Smad1 Interacts with Homeobox DNA-binding Proteins in Bone Morphogenetic Protein Signaling

• Published in: The Journal of biological chemistry Vol. 274; no. 19; pp. 13711 - 13717
• Main Authors: Shi, Xingming, Yang, Xiangli, Chen, Di, Chang, Zhijie, Cao, Xu
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 07.05.1999; American Society for Biochemistry and Molecular Biology
• Subjects: Animals; Base Sequence; Bone Morphogenetic Proteins - metabolism; COS Cells; DNA Primers; DNA-Binding Proteins - metabolism; Homeodomain Proteins - metabolism; Mice; Mice, Inbred C3H; Protein Binding; Signal Transduction; Simian virus 40; Smad Proteins; Smad1 Protein; Trans-Activators - metabolism; Transcription, Genetic
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.274.19.13711

Bone morphogenetic proteins (BMP) transduce their signals into the cell through a family of mediator proteins known as Smads. Upon phosphorylation by the BMP receptors, Smad1 interacts with Smad4 and translocates into the nucleus where the complex recruits DNA-binding protein(s) to activate specific gene transcription. However, the DNA-binding protein(s) involved in BMP signaling has not been identified. Using a yeast two-hybrid approach, we found that Smad1 interacts with Hoxc-8, a homeodomain transcription factor. The interaction between Smad1 and Hoxc-8 was confirmed by a “pull-down” assay and a co-immunoprecipitation experiment in COS-1 cells. Interestingly, purified Smad1 inhibited Hoxc-8 binding to the osteopontin Hoxc-8 site in a concentration-dependent manner. Transient transfection studies showed that native osteopontin promoter activity was elevated upon BMP stimulation. Consistent with the gel shift assay, overexpression of Hoxc-8 abolished the BMP stimulation. When a wild type or mutant Hoxc-8 binding element was linked to an SV40 promoter-driven reporter gene, the wild type but not the mutant Hoxc-8 binding site responded to BMP stimulation. Again, overexpression of Hoxc-8 suppressed the BMP-induced activity of the wild type reporter construct. Our findings suggest that Smad1 interaction with Hoxc-8 dislodges Hoxc-8 from its DNA binding element, resulting in the induction of gene expression.