Characterization of functional domains within Smad4/DPC4

Smad proteins are a family of highly conserved, intracellular proteins that signal cellular responses downstream of transforming growth factor-β (TGF-β) family serine/threonine kinase receptors. One of these molecules, Smad4, originally identified as the candidate tumor suppressor gene dpc-4, reconstitutes TGF-β- and activin-dependent transcriptional responses in Smad4 null cell lines and interacts in a ligand-dependent manner with other Smad family members in both TGF-β, activin, and bone morphogenetic protein- 2/-4 pathways. Here, we used an assay based on the restoration of ligand- dependent transcriptional responses in a Smad4 null cell line to characterize functional domain structures within Smad4. We showed that restoration of TGF- β-induced transcriptional responses by Smad4 was inhibited by co- transfection with a kinase dead TGF-β type II receptor and that constitutive activation was blocked with TGF-β neutralizing antibodies, confirming the essential role of Smad4 in TGF-β signaling. Using a series of Smad4 mutation, deletion, and Smad1/Smad4 chimera constructs we identified a 47- amino acid deletion within the middle-linker region of Smad4 that is essential for the mediation of signaling responses. In addition, we showed that the NH₂-terminal domain of Smad4 augments ligand-dependent activation associated with the middle-linker region, indicating that there is a distinct ligand-response domain within the N terminus of this molecule.