The Smad4 activation domain (SAD) is a proline-rich, p300-dependent transcriptional activation domain

Transforming growth factor-p (TGF-P) family members signal through a unique set of intracellular proteins called Smads, Smad4, previously identified a s the tumor suppressor DPC4, is functionally distinct among the Smad family , and is required for the assembly and transcriptional activation of divers e, Smad-DNA complexes. We previously identified a 48-amino acid proline-ric h regulatory element within the middle linker domain of this molecule, the Smad4 activation domain (SAD), which is essential for mediating these signa ling activities. We now characterize the functional activity of the SAD. Mu tants lacking the SAD are still able to form complexes with other Smad fami ly members and associated transcription factors, but cannot activate transc ription in these complexes. Furthermore, the SAD itself is able to activate transcription in heterologous reporter assays, identifying it as a proline -rich transcriptional activation domain, and indicating that the SAD is bot h necessary and sufficient to activate Smad-dependent transcriptional respo nses. We show that transcriptional activation by the SAD is p300-dependent, and demonstrate that this activity is associated with a physical interacti on of the SAD with the amino terminus of p300, These data identify a novel function of the middle linker region of Smad4, and define the role of the S AD as an important locus determining the transcriptional activation of the Smad complex.