RAP250 Is a Coactivator in the Transforming Growth Factor β Signaling Pathway That Interacts with Smad2 and Smad3

RAP250 is a coactivator for nuclear receptors as well as other transcription factors. Recent studies have established RAP250 as an essential coactivator for many important biological processes, but its exact mechanism of action is not fully understood. To identify novel proteins that can associate with RAP250, we used a yeast two-hybrid system to screen cDNA libraries and identified the intracellular mediators of transforming growth factor-β (TGF-β) response Smad2 and Smad3 as direct interacting proteins. We show that the interaction between RAP250 and Smad2/3 is dependent upon the second LXXLL interaction motif in RAP250 and the MH2 domain in Smad2 and Smad3. Mouse embryonic fibroblasts lacking RAP250 have reduced expression of the TGF-β target gene PAI-1 after stimulation by TGF-β when compared with wild type cells. Furthermore, we demonstrate a cross-talk between TGF-β and liver X receptors (LXR) signaling pathways and show that stimulation of cells with TGF-β and LXR agonists have a synergistic effect on the expression of the LXR target gene ABCG1. Our data identify RAP250 as a new coactivator in the TGF-β signaling pathway that binds Smad2 and Smad3. Our data also suggest that the interaction between RAP250, Smad2, and Smad3 constitutes an important bridging mechanism linking LXR and TGF-β signaling pathways.