Targeted functional analysis of p300 coactivator in Wnt/β-catenin signaling pathway using phosphoproteomic and...

Both p300 and β-catenin are transcriptional activators and phosphoproteins, and play a central role in Wnt/β-catenin-dependent transcriptional regulation. The minimum β-catenin binding domain of p300 has been mapped to the N-terminus 1-111 amino acids. Here, we performed phosphoproteomic analysis for the critical binding region using LC-MS/MS approach to investigate potential phosphosites that may affect the binding affinity. By implementing TiO(2)-based phosphopeptide affinity purification followed by LC-MS/MS analysis with both collision-induced dissociation (CID) and electron transfer dissociation (ETD) methods, two unique phosphosites Ser12 and Ser89 were identified, of which, phosphorylation at Ser12 is novel. Functional studies aided by site-directed mutagenesis, co-immunoprecipitation and mammalian two-hybrid assay have concluded that phosphorylation at Ser12 critically mediates the binding ability of p300 with β-catenin. Further studies utilizing specific MAPK inhibitors suggest that the p38 MAPK activation is the upstream signal required for Ser12 phosphorylation. The transcriptional roles of p300/β-catenin complex in myoblast differentiation are discussed.