Ski acts as a co-repressor with Smad2 and Smad3 to regulate the response to type beta transforming growth factor

The c-ski protooncogene encodes a transcription factor that binds DNA only in association with other proteins. To identify co-binding proteins, we per formed a yeast two-hybrid screen. The results of the screen and subsequent co-immunoprecipitation studies identified Smad2 and Smad3, two transcriptio nal activators that mediate the type beta transforming growth factor (TGF-b eta) response, as Ski-interacting proteins. In Ski-transformed cells, all o f the Ski protein was found in Smad3-containing complexes that accumulated in the nucleus in the absence of added TGF-beta. DNA binding assays showed that Ski, Smad2, Smad3, and Smad4 form a complex with the Smad/Ski binding element GTCTAGAC (SBE). Ski repressed TGF-beta-induced expression of 3TP-Lu x, the natural plasminogen activator inhibitor 1 promoter and of reporter g enes driven by the SEE and the related CAGA element. In addition, Ski repre ssed a TGF-beta-inducible promoter containing AP-1 (TRE) elements activated by a combination of Smads, Fos, and/or Jun proteins. Ski also repressed sy nergistic activation of promoters by combinations of Smad proteins but fail ed to repress in the absence of Smad4. Thus, Ski acts in opposition to TCF- beta-induced transcriptional activation by functioning as a Smad-dependent co-repressor. The biological relevance of this transcriptional repression w as established by showing that overexpression of Ski abolished TGF-beta-med iated growth inhibition in a prostate-derived epithelial cell line.