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

Expression and function of Noxo1c, an alternative splicing
form of the NADPH oxidase organizer 1
Ryu Takeya
1,2
, Masahiko Taura
1
, Tomoko Yamasaki
1
, Seiji Naito
3
and Hideki Sumimoto
1,2
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
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 gp91
phox
, also termed Nox2, is predominantly
expressed in professional phagocytes, and plays a cru-
cial role in host defense; superoxide generation by
gp91
phox
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 mem-
brane-integrated protein p22
phox
to form a functional
heterodimer [15,16].
Activation of gp91
phox
, also complexed with p22
phox
,
absolutely requires the two cytosolic proteins p47
phox
and p67
phox
. Nox organizer 1 (Noxo1) and Nox
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:
(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, gp91
phox
⁄ 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 gp91
phox
⁄ 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.
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)P
2
, phosphatidylinositol 3,5-bisphosphate; PX, phox homology.
FEBS Journal 273 (2006) 3663–3677 ª 2006 The Authors Journal compilation ª 2006 FEBS 3663
activator 1 (Noxa1), novel respective homologs of
p47
phox
and p67
phox
, 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 p47
phox
and Noxo1 each contain two
SH3 domains, arranged tandemly. In p47
phox
, the SH3
domains normally interact intramolecularly with the
autoinhibitory region, which prevents the domains
from binding to the target p22
phox
. A conformational
change in p47
phox
, which can be induced by its
phosphorylation by protein kinase C, enables the pro-
tein to access p22
phox
, leading to superoxide produc-

tion. On the other hand, Noxo1 lacks the
autoinhibitory region [15,17,18]; its SH3 domains are
capable of binding to p22
phox
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 p47
phox
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 p47
phox
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 (p47
phox

or Noxo1) or an oxidase activator
(p67
phox
or Noxa1) [25]. Nox3, like gp91
phox
and Nox1,
forms a functional complex with p22
phox
in transfected
cells; and the organizers p47
phox
and Noxo1 are capable
of enhancing the superoxide production by Nox3 via
the interaction of their SH3 domains with p22
phox
[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. How-
ever, little is known about the expression pattern of
the splicing variants, as they could not be distin-
guished in assays used in previous studies of
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
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)
[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), 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
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 p40
phox
and p47
phox
is directly involved in the interaction with phospho-
inositides [30,31]. The loop region in the PX domain
of p40
phox
faces the phosphoinositide-binding pocket
as shown by the crystal structure of the p40
phox
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 p47
phox
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
Expression and function of Noxo1c R. Takeya et al.
3664 FEBS Journal 273 (2006) 3663–3677 ª 2006 The Authors Journal compilation ª 2006 FEBS

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).
A
C
B
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, p47
phox
, p40
phox
, SNX3, and Vam7p. The alignments take
the secondary structure of the p47
phox
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 p40
phox
and Lys79 in p47
phox
, mentioned in the text, are

underlined.
R. Takeya et al. Expression and function of Noxo1c
FEBS Journal 273 (2006) 3663–3677 ª 2006 The Authors Journal compilation ª 2006 FEBS 3665
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 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
and Noxo1b were almost equally expressed in the tes-
tis, 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 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
[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 andro-
gen-independent prostate cancer PC3 and DU145 cells
and the testicular germ cell 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
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.
Expression and function of Noxo1c R. Takeya et al.
3666 FEBS Journal 273 (2006) 3663–3677 ª 2006 The Authors Journal compilation ª 2006 FEBS
A
B
C

D
E
F G
R. Takeya et al. Expression and function of Noxo1c
FEBS Journal 273 (2006) 3663–3677 ª 2006 The Authors Journal compilation ª 2006 FEBS 3667
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
transfected Chinese hamster ovary (CHO) cells with
pcDNA3.0–Nox1, pEF-BOS–p22
phox
, pEF-BOS–
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
A
B
C
D
Fig. 3. Noxo1c-supported activation of Nox1. (A) CHO cells were
cotransfected with pcDNA3.0–Nox1, pEF-BOS–p22
phox
, pEF-BOS–
myc-Noxa1, and simultaneously with pEF-BOS–HA-Noxo1b, pEF-
BOS–HA-Noxo1c, or pEF-BOS–HA-Noxo1a. The transfected cells
(1 · 10
6
cells) were incubated for 10 min at 37 °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–p22
phox

, pEF-BOS–myc-Noxa1, and simultaneously with
pEF-BOS–HA-Noxo1b, pEF-BOS–HA-Noxo1c, or pEF-BOS–HA-
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–p22
phox
(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–p22
phox
, 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

)at37°C. The graph represents
the mean ± SD of chemiluminescence values obtained from three
independent transfections.
Expression and function of Noxo1c R. Takeya et al.
3668 FEBS Journal 273 (2006) 3663–3677 ª 2006 The Authors Journal compilation ª 2006 FEBS
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 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
p22
phox
, 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-
slips. 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 fur-
ther targeted to the membrane after stimulation with
PMA. On the other hand, Noxo1b was constitutively
A
C
PMA: (–) (+)
1 o x o N
β
β
1 o x o N
γ
γ
1 o x o N
β
β
1 o x o N

γ
γ
Blot: anti-HA
Blot: anti-p22
phox
PMA: (–) (+)
1 o x o N
β
β
1 o x o N
γ
γ
1
o x o
N
β
β
1 o x o N
γ
γ
0
2
1
3
B
Merge
Noxo1 β
β
p22
phox

Noxo1 γ
γ
p22
phox
Merge
n
o i
t
a z i l
a c o l e n a r b m e m
) e
s a
e r c
n
i d
l o f (
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 p22
phox
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 p22
phox
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.
R. Takeya et al. Expression and function of Noxo1c
FEBS Journal 273 (2006) 3663–3677 ª 2006 The Authors Journal compilation ª 2006 FEBS 3669
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.
The mechanism for the PMA-dependent membrane
recruitment of Noxo1c is at present unknown. It is
well established that p47
phox
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
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 glutathione S-transferase (GST)-fused PX
domains. The PX domain of Noxo1b bound to phos-
phatidylinositol 3,5-bisphosphate [PtdIns(3,5)P
2
] with
the highest affinity, which is consistent with the recent
report of Cheng & Lambeth [19]; it also interacted with
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 lipo-
some-binding assay also showed that the Noxo1c PX

domain interacted with phospholipids such as
PtdIns(3,5)P
2
more weakly than that of Noxo1b (data
not shown). Thus the insertion in the PX domain
decreases the affinity for phosphoinositides.
Activation of gp91
phox
and Nox3 by Noxo1c
We next investigated the ability of Noxo1c to activate
gp91
phox
⁄ Nox2 and Nox3. In CHO cells expressing
gp91
phox
⁄ Nox2 with p67
phox
, Noxo1c supported super-
oxide production to a much lesser extent than Noxo1b
(Fig. 6A). When coexpressed with Noxa1, the Noxo1c-
supported superoxide production by gp91
phox
⁄ 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
p22
phox
in Nox1-dependent and Nox3-dependent
superoxide production
It is known that Noxo1 functions via the SH3-mediated
interaction with p22
phox
, which forms a heterodimer
GST-Noxo1
β
β
-PX GST-Noxo1
γ
-PX
AB
PI
PI3P
PI4P
PI5P
PI(3,5)P
2
PI(4,5)P2
PI(3,4)P2
PI(3,4,5)P3
PI
PI3P
PI4P

PI5P
PI(3,5)P
2
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)P
2
, phosphatidylinositol 3,5-bisphosphate;
PI(4,5)P
2
, phosphatidylinositol 4,5-bisphosphate; PI(3,4)P
2
, phosphatidylinositol 3,4-bisphosphate; PI(3,4,5)P
3
, phosphatidylinositol 3,4,5-tris-
phosphate. For details, see Experimental procedures.
Expression and function of Noxo1c R. Takeya et al.
3670 FEBS Journal 273 (2006) 3663–3677 ª 2006 The Authors Journal compilation ª 2006 FEBS
with Nox1, gp91
phox
⁄ Nox2, and Nox3 [25]. It may be
possible that the interaction with p22
phox
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 p22
phox
to the
same extent; the binding was completely abolished by
the P156Q substitution in p22
phox
, a mutation leading
to defective interaction with the SH3 domains of
Noxo1 [15]. In addition, Noxo1c as well as Noxo1b
interacted with p22
phox
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
interaction with p22
phox
. To confirm this, we investi-
gated the dependence of Noxo1c-supported Nox
activation on p22
phox
. 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 p22
phox
cDNA [15], whereas Nox3
activity requires p22

phox
expression under the same
conditions [25]. Similarly, Noxo1c-supported superox-
ide production by Nox1 is partly dependent on p22
phox
(Fig. 7C); on the other hand, the expression of p22
phox
was a requisite for the Noxo1c-supported Nox3
activity (Fig. 7D). Thus Noxo1c probably binds to
p22
phox
in a manner similar to Noxo1b.
Role of phosphoinositide-binding activity of
Noxo1 in Nox3 activation
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 gp91
phox
⁄ 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 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).
A
DC
B
Nox3 + Noxa1
ecnecsenimulimehc
0
1
x

(
6
)
m
pc
5
3
0
1
4
2
gp91
phox
+ Noxa1
ecnecsenimulimehc
01x(
6
)
mpc
1.2
0
0.4
1.6
0.8
gp91
phox
+ p67
phox
ecnecsenimulimehc
01x(

6
)mpc
5
3
0
1
4
2
Noxo1
β
β
Noxo1
γ
γ
Nox3
1.2
ecnecsenimulimehc
01x(
7
)mpc
0
0.4
0.8
PMA(–)
PMA(+)
Noxo1
β
β
Noxo1
γ

γ
Noxo1
β
β
Noxo1
γ
γ
Noxo1
β
β
Noxo1
γ
γ
Fig. 6. Noxo1c-supported activation of gp91
phox
⁄ Nox2 and Nox3.
CHO cells were cotransfected with the following combination of
plasmids: pcDNA3.0–gp91
phox
, pEF-BOS–p22
phox
, pEF-BOS–myc-
p67
phox
, and simultaneously with pEF-BOS–HA-Noxo1b or
pEF-BOS–HA-Noxo1c in (A); pcDNA3.0–gp91
phox
, pEF-BOS–
p22
phox

, pEF-BOS–myc-Noxa1, and simultaneously with pEF-BOS–
HA-Noxo1b or pEF-BOS–HA-Noxo1c in (B); pcDNA3.0–Nox3 and
pEF-BOS–p22
phox
, and simultaneously with pEF-BOS–HA-Noxo1b
or pEF-BOS–HA-Noxo1c in (C); pcDNA3.0–Nox3 and pEF-BOS–
p22
phox
, 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).
R. Takeya et al. Expression and function of Noxo1c
FEBS Journal 273 (2006) 3663–3677 ª 2006 The Authors Journal compilation ª 2006 FEBS 3671
Concluding remarks
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 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 phos-
phoinositides than that of Noxo1b (Fig. 5), which
seems to attenuate the membrane localization in resting
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 gp91
phox
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.
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¢-GCAGGATCC
AT
4

2
0
3
1
ec
n
ecsenimu
l
imehc
01x(
5
)mpc
Nox3 + Noxo1
γ
γ
e
cnecsenimulimehc
01x(
6
)mpc
2.0
1.2
0
0.4
1.6
0.8
Nox1 + Noxa1 + Noxo1
γ
γ
– p22

phox
+ p22
phox
A
B
C
D
p22
phox
-C
(WT)
p22
phox
-C
(P156Q)
1oxoN–TSG
β
-
Δ
C
1oxoN–TSG
γ
-
Δ
C
MBP–p22
phox
-C
Noxo1
γ

-
Δ
C
His
(+) (–)
Noxo1
β
-
Δ
C
p22
phox
-C
(WT)
p22
phox
-C
(P156Q)
His
(+) (–)
1oxoN–TSG
β
-
Δ
C
1oxoN–TSG
γ
-
Δ
C

– p22
phox
+ p22
phox
Fig. 7. Role of the interaction between Noxo1c and p22
phox
in Nox1-dependent and Nox3-dependent superoxide production. (A) Interaction
between Noxo1 and p22
phox
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–p22
phox
-C (amino acids 132–195) or MBP–p22
phox
-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 p22
phox
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 p22
phox
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–p22
phox

in (C); pcDNA3.0–Nox3, pEF-BOS–
myc-Noxa1, pEF-BOS–HA-Noxo1c, and with or without pEF-BOS–p22
phox
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.
Expression and function of Noxo1c R. Takeya et al.
3672 FEBS Journal 273 (2006) 3663–3677 ª 2006 The Authors Journal compilation ª 2006 FEBS
GGCAGGCCCCCGATACCCAG-3¢ and 5¢-CGTCTCGA
G
GAGGCGGCCCGCAGCGCGAGA-3¢; sequences from
the mRNA are underlined. With the two primers, PCR was
performed using Human Multiple Tissue cDNA (MTC
TM
)
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.
A
gp91

phox
+ Noxa1
Nox1 + Noxa1
C
B
PMA(–)
PMA(+)
Noxo1
β Δ
PX vector
Nox3
ecnecsenimulimehc
01x(
7
)mpc
1.6
0
1.2
0.8
0.4
ecnecse
ni
m
uli
me
hc
01x
(
5
)mpc

4.0
0
3.0
2.0
1.0
ecn
ecse
ni
m
uli
me
hc
01x
(
6
)mpc
3.0
0
2.0
1.0
Noxo1
β Δ
PX vector
Noxo1
β Δ
PX vector
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–p22
phox

, 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–
gp91
phox
⁄ Nox2, pEF-BOS–p22
phox
, 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–p22
phox
, 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
).
ecnecsenimulimehc
01x(
6
)mpc
2
0
3
1
Noxo1
β
(R40Q)
Noxo1

γ
(R40Q)
Noxo1
γ
Noxo1
β
A
Nox1 + Noxa1
ec
necsenimulimeh
c
01x(
5
)
m
pc
8
4
0
6
2
Noxo1
β
(R40Q)
Noxo1
γ
(R40Q)
Noxo1
γ
Noxo1

β
B
Nox3
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–p22
phox
, 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–p22
phox
, 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.
R. Takeya et al. Expression and function of Noxo1c
FEBS Journal 273 (2006) 3663–3677 ª 2006 The Authors Journal compilation ª 2006 FEBS 3673
Expression of Noxo1b and Noxo1c in various

human tissues
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
primers: ‘a’, 5¢-TCTCCCAAAGCTTCTCGATGC-3¢ (for-
ward 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 dehydroge-
nase 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 2 lLof
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.
Superoxide-producing activity of CHO cells
expressing Nox1, gp91
phox
, or Nox3
The cDNAs for Nox1, gp91
phox
, and Nox3 were ligated
to the mammalian expression vector pcDNA3.0 (Invitro-
gen), and cDNAs encoding p22
phox
, p47
phox
, p67
phox
,
Noxo1, and Noxa1 were ligated to the mammalian
expression vector pEF-BOS [15,25]. Noxo1 and p47
phox
were constructed for expression as a hemaglutinin (HA)-
tagged protein, Noxa1 and p67
phox
as a myc-tagged pro-
tein, and p22

phox
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
for 1 min at 37 °C, and washed with Hepes-buffered sal-
ine (120 mm NaCl, 5 mm KCl, 5 mm glucose, 1 mm
MgCl
2
, 0.5 mm CaCl
2
and 17 mm Hepes, pH 7.4). Super-
oxide 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 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).
Measurement of superoxide production using
adherent cells undetached from culture dishes
CHO cells were plated on six-well plates (1 · 10
5
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 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].
Localization of Noxo1 c 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 min at 25 °C in 3.7% formaldehyde. The fixed cells
were washed four times with phosphate-buffered saline
(NaCl ⁄ P
i
: 137 mm NaCl, 2.68 mm KCl, 8.1 mm
Na
2
HPO
4
, and 1.47 mm KH

2
PO
4
), and blocked with
NaCl ⁄ P
i
containing 3% BSA for 60 min. The sample was
subsequently incubated with the monoclonal antibody to
HA and probed with Alexa Fluor 488
TM
-labeled goat
anti-mouse IgG (Invitrogen, Carlsbad, CA, USA) as sec-
ondary antibodies. For detection of p22
phox
, the sample
was incubated with polyclonal antibodies to p22
phox
,
which were raised against the C-terminal 20 amino acids
of human p22
phox
[25] and probed with Alexa Fluor
Expression and function of Noxo1c R. Takeya et al.
3674 FEBS Journal 273 (2006) 3663–3677 ª 2006 The Authors Journal compilation ª 2006 FEBS
594
TM
-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).

Membrane translocation of Noxo1
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 · 10
6
cells per ml in NaCl ⁄ P
i
and stimulated for 10 min
at 37 °C with PMA (200 ngÆmL
)1
). After centrifugation,
cells were resuspended in NaCl ⁄ P
i
containing 0.5 mm
EGTA, 20 lm p-amidinophenylmethanesulfonyl fluoride,
80 lgÆmL
)1
leupeptin, 20 lg Æ mL
)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 ⁄ P
i
,
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.
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 (Amer-
sham Bioscience), as previously described [14,21].
An overlay assay was carried out using the PIP array
TM
(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 °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].
In vitro liposome-binding assay was carried out as
previously described [20,23] with minor modifications.
Briefly, liposomes were prepared by mixing phosphatidyl-

ethanolamine (72%) and phosphatidylcholine (18%)
with 10% phosphatidylinositol, PtdIns(3)P, PtdIns(4)P,
PtdIns(5)P, PtdIns(3,4)P
2
, PtdIns(3,5)P
2
, PtdIns(4,5)P
2
or
PtdIns(3,4,5)P
3
, drying the mixture under a stream of
nitrogen, and resuspending in a sample buffer (100 mm
NaCl, 20 mm Hepes, pH 7.2). All synthetic phosphoinosi-
tides with C
16
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.
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 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).
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).
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