Different Smad2 partners bind a common hydrophobic pocket in Smad2 via a defined proline‐rich motif

Transforming growth factor‐β (TGF‐β)/activin‐induced Smad2/Smad4 complexes are recruited to different promoter elements by transcription factors, such as Fast‐1 or the Mix family proteins Mixer and Milk, through a direct interaction between Smad2 and a common Smad interaction motif (SIM) in the transcription factors. Here we identify residues in the SIM critical for Mixer–Smad2 interaction and confirm their functional importance by demonstrating that only Xenopus and zebrafish Mix family members containing a SIM with all the correct critical residues can bind Smad2 and mediate TGF‐β‐induced transcriptional activation in vivo. We identify significant sequence similarity between the SIM and the Smad‐binding domain (SBD) of the membrane‐associated protein SARA (Smad anchor for receptor activation). Molecular modelling, supported by mutational analyses of Smad2 and the SIM and the demonstration that the SARA SBD competes directly with the SIM for binding to Smad2, indicates that the SIM binds Smad2 in the same hydrophobic pocket as does the proline‐rich rigid coil region of the SARA SBD. Thus, different Smad2 partners, whether cytoplasmic or nuclear, interact with the same binding pocket in Smad2 through a common proline‐rich motif.