Novel Cul3 binding proteins function to remodel E3 ligase complexes

BMC Cell Biology

Research Article

Mark West¹  Brittney Davidge¹  Jeffrey D Singer¹  Kebing Yu²  Arthur Salomon³  Wananit Wimuttisuk⁴ 

[1] Department of Biology, Portland State University, Portland, Oregon, USA;Department of Chemistry, Brown University, Providence, RI, USA;Department of Molecular Biology, Cell Biology and Biochemistry, the Center for Genomics and Proteomics Brown University, Providence, RI, USA;Department of Molecular Biology, Cell Biology and Biochemistry, the Center for Genomics and Proteomics Brown University, Providence, RI, USA;National Center for Genetic Engineering and Biotechnology (BIOTEC), Bangkok, Thailand;

关键词: Cullin3;    Tandem-affinity purification;    BTB domain-containing protein;    BCR ubiquitin ligase complex;    Mass spectrometry;    E3 ubiquitin ligase;    Protein purification;    Ubiquitin;    Ubiquitin ligase;   

DOI  :  10.1186/1471-2121-15-28

 received in 2013-11-05, accepted in 2014-07-02,  发布年份 2014

来源: Springer

【 摘 要 】

BackgroundCullins belong to a family of scaffold proteins that assemble multi-subunit ubiquitin ligase complexes to recruit protein substrates for ubiquitination via unique sets of substrate adaptor, such as Skp1 or Elongin B, and a substrate-binding protein with a conserved protein-protein interacting domain, such as l eucine-r ich r epeats (LRR), a WD40 domain, or a zinc-finger domain. In the case of the Cullin3 (Cul3), it forms a B TB-C ul3-R bx1 (BCR) ubiquitin ligase complex where it is believed that a BTB domain-containing protein performs dual functions where it serves as both the substrate adaptor and the substrate recognition protein.ResultsTandem affinity purification and LC/MS-MS analysis of the BCR complex led to the identification of 10,225 peptides. After the SEQUEST algorithm and CDART program were used for protein identification and domain prediction, we discovered a group of C ul3-bound proteins that contain either the L RR or W D40 domain (CLWs). Further biochemical analysis revealed that the LRR domain-containing CLWs could bind both Cul3 and BTB domain-containing proteins. The dual binding role for the LRR domain-containing CLWs results in causing the BTB-domain protein to become a substrate instead of an adaptor.To further distinguish potential substrates from other components that are part of the BCR ubiquitin ligase complex, we altered the parameters in the SEQUEST algorithm to select for peptide fragments with a modified lysine residue. This method not only identifies the potential substrates of the BCR ubiquitin ligase complex, but it also pinpoints the lysine residue in which the post-translational modification occurs. Interestingly, none of the CLWs were identified by this method, supporting our hypothesis that CLWs were not potential substrates but rather additional components of the BCR ubiquitin ligase complex.ConclusionOur study identified a new set of Cul3-binding proteins known as CLWs via tandem affinity purification and LC/MS-MS analysis. Subsequently, our biochemical analysis revealed that some CLWs modify binding of BTB domain-containing proteins to the complex, causing degradation of the BTB domain-containing protein. As these CLWs were excluded from our list of substrates, we propose that CLWs serve as unique Cul3 binding proteins that provide an alternative regulatory mechanism for the complex.

【 授权许可】

Unknown   
© Wimuttisuk et al.; licensee BioMed Central Ltd. 2014. This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.