Noxo1 was determined as previously described [23,43] with minor modifications Briefly, the CHO cells expressing Noxo1b or Noxo1c and other cytosolic factors were suspended at a concentration of · 106 cells per ml in NaCl ⁄ Pi and stimulated for 10 at 37 °C with PMA (200 ngỈmL)1) After centrifugation, cells were resuspended in NaCl ⁄ Pi containing 0.5 mm EGTA, 20 lm p-amidinophenylmethanesulfonyl fluoride, 80 lgỈmL)1 leupeptin, 20 lgỈmL)1 pepstatin A, 20 lgỈmL)1 chymostatin, and lysed by three rounds of 5-s sonication The sonicates were centrifuged for 10 at 10 000 g, and the supernatant was further ultracentrifuged for 45 at 100 000 g The resultant pellet was washed with NaCl ⁄ Pi, suspended in Laemmli sample buffer, and used as the membrane fraction Proteins were analyzed by western blot with the monoclonal antibody to HA and developed by using ECL-plus Lipid-binding assay using recombinant GST–fusion proteins The PX domain of Noxo1b (amino acids 1–153) and its corresponding region of Noxo1c (amino acids 1–158) were expressed as proteins fused to GST in Escherichia coli strain BL21, and purified by glutathione–Sepharose 4B (Amersham Bioscience), as previously described [14,21] An overlay assay was carried out using the PIP arrayTM (Echelon Biosciences, Salt Lake City, UT, USA) following the manufacturer’s protocol Membranes were first incubated with 4% nonfat dry milk in Tris-buffered saline ⁄ Tween (20 mm Tris ⁄ HCl, pH 7.5, 136 mm NaCl, 0.1% Tween-20) at room temperature for h and then overnight at °C with 500 ngỈmL)1 GST fusion protein After being washed three times with Tris-buffered saline ⁄ Tween, the membranes were incubated with : 1000 goat polyclonal antibodies to GST (Amersham Bioscience) Membranes were further incubated with : 2500 donkey anti-goat IgG conjugated to horseradish peroxidase (Santa Cruz Biotechnology, Santa Cruz, CA, USA) The antibodies were detected by chemiluminescence using ECL-plus as previously described [15] In vitro liposome-binding assay was carried out as previously described [20,23] with minor modifications Briefly, liposomes were prepared by mixing phosphatidylethanolamine (72%) and phosphatidylcholine (18%) with 10% phosphatidylinositol, PtdIns(3)P, PtdIns(4)P, PtdIns(5)P, PtdIns(3,4)P2, PtdIns(3,5)P2, PtdIns(4,5)P2 or PtdIns(3,4,5)P3, drying the mixture under a stream of nitrogen, and resuspending in a sample buffer (100 mm Expression and function of Noxo1c NaCl, 20 mm Hepes, pH 7.2) All synthetic phosphoinositides with C16 fatty acids were purchased from Echelon Biosciences Inc; phosphatidylethanolamine, phosphatidylcholine, and phosphatidylinositol from Sigma (St Louis, MO, USA) Liposomes (50 lm) were incubated for 10 on ice with the indicated GST-fusion proteins (80 pmol) in 50 lL of the sample buffer After ultracentrifugation for 30 at 100 000 g, the supernatant was removed carefully, and the liposome pellet resuspended in 50 lL of the sample buffer Samples were analyzed by SDS ⁄ PAGE (10% gel) and stained with Coomassie Brilliant Blue For estimation of the amount of proteins on the gel, densitometric analysis was performed using a LAS-1000plus (Fuji photo film, Tokyo, Japan) image analyzer 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 [15] After the selection for Trp+ and Leu+ phenotype, the transformants were tested for their ability to grow on plates lacking histidine, according to the manufacturer’s recommendation (Clontech) Acknowledgements We are grateful to Yohko Kage (Kyushu University and JST), Miki Matsuo (Kyushu University), Natsuko Yoshiura (Kyushu University), and Namiko Kubo (Kyushu University and JST) for technical assistance, and to Minako Nishino (Kyushu University and JST) for secretarial assistance 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, and CREST and BIRD projects of JST (Japan Science and Technology Agency) References Segal AW (2005) How neutrophils kill microbes Annu Rev Immunol 23, 197–223 Sumimoto H, Miyano K & Takeya R (2005) Molecular composition and regulation of the Nox family NAD(P)H oxidases Biochem Biophys Res Commun 338, 677–686 Lambeth JD (2004) NOX enzymes and the biology of reactive oxygen Nat Rev Immunol 4, 181–189 Geiszt M & Leto TL (2004) The Nox family of NAD(P)H oxidases: host defense and beyond J Biol Chem 279, 51715–51718 FEBS Journal 273 (2006) 3663–3677 ª 2006 The Authors Journal compilation ª 2006 FEBS 3675 Expression and function of Noxo1c R Takeya et al Bokoch GM & Knaus UG (2003) NADPH oxidases: not just for leukocytes anymore! Trends Biochem Sci 28, 502–508 Quinn MT & Gauss KA (2004) Structure and regulation of the neutrophil respiratory burst oxidase: comparison with nonphagocyte oxidases J Leukoc Biol 76, 760–781 Nauseef WM (2004) Assembly of the phagocyte NADPH oxidase Histochem Cell Biol 122, 277–291 Cross AR & Segal AW (2004) The NADPH oxidase of professional phagocytes: prototype of the NOX electron transport chain systems Biochim Biophys Acta 1657, 1–22 Babior BM (2004) NADPH oxidase Curr Opin Immunol 16, 42–47 10 Clark RA, Epperson TK & Valente AJ (2004) Mechanisms of activation of NADPH oxidases Jpn J Infect Dis 57, S22–S23 11 Suh YA, Arnold RS, Lassegue B, Shi J, Xu X, Sorescu D, Chung AB, Griendling KK & Lambeth JD (1999) Cell transformation by the superoxide-generating oxidase Mox1 Nature 401, 79–82 ´ 12 Banfi B, Maturana A, Jaconi S, Arnaudeau S, Laforge T, Sinha B, Ligeti E, Demaurex N & Krause K-H (2000) A mammalian H+ channel generated through alternative splicing of the NADPH oxidase homolog NOH-1 Science 287, 138–142 13 Geiszt M, Lekstrom K, Brenner S, Hewitt SM, Dana R, Malech HL & Leto TL (2003) NAD(P)H oxidase 1, a product of differentiated colon epithelial cells, can partially replace glycoprotein 91phox in the regulated production of superoxide by phagocytes J Immunol 171, 299–306 14 Arnold RS, Shi J, Murad E, Whalen AM, Sun CQ, Polavarapu R, Parthasarathy S, Petros JA & Lambeth JD (2001) Hydrogen peroxide mediates the cell growth and transformation caused by the mitogenic oxidase Nox1 Proc Natl Acad Sci USA 98, 5550–5555 15 Takeya R, Ueno N, Kami K, Taura M, Kohjima M, Izaki T, Nunoi H & Sumimoto H (2003) Novel human homologues of p47phox and p67phox participate in activation of superoxide-producing NADPH oxidases J Biol Chem 278, 25234–25246 16 Ambasta RK, Kumar P, Griendling KK, Schmidt HH, Busse R & Brandes RP (2004) Direct interaction of the novel Nox proteins with p22phox is required for the formation of a functionally active NADPH oxidase J Biol Chem 279, 45935–45941 ´ 17 Banfi B, Clark RA, Steger K & Krause K-H (2003) Two novel proteins activate superoxide generation by the NADPH oxidase NOX1 J Biol Chem 278, 3510– 3513 18 Geiszt M, Lekstrom K, Witta J & Leto TL (2003) Proteins homologous to p47phox and p67phox support superoxide production by NAD(P)H oxidase in colon epithelial cells J Biol Chem 278, 20006–20012 3676 19 Cheng G & Lambeth JD (2004) NOXO1, regulation of lipid binding, localization, and activation of Nox1 by the Phox homology (PX) domain J Biol Chem 279, 4737–4742 20 Ago T, Takeya R, Hiroaki H, Kuribayashi F, Ito T, Kohda D & Sumimoto H (2001) The PX domain as a novel phosphoinositide-binding module Biochem Biophys Res Commun 287, 733–738 21 Kanai F, Liu H, Field SJ, Akbary H, Matsuo T, Brown GE, Cantley LC & Yaffe MB (2001) The PX domains of p47phox and p40phox bind to lipid products of PI (3) K Nat Cell Biol 3, 675–678 22 Wientjes FB & Segal AW (2003) PX domain takes shape Curr Opin Hematol 10, 2–7 23 Ago T, Kuribayashi F, Hiroaki H, Takeya R, Ito T, Kohda D & Sumimoto H (2003) Phosphorylation of p47phox directs phox homology domain from SH3 domain toward phosphoinositides, leading to phagocyte NADPH oxidase activation Proc Natl Acad Sci USA 100, 4474–4479 24 Paffenholz R, Bergstrom RA, Pasutto F, Wabnitz P, Munroe RJ, Jagla W, Heinzmann U, Marquardt A, Bareiss A, Laufs J, et al (2004) Vestibular defects in head-tilt mice result from mutations in Nox3, encoding an NADPH oxidase Genes Dev 18, 486–491 25 Ueno N, Takeya R, Miyano K, Kikuchi H & Sumimoto H (2005) The NADPH oxidase Nox3 constitutively produces superoxide in a p22phox-dependent manner: Its regulation by oxidase organizers and activators J Biol Chem 280, 23328–23339 ´ 26 Banfi B, Malgrange B, Knisz J, Steger K, Dubois-Dauphin M & Krause K-H (2004) NOX3, a superoxide-generating NADPH oxidase of the inner ear J Biol Chem 279, 46065–46072 27 Cheng G, Ritsick D & Lambeth JD (2004) Nox3 regulation by NOXO1, p47phox, and p67phox J Biol Chem 279, 34250–34255 28 Cheng G & Lambeth JD (2005) Alternative mRNA splice forms of NOXO1: differential tissue expression and regulation of Nox1 and Nox3 Gene 356, 118– 126 29 Hiroaki H, Ago T, Ito T, Sumimoto H & Kohda D (2001) Solution structure of the PX domain, a target of the SH3 domain Nat Struct Biol 8, 526–530 30 Bravo J, Karathanassis D, Pacold CM, Pacold ME, Ellson CD, Anderson KE, Butler PJ, Lavenir I, Perisic O, Hawkins PT, et al (2001) The crystal structure of the PX domain from p40phox bound to phosphatidylinositol 3-phosphate Mol Cell 8, 829–839 31 Karathanassis D, Stahelin RV, Bravo J, Perisic O, Pacold CM, Cho W & Williams RL (2002) Binding of the PX domain of p47phox to phosphatidylinositol 3,4-bisphosphate and phosphatidic acid is masked by an intramolecular interaction EMBO J 21, 5057– 5068 FEBS Journal 273 (2006) 3663–3677 ª 2006 The Authors Journal compilation ª 2006 FEBS R Takeya et al 32 Cheng G, Cao Z, Xu X, van Meir EG & Lambeth JD (2001) Homologs of gp91phox: cloning and tissue expression of Nox3, Nox4, and Nox5 Gene 269, 131–140 33 Harper RW, Xu C, Soucek K, Setiadi H & Eiserich JP (2005) A reappraisal of the genomic organization of human Nox1 and its splice variants Arch Biochem Biophys 435, 323–330 34 Arbiser JL, Petros J, Klafter R, Govindajaran B, McLaughlin ER, Brown LF, Cohen C, Moses M, Kilroy S, Arnold RS, et al (2002) Reactive oxygen generated by Nox1 triggers the angiogenic switch Proc Natl Acad Sci USA 99, 715–720 35 Lim SD, Sun C, Lambeth JD, Marshall F, Amin M, Chung L, Petros JA & Arnold RS (2005) Increased Nox1 and hydrogen peroxide in prostate cancer Prostate 62, 200–207 36 Takai Y, Sasaki T & Matozaki T (2001) Small GTPbinding proteins Physiol Rev 81, 153–208 37 Kawahara T, Kohjima M, Kuwano Y, Mino H, Teshima-Kondo S, Takeya R, Tsunawaki S, Wada A, Sumimoto H & Rokutan K (2005) Helicobacter pylori lipopolysaccharide activates Rac1 and transcription of NADPH oxidase Nox1 and its organizer NOXO1 in guinea pig gastric mucosal cells Am J Physiol Cell Physiol 288, C450–C457 38 Motoyama T, Watanabe H, Yamamoto T & Sekiguchi M (1987) Human testicular germ cell tumors in vitro and in athymic nude mice Acta Pathol Jpn 37, 431–448 Expression and function of Noxo1c 39 Yoshida T, Izumi H, Uchiumi T, Sasaguri Y, Tanimoto A, Matsumoto T, Naito S & Kohno K (2006) Expression and cellular localization of dbpC ⁄ Contrin in germ cell tumor cell lines Biochim Biophys Acta 1759, 80–88 40 Ago T, Nunoi H, Ito T & Sumimoto H (1999) Mechanism for phosphorylation-induced activation of the phagocyte NADPH oxidase protein p47phox Triple replacement of serines 303, 304, and 328 with aspartates disrupts the SH3 domain–mediated intramolecular interaction in p47phox, thereby activating the oxidase J Biol Chem 274, 33644–33653 41 Koga H, Terasawa H, Nunoi H, Takeshige K, Inagaki F & Sumimoto H (1999) Tetratricopeptide repeat (TPR) motifs of p67phox participate in interaction with the small GTPase Rac and activation of the phagocyte NADPH oxidase J Biol Chem 274, 25051–25060 42 Takeya R & Sumimoto H (2003) Fhos, a mammalian formin, directly binds to F-actin via a region N-terminal to the FH1 domain and forms a homotypic complex via the FH2 domain to promote actin fiber formation J Cell Sci 116, 4567–4575 43 Kuribayashi F, Nunoi H, Wakamatsu K, Tsunawaki S, Sato K, Ito T & Sumimoto H (2002) The adaptor protein p40phox as a positive regulator of the superoxideproducing phagocyte oxidase EMBO J 21, 6312–6320 FEBS Journal 273 (2006) 3663–3677 ª 2006 The Authors Journal compilation ª 2006 FEBS 3677 ... investigate the physiological relevance of Noxo1c expression in the testis, we examined expression of Nox1 and Noxa1 by PCR analysis and found a small but significant amount of the Nox1 and Noxa1 mRNAs... al Expression and function of Noxo1c A B C Fig Alternative splice forms of human Noxo1 (A) The genomic organization of human NOXO1 gene Translated sequences are shown as black boxes, and untranslated... extent of membrane localization of Noxo1b and Noxo1c The intensities of immunoreactive bands for HA-Noxo1b and HA-Noxo1c in (B) were quantified using a LAS -10 00plus (Fuji film) image analyzer and