Hata A , Lagna G , Massagué J , Hemmati-Brivanlou A .

HD32105-01 NICHD NIH HHS , R01 HD032105 NICHD NIH HHS

	Figure 1.  Structure of Smad6. (A) Alignment of the predicted amino acid sequence of human Smad6 and Smad7. Identical residues are boxed. (B) Smad homology tree and schematic comparison of the structures of Smad6, Smad7, and Smad1. The amino and carboxy-terminal homologous regions (N- and C-domain) are darkly shaded. A region conserved only between Smad6 and Smad7 is lightly shaded. (C) Alignment of C-domain sequences of Smad6 and other Smads, indicating the secondary structure elements of the Smad4 C-domain (Shi et al. 1997). In the crystal structure of Smad4 this region forms an α-helix (α-helix 5) that associates with α-helices 3 and 4 in a three-helix bundle. This structure contributes the formation of a Smad4 homotrimer by interacting with loops 1 and 2 of the adjacent monomer. The region corresponding to loops 1 and 2 in Smad6 is also very divergent from Smad4 and the receptor-regulated Smads. However, most of the components that constitute the β-sandwich core structure of the Smad4 C-domain are conserved in Smad6.
	Figure 2.  Smad6 induces secondary axes and neuralizes ectoderm in Xenopus. (A) Smad6 overexpression induces the formation of a secondary axis. Smad6 RNA (0.5–4 ng) was coinjected with nuclear β-galactosidase(β-gal)RNA (100 pg) into the ventral vegetal blastomeres of eight cell stage embryos. Unlike control embryos injected with β-gal alone (bottom right), Smad6 coinjected embryos develop an ectopic dorsal axis (left and top right). β-Galactosidase staining of the injected embryos reveals that the progeny of the injected blastomere directly contribute to the ectopic axis. Dorsal atleft; anterior at top. Lateral at right; anterior atleft. (B) Smad6 does not affect formation of the primary dorsal axis. Smad6 RNA (1–4 ng) was injected into the dorsal marginal zone of four cell stage embryos. Compared to control embryos (left panel), Smad6-injected tadpoles display enlarged heads and cyclopia, but have normal body axes (right). (C) Secondary axis formation by the activin signaling molecule Smad2 is enhanced by coinjection of Smad6.Smad2 RNA (1 ng), Smad6 RNA (1 ng), or both RNAs (1 ng each) were injected into ventral vegetal blastomeres of eight cell stage embryos. Coinjection of Smad2 and Smad6produces secondary axes (red arrows) that are more complete than those obtained by injection of either Smad alone. Anterior is attop. (D) Smad6 neuralizes ectodermal explants. Smad6 and Smad6 C-domain RNAs were injected at the indicated amount in the animal pole of two-cell stage embryos. At the blastula stage, animal caps were dissected and cultured in saline solution. At the tailbud stages (stage 22), total RNA was harvested and analyzed by RT–PCR for the presence of the indicated transcripts. Full-length human Smad6 (0.5 and 4 ng) induces NRP-1, a pan–neural marker, andXAG-1, a marker of cement gland. Cement gland is induced efficiently even at the lower dose; induction of neural tissue requires a higher dose of Smad6. The isolated C-domain RNA is a more potent inducer of both XAG-1 and NRP-1 than full-length Smad6. Neither construct induced muscle actin, a marker of dorsal (paraxial) mesoderm. EF-1α, ubiquitously expressed, is a loading control. RNA from whole embryos (Embryo) provides the positive control. The RT lane is identical to the embryo lane, except that reverse transcriptase was omitted. (E) Smad6 interferes with blood induction by a constitutively active BMP receptor. BMPR-IB(QD) RNA (1–1000 pg) was injected either alone or together with Smad6 RNA (250 pg) in the animal pole.BMPR-IB(QD)-injected ectodermal explants show induction ofglobin at stage 30, and this response is blocked by coexpressed Smad6.
	Figure 3.  Smad6 specifically inhibits the BMP/Smad1 signaling pathway. (A) Smad6 blocks induction of ventral mesoderm by Smad1 but not induction of dorsal mesoderm by Smad2. An increasing amount of Smad6 RNA (0.5–4 ng) was coinjected with or without a fixed amount of Smad1 or Smad2 RNA (2 ng). Animal caps from injected embryos were collected at gastrula stage (stage 11.5) and subjected to RT–PCR. Xvent-1 andXhox-3 are markers of ventral mesoderm; goosecoid is a dorsal mesoderm marker and brachyury is a pan–mesodermal marker. (B) BMP-dependent transcriptional activation of GAL4–Smad1 fusion protein is blocked by Smad6. R-1B/L17 cells were transfected with the reporter gene (G1E1BCAT, 1 μg), appropriate receptors (TβR-I for TGFβ or BMPR–IB and BMPR–II for BMP2), and either a vector containing the GAL4 DNA-binding domain (DBD) alone or fusion constructs of DBD with Smad1 or Smad2 in the presence or absence of Smad6 (2 μg). Cells were incubated with or without 5 nM BMP2 (B) or 100 pM TGFβ (T) for 18 hr. CAT activity is expressed as the mean ± S.D. of three independent experiments. (C) TGFβ or activin-induced transcriptional activation of the 3TP promoter is not inhibited by Smad6. R-1B/L17 cells were transfected with the reporter gene (p3TP–lux) and type I receptors for TGFβ (TβR-l) or activin (ActR-IB) and were treated with or without 100 pM TGFβ (top) or 2 nM activin (bottom) for 18 hr. The ratios of stimulated to unstimulated levels of luciferase activity are indicated numerically, and their quotients plotted in the bar graph. Data are the mean ± S.D. of triplicate values. Notice that although Smad6 transfection decreased the basal as well as the agonist-induced levels of luciferase activity, it did not decrease the relative induction by TGFβ or activin.
	Figure 4.  Smad6 is a phosphoprotein which binds to type I receptors nonspecifically and inhibits downstream of BMP receptors. (A) Smad6 nonspecifically interacts with type I receptors of the TGFβ superfamily. COS cells were transfected withFlag-tagged Smad6 and HA-tagged wild-type or constitutively active type I receptor (QD and TD) for BMPs (BMPR-IB), TGFβ (TβR-I), or activins (ActR–IB). Cell lysates were subjected to anti-Flag immunoprecipitation using a monoclonal antibody followed by immunoblotting using anti-HA polyclonal antibody (toppanel). Immunoprecipitates of cells transfected with receptor alone did not contain specific proteins (data not shown). Similar levels of Smad6 and receptor expression were confirmed by analyzing aliquots of total cell lysate by SDS-PAGE followed by immunoblotting (middle and bottom panels). (B) The level of phosphorylation of Smad6 is unchanged by BMP2 or TGFβ stimulation. R-1B/L17 cells were transiently transfected with an empty vector (pCMV5) or with theFlag-tagged Smads indicated at the bottom. Smad2- or Smad6-transfected cells (lanes 2–5) were cotransfected with TβR-I; Smad1- or Smad6-transfected cells (lanes 6–9) were cotransfected with BMPR-IB and BMPR-II. Cells were labeled with [32P]phosphate, stimulated with (+) or without (−) 100 pM TGFβ or 5 nM BMP2 for 20 min.Flag-tagged Smads were purified by immunoprecipitation with anti-Flag M2 antibody and analyzed by SDS-PAGE and autoradiography. (C) Smad6 inhibits receptor-independent Smad1 signaling. Expression of dominant-negative BMP type I receptor (tBMPR-IA, 1 ng of RNA) induces neural tissue (NRP-1 marker) in Xenopus animal caps. Smad1 (1 ng of RNA) prevents neuralization when coexpressed with tBMPR-IA, but its activity is inhibited by Smad6 and Smad6(C) (2 ng of RNA).EF1α is the loading control.
	Figure 5.  Smad6 interact with itself and Smad1, but not with Smad2 or Smad4. (A) Specific interaction between Smad1 and Smad6. COS cells were transiently transfected with Flag-tagged Smads as indicated on the top, and HA-tagged Smad6 (toppanel) or Smad6 C-domain (bottom panel). Cells were treated with 5 nM BMP2 or 100 pM TGFβ for 1 hr before harvest. Cell lysates were subjected to immunoprecipitation with anti-Flag antibody and then immunoblotting using the anti-HA monoclonal antibody 12CA5. Expression of Smads was measured by anti-Flag or anti-HA immunoprecipitation of aliquots of cell lysates, followed by anti-Flag or anti-HA immunoblotting (bottompanel). [Ig(H) and Ig(L)] Immunoglobulin heavy and light chain bands, respectively. (B) The interaction between Smad6 C-domain and Smad1 C-domain is specific and direct. The C-domains of Smad1, Smad2, Smad4, and Smad6 fused to the GAL4 activation domain (GAD) were tested for interaction with Smad6 C-domain fused to the LexA DNA-binding domain in yeast. Interaction was monitored by the β-galactosidase assay, which allowed us to score for association by the presence of blue color.

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