Activation of dual oxidases Duox1 and Duox2: Differential regulation mediated by cAMP-dependent protein kinase and protein kinase C-dependent phosphorylation

Abstract

Dual oxidases were initially identified as NADPH oxidases producing H(2)O(2) necessary for thyroid hormone biosynthesis. The crucial role of Duox2 has been demonstrated in patients suffering from partial iodide organification defect caused by bi-allelic mutations in the DUOX2 gene. However, the Duox1 function in thyroid remains elusive. We optimized a functional assay by co-expressing Duox1 or Duox2 with their respective maturation factors, DuoxA1 and DuoxA2, to compare their intrinsic enzymatic activities under stimulation of the major signaling pathways active in the thyroid in relation to their membrane expression. We showed that basal activity of both Duox isoenzymes depends on calcium and functional EF-hand motifs. However, the two oxidases are differentially regulated by activation of intracellular signaling cascades. Duox1 but not Duox2 activity is stimulated by forskolin (EC(50) = 0.1 microm) via protein kinase A-mediated Duox1 phosphorylation on serine 955. In contrast, phorbol esters induce Duox2 phosphorylation via protein kinase C activation associated with high H(2)O(2) generation (phorbol 12-myristate 13-acetate EC(50) = 0.8 nm). These results were confirmed in human thyroid cells, suggesting that Duox1 is also involved in thyroid hormonogenesis. Our data provide, for the first time, detailed insights into the mechanisms controlling the activation of Duox1-2 proteins and reveal additional phosphorylation-mediated regulation.

Full text

Activation of Dual Oxidases Duox1 and Duox2
DIFFERENTIAL REGULATION MEDIATED BY cAMP-DEPENDENT PROTEIN KINASE AND
PROTEIN KINASE C-DEPENDENT PHOSPHORYLATION
*
□
S
Received for publication, September 5, 2008, and in revised form, January 12, 2009 Published, JBC Papers in Press, January 14, 2009, DOI 10.1074/jbc.M806893200
Sabrina Rigutto
‡1,2
, Candice Hoste
‡2
, Helmut Grasberger
§3
, Milutin Milenkovic
‡
, David Communi
‡4
,
Jacques E. Dumont
‡
, Bernard Corvilain
‡¶
, Franc¸oise Miot
‡
, and Xavier De Deken
‡5
From the
‡
Institut de Recherche Interdisciplinaire en Biologie Humaine et Mole´culaire and the
¶
Department of Endocrinology,
Hoˆpital Erasme, Universite´ Libre de Bruxelles, Campus Erasme, 1070 Brussels, Belgium and the
§
Department of Medicine, University
of Chicago, Chicago, Illinois 60637
Dual oxidases were initially identified as NADPH oxidases
producing H
2
O
2
necessary for thyroid hormone biosynthesis.
The crucial role of Duox2 has been demonstrated in patients
suffering from partial iodide organification defect caused by bi-
allelic mutations in the DUOX2 gene. However, the Duox1 func-
tion in thyroid remains elusive. We optimized a functional assay
by co-expressing Duox1 or Duox2 with their respective matura-
tion factors, DuoxA1 and DuoxA2, to compare their intrinsic
enzymatic activities under stimulation of the major signaling
pathways active in the thyroid in relation to their membrane
expression. We showed that basal activity of both Duox isoen-
zymes depends on calcium and functional EF-hand motifs.
However, the two oxidases are differentially regulated by activa-
tion of intracellular signaling cascades. Duox1 but not Duox2
activity is stimulated by forskolin (EC
50
ⴝ0.1
␮
M) via protein
kinase A-mediated Duox1 phosphorylation on serine 955. In
contrast, phorbol esters induce Duox2 phosphorylation via pro-
tein kinase C activation associated with high H
2
O
2
generation
(phorbol 12-myristate 13-acetate EC
50
ⴝ0.8 nM). These results
were confirmed in human thyroid cells, suggesting that Duox1 is
also involved in thyroid hormonogenesis. Our data provide, for
the first time, detailed insights into the mechanisms controlling
the activation of Duox1–2 proteins and reveal additional phos-
phorylation-mediated regulation.
Dual oxidases (Duox1 and Duox2) belong to the family of
NADPH oxidases (Nox), which is composed of five additional
enzymes: Nox1–5 (1–3). These transmembrane proteins are
characterized by a COOH-terminal NADPH oxidase catalytic
core responsible for reactive oxygen species synthesis. The
best characterized NADPH oxidase Nox2 is involved in the
leukocyte respiratory burst and activated by invading patho-
gens (4). The mechanisms controlling the Nox-mediated reac-
tive oxygen species production are multiple and complex. The
activation of Nox1 (5, 6), Nox2 (3, 7), and Nox3 (8, 9) requires
the coordinated assembly of several subunits: the association
with the transmembrane protein p22
phox
and the recruitment
of three cytosolic proteins, the small G protein Rac, p47
phox
(or
NOXO1), and p67
phox
(or NOXA1).
Duox1 and Duox2 isoenzymes are large members of the Nox/
Duox family. In addition to the catalytic core, Duox1 and
Duox2 proteins are NH
2
-terminally extended by an extracellu-
lar peroxidase-like domain followed by a membrane-spanning
segment and an intracellular domain comprising two canonical
EF-hand motifs (2). Duox1 and Duox2 are the sole proteins
directly generating H
2
O
2
(10) outside the cells, whereas small
Nox homologues are mostly superoxide generators (3). Duox
isoforms do not need to be associated with cytosolic factors to
be active but undergo a critical maturation process necessary to
acquire their active conformation at the apical cell surface of
the thyrocytes (11). The immature nonfunctional form (180
kDa) is not properly glycosylated and maintained in the endo-
plasmic reticulum compartment. Only the co-expression of the
Duox maturation factors (DuoxA1 and DuoxA2) allows func-
tional reconstitution. They are N-glycosylated proteins permit-
ting the endoplasmic reticulum exit of properly folded Duox
enzymes (12, 13). The DUOXA genes are localized near their
respective DUOX gene in a head to head orientation on chro-
mosome 15 and co-expressed with their Duox counterpart in
the same tissues (12).
Dual oxidases expressed at the apical side of surface epithelia
exposed to microorganisms, like the airways or the digestive
tract, are supposed to function as components of the innate
host defense system (14–16). However, Duox1 and Duox2
isoenzymes were initially identified as H
2
O
2
-generating thyroid
oxidases (1, 2). The main function of the thyroid is the uptake
and concentration of iodide from the bloodstream to synthesize
thyroid hormones (T3 and T4) in the follicular lumen (17).
H
2
O
2
produced at the apical pole of the thyrocyte is utilized by
thyroperoxidase as an electron acceptor to oxidize iodide,
covalently link oxidized iodide to tyrosines of thyroglobulin,
*This work was supported by the “Fonds National pour la Recherche Me´ di-
cale,” “Actions de Recherches Concerte´ es de la Communaute´ Franc¸aise de
Belgique,” the “Fonds National de la Recherche Scientifique,” and the Fon-
dation Van Buren. The costs of publication of this article were defrayed in
part by the payment of page charges. This article must therefore be hereby
marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to
indicate this fact.
□
S
The on-line version of this article (available at http://www.jbc.org) contains
supplemental Table S1 and Figs. S1–S6.
1
To whom correspondence should be addressed: IRIBHM, Universite´ Libre
de Bruxelles, Campus Erasme, Bat. C., 808 route de Lennik, B-1070
Bruxelles, Belgium. Tel.: 32-2-5554151; Fax: 32-2-5554655; E-mail:
sabrina.rigutto@ulb.ac.be.
2
Recipients of a fellowship from the “Fonds pour la Formation a` la Recherche
dans l’Industrie et l’Agriculture.”
3
Recipient of research grants from the Cancer Research Foundation and the
American Thyroid Association.
4
Senior Research associate at the Fonds National de la Recherche
Scientifique.
5
Postdoctoral researcher at the Fonds National de la Recherche Scientifique.
THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 284, NO. 11, pp. 6725–6734, March 13, 2009
© 2009 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.
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and couple iodinated tyrosyl residues to form protein-bound
iodothyronines (T3 and T4) (18). Under physiological iodide
supply, hormonogenesis is rate-limited by the availability of
hydrogen peroxide (19). Patients presenting iodide organifica-
tion defect caused by mutations in their DUOX2 gene suffer
from transient or permanent hypothyroidism depending on the
mono- or bi-allelic character of the mutation, demonstrating
the crucial function of Duox2 in thyroid hormone synthesis
(20–25). However, the physiological meaning of the co-exist-
ence of the two dual oxidases and their respective maturation
factors in the thyroid tissue remains an open question.
In this study, we analyzed the mechanisms of activation of
Duox1 and Duox2 using Duox/DuoxA co-transfected cells
stimulated by agonists known to regulate the thyroid metabo-
lism (26, 27). Our results indicate that Duox1 and Duox2 activ-
ities are mainly calcium-dependent NADPH oxidases. More-
over, additional mechanisms governing their intrinsic activity
are different; Duox1 is positively regulated by the cAMP-de-
pendent protein kinase A (PKA)
6
cascade, whereas Duox2 is
highly induced by activation of protein kinase C (PKC) with
very low concentrations of PMA.
EXPERIMENTAL PROCEDURES
Plasmids and Mutagenesis—The wild type untagged versions
of human Duox1 (accession number AF230495) and Duox2
(accession number AF230496) cDNA were cloned from ATG to
stop codon into the vector pcDNA3 (Invitrogen). The NH
2
-
terminal hemagglutinin epitope-tagged human Duox2 (HA-
Duox2-pcDNA3.1) and the COOH-terminal c-Myc epitope-
tagged human DuoxA1 and DuoxA2 (DuoxA1-Myc-pcDNA3.1
and DuoxA2-Myc-pcDNA3.1) were described elsewhere (12). We
added an additional Rho tag to Duox1 to be able to distinguish it
from HA-Duox2 in future experiments. The Duox1 native signal
peptide was replaced by the TSH receptor signal peptide to facili-
tate the cloning step. The Rho-HA-Duox1-pcDNA3 construct
was generated as follows. The 23 first amino acids (residue 1 cor-
responding to the initiation methionine) of the human Duox1 pro-
tein (accession number AAF73921) were replaced by the signal
peptide of the human TSH receptor (MRPADLLQLVLLLDL-
PRDLGG) (accession number CAA02195) and the first 19 resi-
dues of the bovine rhodopsin (Rho) (accession number P02699)
(MNGTEGPNFYVPFSNKTGVV; two putative glycosylation sites
are underlined) (28). The cDNA corresponding to the NH
2
-termi-
nal HA-tagged Duox1 protein (24–1551 amino acids) was cloned
just downstream of the Rho tag by insertion of an EcoRI site. The
presence of the TSH receptor signal peptide, Rho, and HA tags at
the NH
2
terminus of Duox1 did not affect its expression nor its
peroxide generating activity (supplemental Fig. S1). Furthermore,
the extra N-linked sugars present on the Rho tag have been very
useful to separate the two glycosylated forms of Duox1 for mass
spectrometry analysis, because it is mainly the mature highly gly-
cosylated form of Duox1 that is phosphorylated upon stimulation.
Mutations in Duox1–2 were introduced by directed mutagenesis
with the QuikChange system (Stratagene, La Jolla, CA) (primers
are described in supplemental Table S1). All of the con-
structs were verified by Big Dye Terminator cycle sequenc-
ing on an automated ABI Prism 3100 sequencer (Applied
Biosystems, Foster City, CA).
Cell Culture and Transfection—Cos-7 cells were cultured in
Dulbecco’s modified Eagle’s medium (Invitrogen) with 10% fetal
bovine serum (Invitrogen), 2% streptomycin-penicillin, 1% fungi-
zone, and 1% sodium pyruvate. For H
2
O
2
assay, adherent cells at
50– 60% confluence were transfected in 6-well plates using
FuGENE 6 reagent (Roche Applied Science) according to the
manufacture’s protocol (ratio: 1
␮
g of DNA for 3
␮
l of FuGENE
6) with 500 ng of Rho-HA-Duox1-pcDNA3 and 500 ng of
DuoxA1-Myc-pcDNA3.1 or with 500 ng of HA-Duox2-
pcDNA3.1 and 500 ng of DuoxA2-Myc-pcDNA3.1. Under opti-
mal conditions, the transfection efficiency reached 20 –30% of cells
expressing Duox proteins at the cell surface detected by FACS. For
immunoprecipitation experiments, the cells seeded in 10-cm-di-
ameter dishes were transfected with 8
␮
g of DNA and 24
␮
lof
FuGENE 6.
Human Thyroid Primary Culture—Human thyroid tissue
was obtained from patients undergoing partial or total thyroid-
ectomy for resection of solitary cold nodules or multinodular
goiters. Only healthy, normal-looking, non-nodular tissue was
used within 30 min after surgical removal. Thyrocytes in pri-
mary culture obtained from follicles isolated by collagenase
digestion and differential centrifugation were cultured in
Dulbecco’s modified Eagle’s medium/Ham’s F-12/MCDB104
(2:1:1) medium (Invitrogen) with 1% sodium pyruvate, 40
␮
g/ml ascorbic acid, 5
␮
g/ml insulin, 2% streptomycin-penicil-
lin, and 1% fungizone (29, 30). Thyrocytes were seeded 5 days
before the assay. The protocol has been approved by the hospi-
tal ethics committee.
H
2
O
2
Measurement and Flow Immunocytometry Analysis—
Production of H
2
O
2
was determined by the sensitive fluorimet-
ric method of Be´nard and Brault (31) slightly modified as pre-
viously described (11). The peroxide released from transfected
cells (Duox1/DuoxA1 or Duox2/DuoxA2) into Krebs-Ringer-
Hepes medium was accumulated in the presence of stimulating
agents for 2.5 h at 37 °C. After removing the medium, cell sur-
face expression of Duox1–2 proteins was measured by flow
cytometry (FACS). Briefly, the cells detached with phosphate-
buffered saline EDTA/EGTA (5 mM) were incubated sequen-
tially with anti-HA antibody (clone 3F10; Roche Applied Sci-
ence) and fluorescein-conjugated anti-rat IgG, both diluted
1/100 in phosphate-buffered saline, 0.1% bovine serum albu-
min. Propidium iodide (5
␮
g/ml) staining in the second incu-
bation step was used to exclude damaged cells from subsequent
analysis. Fluorescence was analyzed using cell sorting (FACS-
can; Becton Dickinson, Erembodegem, Belgium) counting
20,000 events/sample. Relative protein expression was deter-
mined by calculating the differences in total fluorescence inten-
sity (Arbitrary Unit) between the samples and an equal-sized
population of control cells expressing only Duox1 or Duox2
constructs without their respective maturation factors. With-
out the latter, no cell surface expression of Duox could be
detected (11). H
2
O
2
production was normalized to cell surface
6
The abbreviations used are: PKA, cAMP-dependent protein kinase; PKC, pro-
tein kinase C; PMA, phorbol 12-myristate 13-acetate; WT, wild type; HA,
hemagglutinin; TSH, thyroid-stimulating hormone; FACS, fluorescence-ac-
tivated cell sorter; Fsk, forskolin; 6-MB-cAMP, N
6
-monobutyryladenosine-
3⬘,5⬘-cyclic monophosphate.
Activation Mechanisms of Duox1–2 Isoenzymes
6726 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 284• NUMBER 11• MARCH 13, 2009
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expression of each construct and reported as pg of H
2
O
2
/FACS.
For cultures of human thyrocytes, H
2
O
2
released over 90 min in
the presence of various agents was normalized to total proteins
extracted in Laemmli buffer and quantified by paper dye bind-
ing assay (ng of H
2
O
2
/
␮
g of protein) (32).
Immunoprecipitation and Western Blot Analysis—Proteins
were extracted in lysis buffer (10 mMTris-HCl, pH 7.5, 150 mM
KCl, 0.5% Nonidet P-40, 120 mM
␤
-mercaptoethanol, 100 mM
NaF, 2 mMEDTA, pH 8.0, 50 nMokadaic acid, 1 mMvanadate)
supplemented with a mixture of protease inhibitors (Complete;
Roche Applied Science) for1hat4°C.Thelysate was centri-
fuged 15 min at 10,000 rpm, and the supernatant was pre-
cleared with Sepharose beads (GE Healthcare). Duox1 com-
plexes were immunoprecipitated with anti-Duox antibody
(1/100) conjugated to Sepharose beads (2) and Duox2 proteins
with monoclonal anti-HA antibody precoated on agarose beads
(Clone HA-7; Sigma-Aldrich). Proteins of the immunoprecipi-
tate were separated by SDS/PAGE and transferred to nitrocel-
lulose as previously described (2). Phosphorylated Duox pro-
teins were detected using a rabbit polyclonal antibody raised
against phospho-(Ser/Thr) PKA substrate (1/1,000; Cell Signal-
ing, Danvers, MA), which is directed to the phospho motif
RXX(S/T). Fluorescent secondary antibodies (1/10,000; IRDye
800 anti-rabbit from LI-COR, Lincoln, NE) were used for image
acquisition and quantification with the Odyssey infrared imag-
ing system (LI-COR). The membrane was stripped and immu-
noblotted with the anti-Duox antibody (1/16,000) and fluores-
cent secondary antibodies (1/10,000; IRDye 680 anti-rabbit) to
quantify total Duox proteins.
Radioactive Phosphorus Incorporation—Cells maintained
24 h in serum-free phosphate-depleted medium were incu-
bated 2 h with 500
␮
Ci (Cos-7) or 1mCi (thyrocytes) of
[
32
P]orthophosphate. The proteins were prepared as described
above, and phosphorylated proteins were detected and quanti-
fied with a Storage Phosphor Screen (GE Healthcare) scanned
with Typhoon Trio⫹(GE Healthcare). Total Duox proteins for
each condition were measured by Odyssey infrared imaging
system coupled with the polyclonal anti-Duox antibody.
In Vitro PKA Phosphorylation Assay—Cos-7 Duox1/DuoxA1
transfected cells were cultured 24 h without serum. The pro-
teins were prepared in lysis buffer, and Duox1 complexes were
immunoprecipitated overnight with monoclonal anti-HA anti-
body precoated on agarose beads as described above. Immuno-
precipitated proteins were incubated for 10 min at 37 °C in a
50-
␮
l final volume that contained 20 mMHepes, pH 7.4, 0.1 mM
dithiothreitol, 10 mMMgCl
2
, 0.1 mM[
␥
-
32
P]ATP (5
␮
Ci/tube),
and 100 ng of purified catalytic subunit of protein kinase A
(Calbiochem, Gibbstown, NJ). After SDS/PAGE and Western
blotting, radioactive signals were quantified with Typhoon
Trio⫹; Duox proteins were immunodetected with the anti-
Duox antibody and visualized with Odyssey imaging system.
Mass Spectrometry Analysis—Cos-7 cells transfected with
wild type Duox1 and DuoxA1 constructs were cultured 24 h
without serum and stimulated 30 min with 10
␮
Mforskolin.
Duox1 complexes were immunoprecipitated with anti-Duox
antibody as described above. Duox1 proteins were separated by
7%-polyacrylamide gel electrophoresis and stained with colloi-
dal Coomassie Blue. After excision of the Duox gel bands, the
proteins were in-gel digested with trypsin or chymotrypsin, and
the resulting peptides were extracted from the gel (33). The
digested peptides were separated onto a C18 reverse phase 1 ⫻
50 mm column (Vydac; Alltech Associates, Lokeren, Belgium)
and deposited onto a stainless steel target. Mass spectrometry
analysis was performed on a Quadrupole-time of flight Ultima
Global mass spectrometer equipped with a matrix-assisted
laser desorption ionization source (Micromass, Waters, Zellik,
Belgium) calibrated using the monoisotopic masses of tryptic
and chymotryptic peptides from bovine serum albumin.
Statistical Analysis—The data are presented as the means ⫾
S.D. The results were analyzed using the unpaired Student’s t
test, and p⬍0.05 was considered statistically significant (**, p⬍
0.01; ***, p⬍0.001).
RESULTS
DuoxA-based Functional Assay to Study Duox-mediated
H
2
O
2
Generation—Heterologous systems combining DuoxA
expression have already been successfully used to characterize
Duox activity of human Duox2 natural mutants (13) and to
reconstitute a functional H
2
O
2
-generating system in a lung
cancer cell line (34). The originality of our study is to analyze
the specific activity of Duox1 and Duox2 isoenzymes by nor-
malizing the hydrogen peroxide production to the cell surface
expression of the respective proteins. Insertion of an HA tag in
the Duox1–2 ectodomain provides an effective means to reli-
ably estimate Duox membrane expression and compare the
activities of the two enzymes. We first validated our heterolo-
gous system: 1) Measurement of H
2
O
2
produced after 1
␮
M
ionomycin stimulation of cells transfected with Duox/DuoxA
(constant DuoxA quantity, 50 ng) was proportional to the
quantity of transfected Duox plasmids (25–500 ng) with a pla-
teau reached at 250 –500 ng of Duox DNA probably caused by a
limited amount of DuoxA (Fig. 1A). 2) We observed a linear
relationship between Duox-mediated H
2
O
2
generation and the
membrane expression of Duox as measured by FACS (Fig. 1B).
The specific activity of both Duox enzymes was comparable,
and no H
2
O
2
was detected in cells expressing Duox or DuoxA
alone (Fig. 1A). 3) We verified that the addition of the tags did
not modify the expression and activity of Duox1–2 proteins
(supplemental Fig. S1). In all experiments, the specific activity
of the Duox enzymes is represented as the amount of H
2
O
2
produced normalized to their surface expression (pg H
2
O
2
/
FACS).
In humans, the thyroid metabolism is under the control of
the phosphatidylinositol 4,5-bisphosphate cascade and the
cAMP cascade (26, 27). In our functional assay, 1
␮
Mionomycin
increased the activity of both Duox1 and Duox2 enzymes to the
same level, but raising concentrations of ionomycin from 2 to 4
␮
Mresulted in higher activity of Duox1 than Duox2 (Fig. 2A).
The adenylate cyclase agonist, forskolin (Fsk), raised the
amount of H
2
O
2
generated by Duox1 but not by Duox2 with a
50% effective concentration (EC
50
)of0.1
␮
M(Fig. 2B). More-
over PMA, a PKC activator, differentially regulated activities of
Duox1 and Duox2 (Fig. 2, Cand D). Although micromolar con-
centrations of PMA were needed to increase Duox1 activity
with an EC
50
of 1.8
␮
M, a maximal activation of Duox2 enzyme
was already observed with nanomolar PMA concentrations
Activation Mechanisms of Duox1–2 Isoenzymes
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(EC
50
⫽0.8 nM). The addition of Fsk or PMA to ionomycin in
the incubation medium provoked a release of H
2
O
2
corre-
sponding to the sum of the amounts generated by the cells
treated with the agents separately (Iono versus Iono⫹Fsk for
Duox1: p⬍0.01; Iono versus Iono⫹PMA for Duox1: p⬍0.001;
Iono versus Iono⫹PMA for Duox2: p⬍0.01) (Fig. 2E). In con-
trast to Duox1, raising calcium concentration was not sufficient
to activate Duox2 by Fsk. In all of the conditions, 1
␮
Mdiphe-
nyleneiodonium, a flavoprotein inhibitor, reduced the H
2
O
2
level produced by the two enzymes, indicating that it derives
from an NADPH oxidase (data not shown).
Regulation of Duox Activity by Calcium—Duox-mediated
H
2
O
2
generation is presumed to be regulated by binding of
Ca
2⫹
to the two EF-hand motifs, located in the cytosolic por-
tion spanning transmembrane domain 1 and 2 (13, 34, 35).
Indeed, the absence of calcium ions in the H
2
O
2
measurement
assay abolished the Duox1–2 activity (data not shown). The
other calcium-dependent NADPH oxidase, NOX5, possesses
four calcium-binding sites arranged in two functional pairs
(36). To study the role of the two Duox EF-hands, the crucial
glutamate residue was replaced by a glutamine in the twelfth
position of the EF-hand sequence. Duox2 mutants E843Q and
E879Q completely lost the ability to produce H
2
O
2
in basal or
stimulated conditions but maintained a cell surface expression
similar to the wild type (WT) protein (Fig. 3). Similarly, inacti-
vation of one of the two EF-hands in Duox1 (E839Q and E875Q
Duox1 mutants) was sufficient to inactivate Duox1, although a
correct processing to the membrane occurred as for Duox2
(supplemental Fig. S2). These
results strongly suggest that the two
EF-hand motifs operate as one
functional pair necessary for Duox
activation in response to an
increase of intracellular calcium
concentration.
Duox1 Activity Is Positively Modu-
lated through the cAMP Pathway—
As shown in Fig. 2, Duox1-depend-
ent H
2
O
2
generation was positively
controlled by Fsk, contrary to
Duox2. To establish the role of the
protein kinase A, N
6
-monobutyry-
ladenosine-3⬘,5⬘-cyclic monophos-
phate (6-MB-cAMP), a site-selec-
tive PKA agonist, was used. H
2
O
2
produced by Duox1 was signifi-
cantly increased with either 50
␮
M
6-MB-cAMP or 1
␮
MFsk (Fig. 4A).
We also co-transfected a vector
encoding the
␣
isoform of the PKA
catalytic subunit with WT Duox/
DuoxA constructs (37, 38). Overex-
pression of PKA for 48 h, verified by
Western blotting (data not shown),
also induced an increase of Duox1
activity. Co-expression of the PKA
subunit had only a minor effect on
Duox2 activity, and treatment with
the 6-MB-cAMP did not increase H
2
O
2
production by Duox2.
Motif scanning for PKA substrates based on the consensus
sequence (R/K)2X(S/T) identifies three potential PKA phos-
phorylation sites in Duox1 (Ser
955
, Thr
1007
, and Ser
1217
) (sup-
plemental Fig. S3). We analyzed the Duox1 phosphorylation
state with an anti-RXX(pS/pT) antibody that could potentially
recognize phosphorylation on Ser
955
and Ser
1217
but not on
Thr
1007
. Under basal conditions, the mature form of Duox1
(200 kDa) was already phosphorylated in Cos-7 cells (supple-
mental Fig. S4). It is noteworthy that the slower migrating form
of Duox1 (mature form) and the immature form were shifted to
200 and 190 kDa, respectively, instead of the wild type 190- and
180-kDa proteins. This phenomenon can be explained by the
addition of extra N-linked sugars on the two putative N-glyco-
sylation sites present in the Rho tag sequence as previously
described for similar TSH receptor constructs (28). Duox1
phosphorylation was stimulated by Fsk in a time-dependent
way with a maximum reached after 30 min of stimulation (sup-
plemental Fig. S4). Increased Duox1 phosphorylation was also
observed in cells treated 30 min with 6-MB-cAMP or cells over-
expressing the PKA catalytic subunit (Fig. 4B). The stimulatory
effect of Fsk was also observed on H
2
O
2
accumulation for 30
min (data not shown). Constitutive and PKA-induced phos-
phorylation of Duox1 was evident, in
32
P incorporation exper-
iments, with the detection of a major radioactive band corre-
sponding to the high molecular mass form (200 kDa) of Duox1
(supplemental Fig. S5). No increase of Duox2 phosphorylation
FIGURE 1. Intrinsic activity of Duox isoenzymes in reconstituted systems. A, measurement of H
2
O
2
accu-
mulation (ng) produced by Duox1/DuoxA1 or Duox2/DuoxA2 co-transfected Cos-7 cells for 2.5 h in the pres-
ence of 1
␮
Mionomycin. Constant amount of DuoxA-Myc-pcDNA3.1 DNA (50 ng) was transfected with increas-
ing DNA amounts of Rho-HA-Duox1-pcDNA3 (white) or HA-Duox2-pcDNA3.1 (black) (0 –500 ng). Each
measurement corresponds to a transfection experiment performed in duplicate (means ⫾S.D.). B, plot of H
2
O
2
production against the relative Duox expression level at the cell surface. Membrane expression of Rho-HA-
Duox1 (‚) and HA-Duox2 (f) proteins were quantified by FACS using the anti-HA antibody (A.U, arbitrary unit
of fluorescein isothiocyanate fluorescence intensity). Inset, representative histograms of a FACS experiment;
the gray areas represent cells transfected with the Duox construct alone. The percentage of cells expressing
Duox at the cell surface is indicated for each construct.
Activation Mechanisms of Duox1–2 Isoenzymes
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could be detected under Fsk stimulation by
32
P incorporation
experiments (data not shown).
Identification of PKA Target Sites in Duox1—To identify the
phosphorylated sites by PKA, Duox1 protein was isolated from
Fsk-stimulated Cos-7 cells co-transfected with Duox1/DuoxA1
and subjected to mass spectrometry analysis. After trypsin or
chymotrypsin digestions, two phosphorylated peptides were
isolated and microsequenced: the peptide
954
ASYISQDMICP-
SPR
967
, which encloses the serine 955 (underlined), and the
peptide
1214
RRRSFRGF
1221
including the serine 1217. Three
peptides containing the threonine 1007 (
1006
VTSFQPLL-
FTEAHREK
1021
,
1005
KVTSFQPLLFTEAHR
1019
, and
1006
VTS-
FQPLLFTEAHR
1019
) were also isolated but were not
phosphorylated.
To address the role of these potential phosphorylated resi-
dues in Duox1 activity, we replaced them with the nonphos-
phorylatable amino acid, alanine. Three single mutants (S955A,
T1007A, and S1217A) and one double mutant (S955A/S1217A)
were constructed. Cos-7 cell surface expression of T1007A and
S1217A mutants was similar to membrane expression of WT
protein, whereas S955A and S955A/S1217A were less ex-
pressed than the WT Duox1 (Fig. 5A,inset). The basal activity
of S955A Duox1 was severely impaired and was no longer stim-
ulated after Fsk treatment, whereas it still responded to iono-
mycin and PMA (Fig. 5A). Mutant T1007A presented a slightly
lower activity than the WT protein but was still positively reg-
ulated by all agonists. Interestingly, mutation S1217A gener-
ated an enzyme with increased basal activity, and the double
mutant S955A/S1217A produced a similar H
2
O
2
amount as
WT Duox1 in basal and stimulated conditions, except for a loss
of response to Fsk.
Phosphorylation state of Duox1 mutants in basal condition
or after Fsk stimulation was analyzed by radioactive phospho-
rus incorporation (Fig. 5B). Basal phosphorylation of the 200-
kDa mature form of S955A and S1217A Duox1 was highly
decreased compared with the WT Duox1 but was still stimu-
lated by Fsk. The T1007A mutant presented the same Fsk-de-
pendent phosphorylation pattern as WT Duox1. On the other
hand, the S955A/S1217A double mutant showed a very low
basal phosphorylation no longer stimulated by Fsk. The same
experiment was performed using the anti-phospho PKA sub-
strate antibody. Unfortunately, this antibody was unable to
recognize the Ser
1217
and Thr
1007
as demonstrated by the com-
plete absence of phosphorylation of the S955A mutant (supp-
lemental Fig. S6). Nevertheless, the results confirmed the
Fsk-mediated phosphorylation of serine 955. Direct phospho-
rylation of Duox1 was measured in vitro. In the presence of
purified PKA, WT and T1007A Duox1 showed robust phos-
FIGURE 2. H
2
O
2
generation in response to ionomycin, Fsk, or PMA. Meas-
urement of H
2
O
2
produced by Cos-7 cells co-expressing Rho-HA-Duox1/
DuoxA1 (‚) or HA-Duox2/DuoxA2 (f) for 2.5 h normalized to Duox mem-
brane expression. The cells were stimulated during the 2.5-h period with
increasing concentrations of ionomycin (A), Fsk (B), or PMA (Cand D). Each
measurement corresponds to a transfection experiment performed in dupli-
cate (means ⫾S.D.). E,H
2
O
2
production of Cos-7 cells co-expressing Rho-HA-
Duox1/DuoxA1 (white) or HA-Duox2/DuoxA2 (black) stimulated during the
2.5-h period with 1
␮
Mionomycin in combination with 1
␮
MFsk or PMA (5
␮
M
for Duox1 and 0.5 nMfor Duox2). H
2
O
2
produced in basal condition was con-
sidered as 100% (means ⫾S.D., n⫽4). Statistical significances compared with
basal are indicated. **, p⬍0.01; ***, p⬍0.001.
FIGURE 3. Loss of Duox2 activity by mutations in EF-hand motifs. H
2
O
2
accumulation was performed for 2.5 h at 37 °C in the presence of 1
␮
Miono-
mycin (Iono), 1
␮
MFsk, or 1 nMPMA from cells expressing DuoxA2 with wild
type (black), E843Q (gray), or E879Q (white) HA-Duox2. The graphs show the
means ⫾S.D. (n⫽4). Expression of mutated Duox2 constructs at the cell
surface relative to WT is shown in the inset.
Activation Mechanisms of Duox1–2 Isoenzymes
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phorylation with the appearance of an additional highly
phosphorylated form corresponding to the immature 190-
kDa protein (Fig. 5C). As expected, the PKA-dependent
phosphorylation of S955A, S1217A, and S955/S1217A
mutants was clearly decreased. Taken together, these results
demonstrate that Ser
955
and Ser
1217
are two key residues
phosphorylated by PKA controlling Duox1 activity.
Duox2 Activity Is Modulated by the PKC—We have shown
that Duox2 activity was stimulated with nanomolar concentra-
tions of PMA (Fig. 2). To determine whether PMA-dependent
activation of Duox2 involves PKC-dependent phosphorylation,
Cos-7 cells expressing Duox2/DuoxA2 were incubated with
PKC inhibitors. One
␮
MRo318220 or Go¨6976 specifically
inhibited PMA-stimulated H
2
O
2
production by 50% with no
effect in basal and ionomycin stimulated conditions (Fig. 6A).
Analysis of radioactive phosphorus incorporation showed a
time-dependent phosphorylation of Duox2, already visible
after 10 min of exposure to PMA that was prevented by
Ro318220 (Fig. 6, Band C). A 30-min treatment with 1 nMPMA
significantly increased H
2
O
2
generation from Duox2/DuoxA2
co-transfected cells (data not shown). No increase of Duox1
phosphorylation could be observed after PMA treatment, and
the inhibitor Ro318220 did not inhibit PMA-stimulated Duox1
activity (data not shown). These results suggest that physiolog-
ically PKC-
␣
or -
␤
1 isoforms participate in the phosphorylation
and activation of Duox2 and not Duox1.
Regulation of H
2
O
2
Generation in Human Thyroid Primary
Cultures—In the functional assay, we found that Duox1 and
Duox2 activities are both calcium-dependent but differentially
regulated by PKA and PKC. To determine the physiological
relevance of these results, we analyzed the modulation of H
2
O
2
production in human thyrocytes under similar stimulation
conditions. H
2
O
2
measurements from primary cultures
showed that basal H
2
O
2
accumulation (0.31 ng of H
2
O
2
/
␮
gof
protein) was increased by ionomycin (4.08 ng of H
2
O
2
/
␮
gof
protein) and not by Fsk alone (Fig. 7A). Combining Fsk and
ionomycin treatments significantly increased the H
2
O
2
pro-
duction to 7.16 ng of H
2
O
2
/
␮
g of protein (Iono versus
Iono⫹Fsk, p⬍0.001), demonstrating a permissive effect of
calcium rather an additive effect observed in the heterologous
system (Fig. 2E). Ionomycin also increased the H
2
O
2
amount in
response to PMA to a similar extent (Iono versus Iono⫹PMA,
p⬍0.001). The 190-kDa mature form of Duox proteins was
phosphorylated in basal condition and showed increased phos-
phorylation level after stimulation with Fsk or PMA as esti-
mated by
32
P incorporation (Fig. 7B). This Duox phosphoryla-
tion was reproduced by 1 or 10 milliunits/ml of the thyrotropin
hormone (TSH), concentrations described to stimulate either
the adenylate cyclase-cAMP pathway or the phospholipase
C-diacylglycerol-calcium cascade, respectively (27).
DISCUSSION
The biochemical function of hydrogen peroxide in thyroid
hormone synthesis has been known for decades (18). A fla-
voprotein complex was predicted to produce directly H
2
O
2
outside the cell with an activity mediated by NADPH and stim-
ulated by calcium (10, 35, 39, 40). In 2000, the cloning of two
cDNAs encoding novel calcium-dependent NADPH oxidases,
Duox1 and Duox2, revealed the molecular nature of the pre-
sumed thyroid H
2
O
2
-generating system (1, 2, 11). However,
functional studies on dual oxidases have been conducted only
recently thanks to the discovery of their maturation factors,
DuoxA1 and DuoxA2, allowing correct processing of Duox1–2
isoenzymes in heterologous systems (12, 13). In this work, we
improved a DuoxA-based functional assay to compare the specific
activities of Duox1 and Duox2, and we uncovered mechanisms of
activation that discriminate between the two oxidase activities.
Each Duox protein possesses two canonical EF-hand motifs
that are involved in the main regulation step exerted by calcium
(2, 13, 34, 41). Co-expression of Duox/DuoxA proteins in Cos-7
cells reconstitutes an H
2
O
2
generator with an activity acutely
stimulated by an elevation of the intracellular calcium concen-
tration. We have demonstrated that intact EF-hand sequences
are required to maintain functional Duox proteins. Moreover,
FIGURE 4. cAMP-dependent activation of Duox1. A, Cos-7 cells expressing
Rho-HA-Duox1/DuoxA1 (white) or HA-Duox2/DuoxA2 (black) were stimu-
lated for 2.5 h with 1
␮
Mionomycin (Iono), 1
␮
MFsk, or 50
␮
M6-MB-cAMP
corresponding to the time of H
2
O
2
accumulation. In the PKA condition, cells
transfected with a third vector coding for the PKA catalytic subunit. H
2
O
2
production was normalized to Duox cell surface expression, and the resulting
specific activities are expressed relative to the basal condition (set to 100). The
data are shown as the values ⫾S.D. for n indicated measurements (***, p⬍
0.001). B, immunodetection of Duox1 phosphorylation mediated by agents
activating the cAMP cascade. The cells were treated for 30 min with either 1
␮
MFsk or 50
␮
M6-MB-cAMP. After anti-Duox immunoprecipitation, PKA
phosphorylation was immunodetected with the anti-RXX(pS/pT) antibody
(P-PKA) and total Duox1 with anti-Duox polyclonal antibody. In the top panel,
the columns represent PKA-mediated Duox1 phosphorylation corrected to
the total amount of immunoprecipitated Duox1. The level of phosphoryla-
tion is expressed relative to the basal phosphorylation set to 100. The PKA
condition was performed in a different experiment from Fsk and 6-MB-cAMP
(6MB).
Activation Mechanisms of Duox1–2 Isoenzymes
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whereas the two dual oxidases exhibit similar activity at low
ionomycin concentration (1
␮
M), Duox1 seems to be more effi-
cient than Duox2 in cells treated with higher ionomycin con-
centrations. This effect might be
explained by the differences in the
amino acid sequence of the Duox1
second EF-hand motif.
Recent reports have shown that,
in addition to calcium, the Nox5
enzymatic activity is also regulated
through PKC-mediated phosphory-
lations on serine and threonine res-
idues enhancing its sensitivity to
calcium (42). Using radioactive
phosphorus incorporation and anti-
phospho-PKA substrate antibody,
we demonstrated that Duox1 is also
a phosphoprotein. The enzyme is
constitutively phosphorylated, and
activation of the PKA pathway
increases its phosphorylation state
as well as its activity. Immature
Duox1–2 proteins generate essen-
tially superoxide inside the cell and
become H
2
O
2
generators only at the
cell surface (41). In transfected cells,
in vivo
32
P incorporation shows that
mainly the mature form of Duox1 is
phosphorylated in basal and stimu-
lated conditions, even if the imma-
ture form of Duox1 is more abun-
dant (Fig. 5B). These data suggest
that, in intact cells, Duox isoen-
zymes might undergo conforma-
tional changes during their process-
ing from the endoplasmic reticulum
to the plasma membrane modifying
the accessibility of key residues to
the kinase. In in vitro PKA phospho-
rylation experiments, these amino
acids could be artificially demasked
by detergents showing elevated
phosphorylation intensity for the
immature form of Duox1 (Fig. 5C).
Mass spectrometry analyses
revealed Duox1 phosphopeptides
containing the residues Ser
955
and
Ser
1217
in co-transfected Cos-7 cells
in response to Fsk. Systematic
mutations of potential PKA-medi-
ated phosphorylation sites have
identified Ser
955
and Ser
1217
as
major residues responsible for the
basal phosphorylation state of
Duox1. These two serines are con-
served among human, dog, pig,
mouse, and rat species. However,
rendering these amino acids non-
phosphorylatable reveals distinct effects on Duox1 specific
activity. The S1217A variant presents higher constitutive activ-
ity than the WT enzyme and still responds to cAMP cascade
FIGURE 5. Identification of PKA-mediated phosphorylation sites in Duox1. A, Duox1 activity. Cos-7 cells
expressing DuoxA1 in combination with WT (black bars), S955A (vertically hatched bars), T1007A (gray bars),
S1217A (horizontally hatched bars), or S955A/S1217A (open bars) Rho-HA-Duox1 were stimulated for 2.5 h with
1
␮
Mionomycin (Iono), 1
␮
MFsk, or 5
␮
MPMA and H
2
O
2
accumulation normalized to Duox membrane expres-
sion. The inset shows the cell surface expression for each constructs relative to WT Duox1. The graph corre-
sponds to one representative experiment from four independent experiments (means ⫾S.D., n⫽2). B, Duox1
phosphorylation. Cells expressing DuoxA1 with WT or mutant Rho-HA-Duox1 were
32
P-labeled and treated for
30 min with either 1
␮
MFsk (⫹) or solvent (⫺). In the top panel, relative densitometry of phosphorylated Duox1
corrected to the total Duox1 immunoprecipitated (non stimulated WT Duox1 considered as 100%). C,in vitro
phosphorylation of Duox1 by PKA. Each construct was tested in duplicate. The relative Duox1 phosphorylation
corrected to the total amount of Duox1 is showed on the top of the figure.
Activation Mechanisms of Duox1–2 Isoenzymes
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activation, whereas the S955A substitution decreases basal and
Fsk-stimulated Duox1 activity. These results demonstrate that
serine 955 is the crucial residue positively regulating Duox1
activity through PKA-mediated phosphorylation. It could
increase the sensitivity of Duox1 to lower level of intracellular
calcium as has been demonstrated for Nox5 enzyme (42). The
hyperactivity of the mutant S1217A was more surprising and
suggests an inhibitory effect on the overall Duox1 activity
driven by its phosphorylation state. Our hypothesis is that in
resting cells, constitutive phosphorylation on Ser
1217
restrains
the enzyme to limit peroxide generation, whereas PKA-medi-
ated phosphorylation on Ser
955
stimulates Duox1-dependent
H
2
O
2
generation. Absence of phosphorylation on this residue
in the S955A variant would decrease its sensitivity to calcium,
explaining its lower basal activity. Combination of S955A and
S1217A leads to a Duox1 phospho-null mutant with WT-like
constitutive activity but without response to PKA stimulation.
However, we cannot exclude the possibility that these muta-
tions could also be associated with conformational changes,
inactivating the enzyme as suggested by low cell surface expres-
sion of the S955A and S955A/S1217A proteins.
Duox2/DuoxA2 co-transfected cells treated with nanomolar
concentrations of PMA show increased H
2
O
2
generation asso-
ciated with PMA-mediated Duox2 phosphorylations. Duox2
enzyme is 1,000 times more sensitive to PMA than Duox1, sug-
gesting that in physiological conditions, the resting levels of
diacylglycerol combined with basal calcium concentration
would be sufficient to stimulate Duox2 but not Duox1. There-
fore, Duox2 would be the predominant enzyme in the thyroid
conferring constitutive activity to the system. A survey of the
Duox2 primary sequence reveals 11 (S/T)X(R/K) motifs as
potential target sites for PKC. Among them, six are not con-
served in Duox1 and constitute good candidates for PKC-me-
diated phosphorylations. Their implication on Duox2 activity
will be investigated.
The crucial role of Duox2 in the thyroid hormone biosynthe-
sis has been demonstrated in permanent congenital hypothy-
roidism caused by bi-allelic DUOX2 gene mutations (20–25).
Mono-allelic inactivation of DUOX2 gene has also been linked
FIGURE 6. PMA stimulates the activity and phosphorylation of Duox2.
A, cells co-transfected with HA-Duox2/DuoxA2 were preincubated 30 min in
Krebs-Ringer-Hepes medium containing vehicle (black bars) or PKC inhibitors:
1
␮
MRo318220 (gray bars)or1
␮
MGo¨ 6976 (open bars) before 2.5 h of stimu-
lation with 1
␮
Mionomycin (Iono)or1nMPMA. H
2
O
2
accumulation was nor-
malized to Duox2 expression at the plasma membrane. The level of H
2
O
2
is
represented as a percentage of the value obtained in basal condition without
PKC inhibitor (means ⫾S.D., n⫽6). Statistically significant inhibition is indi-
cated. ***, p⬍0.001. B, phosphorylation by
32
P incorporation measured after
10, 20, or 30 min of treatment with 5
␮
MPMA. On the top of the Western blot,
the relative amount of phosphorylated Duox2 corrected to total Duox2 pro-
tein (basal phosphorylation was considered as 100%). C, inhibition of PMA-
mediated Duox2 phosphorylation by Ro318220. The cells were preincubated
or not with 1
␮
MRo318220 before stimulated with 100 nMPMA. Total Duox2
proteins were detected with anti-Duox antibody, and the relative Duox2
phosphorylation corrected to total Duox2 protein is represented at the top of
the figure (basal phosphorylation without PKC inhibitor was considered as
100%).
FIGURE 7. H
2
O
2
measurement and Duox phosphorylation in human thy-
rocytes. A, cells were incubated 90 min at 37 °C with 1
␮
Mionomycin (Iono),
10
␮
MFsk, or 5
␮
MPMA. The peroxide production was normalized to the total
amount of proteins. The graphs show means ⫾S.D. of three independent
experiments performed in duplicate (***, p⬍0.001). B, Duox1–2 phosphoryl-
ation after stimulation by TSH, Fsk, and PMA. Thyrocytes were stimulated 30
min with 1 milliunits/ml TSH or 10
␮
MFsk or 5 min with 10 milliunits/ml TSH or
5
␮
MPMA. Quantification of Duox phosphorylation corrected to total immu-
noprecipitated protein represented at the top. Basal condition performed in
duplicate.
Activation Mechanisms of Duox1–2 Isoenzymes
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to milder and transient cases of neonatal hypothyroidism (43).
Recently, DUOXA2 bi-allelic mutations in a patient suffering
from permanent dyshormonogenesis have been described,
reinforcing the role of Duox2/DuoxA2 in thyroid metabolism
(44). However, the role of existing Duox1/DuoxA1 beside
Duox2 proteins in the same tissue remains obscure. Complete
inactivation of DUOX2 generally leads to partial iodide organi-
fication defect meaning that Duox1/DuoxA1 could compen-
sate the impairment of Duox2 (13, 21, 22, 24, 25). Recently, we
have demonstrated that Duox1 is the main source of hydrogen
peroxide in the rat thyroid cell line: PCCl3 (45). Our experi-
ments in human thyroid cells showing that elevation of intra-
cellular cAMP concentration activates hydrogen peroxide pro-
duction reinforce the concept of a thyroid function for Duox1.
However, this effect is observed only in combination with iono-
mycin treatment. During the completion of this manuscript,
Pacquelet et al. (46) have demonstrated an inhibitory effect on
Duox activity by the Nox1 co-activator, NOXA1, which is
relieved by calcium binding on Duox. They showed that cell
lines devoid of NOXA1 protein exhibit high basal activity of
Duox compared with the airway cells. The absence of Noxa1 in
Cos-7 cells could explain why Fsk and PMA are able to stimu-
late Duox activity independently of ionomycin treatment. The
NOXA1 transcript has been detected in thyrocytes (47), where
it might also act as a down-regulator of the thyroid H
2
O
2
-gen-
erating system.
In humans, thyroid metabolism is under the control of the
thyrotropin hormone (TSH) through its G protein-coupled
receptor linked to adenylate cyclase-cAMP and phospholipase
C-diacylglycerol-calcium cascades (26, 27, 48–50). Because
Duox2 transcript is 2 to 5 times more abundant than Duox1
(51), we propose the following model in which the Duox2 sys-
tem would ensure the constitutive tonic generation of H
2
O
2
to
make use of any available iodide. When thyroid hormone levels
in the blood decrease, TSH concentration augments and trig-
gers the release of both G
s
and G
q
proteins followed by intra-
cellular increase of cAMP, diacylglycerol, and Ca
2⫹
concentra-
tions. The latter constitute the primary activator of the dual
oxidase function, which is sustained by additional phosphoryl-
ations mediated by PKA for Duox1 and PKC for Duox2. Duox1
would represent the emergency program revealed in the case of
hypothyroidism with partial iodide organification defect linked
to inactive mutated Duox2. Nevertheless, Duox1 cannot fully
take over the impairment of Duox2, probably because of too
low protein expression.
In conclusion, we have used molecular approaches to char-
acterize the mechanisms regulating the function of Duox1 and
Duox2 proteins. We have demonstrated that the activity of the
two dual oxidases is controlled via two different phosphoryla-
tion pathways, and we provide the first experimental argument
in favor of a thyroid function for the Duox1 enzyme.
Acknowledgments—We thank Chantal Degraef, Bernadette Bourn-
onville, and Virginie Imbault for excellent technical assistance and
Dr. M. Cappelo for providing the thyroid tissue.
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Activation mechanisms of Duox1-2 isoenzymes
Suppl. Table 1. Primer sequences for mutant constructions. Mutations in Duox1-2 were introduced by
directed mutagenesis using the following sense primers. Position of the mutated amino acid is given
for the wild-type untagged protein and the mutated nucleotides are underlined.
Suppl. Fig. 1. Effect of tags on Duox1-2 activity. A) H2O2 (ng) was accumulated during 2.5h at 37°C
from Cos-7 cells co-expressing DuoxA1 or DuoxA2 (500ng DNA) with Duox1 (white; 500ng), Rho-
HA-Duox1 (horizontal line; 500ng), Duox2 (black; 500ng) or HA-Duox2 (vertical lines; 500ng).
Cells were stimulated during the 2.5h period with 1µM ionomycin, 1µM Fsk or 5µM PMA. Each
measurement was performed in duplicate (mean ± SD). B) Corresponding western showing the
amount of Duox proteins expressed in Cos-7 cells transfected in the same conditions as described
above.
Suppl. Fig. 2. Loss of Duox1 activity by mutations in EF-hand motifs. H2O2 accumulation was
performed during 2.5h at 37°C in the presence of 1µM ionomycin, 1µM Fsk or 5µM PMA from cells
expressing DuoxA1 with WT (black), E839Q (grey) or E875Q (white) Rho-HA-Duox1. Each sample
was tested in duplicate (mean ± SD). Expression of mutated Duox1 constructs at the cell surface
relative to WT is shown in the inset.
Suppl. Fig. 3. Topological model of the Duox1 protein with the substituted amino acids used to
identify the PKA target sites (S955 – T1007 – S1217).
Suppl. Fig. 4. Kinetics of Duox1 phosphorylation by Fsk. Cos-7 cells were incubated with or without
fetal bovine serum (FBS) and stimulated during 10, 20, 30 or 60 min with 10µM Fsk. Phosphorylation
was detected using the anti-RXX(pS/pT) antibody (P-PKA). Relative PKA-mediated Duox1
phosphorylation corrected to the total amount of Duox1 immunoprecipitated is shown on the top of
the figure (non stimulated condition incubated in absence of serum was considered as 100%).
Suppl. Fig. 5. Duox1 phosphorylation is increased through the cAMP pathway. Cos-7 cells co-
transfected with Rho-HA-Duox1/DuoxA1 were incubated with 32P and stimulated 30 min with 10µM
Fsk, 50µM 6-MB-cAMP or co-transfected with the plasmid encoding the PKA catalytic subunit
(PKA). Relative Duox1 phosphorylation corrected to the total amount of Duox1 in the
immunoprecipitate is represented on the top. PKA condition tested in another experiment.
Suppl. Fig. 6. Phosphorylation of Duox1 mutated in potential phosphorylation sites. Co-transfected
cells expressing DuoxA1 together with WT, S955A, T1007A, S1217A or S955A/S1217A Rho-HA-
Duox1 proteins were incubated with (+) or without (-) 10µM Fsk for 30 min. Duox1 phosphorylation
was corrected relative to the total amount of immunoprecipitated Duox1. Phosphorylation was
detected using the anti-RXX(pS/pT) antibody (P-PKA). The mutant S955A/S1217A was tested in
another experiment together with the WT Duox1 protein.

Deken
Corvilain, Françoise Miot and Xavier De
Communi, Jacques E. Dumont, Bernard
Grasberger, Milutin Milenkovic, David
Sabrina Rigutto, Candice Hoste, Helmut
PHOSPHORYLATION
KINASE C-DEPENDENT
PROTEIN KINASE AND PROTEIN
MEDIATED BY cAMP-DEPENDENT
Duox2: DIFFERENTIAL REGULATION
Activation of Dual Oxidases Duox1 and
Enzyme Catalysis and Regulation:
doi: 10.1074/jbc.M806893200 originally published online January 14, 2009
2009, 284:6725-6734.J. Biol. Chem.
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