Comprehensive trapping of polyubiquitinated proteins using the UIM peptide of ASB2a.

Junya Kohroki, Sakiko Kuroda, Eiko Takiguchi, Takaaki Nakamura, Takehiro Nishiyama, Kenta Tsuzuranuki, Takao Kawakami, Yasuhiko Masuho

Faculty of Pharmaceutical Sciences, Tokyo University of Science, Chiba 278-8510, Japan.

Biochemical and biophysical research communications 2011 Oct 22

filter terms:

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. Copyright © 2011 Elsevier Inc. All rights reserved.

Citation

Junya Kohroki, Sakiko Kuroda, Eiko Takiguchi, Takaaki Nakamura, Takehiro Nishiyama, Kenta Tsuzuranuki, Takao Kawakami, Yasuhiko Masuho. Comprehensive trapping of polyubiquitinated proteins using the UIM peptide of ASB2a. Biochemical and biophysical research communications. 2011 Oct 22;414(2):292-7