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Physical and functional interaction of murine and Xenopus Smad7 with bone morphogenetic protein receptors and transforming growth factor-beta receptors.
Souchelnytskyi S
,
Nakayama T
,
Nakao A
,
Morén A
,
Heldin CH
,
Christian JL
,
ten Dijke P
.
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Members of the transforming growth factor-beta (TGF-beta) family transmit signals from membrane to nucleus via intracellular proteins known as Smads. A subclass of Smad proteins has recently been identified that antagonize, rather than transduce, TGF-beta family signals. Smad7, for example, binds to and inhibits signaling downstream of TGF-beta receptors. Here we report that the C-terminal MAD homology domain of murine Smad7 (mSmad7) is sufficient for both of these activities. In addition, we show that mSmad7 interacts with activated bone morphogenetic protein (BMP) type I receptors (BMPR-Is), inhibits BMPR-I-mediated Smad phosphorylation, and phenocopies the effect of known BMP antagonists when overexpressed in ventral cells of Xenopus embryos. Xenopus Smad7 (XSmad7, previously termed Smad8) and mSmad7 are nearly identical within their bioactive C-domain, but have quite distinct N-domains. We found that XSmad7, similar to mSmad7, interacted with BMP and TGF-beta type I receptors and inhibited receptor-mediated phosphorylation of downstream signal-transducing Smads. However, XSmad7 is a less efficient inhibitor of TbetaR-I-mediated responses in mammalian cells than is mSmad7. Furthermore, overexpression of XSmad7 in Xenopus embryos produces patterning defects that are not observed following overexpression of mSmad7, suggesting that mSmad7 and XSmad7 may preferentially target distinct signaling pathways. Our results are consistent with the possibility that the C-domain of antagonistic Smads is an effector domain whereas the N-domain may confer specificity for distinct signaling pathways.
Figure 1
mSmad7 interacts with BMP receptor complexes and inhibits BMP-mediated Smad phosphorylation. A, association of N-terminal Flag-tagged mSmad7 (F-mSmad7) with BMPR-IA and BMPBR-IB in complex with BMPR-II. COS cells were transfected with varioustype I receptors (tagged at C termini with hemaglutinin epitope) with either wild-type (W.T.) or kinase-deficient (K.R.) versions of BMPR-II. The BMP receptors were covalently affinity labeled with 125I-BMP-7, and cell lysates were subjected to immunoprecipitation with Flag antiserum. Equal expression of receptors after transfection was determined directly on aliquots of cell lysates (data not shown). B, mSmad7 inhibits BMPR-IB- and ActR-I-mediated phosphorylation of Smad1. COS cells were transfected with F-Smad1 alone or together with F-mSmad7 in the presence of type I receptors (BMPR-IB or ActR-I) and BMPR-II, after which cells were labeled with [32P]orthophosphate in the absence or presence of BMP-7 and immunoprecipitated with Flag antisera. Expression of F-Smad1 and F-mSmad7 was determined by immunoblotting with Flag on aliquots of cell lysates. C, the32P-label radioactivity associated with Smad1 inpanel B was quantified by using a FujiX Bio-Imager and plotted. D, mSmad7 inhibits BMPR-IB phosphorylation of Smad5. COS cells were transfected with F-Smad5 alone or together with F-mSmad7 in the presence of BMPR-IB and BMPR-II; cells were labeled with [32P]orthophosphate in the absence or presence of BMP-7 and subjected to immunoprecipitation with Flag antisera.E, the 32P-label radioactivity associated with Smad5 in panel D was quantified by using a FujiX Bio-Imager and plotted.
Figure 2
mSmad7 C-domain is sufficient for binding to TGF-β receptor complex and inhibition of TGF-β-induced responses. A, association of wild-type F-mSmad7 (WT) and F-mSmad7C (7C) with TGF-β receptor complex. COS cells were transfected with F-mSmad7 (WT or7C) in combination with TβR-I and TβR-II. The receptors were covalently affinity labeled with 125I-TGF-β1, and cell lysates were subjected to immunoprecipitation with Flag antiserum.B, mSmad7C inhibits TβR-I-mediated phosphorylation of Smad2. COS cells were transfected with F-Smad2 alone or together with F-mSmad7C or wild-type F-mSmad7 in the presence of BMPR-IB and BMPR-II; cells were labeled with [32P]orthophosphate and incubated in the absence or presence of BMP-7, and subjected to immunoprecipitation with Flag antisera. Expression of F-Smad2, F-mSmad7, and F-mSmad7C was determined by immunoblotting with Flag on aliquots of cell lysates.C, transfection of mSmad7C blocks TGF-β-induced p3TPLux transcriptional response but less efficient than wild-type mSmad7.
Figure 3
mSmad7 inhibits TGF-β-mediated signaling responses more effectively than XSmad7. (A) XSmad7 inhibits TβR-I-mediated phosphorylation of Smad2 less efficiently than mSmad7. Cells were transfected with F-Smad2 alone or together with F-mSmad7 or F-XSmad7 in the presence of TβR-I and TβR-II; cells were then incubated in the absence or presence of TGF-β. The level of F-Smad2 phosphorylation was determined by [32P]orthophosphate labeling and immunoprecipitation of cell lysates with Flag antisera. Expression of F-Smads was analyzed by Western blotting with Flag antiserum on aliquots of cell lysates. B, transfection of mSmad7 in Mv1Lu cells blocks the TGF-β-induced p3TPLux response more effectively than XSmad7.
Figure 4
Overexpression of mSmad7 in Xenopusembryos induces formation of a partial secondary axis and eye defects. Photomicrographs of control tadpole (A) and sibling tadpole (B) made to misexpress mSmad7 in ventral cells. Note induction of partial secondary dorsal axis (arrowheads). C and D, control (C) and mSmad7-RNA (D) injected sibling showing immunoreactive muscle in primary and secondary (arrowheads) axes; E, eye defects (arrowhead) and spina bifida (arrow) in an embryo made to overexpress XSmad7 in dorsal cells; F, fusion of eyes (arrowhead) in an embryo made to overexpress mSmad7 in dorsal cells.
