title: Targeted functional analysis of p300 coactivator in Wnt/β-catenin signaling pathway using phosphoproteomic and biochemical approaches.
abstract: 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.
authors.affiliation: Children's Hospital Los Angeles, University of Southern California, CA 90033, USA.
authors.forename: Hong
authors.initials: H
authors.lastname: Ma
authors.forename: Mingquan
authors.initials: M
authors.lastname: Guo
authors.forename: Baoen
authors.initials: B
authors.lastname: Shan
authors.forename: Zanxian
authors.initials: Z
authors.lastname: Xia
mesh_terms: D002454:Cell Differentiation; D050881:E1A-Associated p300 Protein / Q000378:metabolism*; D006801:Humans; D047468:Immunoprecipitation; D032446:Myoblasts / Q000166:cytology; D051787:Myogenic Regulatory Factor 5 / Q000096:biosynthesis; D010766:Phosphorylation; D012694:Serine / Q000378:metabolism; D053719:Tandem Mass Spectrometry; D020798:Two-Hybrid System Techniques; D060449:Wnt Signaling Pathway / Q000502:physiology*; D051176:beta Catenin / Q000378:metabolism*; D048051:p38 Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism
chemical_list: C497417:MYF5 protein, human; D051787:Myogenic Regulatory Factor 5; D051176:beta Catenin; D012694:Serine; D050881:E1A-Associated p300 Protein; C494360:EP300 protein, human; D048051:p38 Mitogen-Activated Protein Kinases