XB-ART-13172J Biol Chem April 23, 1999; 274 (17): 12163-70.
Identification of two Smad4 proteins in Xenopus. Their common and distinct properties.
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.
PubMed ID: 10207044
Article link: J Biol Chem
Species referenced: Xenopus laevis
Genes referenced: acta4 actl6a gsc hba1 myc odc1 smad1 smad2 smad4.1 smad4.2 tbxt tgfb1 ventx2.1 ventx2.2
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|FIG. 2. The temporal and spatial expression of XSmad4a and XSmad4b. A, XSmad4a and XSmad4b transcripts are present during Xenopus early embryogenesis. Equivalent amounts of total RNA isolated from each stage of embryos were analyzed for the expression of XSmad4a and XSmad4b 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 XSmad4a and XSmad4b 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).|
|FIG. 3. XSmad4a and XSmad4b synergize with XSmad1 and XSmad2 to induce expression of mesodermal marker genes. A, effect of co-expression of XSmad4a or XSmad4b 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 XSmad4a or XSmad4b 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 a-globin was analyzed by RT-PCR. Expression of EF-1a was also analyzed as a loading control. No signal was observed in the absence of reverse transcription (2RT). B, effect of carboxylterminal truncated mutants of XSmad4a and XSmad4b (XSmad4a DCand XSmad4bDC) 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 XSmad4a DCmRNA or XSmad4b DCmRNA (1 ng) and were cultured until sibling embryos reached stage 11 or 26. Expression of marker genes was analyzed by RT-PCR.|
|FIG. 4. XSmad4a and XSmad4b cooperate with XSmad1 and XSmad2 to induce the reporter gene expression under the BMPand TGF-b-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; DC, carboxyl-terminal truncated mutant.|
|FIG. 5. Association of XSmad4a and XSmad4b with XSmad1 and XSmad2. A, C2C12 cells were transfected with Myc-tagged wild type (WT) or carboxyl-terminal truncated mutant (DC) of XSmad4a or XSmad4b together with HA-tagged XSmad1 or XSmad2 and stimulated with 10 ng/ml TGF-b (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 XSmad4a and Myc-tagged XSmad4b. Aliquots of the cell lysates were directly analyzed by immunoblotting with anti-HA antibody. B, cells were transfected with Myc-XSmad4a or Myc-XSmad4b combined with HA-XSmad1 or HA-Smad2 and stimulated with BMP or TGF-b 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 XSmad4a and XSmad4b.|
|FIG. 6. Subcellular localization of XSmad4a and XSmad4b proteins. C2C12 cells were transfected with Myc-tagged XSmad4a or XSmad4b together with or without XSmad2 and stimulated with TGF-b by co-transfection with activated TGF-b type I receptor plus treatment with TGF-b (10 ng/ml) for 1 h. Then the cells were fixed and stained with anti-Myc (aMyc) antibody and 49,6-diamidino-2-phenylindole dihydrochloride (DAPI).|
|FIG. 7. Phosphorylation of XSmad4b. C2C12 cells were transfected with Myc-tagged XSmad4a, XSmad4b, or the carboxyl-terminal mutated version of XSmad4b (AAVN). Cells were metabolically labeled with [32P]orthophosphate and further incubated with or without TGF-b (10 ng/ml) for 1 h. Phosphorylation of Myc-tagged XSmad4s was analyzed by immunoprecipitation with anti-Myc (a-Myc) antibody followed by autoradiography. WT, wild type; IB, immunoblotting.|
|FIG. 8. XSmad4b induces ventralization of embryos. A, tadpole stage (stage 35) Xenopus embryos and sibling embryos that have been injected dorsally with XSmad4a mRNA or XSmad4b mRNA at the 4-cell stage at the indicated doses are shown (Dorsal). Embryos injected ventrally with XSmad4a or XSmad4b mRNA (2 ng) are also shown (Ventral). B, semiquantification of ventralization of the embryos by XSmad4b. At the 4-cell stage, two dorsal blastomeres were injected with XSmad4a mRNA or XSmad4b 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 XSmad4a and XSmad4b. Nterminal Myc-tagged XSmad4a and XSmad4b 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 XSmad4a, XSmad4b, 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-1a was also analyzed as a loading control. No signal was observed in the absence of reverse transcription (2RT). gsc, goosecoid; Xbra, Xenopus brachyury.|
|smad4.1 (SMAD family member 4, gene 1 ) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 31, lateral view, anterior left, dorsal up.|