Identification of Two Smad4 Proteins in Xenopus
THEIR COMMON AND DISTINCT PROPERTIES*

Norihisa MasuyamaDagger , Hiroshi HanafusaDagger , Morioh KusakabeDagger , Hiroshi Shibuya§, and Eisuke NishidaDagger

From the Dagger  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
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Smad family proteins have been identified as mediators of intracellular signal transduction by the transforming growth factor-beta (TGF-beta ) 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 (XSmad4beta ) as well as a Xenopus homolog of a well established Smad4 (XSmad4alpha ). XSmad4beta is 70% identical to XSmad4alpha 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-beta , respectively. However, they show distinct properties in terms of their developmental expression patterns, subcellular localizations, and phosphorylation states. Moreover, XSmad4beta , but not XSmad4alpha , 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
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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-beta 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-beta 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-beta 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-beta 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-beta /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 XSmad4alpha and XSmad4beta , 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-beta superfamily. Moreover, XSmad4alpha and XSmad4beta 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 XSmad4beta into dorsal cells of Xenopus embryos leads to strong ventralization, whereas XSmad4alpha 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-beta superfamily signals.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 XSmad4alpha and XSmad4beta , the carboxyl-terminal truncated forms of XSmad4alpha and XSmad4beta , and the carboxyl-terminal serine to alanine mutant of XSmad4beta (AAVN) were amplified by polymerase chain reaction, and the amplified nucleotide sequences were confirmed by DNA sequencing. For the carboxyl-terminal truncated forms of XSmad4alpha and XSmad4beta , the nucleotides corresponding to amino acid residues 1-508 and 1-519 were amplified, respectively. XSmad4alpha cDNA and XSmad4beta 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-SRalpha 456, pSRalpha -HA, or pSRalpha -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 beta -galactosidase activities.

Immunoprecipitation, Immunoblotting, and Metabolic Labeling-- After 12-15 h, cells were treated with or without 10 ng/ml of human TGF-beta 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-beta 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-beta stimulation of C2C12 cells was provided by co-transfecting the activated TGF-beta type I receptor, Tbeta R-I (T204D), and treated with 10 ng/ml TGF-beta 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
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

cDNA Cloning of Xenopus Smad4alpha and Xenopus Smad4beta -- 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 Smad4alpha (XSmad4alpha ) and the latter as Xenopus Smad4beta (XSmad4beta ). We considered XSmad4alpha a Xenopus ortholog of mammalian Smad4 and XSmad4beta the second Smad4 (see below). The nucleotide sequences were predicted to encode proteins of 549 and 560 amino acids for XSmad4alpha and XSmad4beta 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 XSmad4alpha and XSmad4beta from the same cDNA library (data not shown).


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Fig. 1.   cDNA cloning of XSmad4alpha and XSmad4beta . A, alignment of the deduced amino acid sequences of XSmad4alpha , human Smad4/DPC4 (hSmad4), and XSmad4beta 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 XSmad4alpha and XSmad4beta is shown.

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 XSmad4alpha or XSmad4beta and human Smad4 (hSmad4) is shown in Fig. 1A. The MH1 and MH2 domains of XSmad4alpha are 100% identical to those of hSmad4, except that the coding region of XSmad4alpha initiates at the position corresponding to the second methionine residue of hSmad4. The linker region shows 74% identity between XSmad4alpha and hSmad4. In contrast, the identity between XSmad4beta and hSmad4 is about 90% in the MH1 and MH2 domains and 34% in the linker region (Fig. 1B). Another striking difference between XSmad4alpha and XSmad4beta is that the carboxyl-terminal sequence of XSmad4alpha is QPLD, like mammalian Smad4, whereas that of XSmad4beta 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 XSmad4alpha and XSmad4beta 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). XSmad4alpha mRNA was markedly increased by zygotic expression after the blastula stage, whereas XSmad4beta mRNA was highly abundant in eggs and was decreased during the gastrula stage, although both transcripts were detected throughout early embryogenesis (Fig. 2A). Thus, XSmad4alpha mRNA and XSmad4beta mRNA are expressed differently during early development.


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Fig. 2.   The temporal and spatial expression of XSmad4alpha and XSmad4beta . A, XSmad4alpha and XSmad4beta transcripts are present during Xenopus early embryogenesis. Equivalent amounts of total RNA isolated from each stage of embryos were analyzed for the expression of XSmad4alpha and XSmad4beta 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 XSmad4alpha and XSmad4beta 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).

The spatial expression of XSmad4alpha and XSmad4beta was examined by whole mount in situ hybridization with the probes (Fig. 2, B-G). Both XSmad4alpha and XSmad4beta 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). XSmad4beta is expressed more anteriorly than XSmad4alpha in the neural tube.

XSmad4alpha and XSmad4beta Synergize with XSmad1 and XSmad2-- To examine whether XSmad4alpha and XSmad4beta act as co-Smad to cooperate with Smad1 and Smad2 to mediate BMP-like and TGF-beta /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 XSmad4alpha or XSmad4beta combined with Xenopus Smad1 or Xenopus Smad2. Injection of a low dose of XSmad1, XSmad2, XSmad4alpha , or XSmad4beta mRNA alone was not sufficient to induce any noticeable expression of mesodermal genes (Fig. 3A). When XSmad1 mRNA was injected together with either XSmad4alpha or XSmad4beta mRNA, however, strong expression of a ventral mesodermal marker, alpha  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.   XSmad4alpha and XSmad4beta synergize with XSmad1 and XSmad2 to induce expression of mesodermal marker genes. A, effect of co-expression of XSmad4alpha or XSmad4beta 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 XSmad4alpha or XSmad4beta 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 alpha -globin was analyzed by RT-PCR. Expression of EF-1alpha 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 XSmad4alpha and XSmad4beta (XSmad4alpha Delta Cand XSmad4beta Delta 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 XSmad4alpha Delta CmRNA or XSmad4beta Delta CmRNA (1 ng) and were cultured until sibling embryos reached stage 11 or 26. Expression of marker genes was analyzed by RT-PCR.

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 XSmad4alpha and XSmad4beta 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 XSmad4alpha Delta C or XSmad4beta Delta C mRNA was injected along with XSmad1 or XSmad2, the expression of these marker genes was significantly suppressed (Fig. 3B). These results suggest that XSmad4alpha and XSmad4beta 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 XSmad4alpha and XSmad4beta with XSmad1 and XSmad2, we tested the ability of the Smads to induce the reporter gene expression under the BMP- and TGF-beta -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 XSmad4alpha and XSmad4beta , but not XSmad4alpha Delta Cor XSmad4beta Delta C, together with XSmad1 induced a high level of the reporter gene expression (Fig. 4A). Similarly, both XSmad4alpha and XSmad4beta synergized with XSmad2 to induce luciferase expression under the TGF-beta -responsive 3TP reporter, whereas the carboxyl-terminal truncated constructs did not (Fig. 4B). These results demonstrate that XSmad4alpha and XSmad4beta act synergistically with XSmad1 and XSmad2 to induce gene expression in cultured cells.


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Fig. 4.   XSmad4alpha and XSmad4beta cooperate with XSmad1 and XSmad2 to induce the reporter gene expression under the BMP- and TGF-beta -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; Delta C, carboxyl-terminal truncated mutant.

XSmad4alpha and XSmad4beta Associate with XSmad1 and XSmad2-- As XSmad4alpha and XSmad4beta functionally cooperated with XSmad1 and XSmad2, we would expect that XSmad4alpha and XSmad4beta 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-beta stimulation, XSmad2 associates with either of the two XSmad4s (Fig. 5A, lower panel). Thus, both XSmad4alpha and XSmad4beta are able to form heteromeric complexes with XSmad1 or XSmad2 in response to specific stimulation.


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Fig. 5.   Association of XSmad4alpha and XSmad4beta with XSmad1 and XSmad2. A, C2C12 cells were transfected with Myc-tagged wild type (WT) or carboxyl-terminal truncated mutant (Delta C) of XSmad4alpha or XSmad4beta together with HA-tagged XSmad1 or XSmad2 and stimulated with 10 ng/ml TGF-beta (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 XSmad4alpha and Myc-tagged XSmad4beta . Aliquots of the cell lysates were directly analyzed by immunoblotting with anti-HA antibody. B, cells were transfected with Myc-XSmad4alpha or Myc-XSmad4beta combined with HA-XSmad1 or HA-Smad2 and stimulated with BMP or TGF-beta 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 XSmad4alpha and XSmad4beta .

We next assessed whether the extent of the ligand-induced association of XSmad4s with XSmad1 or XSmad2 would differ between XSmad4alpha and XSmad4beta . The association of XSmad2 with XSmad4alpha occurred at the same level as with XSmad4beta in a manner dependent on the TGF-beta concentration (Fig. 5B, lower panel). In contrast, the association of XSmad1 with XSmad4beta in response to BMP stimulation occurred more strongly than with XSmad4alpha (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 XSmad4beta 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 XSmad4alpha and XSmad4beta form a heteromeric complex, we co-expressed both XSmad4alpha and XSmad4beta 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 XSmad4alpha and XSmad4beta form a heteromeric trimer. It may also be possible that after stimulation, XSmad4alpha , XSmad4beta , and one of the pathway-restricted Smads form a heteromeric trimer.

XSmad4beta , but Not XSmad4alpha , Is Constitutively Nuclear-- When expressed in C2C12 cells, XSmad4alpha and XSmad4beta proteins showed different subcellular distribution. XSmad4alpha was present predominantly in the cytoplasm whereas XSmad4beta 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 XSmad4alpha is a Xenopus ortholog of a previously known Smad4. XSmad4alpha became localized to the nucleus in more than 60% of the cells after TGF-beta stimulation when XSmad2 was co-expressed (Fig. 6, top). Hence, the expression of XSmad2 is required for XSmad4alpha to change its subcellular localization in response to TGF-beta under the conditions. In contrast, subcellular localization of XSmad4beta did not change after TGF-beta stimulation, irrespective of co-expression of XSmad2 (Fig. 6, bottom).


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Fig. 6.   Subcellular localization of XSmad4alpha and XSmad4beta proteins. C2C12 cells were transfected with Myc-tagged XSmad4alpha or XSmad4beta together with or without XSmad2 and stimulated with TGF-beta by co-transfection with activated TGF-beta type I receptor plus treatment with TGF-beta (10 ng/ml) for 1 h. Then the cells were fixed and stained with anti-Myc (alpha Myc) antibody and 4',6-diamidino-2-phenylindole dihydrochloride (DAPI).

XSmad4beta , but Not XSmad4alpha , Is a Phosphoprotein-- In addition to the high degree of diversity in the linker region, another striking difference in the amino acid sequence between XSmad4alpha and XSmad4beta is found in their carboxyl termini. The SSVN sequence in XSmad4beta (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 XSmad4beta could be phosphorylated. Myc epitope-tagged XSmads were transfected into C2C12 cells, and the cells were labeled with [32P]orthophosphate and stimulated with TGF-beta . An increase in XSmad2 phosphorylation after TGF-beta treatment was observed, as determined by immunoprecipitation with anti-Myc antibody followed by autoradiography (Fig. 7). No or little phosphorylation of XSmad4alpha was observed before or after TGF-beta stimulation (Fig. 7). On the contrary, phosphorylation of XSmad4beta was observed even before stimulation, and its level did not increase after TGF-beta treatment (Fig. 7). To explore the possible involvement of the SSVN sequence of XSmad4beta in its phosphorylation, we constructed a mutant XSmad4beta having an AAVN sequence instead of SSVN at its carboxyl terminus. This mutant was still phosphorylated irrespective of TGF-beta stimulation, and no decrease in the phosphorylation level was observed by this mutation (Fig. 7). Therefore, the carboxyl-terminal SSVN sequence of XSmad4beta is not phosphorylated, and XSmad4beta may not be a direct substrate of the TGF-beta receptor.


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Fig. 7.   Phosphorylation of XSmad4beta . C2C12 cells were transfected with Myc-tagged XSmad4alpha , XSmad4beta , or the carboxyl-terminal mutated version of XSmad4beta (AAVN). Cells were metabolically labeled with [32P]orthophosphate and further incubated with or without TGF-beta (10 ng/ml) for 1 h. Phosphorylation of Myc-tagged XSmad4s was analyzed by immunoprecipitation with anti-Myc (alpha -Myc) antibody followed by autoradiography. WT, wild type; IB, immunoblotting.

XSmad4beta 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 XSmad4beta mRNA, but not XSmad4alpha 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 XSmad4beta , 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 XSmad4alpha and XSmad4beta were expressed at almost the same level in this series of experiments (Fig. 8C). In addition to the defects in anterior structures, injection of XSmad4beta 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 XSmad4alpha 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.   XSmad4beta induces ventralization of embryos. A, tadpole stage (stage 35) Xenopus embryos and sibling embryos that have been injected dorsally with XSmad4alpha mRNA or XSmad4beta mRNA at the 4-cell stage at the indicated doses are shown (Dorsal). Embryos injected ventrally with XSmad4alpha or XSmad4beta mRNA (2 ng) are also shown (Ventral). B, semiquantification of ventralization of the embryos by XSmad4beta . At the 4-cell stage, two dorsal blastomeres were injected with XSmad4alpha mRNA or XSmad4beta 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 XSmad4alpha and XSmad4beta . N-terminal Myc-tagged XSmad4alpha and XSmad4beta 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 XSmad4alpha , XSmad4beta , 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-1alpha was also analyzed as a loading control. No signal was observed in the absence of reverse transcription (-RT). gsc, goosecoid; Xbra, Xenopus brachyury.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Smad4/DPC4 has been shown to be required as a common partner for pathway-restricted Smads to propagate the TGF-beta family signals from the cell surface receptors to the nucleus by forming heteromeric complexes. Here we have identified a novel Smad4-like molecule, XSmad4beta , as well as a Xenopus homolog of mammalian Smad4 (XSmad4alpha ), 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, XSmad4alpha and XSmad4beta , play both overlapping and distinct roles in transducing a set of TGF-beta superfamily signals in Xenopus.

We reasoned from their subcellular distribution as well as their amino acid sequence similarity to human or mouse Smad4 that XSmad4alpha was a Xenopus ortholog of mammalian Smad4 and XSmad4beta was a novel homolog. XSmad4alpha 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, XSmad4beta 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 XSmad4alpha -XSmad4beta 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 XSmad4alpha and XSmad4beta after the nuclear entry of XSmad4alpha 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-beta 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-beta 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-beta 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 XSmad4alpha , XSmad4alpha may be commonly used as a co-Smad for both pathways, or a putative heteromeric complex between XSmad4alpha and XSmad4beta may be a common partner for both XSmad1 and XSmad2. These considerations may be consistent with our idea that XSmad4alpha and XSmad4beta have overlapping but somewhat distinct functions as co-Smads. Thus the observations of Candia et al. are not inconsistent with our idea that XSmad4beta may be rather preferentially used for the BMP pathway, which was derived from our results showing that XSmad4beta binds to XSmad1 more tightly than does XSmad4alpha and that XSmad4beta has the more potent ventralizing activity.

Although we still do not know the molecular mechanism that defines the distinct ability between XSmad4alpha and XSmad4beta 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 XSmad4beta might have some role in regulating the function of XSmad4beta . We are currently investigating the mechanisms that underlie the difference between XSmad4alpha and XSmad4beta 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-beta , transforming growth factor-beta ; 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.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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