Abstract

Smad family members are newly identified essential intracellular signalling components of the transforming growth factor- (TGF-) superfamily. Smad2 and Smad3 are structurally highly similar and mediate TGF- signals. Smad4 is distantly related to Smads 2 and 3, and forms a heteromeric complex with Smad2 after TGF- or activin stimulation. Here we show that Smad2 and Smad3 interacted with the kinase-deficient TGF- type I receptor (TR)-I after it was phosphorylated by TR-II kinase. TGF-1 induced phosphorylation of Smad2 and Smad3 in Mv1Lu mink lung epithelial cells. Smad4 was found to be constitutively phosphorylated in Mv1Lu cells, the phosphorylation level remaining unchanged upon TGF-1 stimulation. Similar results were obtained using HSC4 cells, which are also growth-inhibited by TGF-. Smads 2 and 3 interacted with Smad4 after TR activation in transfected COS cells. In addition, we observed TR-activation-dependent interaction between Smad2 and Smad3. Smads 2, 3 and 4 accumulated in the nucleus upon TGF-1 treatment in Mv1Lu cells, and showed a synergistic effect in a transcriptional reporter assay using the TGF--inducible plasminogen activator inhibitor-1 promoter. Dominant-negative Smad3 inhibited the transcriptional synergistic response by Smad2 and Smad4. These data suggest that TGF- induces heteromeric complexes of Smads 2, 3 and 4, and their concomitant translocation to the nucleus, which is required for efficient TGF- signal transduction.