Báo cáo khoa học: Expression and function of Noxo1c, an alternative splicing form of the NADPH oxidase organizer 1

Expression and function of Noxo1c, an alternative splicing form of the NADPH oxidase organizer 1 Ryu Takeya1,2, Masahiko Taura1, Tomoko Yamasaki1, Seiji Naito3 and Hideki Sumimoto1,2

1 Medical Institute of Bioregulation, Kyushu University, Fukuoka, Japan 2 CREST, Japan Science and Technology Agency, Saitama, Japan 3 Department of Urology, Kyushu University Graduate School of Medical Sciences, Fukuoka, Japan

Keywords NADPH oxidase (Nox1); Nox organiser 1 (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 phosphoinosi- tide-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 rest- ing cells, Noxo1c is poorly localized to the membrane, and thus less effect- ive 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 transloca- tion of Noxo1c; as a result, Noxo1c is equivalent to Noxo1b in Nox1 acti- vation 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 activa- tion of gp91phox ⁄ Nox2, Noxo1c is less active than Noxo1b even in the presence of PMA, whereas Noxo1c and Noxo1b support the superoxide- producing activity of Nox3 to the same extent in a manner independent of cell stimulation.

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 mem- brane-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 1 (Noxo1) and Nox

Members of the NADPH oxidase (Nox) family pro- duce superoxide from molecular oxygen in conjunction with oxidation of NADPH [1–10]. Superoxide gener- ated serves as a precursor of other reactive oxygen spe- cies, 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 cru- cial role in host defense; superoxide generation by gp91phox leads to subsequent formation of microbicidal reactive oxygen species such as hydroxyl radical and

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.

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expression of the Noxo1 mRNA [15,17,18]. In addi- tion, it remained to be elucidated whether each tran- script possesses the activity to support activation of the Nox enzymes. In this study, we show the expres- sion 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

the

lacks

other

hand, Noxo1

(AY255768)

divergence

among

activator 1 (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 pro- tein to access p22phox, leading to superoxide produc- the tion. On 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 acti- vator of protein kinase C, is not required for Noxo1- dependent 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, mem- brane trafﬁcking, and polarity establishment, and func- tion 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 mem- brane recruitment of the protein and subsequent activation of the phagocyte oxidase [23]. The phospho- inositide-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 forma- tion in mouse inner ears [24], and appears to be consti- tutively 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.

We have previously identiﬁed a transcript (AB097667) of human NOXO1 gene encoding 371 amino acids [15], which is identical with that reported by Ba´ nﬁ et al. (AF539796) and Cheng & Lambeth (AF532984) (Fig. 1A). On the other hand, Geiszt et al. [17,19] reported the alternative transcript [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), cur- rently 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 3 (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 various PX occurs sequence 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 phospho- inositides [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 phospho- inositide-binding region, affecting the afﬁnity for

In the process of cloning of human Noxo1, some the NOXO1 gene have been spliced transcripts of 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. How- ever, little is known about the expression pattern of the splicing variants, as they could not be distin- studies of guished in assays used in previous

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A

B

C

Fig. 1. 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 3 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 hydro- phobic 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 dele- tion 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 3. 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).

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and Noxo1b were almost equally expressed in the tes- tis, whereas Noxo1b was the major form in the colon.

Expression of Noxo1b and Noxo1c in various human tissues

signiﬁcant amount of

testicular germ cell

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 thy- mus, 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 subjec- ted the PCR fragments to PAGE; the two fragments were clearly separated. As shown in Fig. 2E, Noxo1c

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 the Nox1 and Noxa1 mRNAs in the testis (data not shown), which is consis- tent 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 revealed that LNCaP cells [33]. RT-PCR analysis abundantly the mRNA for Noxo1c expressed (Fig. 2F). The Noxo1c mRNA also existed in several Nox1-expressing human cancer cell lines: the andro- gen-independent prostate cancer PC3 and DU145 cells and the tumor NEC8 cells (Fig. 2F). Noxa1, a protein that activates Nox1 in co- operation 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 sys- tem. As shown in Fig. 2G, the Noxo1c mRNA as well as the Noxo1b mRNA was expressed in the occipital

Fig. 2. 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 prim- ers, PCR was performed using cDNAs for Noxo1b or Noxo1c as a template, and the PCR products were subjected to 2% agarose-gel elec- trophoresis, 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 expres- sion 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 lines: androgen-independent prostate cancer cells results. (F) Expression of Noxo1b, Noxo1c, Nox1, and Noxa1 in various human cell (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.

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A

B

C

D

E

F

G

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pEF-BOS–p22phox,

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

is well known that

the classical

It splicing form Noxo1b is essential for activation of Nox1 [15,17–19]. To know the activity of Noxo1c to activate Nox1, we

A

transfected Chinese hamster ovary (CHO) cells with pEF-BOS– pcDNA3.0–Nox1, 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 tran- script, was much less active even in the presence of PMA (Fig. 3B). We further investigated the stimulus- independent activity of Nox1 using CHO cells trans- fected 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

B

C

pEF-BOS–HA-Noxo1c,

D

Fig. 3. 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, pEF- BOS–HA-Noxo1c, or pEF-BOS–HA-Noxo1a. The transfected cells (1 · 106 cells) were incubated for 10 min at 37 (cid:2)C, and then stimu- lated with PMA (200 ngÆmL)1). Chemiluminescence change was continuously monitored with Diogenes, and superoxide dismutase (50 lgÆmL)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- pEF-BOS–HA-Noxo1b, Noxo1a. Superoxide production was assayed by chemilumines- cence using Diogenes in the presence or absence of PMA (200 ngÆmL)1). Each graph represents the mean ± SD of the peak chemiluminescence values obtained from three independent trans- fections. (C) CHO cells were transfected simultaneously with pcDNA3.0–Nox1 (1 lg), pEF-BOS–p22phox (1 lg), pEF-BOS–myc- Noxa1 (1 lg), and the indicated amount of pEF-BOS–HA-Noxo1b (left panel) or pEF-BOS–HA-Noxo1c (right panel). Superoxide pro- duction was assayed by chemiluminescence using Diogenes in the presence or absence of PMA (200 ngÆmL)1), and expressed as the percentage activity relative to that of 1 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 cotransfect- ed with pcDNA3.0–Nox1, pEF-BOS–p22phox, pEF-BOS–myc-Noxa1, and simultaneously with pEF-BOS–HA-Noxo1b, pEF-BOS–HA- Noxo1c. PMA-independent superoxide production was assayed by chemiluminescence using Diogenes in the presence or absence of superoxide dismutase (50 lgÆmL)1) at 37 (cid:2)C. The graph represents the mean ± SD of chemiluminescence values obtained from three independent transfections.

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A

Noxo1 β β

p22 phox

Merge

Noxo1 γ γ

p22 phox

Merge

B

C

3

2 n o i t a z i l a c o

PMA: (–) (+)

) e s a e r c n

i

l

1 d o f (

β β 1 o x o N

γ γ 1 o x o N

β β 1 o x o N

l e n a r b m e m

0 PMA: (–) (+)

Blot: anti-HA

Blot: anti-p22 phox

β β 1 o x o N

γ γ 1 o x o N

β β 1 o x o N

γ γ 1 o x o N

Fig. 4. 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), local- ization 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 ngÆmL)1), the cell lysates were fractionated by centrifugation, and the mem- brane fractions were analyzed by immuno- blot 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.

punctate

intracellular

activation [37]. To test the possibility that cell detach- ment elicits activation of Nox1, we estimated superox- ide production by adherent cells. Adherent CHO cells, coexpressing Nox1 with both Noxo1b and Noxa1, pro- duced a considerable amount of superoxide (Fig. 3D). Under the same conditions, Noxo1c weakly supported superoxide production compared with Noxo1b, con- ﬁrming that Noxo1c is more effective than Noxo1b in activating Nox1 in unstimulated cells.

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

concentrated 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 consis- tent 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 cover- the localization of slips. To biochemically assess 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 fur- ther targeted to the membrane after stimulation with PMA. On the other hand, Noxo1b was constitutively

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A

B

GST-Noxo1ββ-PX

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. 5. 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-tris- phosphate. For details, see Experimental procedures.

phosphatidylinositol

interacted with

phospholipids

such

associated with the membrane fraction (Fig. 4B). As the extent of the membrane localization of Noxo1 cor- related 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.

PtdIns(3)P and 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 lipo- some-binding assay also showed that the Noxo1c PX domain 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

The mechanism for the PMA-dependent membrane recruitment of Noxo1c is at present unknown. It is well established that p47phox undergoes phosphoryla- tion in response to PMA, which is essential for mem- brane translocation of this protein. As Noxo1 also has several potential protein kinase C phosphorylation sites, Noxo1 might become phosphorylated in PMA- stimulated cells, leading to membrane translocation.

Phosphoinositide-binding activity of the PX domains of Noxo1b and Noxo1c

We next investigated the ability of Noxo1c to activate gp91phox ⁄ Nox2 and Nox3. In CHO cells expressing gp91phox ⁄ Nox2 with p67phox, Noxo1c supported super- oxide production to a much lesser extent than Noxo1b (Fig. 6A). When coexpressed with Noxa1, the Noxo1c- supported 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.

(GST)-fused

glutathione

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

The membrane localization of Noxo1b is mediated in part by binding of the PX domain to membrane phos- pholipids [19]. Less association of Noxo1c with the membrane (Fig. 3) raised the possibility that the phos- pholipid-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 phospho- inositide was spotted on the membrane and overlaid with PX S-transferase domains. The PX domain of Noxo1b bound to phos- phatidylinositol 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

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A

B

gp91phox + p67phox

gp91phox + Noxa1

)

PMA(–)

)

1.6 PMA(+)

1.2 m p c

i

0.8 6 0 1

e c n e c s e n m u

l i

6 0 1 x

l i

0.4 x (

(

5 4 3 2 1 0

0 m e h c

Noxo1ββ Noxo1γγ

for

C

D

Nox3

Nox3 + Noxa1

1.2

interaction with p22phox. To conﬁrm this, we investi- gated the dependence of Noxo1c-supported Nox activation on p22phox. It is known that superoxide pro- duction by Nox1 in CHO cells expressing Noxo1b is largely but not completely dependent on the cotrans- fection with the p22phox cDNA [15], whereas Nox3 activity requires p22phox expression under the same conditions [25]. Similarly, Noxo1c-supported superox- ide production by Nox1 is partly dependent on p22phox (Fig. 7C); on the other hand, the expression of p22phox was a requisite the Noxo1c-supported Nox3 activity (Fig. 7D). Thus Noxo1c probably binds to p22phox in a manner similar to Noxo1b.

)

0.8 m p c

i

7 0 1

6 0 1

e c n e c s e n m u

0.4 l i

x

l i

Role of phosphoinositide-binding activity of Noxo1 in Nox3 activation

x (

(

0 5 4 3 2 1 0

m e h c

Noxo1ββ Noxo1γγ

and

simultaneously with

pEF-BOS–HA-Noxo1b

(A);

pcDNA3.0–gp91phox,

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).

Fig. 6. 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–myc- p67phox, or pEF-BOS–HA-Noxo1c 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 ngÆmL)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 mem- brane. 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 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 pro- duction 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-medi- ated 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 suppor- ted 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).

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Expression and function of Noxo1c

B

A

p22phox-C (WT)

p22phox-C (P156Q)

Noxo1β-ΔC

C Δ - γ 1 o x o N – T S G

C Δ - β 1 o x o N – T S G

C Δ - γ 1 o x o N – T S G

C Δ - β 1 o x o N – T S G

Noxo1γ-ΔC

MBP–p22phox-C

(+) (–)

His

p22phox-C (WT)

p22phox-C (P156Q)

C

D

Nox3 + Noxo1γγ

Nox1 + Noxa1 + Noxo1γγ

4

2.0

1.6 )

3 )

m p c

1.2 m p c

i

2 6 0 1

e c n e c s e n m u

0.8 5 0 1 x

l i

(

x (

1

0.4 m e h c

0 – p22phox + p22phox

Fig. 7. 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 histidine- dependent (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 ngÆmL)1). Each graph represents the mean ± SD of the peak chemiluminescence values obtained from three independent transfections.

Concluding remarks

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 pres- ence 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 super- oxide production by all the three Nox enzymes.

cancer PC3

prostate

Experimental procedures

Isolation of cDNA for splice variants of human NOXO1 gene

In this study, we show that Noxo1c, a novel alternat- ive splicing form of human Noxo1 containing an addi- tional 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 Noxa1- expressing human cancer cell lines [the androgen- independent prostate cancer LNCaP cells, and the androgen-independent 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 phos- phoinositides than that of Noxo1b (Fig. 5), which seems to attenuate the membrane localization in resting

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Based on the sequence of mRNA for human NOXO1 (GenBank accession number AB097667), we synthesized the two unique oligonucleotide primers 5¢-GCAGGATCCAT

R. Takeya et al.

Expression and function of Noxo1c

A

Nox1 + Noxa1

3

1.6 PMA(–)

)

PMA(+)

1.2 )

2 m p c

i

0.8 m p c

i

7 0 1

e c n e c s e n m u

l i

e c n e c s e n m u

x (

0.4 6 0 1 x

l i

1 (

m e h c

0 m e h c

Noxo1β ΔPX vector

0 Noxo1β

Noxo1γ

B

gp91phox + Noxa1

Noxo1β (R40Q)

Noxo1γ (R40Q)

4.0 Nox3

)

B

3.0

8 m p c

i

2.0 e c n e c s e n m u

6 )

5 0 1 x

l i

(

1.0 m p c

i

m e h c

4

0 e c n e c s e n m u

Noxo1β ΔPX vector

l i

5 0 1 x (

2 C

Nox3

m e h c

3.0

0 Noxo1β

Noxo1γ

)

Noxo1β (R40Q)

Noxo1γ (R40Q)

2.0 i

e c n e c s e n m u

1.0 l i

m p c 6 0 1 x (

m e h c

0 Noxo1β ΔPX vector

Fig. 9. 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–HA- Noxo1b (R40Q), or pEF-BOS–HA-Noxo1c (R40Q). Superoxide production was assayed by chemiluminescence using Diogenes in the presence of PMA (200 ngÆmL)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 pres- ence of PMA (200 ngÆmL)1). Each graph represents the mean ± SD of the peak chemiluminescence values obtained from three independent transfections.

Fig. 8. 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–myc- Noxa1, 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 sim- ultaneously with pEF-BOS–HA-Noxo1b (wild-type), pEF-BOS–HA- Noxo1-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 ngÆmL)1).

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GGCAGGCCCCCGATACCCAG-3¢ 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. 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.

R. Takeya et al.

Expression and function of Noxo1c

Expression of Noxo1b and Noxo1c in various human tissues

superoxide dismutase-inhibitable

Measurement of superoxide production using adherent cells undetached from culture dishes

for 1 min at 37 (cid:2)C, and washed with Hepes-buffered sal- ine (120 mm NaCl, 5 mm KCl, 5 mm glucose, 1 mm MgCl2, 0.5 mm CaCl2 and 17 mm Hepes, pH 7.4). Super- oxide production by the transfected cells was determined chemiluminescence by with an enhancer-containing luminol-based detection system (Diogenes; National Diagnostics, Atlanta, GA, USA), as pre- viously described [15,23,40,41]. After the addition of the enhanced luminol-based substrate, the cells were stimulated with 200 ngÆmL)1 PMA. The chemiluminescence was assayed using a luminometer (Auto Lumat LB953; Berthold Technologies, Bad Wildbad, Germany).

for the Noxo1

Estimation of expression of cytosolic regulatory proteins

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).

Localization of Noxo1c and Noxo1b in CHO cells

Total cell lysates of transfected CHO cells were used to esti- mate 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].

Superoxide-producing activity of CHO cells expressing Nox1, gp91phox, or Nox3

The expression patterns of the Noxo1b and Noxo1c mes- sengers 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 (Invitro- gen, 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 ‘a’, 5¢-TCTCCCAAAGCTTCTCGATGC-3¢ (for- primers: cDNA); ward primer ‘b’, speciﬁc (forward primer 5¢-CCCAAAGCTTCTCGGTCAGGC-3¢ 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 ‘d’, 5¢-CCGCGT Noxo1c cDNAs, the following primer, 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 dehydroge- nase cDNA. PCR analyses were performed using ABI PRISM(cid:3) 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 2 lL of the ﬁrst-strand cDNA from different human tissues (Human MTC panels I, II, and Fetal MTC panel; Clon- tech) 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.

2.68 mm KCl, 137 mm NaCl,

incubated with polyclonal antibodies

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3674

tested Localization of HA-tagged Noxo1 proteins was using CHO cells as previously described with minor modiﬁcations [42]. Transfected CHO cells were ﬁxed for 15 min at 25 (cid:2)C in 3.7% formaldehyde. The ﬁxed cells were washed four times with phosphate-buffered saline 8.1 mm (NaCl ⁄ Pi: Na2HPO4, and 1.47 mm KH2PO4), and blocked with NaCl ⁄ Pi containing 3% BSA for 60 min. 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 sec- ondary antibodies. For detection of p22phox, the sample to p22phox, was which were raised against the C-terminal 20 amino acids of human p22phox [25] and probed with Alexa Fluor The cDNAs for Nox1, gp91phox, and Nox3 were ligated to the mammalian expression vector pcDNA3.0 (Invitro- gen), 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 pro- tein, and p22phox as a protein without a tag. Transfection of the CHO cells with the cDNAs was performed using (Roche Diagnostics, FuGENE6 Transfection Reagent Mannheim, Germany). After culture for 30 h, adherent cells were harvested by incubating with trypsin ⁄ EDTA

R. Takeya et al.

Expression and function of Noxo1c

Membrane translocation of Noxo1

594TM-labeled goat anti-rabbit IgG (Molecular Probes) as secondary antibodies. Images were visualized with a con- focal laser-scanning microscope LSM5 PASCAL (Carl Zeiss, Oberkochen, Germany).

Two-hybrid experiments

NaCl, 20 mm Hepes, pH 7.2). All synthetic phosphoinosi- tides with C16 fatty acids were purchased from Echelon Biosciences Inc; phosphatidylethanolamine, phosphatidyl- choline, and phosphatidylinositol from Sigma (St Louis, MO, USA). Liposomes (50 lm) were incubated for 10 min on ice with the indicated GST-fusion proteins (80 pmol) in 50 lL of the sample buffer. After ultracentrifugation for 30 min at 100 000 g, the supernatant was removed care- fully, 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, densito- metric analysis was performed using a LAS-1000plus (Fuji photo ﬁlm, Tokyo, Japan) image analyzer.

Acknowledgements

Lipid-binding assay using recombinant GST–fusion proteins

Membrane translocation of Noxo1 was determined as pre- viously described [23,43] with minor modiﬁcations. Brieﬂy, the CHO cells expressing Noxo1b or Noxo1c and other cytosolic factors were suspended at a concentration of 1 · 106 cells per ml in NaCl ⁄ Pi and stimulated for 10 min at 37 (cid:2)C with PMA (200 ngÆmL)1). After centrifugation, cells were resuspended in NaCl ⁄ Pi containing 0.5 mm EGTA, 20 lm p-amidinophenylmethanesulfonyl ﬂuoride, 80 lgÆmL)1 leupeptin, 20 lgÆmL)1 pepstatin A, 20 lgÆmL)1 chymostatin, and lysed by three rounds of 5-s sonication. The sonicates were centrifuged for 10 min at 10 000 g, and the supernatant was further ultracentrifuged for 45 min at 100 000 g. The resultant pellet was washed with NaCl ⁄ Pi, suspended in Laemmli sample buffer, and used as the mem- brane fraction. Proteins were analyzed by western blot with the monoclonal antibody to HA and developed by using ECL-plus. 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 des- cribed [15]. After the selection for Trp+ and Leu+ pheno- type, the transformants were tested for their ability to grow on plates lacking histidine, according to the manufacturer’s recommendation (Clontech).

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 JST (Japan Science and and BIRD projects of Technology Agency).

References

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 (Amer- sham Bioscience), as previously described [14,21].

1 Segal AW (2005) How neutrophils kill microbes. Annu Rev Immunol 23, 197–223. 2 Sumimoto H, Miyano K & Takeya R (2005) Molecular 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 incuba- ted 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 1 h and then overnight at 4 (cid:2)C with 500 ngÆmL)1 GST fusion protein. After being washed three times with Tris-buffered saline ⁄ Tween, the membranes were incubated with 1 : 1000 goat polyclonal antibodies to GST (Amersham Bioscience). Membranes were further incu- bated with 1 : 2500 donkey anti-goat IgG conjugated to horseradish peroxidase (Santa Cruz Biotechnology, Santa Cruz, CA, USA). The antibodies were detected by chemilu- minescence using ECL-plus as previously described [15].

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