BIOLOGICAL OVERVIEW

Smads can be grouped into three classes: receptor-regulated (R-) Smads, common (Co-) Smads, and antagonistic (Anti-) Smads. In Drosophila, four Smads have been identified, the prototypic Mad, an R-Smad; Medea, a Co-Smad; Dad, an Anti-Smad and an R-Smad termed Smad on the X (Smox), described here. The activation mechanism of the Smads has been well defined; first the R-Smads are phosphorylated by the type I receptor of the ligand-activated receptor complex; next, they bind to the Co-Smad and enter the nucleus. Finally, this Smad complex induces the transcriptional activation of target genes, including the Anti-Smads, which act as antagonists of signaling. Smad1, 5 and 8 function in the BMP pathways, while Smad2 and 3, mammalian homologs of Smox, mediate activin and TGFbeta signaling. Smad4 acts as a Co-Smad for the three major TGFbeta signaling pathways and forms hetero-oligomers with the R-Smads (Das, 1999 and references therein).

When expressed alone in cultured cells, Baboon (Babo), the Type 1 activin-A-receptor of Drosophila, is unable to bind TGF-beta, activin, or bone morphogenetic protein 2. However, Baboon binds activin efficiently when coexpressed with the distantly related Drosophila activin receptor (Punt), with which it forms a heteromeric complex. Baboon can also bind activin in concert with mammalian activin type II receptors (ActR-II and ActR-IIB). Maternal Baboon transcripts are abundant in the oocyte and widespread during embryo development and in the imaginal discs of the larva (Brummel, 1999). Prior to the identification of Smad on the X (Smox), Mad and Medea, both of which mediate Dpp signaling, were the only activating Smads that had been identified. Because Babo only appears to induce Smad2/3 responsive promoters in mammalian cell culture, the Drosophila ESTs (Berkeley Genome Project) database was searched for new Smad-like genes. One clone with significant homology to vertebrate Smad2/3 was identified. Using this clone as a probe, a Drosophila ovarian cDNA library was screened to obtain the full-length cDNA, which was sequenced and named Smox (Brummel, 1999, Henderson, 1999 and Das, 1999).

The specificity of Smad interactions with receptors and nuclear targets is dictated by the MH2 domain. This suggests that Smox might function in a Drosophila TGF/Activin-like signaling pathway that involves Babo. To investigate this possibility, the ability of Babo to mediate the phosphorylation of Smox was tested. COS-1 cells were transfected with an epitope-tagged Smox either alone or together with wild-type or constitutively active versions of Babo. Analysis of Smox in the absence of signaling shows some basal phosphorylation of the protein. However, on coexpression of activated Babo, a strong increase in phosphorylation of the protein is observed. Previous work has shown that receptor-dependent phosphorylation of Smads occurs on the last two serines of the protein. To confirm that Babo induces phosphorylation of Smox on these residues, the last two serines were mutated to alanines (dSmad2-2SA). Unlike wild-type Smox, this mutant is not phosphorylated by activated Babo. These data suggest that Smox is a downstream target of Babo and is phosphorylated on the last two serine residues in the carboxyl terminus (Brummel, 1999).

One functional consequence of phosphorylating receptor-regulated Smads is the induction of heteromeric complex formation with the common partner Smad4. In Drosophila, the Smad4 homolog Medea similarly associates with Mad and is required for a subset of Dpp signaling. Thus, an investigation was carried to see whether Babo-dependent phosphorylation of Smox might induce association with Medea. In the absence of signaling, Smox and Medea form some heteromeric complexes, however, the level of complex formation is substantially increased on cotransfection with a constitutively active form of Babo. Furthermore, use of dSmad2-2SA abolishes this receptor-dependent increase in heteromeric complex formation. These data indicate that phosphorylation of Smox on the last two serines is necessary for receptor-dependent induction of heteromeric complexes of Smox and Medea (Brummel, 1999).

Because recognition of type I receptors by Smads requires activation by the type II receptor, a test was carried out to see whether Punt, the Drosophila type II receptor can function to activate Babo. In cultured cells, transient transfection of either Punt or Babo alone has no effect on the 3TP promoter. However, overexpression of Punt together with Babo leads to a strong induction of the promoter. This is consistent with previous observations that type II and type I ser/thr kinase receptors have intrinsic affinity for each other and on overexpression can associate and signal in the absence of ligand. To test whether Smox interacts with Punt/Babo complexes, a kinase-deficient Babo, which functions to stabilize Smad-receptor interactions, was used. When Smox is immunoprecipitated in the presence of wild-type receptors, no complexes can be detected. However, when the kinase-deficient form of Babo is utilized, receptor complexes coprecipitating with Smox are readily detected. In addition, when the phosphorylation site mutant of Smox is tested, stable complex formation is detected between the mutant protein and wild-type receptor complexes. Thus, Smox interacts transiently and specifically with Punt-Babo receptor complexes. Taken together, these functional and biochemical analyses strongly suggest that Smox is a Drosophila homolog of Smad2/Smad3 and functions as a downstream signaling component that directly interacts with Babo (Brummel, 1999).

In order to more directly address the question of whether Drosophila has a activin/TGFbeta pathway, functional assays of the Drosophila genes were performed using Xenopus embryos, since these assays have provided an effective method of classifying TGFbeta superfamily ligands and their signal transduction components into either BMP- or activin-type pathways. These two signals have distinct effects during Xenopus development. When introduced as synthetic mRNA, BMP2/4, the constitutively active (CA-) BMP-type I receptors ALK2, CA-ALK3, CA-ALK6, Smad1 and Smad5 all function to ventralize the mesoderm and promote epidermal fates in the ectoderm. Conversely, Activin, CA-ALK4 (an activin/TGFbeta type I receptor), Smad2 and Smad3 convert ectoderm to mesoderm and induce axial duplications. Therefore, molecules suspected of being involved in transducing TGFbeta superfamily signals can be easily placed into either a BMP- or activin-like pathway (Das, 1999).

mRNA encoding Smox was injected into the animal hemisphere of one-cell Xenopus embryos. The embryos were allowed to develop until the blastula stage, at which time the ectodermal cap was removed and cultured in isolation. The ectodermal explants were harvested at either the gastrula stage or late neurula stage and analyzed by RT-PCR for the presence of mesoderm and muscle-specific markers (Das, 1999).

At the gastrula stage, Smox induces the expression of the mesoderm specific marker, Xbra, while at neurula stages Smox is effective at inducing the expression of the muscle specific marker, cardiac actin (c-actin). Previous studies have shown that the activation of c-actin is characteristic of the Smad2, but not the Smad1 pathway. This result demonstrates that Smox is capable of mediating the vertebrate activin-like signal transduction pathway, and suggests that Drosophila possesses activin-like signals (Das, 1999).

Several lines of evidence demonstrate that Smox and Baboon function in an activin-like pathway in Drosophila. (1) Smox shares many sequence characteristics with the vertebrate activin/TGFbeta Smads -- Smad2 and Smad3 -- including the two residues in the L3 loop that are thought to confer pathway specificity on the Smads. (2) Smox can promote activin-like fates, but not BMP-like fates, in Xenopus animal cap assays, suggesting that the normal role of Smox in Drosophila is in an activin-like pathway. (3) Smox becomes phosphorylated and translocates to the nucleus in response to activin-like signaling, and acts to initiate transcription from activin-stimulated promoters. Smox is not phosphorylated by the Dpp receptor Tkv, while Mad, the Dpp R-Smad, is not activated by Baboon, demonstrating the specificity of Smox signal transduction. (4) Medea is required for Smox activation in vitro, a result which would be expected if the BMP and activin pathways had been conserved during evolution (Das, 1999).

While these results strongly suggest the existence of a functional activin-like pathway in Drosophila, the exact nature of its function will have to await the identification of mutants in components of the pathway, including the two newly identified activin-like ligands. However, the prominent expression of Smox and Baboon in the region of LPC mitoses in the brain, and in the morphogenetic furrow of the eye imaginal disk, implies that one of the roles of the Drosophila activin pathway is in transmitting proliferative signals. The expression of dpp in the OPC would suggest that it has a proliferative role in the development of the brain, and since such a role has also been proposed for Mad, it is possible that the dpp and activin pathways have intersecting functions. Additionally, both pathways may also share the patterning roles that have been shown for dpp in the brain. Interestingly both tolloid (tld) and tolkin (tok), BMP1-related metalloproteases that are implicated in the regulation of TGFbeta-like ligands, are expressed in the OPC of the brain. While a direct role for Tld in the regulation of Dpp activity has been shown, no such function is known for Tok. An interesting possibility is that Tok may be involved in the regulation of the Drosophila activin-like ligands. The identification of mutants in components of the pathway will enable the detailed study of these issues (Das, 1999).

GENE STRUCTURE

cDNA clone length - 3083

Bases in 5' UTR - 792

Bases in 3' UTR - 849

PROTEIN STRUCTURE

Amino Acids - 486

Structural Domains

Smox sequence alignment with other known Smads revealed ~50% overall identity to Mad or human Smad1 and ~70% identity to human Smad2 or Smad3. Furthermore, the MH2 domain represented the region of highest homology with greater than 90% identity between Smox and either Smad2 or Smad3. In the MH1 domain, Smox lacks the two inserts that are found in the MH1 domain of Smad2. In the linker, Smox contains a PY motif that is present in other Smads, but in addition has a glycine, serine, and glutamine-rich insert (amino acid residues 177-251) that is absent in either hSmad2 or hSmad3 (Brummel, 1999).

Smox has 60% overall identity to human SMAD3, 57.1% identity to human SMAD2, and 51.8% identity to Drosophila MAD. Smad proteins have two highly conserved domains, known as MH1 and MH2, separated by a linker region that is less conserved. Smox MH1 has 59.0% identity to the MH1 domain of SMAD3 and 48.9% identity to the MH1 domain of SMAD2. Smox MH2 has 92.9% identity to the MH2 domains of both SMADs2 and 3. Like SMAD3, Smox does not have the two insertions of nine and thirty residues found in the MH1 domain of SMAD2. Smox has an SSXS motif at the C-terminus that has been found in all receptor-regulated SMADs characterized thus far. The two most C-terminal Ser residues of this motif in other class I SMADs are phosphorylated by activated type I receptors (Henderson, 1999).

To determine where Smox may be functioning, the expression of Smox was examined both by Northerns and by in situ hybridization to whole mount embryos. The Northern analyses revealed two Smox transcripts, both approximately 4.5 kb. The larger of the two transcripts is abundant in adult females and in 0-2 hr embryos suggesting that the larger Smox transcript is provided maternally. To determine the spatial expression pattern of Smox, embryos were hybridized with both sense and antisense Smox RNA probes. Global expression was detected throughout embryogenesis with the antisense probe but not with the negative control sense probe, suggesting that Smox is expressed to equivalent levels in all cells of the embryo (Henderson, 1999).

date revised: 22 September 2000

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