title: Comprehensive trapping of polyubiquitinated proteins using the UIM peptide of ASB2a.
abstract: An alternative splicing variant of E3 ubiquitin ligase ASB2, termed ASB2a, has a distinct N-terminal sequence containing a ubiquitin-interacting motif (UIM) consensus sequence. Examination of the minimal essential region for binding to polyubiquitinated proteins indicated that the UIM consensus sequence (residues 26-41) alone is not enough, and that amino acids 12-41 from the N-terminus of ASB2a is essential for binding. ASB2a(12-41) peptide was chemically synthesized and coupled to Sepharose 4B via disulfide bonds. This ASB2a(12-41) peptide-coupled affinity resin bound both K48- and K63-linked polyubiquitinated proteins in cell lysates and comprehensively captured polyubiquitinated proteins, including polyubiquitinated β-catenin, I-κB, and EGF receptor, which were eluted with 2-mercaptoethanol under non-denaturing conditions. These results indicate that this UIM affinity purification (designated as ubiquitin-trapping) is a useful method to discover polyubiquitinated proteins and their associated proteins.
authors.affiliation: Faculty of Pharmaceutical Sciences, Tokyo University of Science, Chiba 278-8510, Japan.
authors.forename: Junya
authors.initials: J
authors.lastname: Kohroki
authors.forename: Sakiko
authors.initials: S
authors.lastname: Kuroda
authors.forename: Eiko
authors.initials: E
authors.lastname: Takiguchi
authors.forename: Takaaki
authors.initials: T
authors.lastname: Nakamura
authors.forename: Takehiro
authors.initials: T
authors.lastname: Nishiyama
authors.forename: Kenta
authors.initials: K
authors.lastname: Tsuzuranuki
authors.forename: Takao
authors.initials: T
authors.lastname: Kawakami
authors.forename: Yasuhiko
authors.initials: Y
authors.lastname: Masuho
mesh_terms: D000595:Amino Acid Sequence; D002846:Chromatography, Affinity / Q000379:methods; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000737:chemistry*; D012685:Sepharose / Q000737:chemistry; D050826:Suppressor of Cytokine Signaling Proteins / Q000737:chemistry* / Q000302:isolation & purification*; D025801:Ubiquitin / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases / Q000737:chemistry* / Q000302:isolation & purification*; D054875:Ubiquitination
chemical_list: C437987:ASB2 protein, human; D009842:Oligopeptides; D050826:Suppressor of Cytokine Signaling Proteins; D025801:Ubiquitin; D012685:Sepharose; D044767:Ubiquitin-Protein Ligases