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-beta (TGF-beta) family serine/threonine kinase receptors. One of these molecules, Smad4, originally identified as the candidate tumor suppressor gene dpc-4, reconstitutes TGF-beta- 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-beta, 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-beta-induced transcriptional responses by Smad4 was inhibited by co-transfection with a kinase dead TGF-beta type II receptor and that constitutive activation was blocked with TGF-beta neutralizing antibodies, confirming the essential role of Smad4 in TGF-beta 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.
de Caestecker
M P
MP
Laboratory of Chemoprevention, NCI, National Institutes of Health, Bethesda, Maryland 20892-5055, USA. decaestm@dce41.nci.nih.gov
Hemmati
P
P
Larisch-Bloch
S
S
Ajmera
R
R
Roberts
A B
AB
Lechleider
R J
RJ
D001665:Binding Sites; D002460:Cell Line; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism; D016147:Genes, Tumor Suppressor; D006801:Humans; D008024:Ligands; D010449:Peptide Mapping; D012333:RNA, Messenger / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D015398:Signal Transduction; D051785:Smad Proteins; D051898:Smad1 Protein; D051901:Smad4 Protein; D015534:Trans-Activators / Q000737:chemistry* / Q000378:metabolism; D016212:Transforming Growth Factor beta / Q000378:metabolism; D014407:Tumor Cells, Cultured
D004268:DNA-Binding Proteins; D008024:Ligands; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; C497399:SMAD1 protein, human; C497796:SMAD4 protein, human; D051785:Smad Proteins; D051898:Smad1 Protein; D051901:Smad4 Protein; D015534:Trans-Activators; D016212:Transforming Growth Factor beta