ELL Protein-associated Factor 2 (EAF2) Inhibits Transforming Growth Factor β Signaling through a Direct Interaction with Smad3*

Background: EAF2 plays an important role in affecting cellular processes, but the mechanisms underlying its effects are poorly understood. Results: EAF2 was identified as a novel repressor of TGF-β signaling through a direct interaction with Smad3. Conclusion: EAF2 specifically interacts with Smad3 to prevent Smad3 nuclear translocation, Smad4-Smad3 complex formation, and p300-Smad3 complex formation. Significance: EAF2 may function by inhibiting TGF-β signaling. A series of in vitro and in vivo studies has shown that EAF2 can affect multiple signaling pathways involved in cellular processes. However, the molecular mechanisms underlying its effects have remained elusive. Here we report the discovery of a new functional link between EAF2 and TGF-β signaling. Promoter reporter assays indicated that EAF2 suppresses Smad3 transcriptional activity, resulting in inhibition of TGF-β signaling. Coimmunoprecipitation assays showed that EAF2 specifically interacts with Smad3 in vitro and in vivo but not with other Smad proteins. In addition, we observed that EAF2 binding does not alter Smad3 phosphorylation but causes Smad3 cytoplasmic retention, competes with Smad4 for binding to Smad3, and prevents p300-Smad3 complex formation. Furthermore, we demonstrated that EAF2 suppresses both TGF-β-induced G1 cell cycle arrest and TGF-β-induced cell migration. This study identifies and characterizes a novel repressor of TGF-β signaling.