title: Functional analysis of novel phosphorylation sites of CREB-binding protein using mass spectrometry and mammalian two-hybrid assays.
abstract: In the Wnt/β-catenin pathway, p300/CBP (CREB-binding protein) is recruited by nuclear β-catenin to regulate a wide array of T-cell factor (TCF)-dependent gene expression. Previous studies have indicated that CBP/β-catenin complex-mediated transcription is critical for cell proliferation. Both CBP and β-catenin are phosphoproteins. The interaction domain has been mapped to the N-terminal region of CBP (amino acids 1-111) and the C-terminal region of β-catenin, but it is unclear whether phosphorylation on specific residues of these regions is required for the interaction. To address this unmet challenge, phosphoproteomic profile of the critical N-terminus of CBP was determined by utilizing TiO(2) affinity chromatography followed by LC-MS/MS analysis. Two unique and novel phosphorylation sites Ser77 and Ser92 were identified. Further studies aided by site-directed mutagenesis, immunoprecipitation and mammalian two-hybrid assay have concluded that the phosphorylation of a Proline-directed Ser92 residue modulates the selective binding ability of CBP with β-catenin. The specific Mitogen-activated protein kinase inhibitor PD98059, which promotes cell cycle G1 arrest, concomitantly inhibits the interaction, and the evidences suggest that the MEK/ERK (extracellular signal-regulated kinase) cascade activation is the upstream signal required for Ser92 phosphorylation, leading to enhancement of the interaction of CBP with β-catenin.
authors.affiliation: Keck School of Medicine, University of Southern California, Los Angeles, CA, USA.
authors.forename: Zanxian
authors.initials: Z
authors.lastname: Xia
authors.forename: Mingquan
authors.initials: M
authors.lastname: Guo
authors.forename: Hong
authors.initials: H
authors.lastname: Ma
mesh_terms: D000818:Animals; D050882:CREB-Binding Protein / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002453:Cell Cycle; D002454:Cell Differentiation; D002460:Cell Line; D013058:Mass Spectrometry; D051379:Mice; D016297:Mutagenesis, Site-Directed; D010710:Phosphates / Q000032:analysis; D010766:Phosphorylation; D060066:Protein Interaction Maps; D020798:Two-Hybrid System Techniques; D051176:beta Catenin / Q000378:metabolism
chemical_list: D010710:Phosphates; D051176:beta Catenin; D050882:CREB-Binding Protein