Characterization of a bone morphogenetic protein-responsive Smad-binding element

Bone morphogenetic proteins (BMPs) are pleiotropic growth and differentiati on factors belonging to the transforming growth factor-beta (TGF-beta) supe rfamily. Signals of the TGF-beta-like ligands are propagated to the nucleus through specific interaction of transmembrane serine/threonine kinase rece ptors and Smad proteins. GCCGnCGC has been suggested as a consensus binding sequence for Drosophila Mad regulated by a BMP-like ligand, Decapentaplegi c. Smad1 is one of the mammalian Smads activated by BMPs. Here we show that Smad1 binds to this motif upon BMP stimulation in the presence of the comm on Smad, Smad4. The binding affinity is likely to be relatively low, becaus e Smad1 binds to three copies of the motif weakly, but more repeats of the motif significantly enhance the binding. Heterologous reporter genes (GCCG- Lux) with multiple repeats of the motif respond to BMP stimulation but not to TGF-beta or activin. Mutational analyses reveal several bases critical f or the responsiveness. A natural BMP-responsive reporter, pT1x-Lux, is acti vated by BMP receptors in P19 cells but not in mink lung cells. In contrast , GCCG-Lux responds to BMP stimulation in both cells, suggesting that it is a universal reporter that directly detects Smad phosphorylation by BMP rec eptors.