Alternatively Spliced Variant of Smad2 Lacking Exon 3
COMPARISON WITH WILD-TYPE Smad2 AND Smad3*
Ken
Yagi
,
Daisuke
Goto
,
Toshiaki
Hamamoto
,
Seiichi
Takenoshita§,
Mitsuyasu
Kato
¶, and
Kohei
Miyazono
From the
Department of Biochemistry, Cancer
Institute, Japanese Foundation for Cancer Research, and Research for
the Future Program, Japan Society for the Promotion of Science, 1-37-1 Kami-ikebukuro, Toshima-ku, Tokyo 170-8455 and the § First
Department of Surgery, Gunma University School of Medicine, 3-39-22 Showa-machi, Maebashi, Gunma 371-8511, Japan
 |
ABSTRACT |
An alternatively spliced variant of Smad2 with a
deletion of exon 3 (Smad2
exon3) is found in various cell types.
Here, we studied the function of Smad2
exon3 and compared it with
those of wild-type Smad2 containing exon 3 (Smad2(wt)) and Smad3. When transcriptional activity was measured using the p3TP-lux construct, Smad2
exon3 was more potent than Smad2(wt), and had activity similar to Smad3. Transcriptional activation of the activin-responsive element
(ARE) of Mix.2 gene promoter by Smad2
exon3 was also
similar to that by Smad3, and slightly less potent than that by
Smad2(wt). Phosphorylation by the activated transforming growth
factor-
type I receptor and heteromer formation with Smad4 occurred
to similar extents in Smad2
exon3, Smad2(wt), and Smad3. However, DNA
binding to the activating protein-1 sites of p3TP-lux was observed in
Smad2
exon3 as well as in Smad3, but not in Smad2(wt). In contrast,
Smad2(wt), Smad2
exon3, and Smad3 efficiently formed ARE-binding
complexes with Smad4 and FAST1, although Smad2(wt) did not directly
bind to ARE. These results suggest that exon 3 of Smad2 interferes with
the direct DNA binding of Smad2, and modifies the function of Smad2 in
transcription of certain target genes.
 |
INTRODUCTION |
Members of the transforming growth factor-
(TGF-
)1 superfamily
transduce signals through two different types of serine/threonine kinase receptors, known as type II and type I receptors (1). In the
TGF-
receptor system, ligand binds to the TGF-
type II receptor
(T
R-II), which has a constitutively active kinase. TGF-
type I
receptor (T
R-I) is then recruited into the TGF-
·T
R-II complex, and phosphorylated mainly at the glycine/serine-rich domain
(GS domain), which results in the activation of T
R-I kinase (2). The
T
R-I kinase transduces intracellular signals by activation of
various proteins, including Smad proteins. T
R-I thus acts as a
downstream component of T
R-II. Mutation in Thr-204 of T
R-I to
aspartic acid (T
R-I(TD)) results in the constitutive activation of
the T
R-I kinase, which has signaling activity in the absence of
TGF-
and T
R-II (3).
Smad proteins have recently been shown to comprise a family of proteins
that mediate signals for members of the TGF-
superfamily (4-6).
Thus far, eight mammalian Smad proteins have been identified, termed
Smad1 through Smad8. Smads are classified into three subgroups based on
their structure and function, i.e. pathway-restricted Smads,
common mediator Smads, and inhibitory Smads. Pathway-restricted Smads
can be further subdivided into those involved in the TGF-
and
activin signaling pathways, and those activated by the bone morphogenetic protein pathway. Smad2 and Smad3 serve as
pathway-restricted Smads for the TGF-
/activin signaling pathways.
Smad1, Smad5, and possibly Smad8/MADH6 are activated by bone
morphogenetic protein receptors. Smad4 is a common mediator Smad; thus
far only one common mediator Smad has been identified in mammals. Smad6
and Smad7 are inhibitory Smads.
Pathway-restricted Smads are phosphorylated by serine/threonine kinase
receptors, and form hetero-oligomers with Smad4. Phosphorylation of
pathway-restricted Smads occurs at the C-terminal
Ser-Ser-X-Ser motif (7-9). The heteromers then translocate
into the nucleus and activate the transcription of various target
genes. Smad proteins have been shown to interact with DNA-binding
proteins, such as winged-helix transcription factors,
Xenopus, and human FAST1 and mouse FAST2 (10-12), and also
to directly bind to specific DNA sequences (13-17).
Smads have conserved N- and C-terminal regions known as Mothers against
decapentaplegic (Mad) homology domain-1 (MH1) and -2 (MH2),
respectively, which are linked by a linker region of variable length
and amino acid sequence. The MH2 domain is a functional domain, which
has transactivation activity when fused to the Gal4-DNA binding domain
(18, 19). The MH2 domain also plays important roles in interaction with
type I receptors (20), homo- and hetero-oligomerization by Smad
proteins (21, 22), interaction with a transcription factor, FAST1 (23,
24), and association with transcriptional co-activators, p300/CBP
(25-28). The MH1 domain has been shown to physically interact with the
MH2 domain, and thereby inhibits the activity of the latter (29).
However, it has been shown that the MH1 domain has an intrinsic
function in signal transduction, i.e. direct binding to
specific DNA sequences. Mad in Drosophila was shown to bind
to the quadrant enhancer of vestigial gene (13). Smad3
and Smad4 have also been shown to bind to specific DNA sequences through their MH1 domains (13-17).
Although Smad2 and Smad3 are 91% identical in amino acid sequence,
they have certain differences in biological activity. In contrast to
Smad3 and Smad4, Smad2 does not directly bind to DNA (12-17). Binding
to a transcriptional regulator, Evi-1, is observed for Smad3 but not
for Smad2 (30). A functional difference between Smad2 and Smad3 has
also been suggested in the effects of TGF-
and activin on the HaCaT
keratinocyte cells (31). TGF-
and activin inhibit the growth of
HaCaT cells, but TGF-
is much more potent than activin. TGF-
induces the phosphorylation of both Smad2 and Smad3, whereas activin A
preferentially activates Smad3 (31).
Smad2 has a region with 30 amino acid residues, which is not found in
Smad3 or other Smads in mammals. The Smad2 gene is composed of 11 exons, and this short 30-amino acid region is translated by exon
3. Recently, we found that a Smad2 transcript which lacks exon 3 is
present in certain tissues and cells, although the amount of the
transcript is about 1/10 of that containing exon 3 (32). Here we
studied the function of Smad2 without exon 3 (Smad2
exon3) and
compared it with those of wild-type Smad2 (Smad2(wt)) and Smad3. In the
transcriptional activation assay using the p3TP-lux construct,
Smad2
exon3 was more potent than Smad2(wt), and had activity almost
similar to Smad3. Phosphorylation by T
R-I and heteromer formation
with Smad4 did not differ between Smad2
exon3 and Smad2(wt). However,
Smad2
exon3, but not Smad2(wt), was able to bind to the activating
protein (AP)-1 sites from p3TP-lux. In contrast, both Smad2(wt) and
Smad2
exon3 could form the activin-responsive factor (ARF) with FAST1
and Smad4, and transactivate the Xenopus Mix.2 gene promoter.
 |
EXPERIMENTAL PROCEDURES |
cDNA Constructs--
The original constructions of
Flag-pcDNA3, Myc-pcDNA3, HA-pcDNA3, T
R-I(TD)-HA,
Smad2(wt), and Smad3 have been described previously (33, 34).
Smad2
exon3 was prepared by inserting the 330-bp polymerase chain
reaction (PCR) product of Smad2
exon3 into Smad2(wt). In order to
obtain efficient expression levels of proteins, some constructs were
subcloned into another expression vector, pcDEF3 (35). Constructs for
GST-fused Smad2(wt)
MH2, Smad3
MH2, and Flag-tagged
Smad2
exon3
MH2 lacking the MH2 domains were obtained by subcloning
the corresponding cDNAs into pGEX-4T-1 (Amersham Pharmacia
Biotech). Xenopus FAST1 cDNA (provided by M. Whitman) was subcloned into HA-pcDEF3. All of the PCR products were sequenced.
Reverse Transcriptase-PCR of Smad2--
Poly(A)+
RNAs were prepared from various cell lines and reverse-transcribed. The
resulting cDNAs were amplified by PCR using primers 2A (5'-TTT TCC
TAG CGT GGC TTG-3') and 4A (5'-TCA GAG AGT TGA GAC ACC AG-3') under
conditions as described (32). The PCR products were subjected to the
second-round PCR using primers 2B (5'-GAA GAG ACT GCT GGG ATG GAA GAA
GT-3') and 4B (5'-CAA GGC AAT TGA AAA CTG CGA ATA TGC-3'). The PCR
fragment was run on a 2% agarose gel, and a 330-bp product was cloned
using a TA cloning kit (Promega).
Cell Culture and cDNA Transfection--
COS7 cells were
obtained from American Type Culture Collection. R mutant Mv1Lu cells
were provided by J. Massagué. The cells were cultured in
Dulbecco's modified Eagle's medium with 10% fetal bovine serum, 100 units/ml penicillin, and 10 µg/ml gentamycin. For transient
transfection, 60-80% confluent cells in six-well plates or 10-cm cell
culture dishes were transfected using FuGENE6 transfection reagent
(Boehringer Mannheim) following the manufacturer's protocol.
Luciferase Assay--
R mutant Mv1Lu cells were transiently
transfected with p3TP-lux or pAR3-lux (provided by J.L. Wrana) (36) in
the presence of various combinations of Smad constructs, T
R-I(TD),
and FAST1. For normalization of transfection efficiency, the
Renilla luciferase reporter gene in the pRL-CMV vector
(Promega) was co-transfected in each transfection. After transfection,
cells were incubated for 36 h, and luciferase activity in the cell
lysates was determined with a dual luciferase assay system (Promega)
using a luminometer (Lumat LB 9501, EG & G Berthold) according to the
manufacturer's recommendations. The luciferase activities of p3TP-lux
and pAR3-lux constructs were measured as luminescence of firefly
luciferase. As an internal control, Renilla luciferase was
measured immediately afterward by quenching the firefly luminescence.
Immunoprecipitation and Immunoblotting--
Immunoprecipitation
and immunoblotting have been described previously (33). Briefly, COS7
cells were transfected with expression constructs for Smads,
T
R-I(TD), and FAST1. Forty-eight hours after transfection, the cells
were solubilized, and the cell lysates were incubated with the
anti-Flag M2 (Eastman Kodak Co.) or anti-Myc 9E10 antibodies (Santa
Cruz Biotechnology), followed by incubation with protein G-Sepharose
beads. The immunocomplexes were then eluted by boiling for 3 min in the
SDS sample buffer containing 10 mM dithiothreitol and
subjected to SDS-polyacrylamide gel electrophoresis (PAGE). Aliquots of
the cell lysates were directly subjected to SDS-PAGE. Proteins were
electrotransferred to polyvinylidene difluoride membrane (ProBlott
membranes, Applied Biosystems) and immunoblotted with the anti-Flag M2,
anti-Myc 9E10, anti-HA 3F10 (Boehringer Mannheim), or
anti-phosphoserine antibodies (Zymed Laboratories Inc.) and developed using an enhanced chemiluminescence detection system (Amersham Pharmacia Biotech). For re-blotting, the
polyvinylidene difluoride membranes were stripped following the
manufacturer's protocol.
Gel-mobility Shift Assay--
Gel-mobility shift assay was
performed as described previously (14). Briefly, whole cell extracts
were prepared from the COS7 cells transfected with Smad, T
R-I(TD),
and FAST1 expression constructs. Cells were lysed in a lysis buffer
containing 50 mM Tris-HCl, pH 7.5, 150 mM NaCl,
0.5% Nonidet P-40, 50 mM NaF, 1 mM
NaPO4, 1 mM dithiothreitol, and protease
inhibitors. Glutathione S-transferase (GST) fusion proteins
were prepared as described (16). For supershift analysis, anti-Flag
and/or anti-Myc antibodies (1-2 µl each) or antisera against Smad2
or Smad3 (2 µl each, provided by P. ten Dijke) (33) were added to the
whole cell lysates or GST-fused Smad proteins. The probe containing
AP-1 sites (77 bp) was created by digestion of the p3TP-lux with
NdeI and SphI, and that containing ARE was
prepared by digestion of the pAR3-lux with Acc I and
BamHI. The probes were then subjected to
[
-32P]dCTP Klenow labeling. Whole cell lysates (3 µl, containing 3 µg of protein) or GST fusion proteins (150 ng
each) were added with a premix solution (13.4 µl) containing 1 µg
of poly(dI-dC) and 1 µl of the probe labeled to an activity of
2.0 × 104 cpm/µl (37). The final concentration of
NaCl in the samples was adjusted to 110 mM by hypotonic and
hypertonic lysis buffers. Complexes were then resolved on a 4%
polyacrylamide gel and analyzed by autoradiography.
 |
RESULTS |
Construction of Smad2
exon3 Plasmid--
Smad2 has a 30-amino
acid region in the middle of the MH1 domain (amino acid 79-108), which
is not found in other Smads in mammals (Fig.
1A). mRNAs were prepared
from various human cell lines, i.e. the HaCaT keratinocyte
cell line, HEL human erythroleukemia cell line, U937 human monocytic
leukemia cell line, and Molt-4 human T cell leukemia cell line. PCR was
performed using primers (2A and 4A) corresponding to the sequences in
exons 2 and 4, and the PCR products were subjected to second-round PCR
using primers 2B and 4B. Similar to previous results obtained with
mRNAs from placenta and HaCaT keratinocytes (32), we observed two
bands in HaCaT and Molt-4 cells, i.e. major bands of 420 bp
and faint bands of 330 bp (Fig. 1B). The 330-bp bands were
only very weakly detected in the other two cell lines. By DNA
sequencing, we confirmed that the 330-bp products correspond to Smad2
lacking exon 3. By inserting the 330-bp product into Smad2(wt) at
PvuII-RcaI sites, we prepared a construct for
Smad2
exon3.

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Fig. 1.
Comparison of the structures of Smad2(wt),
Smad2 exon3, and Smad3. A, schematic representation
of the structures of Smad2(wt), Smad2 exon3, and Smad3. Exon
boundaries of Smad2 are indicated. Exon 1 of Smad2 is a noncoding exon.
Primers used for PCR are shown by arrows. B,
reverse transcriptase-PCR and gel electrophoresis analyses of Smad2
using the primers in exon 2 and exon 4. cDNAs for Smad3 and
Smad2(wt) were used as controls.
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The Smad constructs were transfected into COS7 cells, and the
expression of proteins was analyzed by immunoblotting. N-terminally Flag-tagged Smad2
exon3 was efficiently expressed in COS7 cells, with
protein levels similar to those with Smad2(wt) and Smad3 (data not
shown). The size of Smad2
exon3 is smaller than that of Smad2(wt),
and larger than that of Smad3, consistent with their estimated
molecular weights.
Smad2
exon3 Has Transcriptional Activity Different from That of
Smad2(wt)--
Next, we studied the transcriptional activity of
Smad2
exon3 and compared it with those of Smad2(wt) and Smad3 using
the p3TP-lux promoter reporter construct, which contains the promoter
region of plasminogen activator inhibitor (PAI)-1 and three tandemly linked AP-1 sites (38) (Fig.
2A). The R mutant Mv1Lu cells, which lack functional T
R-I, were used to determine the
transcriptional activity of Smads. In the presence of small amounts of
T
R-I(TD) plasmid, a slight increase in the transcriptional activity
on p3TP-lux was observed. Smad3 efficiently induced transcription even
in the absence of exogenous Smad4, while Smad2(wt) was less efficient
in transcriptional activation than Smad3. Co-expression of Smad4 led to
higher transcriptional activity of Smad2(wt), although it was less than
that of Smad3. Interestingly, Smad2
exon3 had transcriptional
activity similar to that of Smad3 in the absence or presence of Smad4,
which was more potent than that of Smad2(wt).

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Fig. 2.
Signaling activity of Smad2(wt),
Smad2 exon3, and Smad3. A, R mutant Mv1Lu cells which
lack functional T R-I were co-transfected with p3TP-lux together with
various combinations of T R-I(TD) and Smad cDNAs. Luciferase
activity in cell lysate was measured using the dual luciferase reporter
assay system. Data are presented after normalization of transfection
efficiency using the Renilla luciferase reporter gene.
B, R mutant Mv1Lu cells were co-transfected with pAR3-lux
(36) together with Xenopus FAST1, T R-I(TD), and the
indicated combinations of Smad constructs.
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The transcriptional activity of Smad2
exon3 was also tested in the
reporter gene pAR3-lux containing the ARE of Mix.2 promoter in Xenopus (36) in the presence or absence of the specific
DNA-binding protein, Xenopus FAST1 (10). In contrast to the
results obtained using p3TP-lux, Smad2 was slightly more potent than
Smad3 in inducing luciferase activity of pAR3-lux (Fig. 2B).
The luciferase induction by Smad2
exon3 was similar to that by Smad3,
and slightly less effective than that by Smad2.
Phosphorylation by T
R-I and Complex Formation with
Smad4--
In order to understand mechanism of the higher
transcriptional activity in p3TP-lux of Smad2
exon3 than of
Smad2(wt), we examined the phosphorylation of Smad2 and Smad3 by
T
R-I. Flag-tagged Smad constructs were transfected into COS7 cells
together with various amounts of T
R-I(TD), and immunoprecipitated
with anti-Flag antibody, followed by immunoblotting using
anti-phosphoserine antibodies (Fig.
3A). Although Smad2
exon3
and Smad3 were more potent than Smad2(wt) in inducing transcriptional
activity on p3TP-lux, there was no significant difference between the
levels of phosphorylation in Smad2
exon3, Smad2(wt), and Smad3.

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Fig. 3.
Phosphorylation of Smad2(wt), Smad2 exon3,
and Smad3 and heteromer formation with Smad4. A,
phosphorylation of Smad proteins by T R-I(TD). COS7 cells were
transfected with Smad constructs together with increasing amounts of
the T R-I(TD) plasmid. Cell lysates were immunoprecipitated with the
anti-Flag antibody, followed by immunoblotting using anti-phosphoserine
(anti-P-serine) antibody. Aliquots of the cell lysates were
directly subjected to SDS-PAGE and immunoblotted with the anti-Flag or
anti-HA antibody to detect the expression of Smad proteins and
T R-I(TD), respectively. B, Complex formation of Smad2,
Smad2 exon3, and Smad3 with Smad4. COS7 cells were transfected with
Smad2/3 and Smad4 constructs with or without T R-I(TD). Cell lysates
were immunoprecipitated with the anti-Myc antibody followed by
immunoblotting with the anti-Flag antibody. In order to detect the
phosphorylation of Smad2/3, aliquots of the cell lysates were subjected
to immunoprecipitation using the anti-Flag antibody followed by
immunoblotting using the anti-phosphoserine antibodies, as described in
A. Expression of Flag-Smad2/3 was detected after stripping
the membrane and immunoblotting using the anti-Flag antibody.
Expression of T R-I(TD) was detected by immunoblotting using the
anti-HA antibody.
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Heteromer formation with Smad4 was also studied using Smad2
exon3,
Smad2(wt), and Smad3 (Fig. 3B). Weak interaction with Smad4 was
observed in Smad2
exon3 and Smad2(wt) even without T
R-I(TD), and
was correlated with the weak phosphorylation of the Smad2 proteins.
Strong heteromer formation was induced in all Smad2 and Smad3
constructs by T
R-I(TD), and we found no differences in between
Smad2
exon3, Smad2(wt), and Smad3.
Smad2
exon3 but Not Smad2(wt) Binds to the AP-1 Sites of
p3TP-lux--
We then studied the formation of DNA-binding complexes
by Smad2
exon3 at the AP-1 sites of p3TP-lux. It was previously shown that Smad3 and Smad4, but not Smad2, bind to probe prepared from p3TP-lux DNA (13). Whole cell extracts were prepared from COS7 cells
transfected with the indicated Smad constructs and T
R-I(TD), and
subjected to gel-mobility shift analysis (Fig.
4A). None of the Smad proteins
formed DNA-binding complexes in the absence of T
R-I(TD) (data not
shown). In the presence of T
R-I(TD), Smad3 and Smad2
exon3, but
not Smad2(wt), formed DNA-binding complexes. The complexes supershifted
in the presence of the anti-Flag antibody. Smad4 alone did not form a
DNA-binding complex in the presence of T
R-I(TD), but it did
participate in DNA-binding complexes in the presence of Smad2
exon3
or Smad3, but not of Smad2(wt). The bands shifted in the presence of
anti-Myc antibody, although they were weak compared with those obtained
with the anti-Flag antibody. Moreover, addition of both anti-Myc and
anti-Flag antibodies led to shift with a slower mobility, indicating
that the DNA-binding complexes contained Smad4. These results indicate
that by deletion of exon 3, Smad2 acquired the ability to participate
in DNA-binding complexes.

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Fig. 4.
Smad2 exon3 and Smad3 bind to the AP-1
sites derived from p3TP-lux. A, Flag-Smad2(wt),
Flag-Smad2 exon3, or Flag-Smad3 and Myc-Smad4 were transfected into
COS7 cells in the presence of T R-I(TD)-HA. Whole cell lysates were
prepared, and binding to the 32P-labeled probe containing
the AP-1 sites was analyzed by gel-mobility shift assay. For supershift
analysis, whole cell lysates were incubated with the anti-Flag
(F) and/or anti-Myc (M) antibodies, and subjected
to gel-shift assay. B, binding of purified GST-Smad proteins
to DNA. GST-fused Smad2(wt) MH2, Flag-Smad2 exon3 MH2, and
Smad3 MH2 lacking MH2 domains were prepared, and gel shifts were
performed using the probe containing the AP-1 sites. For supershift
analysis, the antiserum to Smad2 (2) or Smad3
(3), or anti-Flag (F) antibody was used. For
competition (competitor; C) of the DNA binding, excess
amounts of cold probe was added.
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|
We next examined whether Smad2
exon3
MH2 can directly bind to the
AP-1 sites of p3TP-lux. Smad3 lacking the MH2 domain was previously
shown to directly bind to DNA (12, 14-17). GST-fused Smad2(wt)
MH2,
Smad3
MH2, and Smad2
exon3
MH2 lacking MH2 domains were used for
gel shift analysis (Fig. 4B). Similar to the results obtained with whole cell extracts transfected with Smads and
T
R-I(TD), both Smad2
exon3 and Smad3 were able to bind the AP-1
sites, but Smad2(wt) did not. The bands shifted in the presence of
corresponding Smad antisera, although the shift bands (lane
15 and 18, Fig. 4B) were weak compared
with that obtained with the anti-Flag antibody (lane
19), probably because of the lower affinities of the Smad antisera.
Smad2(wt), Smad3, and Smad2
exon3 Participate in ARF--
Smad2
associates with Smad4 and FAST1 to form ARF, which binds to ARE of the
Mix.2 gene promoter (23, 24). Smad2(wt) was slightly more
potent in inducing transcriptional activity on pAR3-lux than Smad3 or
Smad2
exon3; however, complex formation with Xenopus FAST1
was observed similarly in between Smad2(wt), Smad2
exon3, and Smad3
in the presence and absence of T
R-I(TD) (Fig.
5A). We, therefore, examined
whether there are any differences in the DNA-binding abilities of the
ARF complexes containing different Smads. ARF containing Smad2(wt),
Smad4, and FAST1 efficiently bound ARE in response to TGF-
receptor
activation, similar to those containing Smad3 or Smad2
exon3 (Fig.
5B). Addition of anti-Flag, anti-Myc, or anti-HA antibodies
led to shifts of the bands, indicating that the DNA-binding ARF
complexes contained Smad2, Smad3, or Smad2
exon3, together with Smad4
and FAST1. FAST1 was shown to interact with Smad2 through the MH2
domain (23, 24). These findings indicate that the presence of exon 3 of
Smad2 does not interfere with the formation of DNA-binding ARF complex
containing FAST1.

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Fig. 5.
Smad2(wt), Smad2 exon3, as well as Smad3
participate in the ARF complexes. A, complex formation
of Smad2(wt), Smad2 exon3, and Smad3 with Xenopus FAST1.
COS7 cells were transfected with Smad constructs with or without
T R-I(TD) and Xenopus HA-FAST1. Cell lysates were
immunoprecipitated with the anti-Flag antibody followed by
immunoblotting with the anti-HA antibody. Expression of Flag-Smad2/3
was detected after stripping the membrane and immunoblotting using the
anti-Flag antibody. For the detection of the phosphorylated Smad2/3,
the membrane was subjected to immunoblotting using the
anti-phosphoserine antibodies. Expression of T R-I(TD) and FAST1 was
detected by immunoblotting using the anti-HA antibody. B,
Flag-Smad2(wt), Flag-Smad2 exon3, or Flag-Smad3 and Myc-Smad4 were
transfected into COS7 cells in the presence of T R-I(TD) and
HA-tagged Xenopus FAST1. Whole cell lysates were prepared,
and binding to the 32P-labeled probe containing ARE from
the Mix.2 promoter was analyzed by gel-mobility shift assay.
For supershift analysis, whole cell lysates were incubated with the
anti-Flag (F), anti-Myc (M), or anti-HA
(H) antibodies and subjected to gel-shift assay.
C, binding of purified GST-Smad proteins to the
Mix.2 promoter. GST-fused Smad2(wt) MH2,
Flag-Smad2 exon3 MH2, and Smad3 MH2 lacking MH2 domains were
prepared, and gel shifts were performed using the probe containing ARE
of the Mix.2 promoter. For supershift analysis, the
antiserum to Smad2 (2) or Smad3 (3), or anti-Flag
(F) antibody was used.
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We further investigated the ability of different GST-fused Smad
proteins to directly bind to DNA containing the ARE sequence in the
absence of FAST1. Similar to binding to the AP-1 sites (Fig.
4A), Smad3
MH2 and Smad2
exon3
MH2 recognized the ARE
sequence, but Smad2(wt)
MH2 did not (Fig. 5C). The bands
shifted in the presence of Smad antisera (lanes 6 and 8). Shift of the bands was more remarkable with the
anti-Flag antibody (lane 9), probably because of
the higher affinity of the anti-Flag antibody and possible induction of
oligomerization. These data indicate that in addition to indirectly
binding to DNA as ARF complexes, Smad3 and Smad2
exon3, but not
Smad2(wt), have abilities to directly bind to ARE .
 |
DISCUSSION |
Smad proteins have MH1 and MH2 domains, which are linked by a
linker region. Inhibitory Smads have MH2 domains, but have divergent MH1-like regions. The MH2 domain plays important roles in various functions of Smads, i.e. interaction with type I receptors,
homo- and hetero-oligomer formation, association with DNA-binding
proteins, and interaction with transcriptional coactivators,
e.g. p300/CBP (4-6, 25-28). Smad2, which lacks the MH1
domain, is constitutively located in the nucleus and activates target
genes (39). The MH2 domain interacts with type I receptors, and the L3
loop, a 17-amino acid region in the MH2 domain, plays a critical role in this interaction (20). In addition,
-helix 2 of the MH2 domain
has recently been shown to determine the binding specificity to
DNA-binding proteins such as FAST1 (40).
The MH1 domain plays an important role as a repressor of the function
of the MH2 domain. In addition, the MH1 domain has intrinsic activity,
i.e. binding to specific DNA sequences.
Drosophila Mad, an ortholog of mammalian Smad1 and 5, directly binds to the quadrant enhancer of vestigial gene
(13). Smad3 and Smad4, but not Smad2, have been shown to bind to the
AP-1 sites of p3TP-lux (14, 41). More recently, Smad3 and Smad4 have
been shown to bind to specific DNA sequences, and luciferase reporter
constructs containing multiple copies of these specific DNA sequences
have been shown to be activated by Smad3 and Smad4 (15-17).
Smad2 and Smad3 are structurally very similar, and serve as
pathway-restricted Smads in the TGF-
and activin signaling pathways. However, functional differences between Smad2 and Smad3 have been suggested. In addition to the difference between them in DNA-binding ability, Smad3, but not Smad2, binds to a transcriptional regulator Evi-1, which consequently suppresses the activity of Smad3 (30). In the
HaCaT keratinocyte cell line, TGF-
induces phosphorylation of both
Smad2 and Smad3, while activin A preferentially activates Smad3 (31).
Since TGF-
and activin have different activities in the growth
inhibition and differentiation of this cell
type,2 the difference in
phosphorylation between Smad2 and Smad3 may be, at least in part,
responsible for their distinct biological effects.
Smad2 differs from Smad3 in having a 30-amino acid region in the middle
of the MH1 domain. We have shown in the previous report (32) and in the
present study that mRNA lacking exon 3 is present in various
tissues and cells. Amounts of the Smad2 transcript lacking exon 3 appear to differ between cell types. It would be of great interest to
determine whether the mRNA encoding Smad2
exon3 is preferentially
induced under certain physiological and pathological conditions.
In the present study, we have shown that Smad2 lacking exon 3 has
higher transcriptional activity on p3TP-lux than does Smad2(wt). The
MH1 domain represses the function of the MH2 domain. Mutation of
Arg-133 in Smad2 resulted in increase of the affinity between MH1 and
MH2 domains, and this mutant was less active than the wild-type Smad2
(29). Similar results were reported for Smad4 with mutation at Arg-100
(29). In the present study, however, deletion of exon 3 of Smad2 did
not lead to increase in phosphorylation by T
R-I, or heteromer
formation with Smad4, suggesting that exon 3 is not directly involved
in the repressor activity of the MH1 domain.
Smad2
exon3 is able to bind to DNA containing the AP-1 sites, a
finding not observed for Smad2(wt). These findings strongly suggest
that the direct binding of Smad2/3 to DNA is a crucial step in the
transcriptional activation of the p3TP-lux reporter. In whole cell
extracts, DNA-binding complex formation was observed in both the
presence and absence of transfected Smad4. Direct DNA binding of Smad3
and Smad2
exon3 was detected when GST-Smad proteins lacking MH2
domains were used. These findings together with those of previous
reports (12-17) suggest that the interaction may occur through the MH1
domain, when repression by the MH2 domain is released. Since
Smad2
exon3 has higher transcriptional activity than Smad2(wt),
direct DNA binding of Smads may positively regulate their
transcriptional activity on p3TP-lux promoter. However, since Smad2(wt)
can, to some extents, induce transcriptional activation of p3TP-lux,
Smad proteins can partially activate target genes through a mechanism
which does not require the direct DNA binding of Smads.
In contrast to the possible role of DNA binding of Smad2/3 in the
transcriptional activation of p3TP, transcription of the Mix.2 gene appeared to be less strongly affected by direct
DNA binding of Smad2/3. The Mix.2 promoter contains a
sequence similar to the Smad-binding element (15, 17), to which Smad3,
and possibly Smad2
exon3, may bind. Smad2(wt), which failed to
directly bind the Mix.2 promoter, was efficient in inducing
transcriptional activation of pAR3-lux. Since the binding of Smad2 to
FAST1 occurs through the MH2 domain, differences in the structure of
the MH1 domain may have less important effects on the transcription of the Mix.2 gene. Interestingly, Smad2 activated the
goosecoid promoter together with Smad4 and mouse FAST2, a
winged-helix transcription factor related to Xenopus FAST1,
whereas Smad3 strongly suppressed transactivation of this promoter
(12). Smad3 and Smad4, but not Smad2, directly bound to the GC-rich
sequences of the goosecoid promoter. These findings suggest
that direct DNA binding of Smads thorough the MH1 domain as well as
indirect binding via other DNA-binding factors, e.g. FAST1
and FAST2, may cooperatively regulate the transcription of target
genes. Direct DNA binding of Smads may play important roles in
transcription of certain target genes, such as PAI-1, while
it is less critical or acts negatively for other genes, i.e.
Mix.2 and goosecoid.
Expression profile of Smad3 appears to be limited compared with that of
Smad2 (32). In certain cell types that lack the expression of Smad3,
Smad2
exon3 may function as a Smad3-like molecule. The region like
exon 3 is observed only in Smad2 but not in other Smads in mammals.
Exon 3 of Smad2 may play a crucial role in modulating the function of
Smad2 by interfering with the direct DNA binding to target genes.
 |
ACKNOWLEDGEMENTS |
We are grateful to H. Hoshikawa, H. Inoue, M. Kawabata, C.-H. Heldin, and P. ten Dijke for valuable discussion and
comments, and A. Hanyu and Y. Yuuki for technical assistance. We also
thank J. A. Langer for pcDEF3, J. L. Wrana for pAR3-lux, and
M. Whitman for Xenopus FAST1.
 |
FOOTNOTES |
*
This work was supported in part by Grants-in-aid for
Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan, and Special Coordination Funds for Promoting Science
and Technology from the Science and Technology Agency of Japan.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.
¶
To whom correspondence should be addressed. Tel./Fax:
81-3-3918-0342; E-mail: mit-ind{at}umin.ac.jp.
Supported by the Suzuken Memorial Foundation and Yamanouchi
Foundation for Research on Metabolic Disorders.
The abbreviations used are:
TGF-
, transforming growth factor-
; T
R, TGF-
receptor; Mad, Mothers
against decapentaplegic; MH, Mad homology; wt, wild-type; AP, activating protein; ARF, activin-responsive factor; PCR, polymerase
chain reaction; PAGE, polyacrylamide gel electrophoresis; PAI, plasminogen activator inhibitor; GST, glutathione
S-transferase; ARE, activin-responsive element; bp, base pair(s).
2
A. Shimizu and M. Kato, unpublished data.
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