ATF-2 Is a Common Nuclear Target of Smad and TAK1 Pathways in
Transforming Growth Factor-
Signaling*
Yuji
Sano
§,
Jun
Harada
,
Shigeki
Tashiro
,
Ryoko
Gotoh-Mandeville
,
Toshio
Maekawa
¶, and
Shunsuke
Ishii
¶
From the
Laboratory of Molecular Genetics, Tsukuba
Life Science Center, RIKEN, 3-1-1 Koyadai, Tsukuba, Ibaraki 305-0074 and the ¶ Core Research for Evolutional Science and Technology,
Japan Science and Technology Corporation, Japan
 |
ABSTRACT |
Upon transforming growth factor-
(TGF-
)
binding to its cognate receptor, Smad3 and Smad4 form heterodimers and
transduce the TGF-
signal to the nucleus. In addition to the Smad
pathway, another pathway involving a member of the mitogen-activated
protein kinase kinase kinase family of kinases, TGF-
-activated
kinase-1 (TAK1), is required for TGF-
signaling. However, it is
unknown how these pathways function together to synergistically amplify TGF-
signaling. Here we report that the transcription factor ATF-2
(also called CRE-BP1) is bound by a hetero-oligomer of Smad3 and Smad4
upon TGF-
stimulation. ATF-2 is one member of the ATF/CREB family
that binds to the cAMP response element, and its activity is enhanced
after phosphorylation by stress-activated protein kinases such as c-Jun
N-terminal kinase and p38. The binding between ATF-2 and Smad3/4 is
mediated via the MH1 region of the Smad proteins and the basic leucine
zipper region of ATF-2. TGF-
signaling also induces the
phosphorylation of ATF-2 via TAK1 and p38. Both of these actions are
shown to be responsible for the synergistic stimulation of ATF-2
trans-activating capacity. These results indicate that
ATF-2 plays a central role in TGF-
signaling by acting as a common
nuclear target of both Smad and TAK1 pathways.
 |
INTRODUCTION |
Members of the Smad group of proteins mediate
TGF-
,1 BMP (bone
morphogenetic protein), and activin signaling from receptors to nuclei
(for review, see Refs. 1 and 2). Smad2 and Smad3 are substrates and
mediators of the related TGF-
and activin receptors in vertebrates
(3-7). TGF-
first directly binds to the TGF-
type II receptor
and leads to the formation of an oligomeric complex of the type I and
type II receptors (8). Upon ligand binding, the C-terminal ends of
these Smad proteins, which bind directly to the type I receptor, are
phosphorylated by the type I receptor. This results in their release
(7) and hetero-oligomerization with Smad4, a common-mediator of Smad
(9-11). Hetero-oligomers of Smad move into the nucleus and directly
participate in TGF-
- and activin-dependent
transcriptional activation (12-14). Smad2 and Smad4 interact with
FAST-1, a member of the winged-helix transcription factor family, and
mediate activin-dependent transcriptional activation (13,
14). Recently, the N-terminal regions of Drosophila Mad and
mammalian Smad3 and Smad4, which are conserved in the Smad gene family,
were shown to interact with specific DNA sequences, and the
direct binding of Smad3/4 to DNA is critical for the
TGF-
-induced transcriptional activation (15-18).
In addition to the Smad group of proteins, another pathway involving a
member of the MAPKKK family of kinases, TAK1 (TGF-
-activated kinase), is also known to be involved in TGF-
signaling (19). TAB1
and TAB2 were identified as proteins that directly bind to TAK1 (20).
Overexpression of TAB1 enhances the activity of the plasminogen
activator inhibitor 1 (PAI-1) gene promoter, which is regulated by
TGF-
, and increases the kinase activity of TAK1, suggesting that
TAB1 is an upstream regulator of TAK1. Furthermore, TAK1 activates
stress-activated protein kinases (SAPKs), p38 through MKK6 or MKK3 (21)
and c-Jun N-terminal kinases (JNKs) via MKK4 (22). Since MKK4 can also
activate p38 (23, 24), TAK1 may activate p38 via MKK4. However, it is
unknown how the Smad and TAK1 pathways function together to
synergistically amplify TGF-
signaling.
Recently, the cAMP response element (CRE) in the
Ultrabithorax (Ubx) gene enhancer was shown to
mediate transcriptional activation by Dpp, a Drosophila
homologue of TGF-
/BMP (25). In addition, mutation of the AP-1 sites
of the collagenase promoter eliminated TGF-
-dependent
transcriptional activation (16). The sequences of the CRE and AP-1
sites (12-O-tetradecanoylphorbol-13-acetate response
element,) are similar to each other, and ATF/CREB and members of the
Jun family of proteins bind to these sites, respectively (26). So far,
a number of transcription factors of the ATF/CREB family have been
identified. All members of this family contain a DNA binding domain
consisting of a cluster of basic amino acids and a leucine zipper
region, the so-called b-ZIP (for review, see Ref. 27). They form
homodimers or heterodimers through their leucine zipper regions and
bind to CRE. Among many of the transcription factors of the ATF/CREB
family, two factors, CREB (28, 29) and ATF-2 (also called CRE-BP1)
(30-32), are the best characterized. CREB is activated via direct
phosphorylation by cAMP-dependent protein kinase (33). On
the other hand, SAPKs such as JNKs and p38 phosphorylate ATF-2 at
Thr-69, Thr-71, and Ser-90 which lie close to the N-terminal
transcriptional activation domain and stimulate its
trans-activating capacity (34-36). Thus, these two groups
of factors, CREB and ATF-2, are linked to distinct signaling cascades
involving the cAMP-dependent protein kinase and SAPK pathways. ATF-2, ATF-a, and CRE-BPa form a subgroup (30, 37, 38) and
have a transcriptional activation domain containing the metal finger
structure located in their N-terminal regions (38, 39). These factors
bind to CRE with high affinity as a homodimer or heterodimer with c-Jun
(26, 40). Among these three factors, ATF-2 has been more extensively
studied, and shown to be ubiquitously expressed, with the highest level
of expression being observed in the brain (41). Mutant mice generated
by gene targeting exhibited lowered postnatal viability and growth, in addition to a defect in endochondrial ossification and a reduced number
of cerebellar Purkinje cells (42).
The fact that ATF-2 activity is enhanced by SAPK whose activity in turn
is stimulated by TAK1 allowed us to hypothesize that ATF-2 might play
an important role in the TGF-
signal transduction pathway. Our
results indicate that ATF-2 not only directly binds to Smad3/4
hetero-oligomers but also that ATF-2 is phosphorylated by TGF-
signaling via TAK1 and p38. The two pathways, Smad and TAK1,
synergistically enhance the activity of ATF-2 which acts as their
common nuclear target.
 |
EXPERIMENTAL PROCEDURES |
In Vitro Binding Assay--
The plasmids used to express the
GST-Smad fusion proteins containing various forms of Smad3/4 were
constructed by using appropriate enzyme sites or the polymerase chain
reaction (PCR)-based method with the pGEX vectors (Amersham Pharmacia
Biotech). Each construct contained one of the following regions: Smad3
CT1 (amino acids 1-105), Smad3 CT2 (aa 1-147), Smad3 CT3 (aa 1-266),
Smad3 CT4 (aa 1-341), Smad3 NT1 (aa 148-424), Smad4 CT1 (aa 1-65),
Smad4 CT2 (aa 1-227), Smad4 CT3 (aa 1-427), Smad4 NT1 (aa 228-552), or Smad4 NT2 (aa 152-552). For the in vitro translation of
ATF-2, plasmids encoding a series of mutants of ATF-2, which were
described previously (39), were similarly constructed using the pSPUTK vector (Stratagene). The GST pull-down assays were performed as described (43) except for the use of about 20 µg of GST fusion protein per assay. To avoid the nonspecific interaction via DNA, the
resins bound to GST fusion proteins were washed with TE buffer (50 mM Tris-HCl, pH 7.5, 0.1 mM EDTA) containing
0.6 M NaCl to remove bacterial DNA. The binding buffer
consisting of 20 mM Hepes, 75 mM KCl, 0.1 mM EDTA, 2.5 mM MgCl2, 1% skim
milk, 1 mM dithiothreitol, 0.05% Nonidet P-40, and a
protease inhibitor mixture (Boehringer Mannheim) was used for binding assays.
Co-immunoprecipitation--
A mixture of 2 µg of the ATF-2
expression plasmid, pact-CRE-BP1, 2 µg of the Flag-Smad3 expression
plasmid, pact-Flag-Smad3, and 2 µg of the Flag-Smad4 expression
plasmid, pact-Flag-Smad4, was transfected into the TGF-
-responsive
293 cells using LipofectAMINE (Life Technologies, Inc.). In some
assays, the plasmid to express the constitutively active form of the
TGF-
type I receptor, pact-ALK5-T204D, was also co-transfected. The
total amount of plasmid DNA was adjusted to 8 µg by adding the
control effector plasmid, pact1, lacking the cDNA to be expressed.
TGF-
treatment at a final concentration of 7.2 ng/ml was performed
for 1 h before lysate preparation. Forty hours after transfection,
cells were lysed in lysis buffer (50 mM Hepes, pH 7.5, 250 mM NaCl, 0.2 mM EDTA, 0.5% Nonidet P-40, 50 mM NaF, 2 mM Na3VO4,
0.1 µM okadaic acid, 25 mM
-glycerophosphate, and protease inhibitor mixture), and whole-cell
lysates were prepared. After decreasing the NaCl concentration to 100 mM by adding the lysis buffer lacking NaCl, lysates were
immunoprecipitated using anti-ATF-2 polyclonal antibodies (N-96, Santa
Cruz Biotechnology) or normal rabbit IgG as a control. The immune
complex was analyzed by Western blotting to detect the co-precipitated
Smad3 and Smad4 using anti-Flag monoclonal antibody (Eastman Kodak Co.)
and LumiGO chemiluminescent detection reagent (New England Biolabs). To
examine the Smad3/4 and ATF-2 proteins expressed, aliquots of cell
lysates were also directly used for Western blotting with anti-Flag and anti-ATF-2 antibodies.
Mammalian Two-hybrid Assay--
The plasmids used to express the
Gal4-Smad fusion protein containing the Gal4 DNA-binding domain (amino
acids 1-147) joined to the N-proximal region of Smad3 (amino acids
1-189) or Smad4 (amino acids 1-265) were made by the PCR-based method
with the use of the cytomegalovirus promoter-containing expression
vector. The plasmids encoding the VP16-ATF-2 fusion protein containing the C-proximal region of ATF-2 (amino acids 291-505) were constructed similarly using the pcDNA3 vector (Invitrogen). The plasmids used to express the Gal4-ATF-2 fusion protein containing the Gal4
DNA-binding domain joined to the C-proximal region of ATF-2 (amino
acids 291-414) were made by the PCR-based method with the use of the
cytomegalovirus promoter-containing expression vector. The plasmids
encoding the VP16-Smad fusion protein containing the full-length form
of Smad3 or Smad4 were constructed similarly using the pcDNA3
vector. Co-transfection assays were performed as described (44) using
the firefly luciferase reporter plasmid containing three copies of the
Gal4-binding site. A mixture containing 1 µg of the luciferase
reporter plasmid, 3 µg of either the Gal4-Smad3N, Gal4-Smad4N, or
Gal4-ATF-2 expression plasmid, 4 µg of either the VP16-ATF-2,
VP16-Smad3FL, VP16-Smad4FL, or VP16 expression plasmid, and 0.5 µg of
the internal control plasmid pRL-TK (Promega) was transfected into
HepG2 cells. The total amount of plasmid DNA was adjusted to 8.5 µg
by the addition of the control plasmid lacking the cDNA to be
expressed. Luciferase assays were performed using the dual-luciferase
assay system (Promega). Experiments were repeated three times, and the
data were averaged.
Detection of Phosphorylated Proteins--
To examine the
phosphorylation of endogenous protein, 293 cells were serum-starved and
incubated with TGF-
(3 ng/ml). The cells were disrupted in RIPA
buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl,
1% Triton X-100, 1% deoxycholate, 0.1% SDS, 50 mM NaF, 2 mM Na3VO4, 0.1 µM okadaic
acid, 25 mM
-glycerophosphate, and protease inhibitor
mixture). After centrifugation, the supernatant was analyzed by 10%
SDS-PAGE, followed by Western blotting. The phosphorylation of ATF-2,
p38, JNK1, and JNK2 were examined using PhosphoPlus ATF-2 (Thr-71),
p38MAPK (Thr-180/Tyr-182), and JNK1/2(Thr-183/Tyr-185) antibody kits,
respectively (New England Biolab.). To inhibit p38 activity, 293 cells
were treated with SB203580 (Calbiochem) for 45 min before the
preparation of cell lysates. To analyze the phosphorylated state of
ATF-2 expressed by the transfected DNA, 293 cells were transfected
using LipofectAMINE (Life Technologies, Inc.) and a mixture made up of
1.5 µg of the plasmid expressing the wild type or CT91 mutant lacking
the C-terminal 91 amino acids and 1.5 µg of a plasmid expressing the
activated form of TAK1 (TAK1
N) or no protein at all. About 45 h
after transfection, cell lysates were prepared, and the phosphorylated
state of ATF-2 was examined again as described above.
Co-transfection Assay--
The plasmids to express Smad3 and
Smad4 were constructed by inserting the corresponding cDNAs
downstream of the cytomegalovirus promoter. The CRE-containing
luciferase reporter was constructed using the previously reported
CRE-CAT reporter plasmid (39). The ATF-2 and c-Jun expression plasmids
containing the chicken cytoplasmic
-actin promoter were described
previously (39). In the experiments using the CRE-containing reporter,
a mixture containing 2 µg of the reporter plasmid, 2 µg of the
ATF-2 expression plasmid, or 1 µg of the plasmid to express ATF-2 or
c-Jun, 1.5 µg of the plasmid to express Smad3 or Smad4, 1.5 µg of
the activated TAK1 (TAK1
N) expression plasmid, and 0.5 µg of the
internal control plasmid pRL-TK was transfected into HepG2 cells. The
total amount of plasmid DNA was adjusted to 9 µg by the addition of
the control plasmid lacking the cDNA to be expressed. In the
experiments using the p3TP-Lux reporter plasmid, a mixture containing
1.5 µg of the reporter plasmid, 2 µg of the ATF-2 expression
plasmid, 1.5 µg of the plasmid to express Smad3 or Smad4, 1.5 µg of
the activated TAK1 expression plasmid, and 0.5 µg of the internal
control plasmid pRL-TK was transfected.
To examine the effect of various dominant negative forms of ATF-2,
Smad3/4, and TAK1, the following mutants were used. The Ala mutant
(ATF-2Ala) in which the three SAPK phosphorylation sites (Thr-69,
Thr-71, and Ser-90) were replaced by alanine was constructed using the
PCR-based method. The N-truncated mutant of ATF-2 (ATF-2
107) lacking
the N-terminal 107 amino acids was described previously (39). The
C-truncated mutant of Smad3 (Smad3
C) or Smad4 (Smad4
C) lacking
the C-terminal 40 or 38 amino acids were made by using the PCR-based
method. The Smad3 mutant in which all the three serine residues of the
SSXS motif are mutated to alanine (Smad3AAVA) was also
constructed by using the PCR-based method. The dominant negative form
of TAK1, in which Lys-63 of the ATP-binding site was replaced by
tryptophan (TAK1K63W), was a gift from Dr. K. Matsumoto (19). To
examine the effect of various dominant negative forms on the
TGF-
-induced activity of 3TP-Lux promoter, a mixture containing 1.5 µg of the 3TP-Lux reporter plasmid, 2 µg of the plasmid to express
various forms of dominant negative forms of ATF-2, Smad3/4, or TAK1,
and 0.5 µg of the internal control plasmid pRL-TK was transfected
into HepG2 cells by using the CaPO4 method. The total
amount of plasmid DNA was adjusted to 9 µg by the addition of the
control plasmid lacking the cDNA to be expressed. To examine the
effect of dominant negative form of ATF-2 on the Smad- and/or
TAK1-induced activity of 3TP-Lux promoter, a mixture containing 1.5 µg of the 3TP-Lux reporter plasmid, 1 µg of the plasmid to express
Smad3 or Smad4, 1.5 µg of the activated TAK1 (TAK1
N) expression
plasmid, 1 µg of the plasmid to express the dominant negative forms
of ATF-2, and 0.5 µg of the internal control plasmid pRL-TK was
transfected into HepG2 cells by using the CaPO4 method. The
total amount of plasmid DNA was adjusted to 10 µg. TGF-
treatment
was performed for 12 h at a final concentration of 2.4 ng/ml
before lysate preparation. Luciferase assays were performed using the
dual luciferase assay system (Promega). Experiments were repeated 2-4
times, and the data were averaged.
 |
RESULTS |
ATF-2 Binds to the MH1 Region of Smad3 and Smad4--
To
investigate whether ATF-2 functions in the Smad pathway, we first of
all examined for a direct interaction between Smad3/4 and ATF-2 (Fig.
1). Protein affinity resins in which the
GST, GST-Smad3, or GST-Smad4 fusion protein containing the full-length form of Smad3 or Smad4 was used as a ligand were prepared (Fig. 1,
A and B). The full-length form of human ATF-2 was
synthesized using the in vitro transcription/translation
system and was mixed with this affinity resin. Approximately 17 and
20% of ATF-2 were bound to the resin containing the GST-Smad3 and the
GST-Smad4 fusion protein, respectively, but none was bound by the GST
resin alone (Fig. 1C). We further examined which region of
Smad3 and Smad4 binds to ATF-2. In addition to the GST fusion protein
containing the full-length form of Smad3 and Smad4, five fusion
proteins containing a series of truncated Smad3 or Smad4 protein were
prepared and used in the binding assays (Fig. 1, A and
B). Among Smad proteins, there are two homologous regions,
the N-terminal MH1 (mad homology domain 1) and the C-terminal MH2,
which are conserved in Smad-related proteins in various species ranging
from insects to vertebrates (Fig. 1A). The truncated mutants
of Smad3 and Smad4 that lacked the region downstream of the MH1 region
still retained the ability to interact with ATF-2 (Fig. 1, A
and C). However, the mutants of Smad3 and Smad4 that lacked
a part or the whole region of MH1 could not bind to ATF-2 (see CT1 and
NT1 of Smad3 and CT1, NT1, and NT2 of Smad4). These results indicate
that the N-terminal MH1 region binds to ATF-2.

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Fig. 1.
ATF-2 binds to the MH1 region of Smad3/4
in vitro. A, structures of the
GST-Smad3 and GST-Smad4 fusion proteins used in the binding assays. The
GST fusion proteins containing various forms of Smad3 or Smad4 are
schematically shown. The regions, MH1 and MH2,
conserved among members of the Smad gene family are indicated by
shaded boxes. The results of binding assays are shown to the
right. + and indicate the binding of 17-40% and
less than 0.6% of the input protein, respectively. B,
analysis of GST-Smad3/4 fusion proteins. The bacterial lysates
containing 3-5 µg of various GST-Smad3 or GST-Smad4 fusion proteins
or control GST were mixed with the glutathione-Sepharose resin and
washed. The bound proteins were analyzed on 10% SDS-PAGE followed by
Coomassie Brilliant Blue staining. C, binding of ATF-2 to
GST-Smad3 (left panel) or GST-Smad4 (right panel)
fusion proteins. The Sepharose resin containing GST-Smad3, GST-Smad4,
or GST as a ligand were mixed with the in vitro translated
35S-ATF-2 protein. After washing, the bound proteins were
released and analyzed on 10% SDS-PAGE followed by autoradiography. In
the input lanes, the amount of 35S-ATF-2 protein
was 10% that used for the binding assay.
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Smad4 Binds to the b-ZIP Region of ATF-2--
To determine further
which region of ATF-2 interacts with Smad4, we made various mutants of
ATF-2 by an in vitro transcription/translation system and
used them in the GST pull-down assay (Fig.
2). Among the six mutants, the two
mutants lacking the basic region (
BR) or containing a mutated
leucine zipper (L34V), in which the third and fourth leucine residues
were mutated to valine, failed to bind to GST-Smad4. In contrast, all
the other ATF-2 mutants bound to GST-Smad4 with an efficiency similar
to that of the wild type (approximately 15-30% of the input ATF-2
protein was bound). These results indicated that the b-ZIP region
of ATF-2 interacts with Smad4.

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Fig. 2.
Smad3/4 bind to the b-ZIP region of
ATF-2. On the top, the functional domains of ATF-2 are
schematically shown. The structures of the various forms of ATF-2 used
are shown below. The results of binding assays shown below
are indicated on the right. The relative binding activities
of the mutants are designated + and , which indicate the binding of
14-40% and less than 0.5% of the input protein, respectively. In the
input lanes, various forms of ATF-2 indicated
above each lane were synthesized in vitro and
analyzed by 10% SDS-PAGE. In the right panel, the
35S-ATF-2 proteins indicated above each lane
were mixed with the GST-Smad4 affinity resin, which contains
full-length Smad4, and the bound proteins were analyzed on 10%
SDS-PAGE followed by autoradiography. In the input lanes,
the amount of protein was 10% that used in the binding assay. Less
than 0.5% of the input ATF-2 proteins bound to the control GST resin
(data not shown). WT, wild type.
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|
TGF-
Signaling-induced Association between ATF-2 and
Smad3/4--
We investigated the interaction between ATF-2 and Smad3/4
in mammalian cells by co-immunoprecipitation (Fig.
3A). The ATF-2 expression
plasmid was co-transfected into the TGF-
-responsive 293 cells with
the two plasmids expressing Flag-linked Smad3 and Smad4. The cell
lysates were immunoprecipitated with the anti-ATF-2 polyclonal
antibody, and the co-precipitated Smad3 and Smad4 proteins were
detected using anti-Flag antibody. Both Smad3 and Smad4 were co-precipitated with anti-ATF-2 antibody (Fig. 3A, lane 2).
The Smad3 proteins overexpressed from the transfected DNA were reported to be localized in the nuclei even in the absence of ligand (45), and
ATF-2 is constitutively in the nuclei. These facts are consistent with
the data described above. To examine whether TGF-
signaling enhances
the association between ATF-2 and Smad3/4, the constitutively active
TGF-
type I receptor, in which Thr-204 was replaced by aspartic
acid, was co-transfected, and the cells were treated with TGF-
.
Under these conditions higher amounts of Smad3/4 were co-precipitated
with the anti-ATF-2 antibody than in the absence of the exogenous
TGF-
type I receptor and TGF-
stimulation (Fig. 3A,
compare lanes 2 and 3). The control IgG did not
co-precipitate Smad3/4 at all (Fig. 3A, lane 1). These
results show that ATF-2 associates with Smad3/4 upon TGF-
stimulation in vivo.

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Fig. 3.
Interaction between ATF-2 and Smad3/4 in
mammalian cells. A, co-immunoprecipitation
(Co-IP). Whole-cell lysates were prepared from 293 cells
transfected with a mixture of plasmids to express ATF-2 and Flag-linked
Smad3 and Smad4, and samples from the lysates were directly used for
Western blotting with the anti-Flag or anti-ATF-2 antibodies
(Direct Western). Whole-cell lysates were also
immunoprecipitated by anti-ATF-2 antibody (Ab), and the
immunocomplexes were analyzed by Western blotting using anti-Flag
antibodies. In lanes 1 and 3, the plasmid to
express the constitutively active TGF- type I receptor was also
co-transfected, and the transfected cells were stimulated by TGF-
for 1 h before preparation of cell lysates. In lane 1,
normal IgG was used as a control for immunoprecipitation. B,
mammalian two-hybrid interaction. Left, HepG2 cells were
co-transfected with the Gal4 site-containing reporter, the plasmid to
express Gal4-Smad3 or the Gal4-Smad4 fusion containing the MH1 region
of Smad3/4, and the expression plasmid for VP16-ATF-2 containing the
b-ZIP region of ATF-2. The degree of activation is indicated (means
S.E.). Right, HepG2 cells were co-transfected with the Gal4
site-containing reporter, the expression plasmid for the Gal4-fusion
protein containing the b-ZIP region of ATF-2, and the VP16-Smad3 or
VP16-Smad4 expression plasmid containing the full length Smad3/4 or
VP16 alone.
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To confirm the interaction in mammalian cells between the b-ZIP region
of ATF-2 and the MH1 region of Smad3/4, the two-hybrid assay was
performed using HepG2 cells (Fig. 3B). In the first experiment, two types of chimeric proteins were employed. In one, the
N-proximal region of Smad3 or Smad4 was fused to the DNA binding domain
of Gal4, and in the other, the C-proximal region of ATF-2 containing
the b-ZIP structure was fused to the transcriptional activation domain
of VP16, and the degree of transcriptional activation in HepG2 cells
was examined (Fig. 3B, left panel). The VP16-ATF-2 fusion
proteins stimulated Gal4-Smad3N and Gal4-Smad4N activity by 8.9- and
11-fold, respectively, whereas VP16 alone had no effect. In the second
experiment, the C-proximal region of ATF-2 containing the b-ZIP
structure was fused to the DNA-binding domain of Gal4, and the
full-length form of Smad3 or 4 was fused to the strong transcriptional
activation domain of VP16 (Fig. 3B, right panel). The
VP16-Smad3 and VP16-Smad4 fusion proteins stimulated Gal4-ATF-2 activity by 17- and 7-fold, respectively, whereas VP16 alone had no
effect. These results indicate that the b-ZIP domain of ATF-2 interacts
in mammalian cells with the MH1 region of Smad3 and Smad4.
TGF-
Signaling Induces Phosphorylation of ATF-2 via
TAK1--
We next examined whether phosphorylation of ATF-2 is
enhanced by TGF-
treatment (Fig. 4).
The TGF-
-responsive 293 cells were treated with TGF-
, and ATF-2
phosphorylated at Thr-71 was detected by the phospho-ATF-2-specific
antibody at various intervals after TGF-
treatment (Fig.
4A). The Thr-71 residue is known to be the phosphorylation
site of SAPK (34-36). The degree of phosphorylation of ATF-2 increased
up to a maximum of 4-fold at 15 min after TGF-
treatment, whereas
the amount of ATF-2 was not affected by TGF-
treatment. To confirm
that TGF-
signaling phosphorylates ATF-2 at the same sites as SAPK,
the ATF-2 mutant, whose three SAPK phosphorylation sites (Thr-69,
Thr-71, and Ser-90) were replaced by alanine, was used. Since this
alanine mutant cannot be recognized by the antibody raised against the
peptide containing these phosphorylation sites, we used the C-truncated
form of ATF-2 to discriminate from the endogenous protein, and we
judged the phosphorylation status of the mutants by their altered
migration during SDS-PAGE. The C-truncated form of ATF-2, which was
phosphorylated by TGF-
signaling via SAPK, migrated more slowly
during SDS-PAGE than the non-phosphorylated form (Fig. 4B,
compare lanes 1 and 2). However, the migration of
the alanine mutant was the same as that of the wild type even in the
presence of TGF-
treatment, confirming that at least one of these
three sites was phosphorylated by TGF-
signaling. To investigate
further whether phosphorylation of ATF-2 is mediated by TAK1, we
examined the effect of activated TAK1 on the phosphorylation of ATF-2
(Fig. 4C). Co-transfection of the plasmid to express the
activated form of TAK1, which lacked its N-terminal 22 amino acids,
with the C-truncated ATF-2 expression plasmid increased the amount of
ATF-2 phosphorylated at Ser-71, as detected by the phospho-ATF2-specific antibody (Fig. 4C, compare lanes
1 and 3). In addition, the activated form of TAK1
further enhanced the phosphorylation of ATF-2 in the presence of
TGF-
treatment (Fig. 4C, compare lanes 2 and
4). These results suggest that TGF-
signaling induces the
phosphorylation of ATF-2 at the SAPK phosphorylation sites via
TAK1.

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Fig. 4.
Induction of ATF-2 phosphorylation by
TGF- signaling via TAK1. A,
time course of phosphorylation of endogenous ATF-2. Total cell lysates
were prepared from TGF- -treated or untreated 293 cells and used for
Western blotting. In the upper and lower panels,
the ATF-2 proteins phosphorylated at Thr-71, and both the
phosphorylated and non-phosphorylated forms are indicated,
respectively. B, phosphorylation of exogenous ATF-2 at
JNK/p38 phosphorylation sites by TGF- signal. The plasmid to express
the ATF-2 protein lacking the C-terminal 91 amino acids but containing
either the normal three JNK/p38 phosphorylation sites or these sites
mutated to alanines was transfected into 293 cells. Cell lysates were
prepared, and the C-truncated ATF-2 was detected by Western blotting
using anti-ATF-2 antibody which recognizes both the phosphorylated and
non-phosphorylated forms. Since the phospho-ATF-2-specific antibody
cannot react with the alanine mutant, the phosphorylated form was
detected as a slower migrating band on a long SDS-PAGE gel.
C, phosphorylation of ATF-2 through TAK1. The two plasmids
to express ATF-2 lacking the C-terminal 91 amino acids and the
activated form of TAK1 (TAK1 N) or no protein were transfected into
293 cells. Phospho-ATF-2 and ATF-2 were detected as described in
A.
|
|
Involvement of p38 in TGF-
-induced Phosphorylation of
ATF-2--
The results of the ATF-2 phosphorylation assays and the
fact that TAK1 activates SAPKs, JNKs, and p38 (21, 22) suggest that
SAPKs phosphorylate ATF-2 upon TGF-
stimulation. To investigate which SAPK is activated by TGF-
signaling, we examined the
phosphorylation of p38, JNK1, and JNK2. The 293 cells were treated with
TGF-
, and p38 phosphorylated at Thr-180/Tyr-182 and JNK1/JNK2
phosphorylated at Thr-183/Tyr-185 were detected by the phosphorylated
form-specific antibody at various intervals after TGF-
treatment
(Fig. 5A). The degree of
phosphorylation of p38, which displayed a timing similar to that of
ATF-2, increased up to 4-fold. In contrast, the phosphorylation of JNK1
and JNK2 remained unchanged, suggesting that TGF-
signaling leads to
phosphorylation of ATF-2 through mainly p38 rather than JNK. To confirm
these results further, the effect of the specific inhibitor of p38, the
pyridinyl imidazole derivative SB203580 which cannot inhibit JNKs (46,
47), on the TGF-
-induced phosphorylation of ATF-2 was examined (Fig. 5B). SB203580 almost completely blocked TGF-
-induced
phosphorylation of ATF-2. These results indicate that TGF-
induces
the phosphorylation of ATF-2 through the action of TAK1 and p38.

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Fig. 5.
Phosphorylation of ATF-2 by
TGF- signaling via p38. A,
activation of p38 by TGF- signaling. Total cell lysates were
prepared from TGF- -treated or untreated 293 cells and used for
Western blotting with the phosphorylated form-specific or nonspecific
antibody against ATF-2, p38, JNK1, or JNK2. B, inhibition of
the TGF- -induced phosphorylation of ATF-2 by the p38 inhibitor. In
the presence or absence of TGF- treatment, 293 cells were treated
with SB203580. +, 0.1 µM; ++, 10 µM. Whole
cell lysates were prepared and used for Western blotting to detect
phosphorylated ATF-2 and both phosphorylated and non-phosphorylated
ATF-2 as described in A.
|
|
Synergistic Activation of ATF-2 Activity by Smad and TAK1
Pathways--
To investigate whether the trans-activating
capacity of ATF-2 is enhanced by Smad3/4 and TAK1 pathways,
co-transfection assays were performed using a reporter plasmid
containing four copies of the consensus CRE sequence (Fig.
6). This artificial promoter was weakly
responsive to TGF-
in HepG2 cells (2-fold). When present separately,
ATF-2, Smad3/4, and the activated form of TAK1 stimulated this promoter
activity by 2-, 10-, and 2-fold, respectively, in the absence of
TGF-
treatment, and by 5-, 17-, and 7-fold, respectively, in the
presence of TGF-
treatment. The degree of activation of this
promoter by ATF-2 was synergistically increased by co-expression of
Smad3/4 or the activated form of TAK1. Furthermore, promoter activity
could be strongly enhanced by co-expression of all the three effectors
together, resulting in a 145- and 203-fold stimulation in the absence
and presence of TGF-
treatment, respectively. These results support
the idea that both the Smad3/4 pathway and TAK1 pathway synergistically
activate ATF-2. CRE is recognized by the ATF-2/c-Jun heterodimer with
high affinity and the c-Jun homodimer with lower affinity (40). To
determine which of these actually contributes to
CRE-dependent activation, we transfected the cells with
plasmids expressing both ATF-2 and c-Jun or with a plasmid expressing
c-Jun alone. As reported previously (39), the ATF-2/c-Jun heterodimer
activated more strongly the CRE-containing promoter (8-fold) compared
with the ATF-2 homodimer. However, further stimulation of ATF-2/c-Jun
heterodimerdependent activation by co-expression of both
Smad3/4 and the activated form of TAK1 was inefficient compared with
that seen with ATF-2 alone. The trans-activating capacity of
the c-Jun homodimer was also not so strongly enhanced by Smad3/4 and
TAK1 compared with the marked increase in the capacity of the ATF-2
homodimer. These results suggest that the ATF-2 homodimer is the
preferred target for the Smad and TAK1 pathways at least in HepG2
cells, although the activity of the ATF-2/c-Jun heterodimer and the
c-Jun homodimer are also stimulated to some extent by both
pathways.

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Fig. 6.
Synergistic enhancement of ATF-2 activity by
Smad3/4, and TAK1. Transcriptional activation of the
CRE-containing reporter by ATF-2. HepG2 cells were transfected by a
mixture containing the CRE-containing luciferase reporter, the ATF-2
expression plasmid, the Smad3 and Smad4 expression plasmids, the
plasmid to express the activated form of TAK1, and the internal control
plasmid. The degree of activation is indicated (means S.E.). The data
obtained after TGF- treatment is indicated by shaded
bars. In some cases, the c-Jun expression plasmid or a mixture of
the ATF-2 and c-Jun expression plasmids was used instead of the ATF-2
expression plasmid.
|
|
Involvement of ATF-2 in TGF-
-inducible Promoter
Activation--
To examine the role of ATF-2 in the regulation of the
TGF-
-inducible promoters, co-transfection experiments were performed using a fusion promoter (p3TP-Lux reporter) consisting of PAI-1 and
collagenase promoters (48) (Fig.
7A). This promoter was highly
responsive to TGF-
in HepG2 cells (38-fold). Smad3/4 stimulated this
promoter activity by 251-fold in the absence of TGF-
treatment and
by 409-fold in the presence of TGF-
treatment. The degree of
activation of this promoter by Smad3/4 was slightly enhanced by
co-expression of ATF-2 or the activated form of TAK1. Furthermore, promoter activity could be strongly enhanced by co-expression of all
the three effectors together, resulting in a 791- and 1104-fold stimulation in the absence and presence of TGF-
treatment,
respectively. These results support the idea that co-expression of
ATF-2, Smad3/Smad4, and the activated form of TAK1 synergistically
activated this promoter activity. When these results are compared with
those with the CRE-containing promoter described above, however, some difference is evident. Unlike the case of CRE-containing promoter, Smad3/4 strongly activated this promoter. In addition, ATF-2 alone did
not enhance this promoter activity, and the synergism between ATF-2 and
the activated form of TAK1 was not observed using this promoter. This
could be due to the fact that Smad3/4 can activate this promoter not
only via a complex formation with ATF-2 but also via direct binding to
the specific sites in the PAI-I promoter (see "Discussion").

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Fig. 7.
Involvement of ATF-2 in
TGF- -induced transcriptional activation.
A, transcriptional activation of the p3TP-Lux reporter by
ATF-2. HepG2 cells were transfected by a mixture containing the
p3TP-Lux reporter plasmid, the ATF-2 expression plasmid, the Smad3 and
Smad4 expression plasmids, the plasmid to express the activated form of
TAK1, and the internal control plasmid. The degree of activation is
indicated (means S.E.). The data obtained after TGF- treatment is
indicated by shaded bars. B, effect of dominant
negative forms of ATF-2, TAK1, and Smad3/4 on the TGF- -induced
activity of 3TP-Lux promoter. HepG2 cells were transfected by a mixture
containing the p3TP-Lux reporter, the plasmid to express various
dominant negative forms of ATF-2, TAK1, or Smad3/4, and the internal
control plasmid. The degree of activation is indicated (means S.E.).
The data obtained after TGF- treatment is indicated by shaded
bars. C, effect of dominant negative forms of ATF-2 on
the Smad3/4 and/or TAK1-induced activity of 3TP-Lux promoter. HepG2
cells were transfected by a mixture containing the p3TP-Lux reporter,
the Smad3/4 expression plasmid and/or the activated TAK1 expression
plasmid, the plasmid to express either of two dominant negative forms
of ATF-2, and the internal control plasmid.
|
|
To confirm that ATF-2 plays an important role for TGF-
-induced
activation of the p3TP-Lux promoter, we used two ATF-2 mutants as
follows: the Ala mutant (ATF-2Ala) in which the three SAPK phosphorylation sites (Thr-69, Thr-71, and Ser-90) (34-36) were replaced by alanine and the N-truncated mutant (ATF-2
107) lacking the N-terminal 107 amino acids including the SAPK phosphorylation sites
(Fig. 7B). These two mutants cannot be phosphorylated by TGF-
signaling via p38 and were expected to act as a dominant negative form. Co-transfection of either of these two mutants strongly
inhibited the TGF-
-induced activity of the p3TP-Lux promoter,
indicating that ATF-2 is involved in the activation of 3TP-Lux promoter
by TGF-
signaling. In addition, the dominant negative form of TAK1,
in which Lys-63 of the ATP-binding site was replaced by tryptophan
(TAK1K63W), inhibited the TGF-
-induced activity of the p3TP-Lux
promoter. To confirm the role of Smad3/4 in the TGF-
-induced
activity of the p3TP-Lux promoter, we used two types of mutants. The
C-truncated mutant of Smad3 (Smad3
C) or Smad4 (Smad4
C) lacking
the C-terminal transcriptional activation domain was reported to act as
a dominant negative form (6). The TGF-
type I receptor
phosphorylates Smad2 at Ser-465 and Ser-467 in the SSXS
motif, and the mutant in which all the three serine residues in the
SSXS motif were replaced by alanines acts as a dominant
negative form, because this mutant stably binds to the TGF-
type I
receptor (7, 49, 50). In addition, the alanine mutant of Ser-464 of
Smad2 also act as a dominant negative form, although this site is not
directly phosphorylated. Therefore, we constructed second type of
putative dominant negative form of Smad3 by replacing all the three
serine residues of the corresponding SSXS motif to alanine
(Smad3AAVA). Co-transfection of the C-truncated mutant of Smad3
(Smad3
C) or Smad4 (Smad4
C) or the Smad3 alanine mutant
(Smad3AAVA) inhibited the TGF-
-dependent p3TP-Lux
promoter activity. These results indicate that that both Smad and TAK1
pathway are required for the TGF-
-induced activation of the 3TP-Lux promoter.
To confirm the role of ATF-2 further, we examined the effect of two
ATF-2 mutants (ATF-2Ala and ATF-2
107) on the Smad3/4- and/or
activated TAK1-induced promoter activity of 3TP-Lux (Fig. 7C). Either of these two mutant significantly inhibited the
3TP-Lux promoter activity enhanced by Smad3/4, activated TAK1, or both Smad3/4 and activated TAK1. Thus, a dominant negative form of ATF-2 can
inhibit the stimulatory effect of either Smad and TAK1 pathways on the
of 3TP-Lux promoter.
 |
DISCUSSION |
Our results indicate that ATF-2 is a common nuclear target of the
Smad and TAK1 pathways (Fig. 8). Upon
binding of TGF-
to the type II receptor, the TGF-
-bound type II
receptor makes a heteromeric complex with the type I receptor,
resulting in the activation of the latter's serine/threonine kinase
activity. The activated serine/threonine kinase of the type I receptor
then phosphorylates the bound Smad3 or Smad2 protein, which results in
its release from the type I receptor. The released Smad3 forms a
hetero-oligomer with Smad4, which is thought to be localized in the
cytosol in the absence of TGF-
stimulation, and the hetero-oligomer moves into the nucleus. This hetero-oligomer directly binds to ATF-2
through the MH1 region of Smad3/4 and the b-ZIP region of ATF-2,
although the exact number of ATF-2 and Smad3/4 molecules in this
complex remains unknown. Binding of the Smad3/4 complex to ATF-2
enhances ATF-2 activity, as suggested by the observation that
overexpression of Smad3/4 enhances the trans-activating
capacity of ATF-2 (Fig. 6). In this sense, Smad3/4 resembles adenovirus E1A, which stimulates CRE-dependent transcription via
binding to the b-ZIP region of ATF-2 (51). In addition to this Smad pathway, another pathway, the TAK1 pathway, is required for TGF-
signal transduction. The expression of the dominant negative form of
TAK1 inhibits the TGF-
-induced activation of the PAI-1 promoter (19). Upon TGF-
stimulation, the TAB1 protein is thought to be
activated, an event that results in its binding to the serine/threonine kinase domain of TAK1 (20). However, the precise mechanism
of signal transduction from the TGF-
receptor to TAB1 remains
unknown. TAK1 is a member of the MAPKKK family and activates MKK3 and
MKK6 of the MAPKK family, both of which share striking homology with each other (21). TAK1 also activates MKK4, another member of MAPKK
(22). TAK1 activates p38, one member of the SAPK family via MKK6/MKK3
(21), and also possibly through MKK4 (22-24). p38 directly
phosphorylates ATF-2 at Thr-69, Thr-71, and Ser-90, resulting in
stimulation of its trans-activating capacity. In fact,
co-expression of activated TAK1 enhanced this
trans-activating capacity of ATF-2 (Fig. 6). Thus, the Smad
and TAK1 pathways synergistically stimulate TGF-
-induced
transcription by acting on the common nuclear target ATF-2. TGF-
has
an important role in the regulation of genes involved in cell cycle
control and genes encoding the extracellular matrix, and many of them
have a CRE in their transcriptional control regions. Recently we found
that the expression level of some TGF-
-inducible genes encoding
extracellular matrix was decreased in mouse embryonic fibroblasts
lacking ATF-2 and its related gene
CRE-BPa2
supporting that the ATF-2 family is important for the TGF-
-mediated transcriptional activation. The identification of ATF-2 as a common nuclear target of Smad and TAK1 pathways may provide a clue as to how
the signal transduction of TGF-
is regulated. Recent studies using
the Xenopus system indicated that TAK1 and TAB1 also
function in the BMP signal transduction pathway in Xenopus
embryos (52). Therefore, ATF-2 could also play an important role in BMP
signal transduction. In fact, ATF-2 binds to Smad1 in the GST pull-down assay which is a mediator of BMP signal
transduction.2

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Fig. 8.
Schematic representation of synergistic
action of the Smad and TAK pathways via the common nuclear target,
ATF-2.
|
|
The b-ZIP region of ATF-2 directly interacts with the N-terminal MH1
region of Smad3/4. The b-ZIP region of ATF-2 and the MH1 region of
Smad3/4 can also directly bind to CRE and the recently identified
Smad-recognition sequence, respectively (17, 30). Therefore, the
interaction between ATF-2 and Smad3/4 is mediated by a different type
of DNA binding domain. There exist numerous examples of DNA binding
domains that can additionally serve as interaction sites for specific
proteins. The adenovirus 13SE1A protein binds to multiple DNA binding
domains including not only the b-ZIP region of ATF-2 but also the metal
finger of Sp1 and the basic-helix-loop-helix region of upstream factor
(51). Similarly, the helical structure of the b-ZIP region of ATF-2,
which would be exposed to the solvent, may serve as a protein surface
for interaction with Smads. Several examples of protein-protein
interaction being mediated via different DNA binding domains have also
been reported (44, 53).
Two SAPK family members have been identified in mammalian cells, JNKs
(54, 55) and p38 homologues (also termed p40, RK, and CSBP) (56-59).
Among these two type of SAPKs, only p38 was activated up to a maximum
at 15 min after TGF-
treatment via TAK1 in 293 cells (Fig. 5). In
contrast to this, TAK1 stimulated by ceramide was reported to activate
JNK in COS7 cells at the similar timing (22). Although JNK was also
reported to be activated by TGF-
stimulation in 293T cells, this
activation of JNK activity was observed at 12 h after TGF-
treatment (60). This contradiction could be due to the difference of
cells used, possibly due to the the cell type-specific expression of
some co-factor(s). ATF-2 is a good substrate of both SAPKs, whereas
c-Jun is phosphorylated by JNKs but not by p38 (61), suggesting that
ATF-2 is a preferable nuclear target of the TAK1 signal transduction
pathway at least in 293 cells. This may be consistent with our
observation that the trans-activating capacity of the c-Jun
homodimer is not so strongly enhanced by Smad3/4 and TAK1 compared with
the marked increase in the capacity of the ATF-2 homodimer (Fig. 6).
However, we observed that c-Jun directly binds to Smad3 via its b-ZIP
region like in the case of ATF-2.2 Therefore, c-Jun may
play a role in the Smad pathway in some types of cells. This could be
consistent with the recent report that Jun/Fos interacts with Smad3 and
is involved in TGF-
signaling (62).
Synergistic activation of the CRE-containing promoter by ATF-2 and
Smad3/4 suggests the interaction between ATF-2 bound to CRE and Smad3/4
unbound to DNA in this case. Recently, however, direct binding of Smad3
and Smad4 to a specific DNA sequence was reported (17). In addition,
the putative Smad-binding sites in the PAI-1 promoter were demonstrated
to be critical for the TGF-
inducibility of the promoter activity
(18, 63). Consistent with these reports, we observed that co-expression
of Smad3/4 alone enhanced the 3TP-Lux promoter, which contains the
PAI-1 promoter segment, more strongly than the case of CRE-containing promoter. On the other hand, expression of the dominant negative form
of TAK1 (19) or ATF-2 (Fig. 7, B and C) also
lowered the TGF-
-induced activity of the 3TP-Lux promoter,
indicating that the ATF-2-TAK1 pathway is also important for TGF-
responsiveness of this promoter. These results may suggest that both
ATF-2 and Smad3/4 directly bind to their own target sequences in the
PAI-1 promoter and that ATF-2 and Smad3/4 may interact via
protein-protein interaction. This may give a high TGF-
responsiveness to this promoter. Synergism of promoter activation by
multiple DNA binding transcription factors is well known (64).
Promoters containing four copies of CRE (Fig. 6) or two copies of
Smad3/4-binding sites (17) exhibited only weak TGF-
responsiveness,
supporting the idea that only one type of DNA binding factor may not
sufficient to exhibit the TGF-
responsiveness. Transcription factors
other than ATF-2 may also function in TGF-
-induced activation of
many other target promoters of TGF-
signaling. In fact, recently, Sp1 was demonstrated to activate p21/WAF1/Cip1 promoter activity by
interacting with Smad3/4 (65).
 |
ACKNOWLEDGEMENTS |
We thank K. Matsumoto for the TAK1 expression
plasmids and communication of unpublished results; R. Derynck for the
Smad3 cDNA; S. Kern for the Smad4 cDNA; J. Wrana for the
p3TP-Lux reporter; Masahiro Kawabata and Kohei Miyazono for the plasmid
to express the constitutively active TGF-
type I receptor; and T. Yamamoto for encouragement.
 |
FOOTNOTES |
*
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
§
On leave from the Institute of Medical Science, University of
Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-0071, Japan.
To whom correspondence should be addressed: Laboratory of
Molecular Genetics, Tsukuba Life Science Center, RIKEN, 3-1-1 Koyadai, Tsukuba, Ibaraki 305-0074, Japan. Tel.: 81-298-36-9031; Fax:
81-298-36-9030; E-mail: sishii{at}rtc.riken.go.jp.
2
Y. Sano and S. Ishii, unpublished data.
 |
ABBREVIATIONS |
The abbreviations used are:
TGF-
, transforming growth factor-
;
BMP, bone morphogenetic protein;
CRE, cAMP response element;
JNK, c-Jun N-terminal kinase;
SAPK, stress-activated kinase;
TAK1, TGF-
-activated kinase;
b-ZIP, basic
leucine zipper;
MAPKK, mitogen-activated protein kinase kinase;
MAPKKK, mitogen-activated protein kinase kinase kinase;
aa, amino acid;
GST, glutathione S-transferase;
PCR, polymerase chain reaction;
PAGE, polyacrylamide gel electrophoresis;
PAI-1, plasminogen activator
inhibitor 1.
 |
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