Identification of Two Smad4 Proteins in Xenopus
THEIR COMMON AND DISTINCT PROPERTIES*
Norihisa
Masuyama
,
Hiroshi
Hanafusa
,
Morioh
Kusakabe
,
Hiroshi
Shibuya§, and
Eisuke
Nishida
¶
From the
Department of Biophysics, Graduate School of
Science, Kyoto University, Sakyo-ku, Kyoto 606-8502 and the
§ Division of Morphogenesis, Department of Developmental
Biology, National Institute for Basic Biology, Okazaki 444-8585, Japan
 |
ABSTRACT |
Smad family proteins have been identified as
mediators of intracellular signal transduction by the transforming
growth factor-
(TGF-
) superfamily. Each member of the
pathway-restricted, receptor-activated Smad family cooperates and
synergizes with Smad4, called co-Smad, to transduce the signals. Only
Smad4 has been shown able to function as a common partner of the
various pathway-restricted Smads in mammals. Here we have identified a
novel Smad4-like molecule in Xenopus (XSmad4
) as well as
a Xenopus homolog of a well established Smad4 (XSmad4
).
XSmad4
is 70% identical to XSmad4
in amino acid sequence. Both
of the Xenopus Smad4s can cooperate with Smad1 and Smad2,
the pathway-restricted Smads specific for bone morphogenetic protein
and TGF-
, respectively. However, they show distinct properties in
terms of their developmental expression patterns, subcellular localizations, and phosphorylation states. Moreover, XSmad4
, but not
XSmad4
, has the potent ability to induce ventralization when
microinjected into the dorsal marginal region of the 4-cell stage of
the embryos. These results suggest that the two Xenopus Smad4s have overlapping but distinct functions.
 |
INTRODUCTION |
Recent studies identified a family of proteins, termed Smads, as
essential components in intracellular signaling pathways downstream of
serine/threonine kinase receptors for the
TGF-
1 superfamily (1-5).
In vertebrates, at least nine different kinds of Smads have been
identified. Smad proteins share a high degree of homology in their
amino- and carboxyl-terminal domains, the MH1 and MH2 domains,
respectively, connected with a divergent proline-rich linker region.
Smad proteins can be classified into three subtypes according to
structure and function (1-5). One subtype is the pathway-restricted
Smad, which is a direct substrate of type I receptors for the TGF-
superfamily. The second subtype is the co-Smad, which is not a direct
substrate of receptors but participates in signaling by associating
with pathway-restricted Smads. The third subtype consists of those
Smads that inhibit the activation of pathway-restricted Smads and are
referred to as anti-Smads (6-8).
Pathway-restricted Smads contain a consensus phosphorylation motif,
SS(V/M)S, for the type I serine/threonine receptors at their carboxyl
termini. This class of Smads interacts transiently with specific
activated type I receptors and thus becomes phosphorylated following
ligand stimulation. Smad2 and Smad3 are specific mediators of TGF-
and activin signaling, whereas Smad1, Smad5, and Smad8 are involved in
the bone morphogenetic protein (BMP) pathway (9-22). Signaling by
pathway-restricted Smads requires an association with co-Smad. Only
Smad4 is known as co-Smad in mammals, where its structure is divergent
from that of pathway-restricted Smads. Smad4 lacks a carboxyl-terminal
phosphorylation motif and does not associate with the TGF-
receptors
(14, 15, 23). Each member of the various pathway-restricted Smads forms
a complex with Smad4 upon ligand stimulation. Then, the heteromeric
complex is translocated to the nucleus where it participates in the
transcriptional activation of specific target genes (9, 14, 15, 19, 20, 23-26).
Smad4 was originally identified as the product of the Dpc4
(deleted in pancreatic cancer) tumor suppressor gene that is mutated or
deleted in a high proportion of pancreatic cancers and in a smaller
proportion of other cancers (27). A general requirement of Smad4 is
suggested not only in mammalian cells but also in Xenopus
embryos, as a dominant-negative Smad4 construct interferes with Smad1
and Smad2 signaling (15, 23, 28), although cDNA cloning of a
full-length Xenopus Smad4 has not yet been performed. It has
been reported that Smad4-deficient mice die early in embryogenesis (29,
30). These mice exhibit severe defects in cellular proliferation, gastrulation, and mesoderm differentiation (29, 30). Thus, Smad4 has
been supposed to be a shared and obligate partner, participating in
both TGF-
/activin and BMP signaling pathways.
Here we report the identification, cDNA cloning, and
characterization of two kinds of Smad4 in Xenopus. Both of
the two Xenopus Smad4s, termed XSmad4
and XSmad4
, are
shown able to function as common partners of both Smad1 and Smad2 in
Xenopus embryos as well as in mammalian cells to transduce
respective signals from the TGF-
superfamily. Moreover, XSmad4
and XSmad4
themselves form heteromeric as well as homomeric
complexes. In addition to their amino acid sequence diversity, these
two XSmad4s differ in their expression profiles during early
Xenopus development and in their subcellular localization
and phosphorylation states when expressed in cultured cells.
Furthermore, microinjection of mRNA encoding XSmad4
into dorsal
cells of Xenopus embryos leads to strong ventralization,
whereas XSmad4
has little ventralizing activity. These results
suggest that the two Xenopus Smad4s may function as co-Smads
with distinct properties in transducing a set of TGF-
superfamily signals.
 |
EXPERIMENTAL PROCEDURES |
Molecular Cloning and Plasmid Construction--
A
Xenopus oocyte cDNA library
(CLONTECH) was screened using the human Smad4
coding region as a probe. RNase protection assay was carried out using
an Ambion HybSpeed RPA kit (Ambion) according to the manufacturer's
instruction. The entire coding regions of XSmad4
and XSmad4
, the
carboxyl-terminal truncated forms of XSmad4
and XSmad4
, and the
carboxyl-terminal serine to alanine mutant of XSmad4
(AAVN) were
amplified by polymerase chain reaction, and the amplified nucleotide
sequences were confirmed by DNA sequencing. For the carboxyl-terminal
truncated forms of XSmad4
and XSmad4
, the nucleotides
corresponding to amino acid residues 1-508 and 1-519 were amplified,
respectively. XSmad4
cDNA and XSmad4
cDNA were ligated
into pSP64T or into a Myc tag fused version of pSP64T plasmids to
synthesize mRNAs (31). In vitro synthesis of capped mRNA was performed using mMESSAGE mMACHINE (Ambion) according to
the manufacturer's instruction. Other constructs for cell transfection were inserted into pcDL-SR
456, pSR
-HA, or pSR
-Myc.
Xenopus Embryo Manipulation, In Situ Hybridization, and Animal
Cap Assay--
Xenopus embryos were obtained by in
vitro fertilization of eggs with testes homogenates. Embryos were
staged according to Nieuwkoop and Faber (32). The animal cap assay was
performed as described elsewhere (33). Dorsal marginal zone explants
were dissected at the gastrula stage (stage 10) and were cultured until sibling embryos reached stage 13. The primer pairs used here for reverse transcription-coupled polymerase chain reaction (RT-PCR) were
reported elsewhere (23, 33, 34). Whole mount in situ hybridization was performed essentially as described (35).
Cell Culture, Transfection, and Transcriptional Reporter
Assay--
C2C12 cells were maintained in Dulbecco's modified
Eagle's medium supplemented with 10% fetal calf serum. Cells were
transfected at 24 h after seeding using the LipofectAMINE Plus
reagent (Life Technologies, Inc.). After 48 h, lysates were
prepared, and the luciferase activity was determined with the
luciferase assay system (Promega). Relative luciferase activities were
normalized by co-expressed
-galactosidase activities.
Immunoprecipitation, Immunoblotting, and Metabolic
Labeling--
After 12-15 h, cells were treated with or without 10 ng/ml of human TGF-
1 (purchased from Austral
Biologicals) or 300 ng/ml BMP (Xenopus BMP4 (36)) for 1 h, and subsequently lysates were prepared as described (37).
Immunoprecipitation was performed by incubation with the 9E10 anti-Myc
antibody (Santa Cruz Biotechnology) and protein G-Sepharose (Amersham
Pharmacia Biotech). The immunoprecipitates and the aliquots of total
lysates were separated in SDS-polyacrylamide gel electrophoresis and
transferred to a polyvinylidene difluoride membrane (Millipore).
Membranes were incubated with antibodies against Myc or HA (Santa Cruz
Biotechnology) and subsequently with horseradish peroxidase-conjugated
sheep anti-mouse antibody or donkey anti-rabbit antibody (Amersham).
Immunoreactive bands were detected by the ECL Western blotting
detection system (Amersham).
For metabolic labeling of C2C12 cells, 24 h post-transfection
cells were incubated with [32P]orthophosphate for 3 h and treated with or without 10 ng/ml of TGF-
1 and
lysed in TNE buffer (10 mM Tris-HCl, pH 7.8, 150 mM NaCl, 1% Nonidet P-40, and 1 mM EDTA) with
protease inhibitors. Myc-tagged Smads were immunoprecipitated with the
9E10 anti-Myc antibody (Santa Cruz Biotechnology) and protein
G-Sepharose (Amersham Pharmacia Biotech). The precipitates and the
aliquots of cell lysates were resolved by SDS-polyacrylamide gel
electrophoresis and visualized by autoradiography.
Immunofluorescence--
TGF-
stimulation of C2C12 cells was
provided by co-transfecting the activated TGF-
type I receptor,
T
R-I (T204D), and treated with 10 ng/ml TGF-
1 for
1 h (25). Cells were then fixed by formaldehyde. Immunostaining
was performed by incubation with the 9E10 anti-Myc antibody (Santa Cruz
Biotechnology) for 2 h followed by incubation with the fluorescein
isothiocyanate-conjugated goat anti-mouse antibody (1:400) for 1 h.
 |
RESULTS |
cDNA Cloning of Xenopus Smad4
and Xenopus
Smad4
--
By screening a Xenopus oocyte cDNA
library with human Smad4 as a probe under low stringency, we isolated
several positive clones. Sequence analysis of these cDNA clones
revealed that one of them has a very high homology (91% identity) to
human Smad4/DPC4 in the coding region and the other has a relatively
lower (71% identity), but still the highest, homology to human Smad4
among the other Smad family proteins reported to date. Therefore, we referred to the former as Xenopus Smad4
(XSmad4
) and
the latter as Xenopus Smad4
(XSmad4
). We considered
XSmad4
a Xenopus ortholog of mammalian Smad4 and
XSmad4
the second Smad4 (see below). The nucleotide sequences were
predicted to encode proteins of 549 and 560 amino acids for XSmad4
and XSmad4
with calculated molecular masses of 60 and 61 kDa,
respectively (Fig. 1A). It is
unlikely that these two Smad4-related Xenopus cDNA
clones were derived from pseudoalleles resulting from the
pseudotetraploid nature of Xenopus laevis genome, because we
obtained several cDNA clones encoding pseudogenes for both
XSmad4
and XSmad4
from the same cDNA library (data not
shown).

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Fig. 1.
cDNA cloning of XSmad4
and XSmad4 . A, alignment
of the deduced amino acid sequences of XSmad4 , human Smad4/DPC4
(hSmad4), and XSmad4 is shown. Residues that are identical in three
proteins are shaded black and those identical in two of
three proteins are shaded gray. Dashes denote
gaps in the alignments. The sequences were aligned using the ClustalW
1.6 program. B, the schematic diagram of overall structures
of XSmad4 and XSmad4 is shown.
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The Smad proteins typically consist of three modules: highly conserved
amino-terminal (MH1) and carboxyl-terminal (MH2) domains and a poorly
conserved linker region. The amino acid sequence comparison between
XSmad4
or XSmad4
and human Smad4 (hSmad4) is shown in Fig.
1A. The MH1 and MH2 domains of XSmad4
are 100% identical
to those of hSmad4, except that the coding region of XSmad4
initiates at the position corresponding to the second methionine
residue of hSmad4. The linker region shows 74% identity between
XSmad4
and hSmad4. In contrast, the identity between XSmad4
and
hSmad4 is about 90% in the MH1 and MH2 domains and 34% in the linker
region (Fig. 1B). Another striking difference between
XSmad4
and XSmad4
is that the carboxyl-terminal sequence of
XSmad4
is QPLD, like mammalian Smad4, whereas that of XSmad4
is
SSVN, which resembles the carboxyl-terminal SS(V/M)S phosphorylation motif of pathway-restricted Smads (Fig. 1B).
To examine the temporal expression of XSmad4
and XSmad4
during
early development, an RNase protection analysis was performed with
probes corresponding to each of the linker region of XSmad4s. Each
probe was confirmed not to cross-hybridize to the other (data not
shown). XSmad4
mRNA was markedly increased by zygotic expression after the blastula stage, whereas XSmad4
mRNA was highly
abundant in eggs and was decreased during the gastrula stage, although both transcripts were detected throughout early embryogenesis (Fig.
2A). Thus, XSmad4
mRNA
and XSmad4
mRNA are expressed differently during early
development.

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Fig. 2.
The temporal and spatial expression of
XSmad4 and
XSmad4 . A, XSmad4 and
XSmad4 transcripts are present during Xenopus early
embryogenesis. Equivalent amounts of total RNA isolated from each stage
of embryos were analyzed for the expression of XSmad4 and XSmad4
in an RNase protection assay. Numbers represent the
developmental stages (st.) (32): stages 1 and
6, maternal; stage 11, gastrula; stage
16, neurula; stage 23, tailbud; stages 28, 34, and 39, tadpole. The expression of ornithine
decarboxylase (ODC) was also examined as a control for equal
loading of RNA. B-G, the spatial expression patterns of
XSmad4 and XSmad4 in developing Xenopus embryos are
analyzed by whole mount in situ hybridization: B
and E, animal view of early gastrula stage embryos (stage
10, ventral, at the top); C and F,
dorsal view of neurula stage embryos (stage 20, anterior to the
left); D and G, lateral view of
tadpoles (stage 31, anterior to the left).
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The spatial expression of XSmad4
and XSmad4
was examined by whole
mount in situ hybridization with the probes (Fig. 2,
B-G). Both XSmad4
and XSmad4
mRNAs are
ubiquitously expressed in the ectoderm at the early gastrula stage
(Fig. 2, B and E) but become restricted to the
neuroectoderm at the neurula stage (Fig. 2, C and
F). In the tadpole stage, both of the XSmad4s are expressed in the central nervous system, eye, and otic vesicle (Fig. 2, D and G). XSmad4
is expressed more anteriorly
than XSmad4
in the neural tube.
XSmad4
and XSmad4
Synergize with XSmad1 and XSmad2--
To
examine whether XSmad4
and XSmad4
act as co-Smad to cooperate
with Smad1 and Smad2 to mediate BMP-like and TGF-
/activin-like responses, respectively, we first analyzed the expression of several marker genes in animal caps obtained from Xenopus embryos
injected with synthetic mRNAs encoding XSmad4
or XSmad4
combined with Xenopus Smad1 or Xenopus Smad2.
Injection of a low dose of XSmad1, XSmad2, XSmad4
, or XSmad4
mRNA alone was not sufficient to induce any noticeable expression
of mesodermal genes (Fig. 3A).
When XSmad1 mRNA was injected together with either XSmad4
or
XSmad4
mRNA, however, strong expression of a ventral mesodermal
marker,
globin, was induced. Similarly, when XSmad2 mRNA was
injected with either of the XSmad4 mRNAs, dorsal mesodermal
markers, goosecoid and muscle actin, and a pan-mesodermal marker,
Xenopus brachyury, were strongly induced (Fig.
3A).

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Fig. 3.
XSmad4 and
XSmad4 synergize with XSmad1 and XSmad2 to
induce expression of mesodermal marker genes. A, effect
of co-expression of XSmad4 or XSmad4 on XSmad1- or XSmad2-induced
expression of mesodermal maker genes in isolated animal caps. Animal
caps were dissected at the blastula stage from embryos that had been
injected with XSmad4 or XSmad4 mRNA (0.2 ng) together with
XSmad1 or XSmad2 mRNA (0.2 ng) at the 2-cell stage and were
cultured until sibling embryos reached stage 11 (upper
panel) or 26 (lower panel). Expression of
Xenopus brachyury (Xbra), goosecoid
(gsc), muscle-actin, and -globin was analyzed by RT-PCR.
Expression of EF-1 was also analyzed as a loading control. No signal
was observed in the absence of reverse transcription ( RT).
B, effect of carboxyl-terminal truncated mutants of
XSmad4 and XSmad4 (XSmad4 Cand XSmad4 C) on the
mesodermal gene expression induced by XSmad1 or XSmad2 in animal caps.
Animal caps were dissected at the blastula stage from embryos that had
been injected with XSmad1 mRNA or XSmad2 mRNA (1 ng) together
with XSmad4 CmRNA or XSmad4 CmRNA (1 ng) and were
cultured until sibling embryos reached stage 11 or 26. Expression of
marker genes was analyzed by RT-PCR.
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Because the carboxyl-terminal truncated form of human Smad4 has been
shown to act as a dominant-negative inhibitor for signal transductions
(15, 28), we next examined the action of carboxyl-terminal truncated
constructs of XSmad4
and XSmad4
in the animal cap assay.
Injection of a high dose of XSmad1 or XSmad2 mRNA was sufficient to
induce expression of ventral or dorsal mesodermal marker genes, respectively. When either XSmad4
C or XSmad4
C mRNA was
injected along with XSmad1 or XSmad2, the expression of these marker
genes was significantly suppressed (Fig. 3B). These results
suggest that XSmad4
and XSmad4
can function as common partners
for pathway-restricted Smads to induce expression of specific marker
genes in Xenopus animal caps.
To elucidate more directly the cooperativity of XSmad4
and XSmad4
with XSmad1 and XSmad2, we tested the ability of the Smads to induce
the reporter gene expression under the BMP- and TGF-
-responsive promoters in cultured cells. Transfection of XSmad1 alone into C2C12
mouse myoblast cells induced a low level of luciferase expression under
the promoter of Xvent-2, a BMP-inducible Xenopus
homeobox gene (38, 39). However, co-expression of XSmad4
and
XSmad4
, but not XSmad4
Cor XSmad4
C, together with XSmad1
induced a high level of the reporter gene expression (Fig.
4A). Similarly, both XSmad4
and XSmad4
synergized with XSmad2 to induce luciferase expression
under the TGF-
-responsive 3TP reporter, whereas the carboxyl-terminal truncated constructs did not (Fig. 4B).
These results demonstrate that XSmad4
and XSmad4
act
synergistically with XSmad1 and XSmad2 to induce gene expression in
cultured cells.

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Fig. 4.
XSmad4 and
XSmad4 cooperate with XSmad1 and XSmad2 to
induce the reporter gene expression under the BMP- and
TGF- -responsive promoters. C2C12 cells
were transiently transfected with the Xvent2-Luc
reporter plasmid and an expression vector encoding XSmad1 with or
without either of the XSmad4s (A) or with the 3TP-Lux
reporter plasmid and an expression vector encoding XSmad2 with or
without either of the XSmad4s (B). Cells were harvested
48 h after transfection and assayed for luciferase activity. These
results are the averages of three separate experiments. WT,
wild type; C, carboxyl-terminal truncated mutant.
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XSmad4
and XSmad4
Associate with XSmad1 and XSmad2--
As
XSmad4
and XSmad4
functionally cooperated with XSmad1 and XSmad2,
we would expect that XSmad4
and XSmad4
bind to XSmad1 and XSmad2
to form heteromeric complexes. To examine this hypothesis, we
co-expressed Myc-tagged XSmad4s with HA-tagged XSmad1 or XSmad2 in
C2C12 cells and subjected the obtained cell lysates to
immunoprecipitation with anti-Myc antibody followed by immunoblotting
with anti-HA antibody. The results showed that in response to BMP
treatment, XSmad1 associates with either of the two XSmad4s (Fig.
5A, upper panel)
and that in response to TGF-
stimulation, XSmad2 associates with
either of the two XSmad4s (Fig. 5A, lower panel).
Thus, both XSmad4
and XSmad4
are able to form heteromeric
complexes with XSmad1 or XSmad2 in response to specific
stimulation.

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Fig. 5.
Association of XSmad4
and XSmad4 with XSmad1 and XSmad2.
A, C2C12 cells were transfected with Myc-tagged wild type
(WT) or carboxyl-terminal truncated mutant ( C)
of XSmad4 or XSmad4 together with HA-tagged XSmad1 or XSmad2 and
stimulated with 10 ng/ml TGF- (T) or 300 ng/ml BMP
(B) for 1 h. Complex formation was detected by
immunoprecipitation (IP) with the anti-Myc antibody followed
by immunoblotting (IB) with the anti-HA antibody, and the
aliquots were also blotted with anti-Myc antibody to detect the
expression of Myc-tagged XSmad4 and Myc-tagged XSmad4 . Aliquots
of the cell lysates were directly analyzed by immunoblotting with
anti-HA antibody. B, cells were transfected with
Myc-XSmad4 or Myc-XSmad4 combined with HA-XSmad1 or HA-Smad2 and
stimulated with BMP or TGF- at indicated concentrations for 1 h. Oligomerization was detected by immunoprecipitation followed by
immunoblotting. C, homomeric or heteromeric oligomer
formation was detected by immunoprecipitation followed by
immunoblotting from the lysates of C2C12 cells transfected with HA- or
Myc-tagged XSmad4 and XSmad4 .
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We next assessed whether the extent of the ligand-induced association
of XSmad4s with XSmad1 or XSmad2 would differ between XSmad4
and
XSmad4
. The association of XSmad2 with XSmad4
occurred at the
same level as with XSmad4
in a manner dependent on the TGF-
concentration (Fig. 5B, lower panel). In
contrast, the association of XSmad1 with XSmad4
in response to BMP
stimulation occurred more strongly than with XSmad4
(Fig.
5B, upper panel). This may suggest that although
both of the XSmad4s can function as common partners of XSmad1 and
XSmad2, they do not have completely redundant functions, and XSmad4
may play a major role in BMP signaling.
The crystallographic structure analysis of the MH2 domain of human
Smad4 implied that Smad4 was able to form a homotrimer (40). To examine
whether XSmad4
and XSmad4
form a heteromeric complex, we
co-expressed both XSmad4
and XSmad4
tagged with different
epitopes in C2C12 cells. Stimulation-independent hetero-oligomerization as well as homo-oligomerization was observed (Fig. 5C).
Therefore, it is possible that XSmad4
and XSmad4
form a
heteromeric trimer. It may also be possible that after stimulation,
XSmad4
, XSmad4
, and one of the pathway-restricted Smads form a
heteromeric trimer.
XSmad4
, but Not XSmad4
, Is Constitutively Nuclear--
When
expressed in C2C12 cells, XSmad4
and XSmad4
proteins showed
different subcellular distribution. XSmad4
was present predominantly
in the cytoplasm whereas XSmad4
was predominantly in the nucleus
(Fig. 6). Because it has been
demonstrated that human Smad4 and a Drosophila co-Smad,
Medea, are present in the cytoplasm in the absence of stimulation (25,
41), the above result may again indicate that XSmad4
is a
Xenopus ortholog of a previously known Smad4. XSmad4
became localized to the nucleus in more than 60% of the cells after
TGF-
stimulation when XSmad2 was co-expressed (Fig. 6,
top). Hence, the expression of XSmad2 is required for
XSmad4
to change its subcellular localization in response to TGF-
under the conditions. In contrast, subcellular localization of
XSmad4
did not change after TGF-
stimulation, irrespective of
co-expression of XSmad2 (Fig. 6, bottom).

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Fig. 6.
Subcellular localization of
XSmad4 and XSmad4
proteins. C2C12 cells were transfected with Myc-tagged
XSmad4 or XSmad4 together with or without XSmad2 and stimulated
with TGF- by co-transfection with activated TGF- type I receptor
plus treatment with TGF- (10 ng/ml) for 1 h. Then the cells
were fixed and stained with anti-Myc ( Myc) antibody and
4',6-diamidino-2-phenylindole dihydrochloride (DAPI).
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XSmad4
, but Not XSmad4
, Is a Phosphoprotein--
In addition
to the high degree of diversity in the linker region, another striking
difference in the amino acid sequence between XSmad4
and XSmad4
is found in their carboxyl termini. The SSVN sequence in XSmad4
(see
Fig. 1B) is similar to the carboxyl-terminal phosphorylation
motif (SS(V/M)S) of pathway-restricted Smads in which the last two
serine residues undergo phosphorylation upon stimulation (42, 43). We
then examined whether XSmad4
could be phosphorylated. Myc
epitope-tagged XSmads were transfected into C2C12 cells, and the cells
were labeled with [32P]orthophosphate and stimulated with
TGF-
. An increase in XSmad2 phosphorylation after TGF-
treatment
was observed, as determined by immunoprecipitation with anti-Myc
antibody followed by autoradiography (Fig.
7). No or little phosphorylation of
XSmad4
was observed before or after TGF-
stimulation (Fig. 7). On
the contrary, phosphorylation of XSmad4
was observed even before
stimulation, and its level did not increase after TGF-
treatment
(Fig. 7). To explore the possible involvement of the SSVN sequence of
XSmad4
in its phosphorylation, we constructed a mutant XSmad4
having an AAVN sequence instead of SSVN at its carboxyl terminus. This
mutant was still phosphorylated irrespective of TGF-
stimulation,
and no decrease in the phosphorylation level was observed by this
mutation (Fig. 7). Therefore, the carboxyl-terminal SSVN sequence of
XSmad4
is not phosphorylated, and XSmad4
may not be a direct
substrate of the TGF-
receptor.

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Fig. 7.
Phosphorylation of
XSmad4 . C2C12 cells were transfected with
Myc-tagged XSmad4 , XSmad4 , or the carboxyl-terminal mutated
version of XSmad4 (AAVN). Cells were metabolically labeled with
[32P]orthophosphate and further incubated with or without
TGF- (10 ng/ml) for 1 h. Phosphorylation of Myc-tagged XSmad4s
was analyzed by immunoprecipitation with anti-Myc ( -Myc)
antibody followed by autoradiography. WT, wild type;
IB, immunoblotting.
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XSmad4
Induces Ventralization of Xenopus Embryos--
To find
the functional difference between the two XSmad4s, we tested the effect
of expression of XSmad4 in Xenopus embryos. Injection of
XSmad4
mRNA, but not XSmad4
mRNA, into two dorsal cells
of the 4-cell stage embryos led to strong ventralization, as revealed
by the defects in anterior structures at the late stage (Fig.
8A). Similar phenotypes were
reported to be induced by expressing Smad1 or Smad5 (16, 22). Injection
of either of the mRNAs into the ventral side had little or no
effect on the embryonic development (Fig. 8A). The
ventralizing effect of XSmad4
, when expressed in the dorsal marginal
region, was dose-dependent. This was clearly demonstrated
by semiquantification by scoring the dorsoanterior index (DAI) of the
embryos (44), where 5 represents a normal embryo and 0 indicates an
embryo lacking axial structures (Fig. 8B). We confirmed that
XSmad4
and XSmad4
were expressed at almost the same level in this
series of experiments (Fig. 8C). In addition to the defects
in anterior structures, injection of XSmad4
mRNA into dorsal
marginal cells induced an increase in the expression of a ventral
mesodermal marker, Xvent-2, and a decrease in the expression of a
dorsal mesodermal marker, goosecoid, more strongly than did XSmad4
mRNA, as analyzed in isolated dorsal marginal zone explants (Fig.
8D). These results indicate that the two XSmad4 proteins
have distinct abilities to induce ventralization in Xenopus
embryos.

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|
Fig. 8.
XSmad4 induces
ventralization of embryos. A, tadpole stage (stage 35)
Xenopus embryos and sibling embryos that have been injected
dorsally with XSmad4 mRNA or XSmad4 mRNA at the 4-cell
stage at the indicated doses are shown (Dorsal). Embryos
injected ventrally with XSmad4 or XSmad4 mRNA (2 ng) are also
shown (Ventral). B, semiquantification of
ventralization of the embryos by XSmad4 . At the 4-cell stage, two
dorsal blastomeres were injected with XSmad4 mRNA or XSmad4
mRNA at the indicated doses. The DAI of the embryos was scored
after 2 days, and the average DAI for each sample is shown. Numbers of
embryos examined are indicated above the figure. Embryos with a DAI of
0 lack dorsal structures completely and those with a DAI of 5 are normal. C,
immunoblotting analysis of the exogenously expressed XSmad4 and
XSmad4 . N-terminal Myc-tagged XSmad4 and XSmad4 mRNAs were
injected at the 4-cell stage embryos at the indicated doses, and
extracts were obtained at the blastula stage (stage 9). Expressed
proteins were detected by immunoblotting with anti-Myc antibody.
Myc-tagged XSmad4s had essentially the same effect as nontagged
constructs on the phenotypes of embryos (data not shown). D,
expression of marker genes in dorsal marginal zone explants. Dorsal
marginal zone explants were dissected at the early gastrula stage from
embryos that had been injected dorsally with XSmad4 , XSmad4 ,
XSmad1, or XSmad2 mRNA (each at 2 ng) at the 4-cell stage and were
cultured until sibling embryos reached the midgastrula stage.
Expression of indicated marker genes was analyzed by RT-PCR. Expression
of EF-1 was also analyzed as a loading control. No signal was
observed in the absence of reverse transcription ( RT).
gsc, goosecoid; Xbra, Xenopus
brachyury.
|
|
 |
DISCUSSION |
Smad4/DPC4 has been shown to be required as a common partner for
pathway-restricted Smads to propagate the TGF-
family signals from
the cell surface receptors to the nucleus by forming heteromeric complexes. Here we have identified a novel Smad4-like molecule, XSmad4
, as well as a Xenopus homolog of mammalian Smad4
(XSmad4
), and shown that both of the XSmad4s are able to function as
co-Smad to cooperate and synergize with XSmad1 and XSmad2. However,
they have distinct properties in terms of their temporal expression during early embryogenesis, subcellular distribution, phosphorylation state, and ventralizing activity in Xenopus embryos.
Therefore, it is likely that two co-Smads, XSmad4
and XSmad4
,
play both overlapping and distinct roles in transducing a set of
TGF-
superfamily signals in Xenopus.
We reasoned from their subcellular distribution as well as their amino
acid sequence similarity to human or mouse Smad4 that XSmad4
was a
Xenopus ortholog of mammalian Smad4 and XSmad4
was a
novel homolog. XSmad4
localized predominantly in the cytoplasm and
translocated to the nucleus after stimulation when a pathway-restricted Smad was co-expressed (Fig. 6). This behavior is the same as that previously reported for human Smad4 (25). In contrast, XSmad4
was
present predominantly in the nucleus before or after stimulation. The
subcellular distribution of either of the XSmad4s was not affected when
the other XSmad4 was co-expressed in cells (data not shown), although
they could bind to each other to form the XSmad4
-XSmad4
complex
in vitro (Fig. 5C). Although we do not know the
functional significance of their different subcellular localizations,
the possible formation of the heteromeric complex between XSmad4
and
XSmad4
after the nuclear entry of XSmad4
in response to
stimulation might have some physiological significance.
An intriguing question is whether or not the existence of the second
co-Smad is specific to Xenopus. At present, there is no
evidence for the existence of another Smad4-like gene in mammals. We
could not find any related genes in the data base of expressed sequence
tags (dbEST). Smad4 knockout mice were shown to die early in their
development (29, 30), and the Smad4-deficient cells lost responsiveness
to the TGF-
superfamily (45). Moreover, lack of Smad4 in colon
cancer cells leads to an increase in metastasis and malignancy, which
suggests that Smad4 has a tumor-suppressing function (46). These
observations suggest that if mammals have the second co-Smad, its
function may be distinct from that of Smad4/DPC4.
The Xenopus system has been extensively used to elucidate
the cellular signaling mechanism of growth factors, including the TGF-
superfamily, that control cell differentiation and pattern formation during early embryogenesis (47-52). It has been suggested that activin, nodal, and Vg1, members of the TGF-
superfamily, are
involved in the differentiation of dorsal mesodermal tissues, whereas
the BMP family ligands regulate the ventral mesoderm differentiation in
Xenopus early development. Xenopus homologs of
Smad1 and Smad2 have been identified and shown to mediate activin and
BMP signaling pathways, respectively (10, 12, 16, 53). Interestingly, Candia et al. (39) observed that the expression of an excess amount of human Smad4 in Xenopus embryos compromised the
antagonism between the activin/Vg1 and BMP pathways. They proposed an
attractive model, which supports the theory that the activin/Vg1 and
BMP pathways modulate each other's activity by sequestering a limited pool of Smad4, which commonly participates in both pathways by associating with Smad2 and Smad1, respectively. As human Smad4 corresponds to XSmad4
, XSmad4
may be commonly used as a co-Smad for both pathways, or a putative heteromeric complex between XSmad4
and XSmad4
may be a common partner for both XSmad1 and XSmad2. These
considerations may be consistent with our idea that XSmad4
and
XSmad4
have overlapping but somewhat distinct functions as co-Smads.
Thus the observations of Candia et al. are not inconsistent with our idea that XSmad4
may be rather preferentially used for the
BMP pathway, which was derived from our results showing that XSmad4
binds to XSmad1 more tightly than does XSmad4
and that XSmad4
has
the more potent ventralizing activity.
Although we still do not know the molecular mechanism that defines the
distinct ability between XSmad4
and XSmad4
in cooperation with
XSmad1 and XSmad2, it is likely that some factors interact specifically
with each of the XSmad4s. It is also possible that phosphorylation of
XSmad4
might have some role in regulating the function of XSmad4
.
We are currently investigating the mechanisms that underlie the
difference between XSmad4
and XSmad4
in their cooperativity with
pathway-restricted Smads, as well as the molecular basis for
determining their subcellular localization.
 |
ACKNOWLEDGEMENTS |
We thank Dr. Douglas A. Melton for providing
Xenopus Smad1 and Smad2 plasmids and Dr. Ken W. Y. Cho
for the Xvent2-Luc plasmid. We also thank K. Kawachi and M. Watanabe for technical assistance.
 |
FOOTNOTES |
*
This work was supported by grants from the Ministry of
Education, Science and Culture of Japan (to E. N.).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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AB022721 and AB022722.
¶
To whom correspondence should be addressed. Fax:
81-75-753-4235; E-mail: L50174{at}sakura.kudpc.kyoto-u.ac.jp.
 |
ABBREVIATIONS |
The abbreviations used are:
TGF-
, transforming growth factor-
;
BMP, bone morphogenetic protein;
HA, hemagglutinin;
RT-PCR, reverse transcription-coupled polymerase chain
reaction;
XSmad, Xenopus Smad;
hSmad, human Smad;
MH1, mad homology 1 (amino-terminal domain);
DAI, dorsoanterior
index;
MH2, mad homology 2 (carboxyl-terminal domain);
DPC, deleted in pancreatic cancer.
 |
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