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Targeted functional analysis of p300 coactivator in Wnt/beta-catenin signaling pathway using phosphoproteomic and biochemical approaches

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机构： [1]Children's Hospital Los Angeles, University of Southern California, CA, USA [2]Keck School of Medicine, University of Southern California, CA, USA [3]Research Center, the Fourth Hospital of Hebei Medical University and Hebei Cancer Institute, Shijiazhuang 050011, China [4]School of Biological Science and Technology, Central South University, Changsha, Hunan 410078, China

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关键词： Collision-induced dissociation Electron transfer dissociation Mammalian two-hybrid assay p300 Phosphorylation Site-directed mutagenesis

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Both p300 and beta-catenin are transcriptional activators and phosphoproteins, and play a central role in Wnt/beta-catenin-dependent transcriptional regulation. The minimum beta-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 TiO2-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 beta-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/beta-catenin complex in myoblast differentiation are discussed. (C) 2012 Elsevier B.V. All rights reserved.

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出版当年[2012]版：

大类 | 2 区生物

小类 | 2 区生化研究方法

最新[2025]版：

大类 | 3 区生物学

小类 | 2 区生化研究方法

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出版当年[2012]版：

Q1 BIOCHEMICAL RESEARCH METHODS

最新[2024]版：

Q2 BIOCHEMICAL RESEARCH METHODS

影响因子： 2.8 最新[2024版] 3 最新五年平均 4.088 出版当年[2012版] 4.302 出版当年五年平均 4.878 出版前一年[2011版] 3.929 出版后一年[2013版]

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第一作者机构： [1]Children's Hospital Los Angeles, University of Southern California, CA, USA

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通讯机构： [2]Keck School of Medicine, University of Southern California, CA, USA [4]School of Biological Science and Technology, Central South University, Changsha, Hunan 410078, China [*1]Keck School of Medicine, University of Southern California, 1501 San Pablo Street, Los Angeles, CA 90033, USA. [*2]School of Biological Science and Technology, Central South University, Changsha, Hunan 410078, China.

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