Characterization of the DNA-binding property of Smad5.
Activation of TGF-beta superfamily receptors leads to phosphorylation of Smad proteins which function as transcription factors to regulate gene expression. Previous studies have indicated that Smad5, together with Smad1 and Smad8, participates in signaling downstream of BMP receptors. To characterize the DNA-binding characteristics of Smad5, we used the GST-Smad5 N-terminal fusion protein to select for random oligonucleotide sequences that were able to binds the protein. As a result, we found that Smad5 is able to bind a consensus sequence TGTGC. We further used the Smad7 promoter sequence that contains a Smad-binding element (SBE), GTCTAGAC to determine how mutations in each nucleotide in the SBE affects the binding with Smad5, compared with the binding with Smad1, Smad2, Smad3, Smad4, and Smad8. Interestingly, Smad5, but not Smad1 and Smad8, was able to bind the SBE, at a level similar to the binding by Smad3 and Smad4. However, mutations at the SBE had different effect on the binding with Smad5, compared to that with Smad3 and Smad4. These studies suggest that even though Smad5 falls into the same subfamily with Smad1 and Smad8 in mediating the signaling by BMP receptors, it has an unique DNA-binding property that is similar to Smad3, which specifically transduces signaling for TGF-beta and activin receptors.
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Department of Medical and Molecular Genetics, Indiana University School of Medicine, Indianapolis, Indiana 46202, USA.
Chen
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Nagarajan
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Liu
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Chen
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D000818:Animals; D001483:Base Sequence; D052004:Bone Morphogenetic Protein Receptors; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D005982:Glutathione Transferase / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D010750:Phosphoproteins / Q000737:chemistry*; D010957:Plasmids / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D051381:Rats; D011956:Receptors, Cell Surface / Q000378:metabolism; D017978:Receptors, Growth Factor; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D015398:Signal Transduction; D051902:Smad5 Protein; D051906:Smad7 Protein; D015534:Trans-Activators / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D004268:DNA-Binding Proteins; D010750:Phosphoproteins; D011956:Receptors, Cell Surface; D017978:Receptors, Growth Factor; D011993:Recombinant Fusion Proteins; C497797:SMAD5 protein, human; C497806:SMAD7 protein, human; D051902:Smad5 Protein; C497799:Smad5 protein, rat; D051906:Smad7 Protein; C497808:Smad7 protein, rat; D015534:Trans-Activators; D005982:Glutathione Transferase; D052004:Bone Morphogenetic Protein Receptors