Characterization of a bone morphogenetic protein-responsive Smad-binding element.
Bone morphogenetic proteins (BMPs) are pleiotropic growth and differentiation factors belonging to the transforming growth factor-beta (TGF-beta) superfamily. Signals of the TGF-beta-like ligands are propagated to the nucleus through specific interaction of transmembrane serine/threonine kinase receptors and Smad proteins. GCCGnCGC has been suggested as a consensus binding sequence for Drosophila Mad regulated by a BMP-like ligand, Decapentaplegic. 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 common Smad, Smad4. The binding affinity is likely to be relatively low, because 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 for the responsiveness. A natural BMP-responsive reporter, pTlx-Lux, is activated 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 receptors.
Kusanagi
K
K
Department of Biochemistry, The Cancer Institute of Japanese Foundation for Cancer Research, and Research for the Future Program, Japan Society for Promotion of Science, Tokyo 170-8455, Japan.
Inoue
H
H
Ishidou
Y
Y
Mishima
H K
HK
Kawabata
M
M
Miyazono
K
K
D028341:Activins; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D019485:Bone Morphogenetic Proteins / Q000494:pharmacology*; D002460:Cell Line; D016384:Consensus Sequence / Q000235:genetics; D004247:DNA / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D029721:Drosophila Proteins; D017930:Genes, Reporter / Q000235:genetics; D007265:Inhibins / Q000494:pharmacology; D019476:Insect Proteins / Q000494:pharmacology; D051379:Mice; D009154:Mutation / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012091:Repetitive Sequences, Nucleic Acid / Q000235:genetics; D020218:Response Elements / Q000235:genetics*; D015398:Signal Transduction / Q000187:drug effects; D051785:Smad Proteins; D051898:Smad1 Protein; D051901:Smad4 Protein; D015534:Trans-Activators / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014157:Transcription Factors; D015533:Transcriptional Activation / Q000187:drug effects*; D016212:Transforming Growth Factor beta / Q000494:pharmacology
D019485:Bone Morphogenetic Proteins; D004268:DNA-Binding Proteins; D029721:Drosophila Proteins; D019476:Insect Proteins; C112506:MAD protein, Drosophila; C112504:Med protein, Drosophila; D011993:Recombinant Fusion Proteins; D051785:Smad Proteins; D051898:Smad1 Protein; C497401:Smad1 protein, mouse; D051901:Smad4 Protein; C490810:Smad4 protein, mouse; D015534:Trans-Activators; D014157:Transcription Factors; D016212:Transforming Growth Factor beta; C064196:dpp protein, Drosophila; D028341:Activins; D007265:Inhibins; D004247:DNA