TGF-beta-induced phosphorylation of Smad3 regulates its interaction with coactivator p300/CREB-binding protein

Smads are intermediate effector proteins that transduce the TGF-beta signal from the plasma membrane to the nucleus, where they participate in transac tivation of downstream target genes. We have shown previously that coactiva tors p300/CREB-binding protein are involved in TGF-beta-mediated transactiv ation of two Cdk inhibitor genes, p21 and p15. Here we examined the possibi lity that Smads function to regulate transcription by directly interacting with p300/CREB-binding protein. We show that Smad3 can interact with a C-te rminal fragment of p300 in a temporal and phosphorylation-dependent manner. TGF-beta-mediated phosphorylation of Smad3 potentiates the association bet ween Smad3 and p300, likely because of an induced conformational change tha t removes the autoinhibitory interaction between the N- and C-terminal doma ins of Smad3. Consistent with a role for p300 in the transcription regulati on of multiple genes, overexpression of a Smad3 C-terminal fragment causes a general squelching effect on multiple TGF-beta-responsive reporter constr ucts. The adenoviral oncoprotein E1A can partially block Smad-dependent tra nscriptional activation by directly competing for binding to p300. Taken to gether, these findings define a new role for phosphorylation of Smad3: in a ddition to facilitating complex formation with Smad4 and promoting nuclear translocation, the phosphorylation-induced conformational change of Smad3 m odulates its interaction with coactivators, leading to transcriptional regu lation.