Structural and functional characterization of the transforming growth factor-beta -induced Smad3/c-Jun transcriptional cooperativity

Smads are intracellular proteins that act as central effectors for transforming growth factor-beta (TGF-beta) and related proteins from the activated receptor into the nucleus, where they regulate ligand-induced gene expression. AP-1 binding sites have been functionally linked to the transcriptional activation of various genes in response to TGF-beta. Accordingly, we have previously shown that the heteromeric complex of SMAD3 and SMAD4 synergizes with c-Jun/c-Fos at the AP-1 binding site of the collagenase I promoter to induce transcriptional activation in response to TGF-beta. Using the collagenase I promoter as model system, we have now investigated the role of the c-Jun and SMAD3 interactions with the promoter DNA and have further characterized the physical basis of the c-Jun/SMAD3 interaction in the transcriptional response. Mutational analyses of the c-Jun protein and the AP-1 binding site in the promoter revealed that the interaction of c-Jun with DNA is necessary for transcriptional activation by TGF-beta and SMAD3. Similar analyses of SMAD3 and the Smad binding sites revealed that binding of SMAD3 to DNA is also required, but that its DNA sequence-specific recognition is not essential. We also found that the basic leucine zipper domain of c-Jun and a short sequence close to the N terminus of SMAD3 mediate their physical interaction, and that these regions are critical for their DNA-binding function. Our studies provide a basis for understanding the functional cooperativity of Smads with the diversity of transcription factors, which underlies the Smad-induced transcriptional activation in response to TGF-beta and related factors.