Guo, Zhengguang Song, Eli Ma, Sucan Wang, Xiaorong Gao, Shijuan Shao, Chen Hu, Siqi Jia, Lulu Tian, Rui Xu, Tao Gao, Youhe Proteomics Strategy to Identify Substrates of LNX, a PDZ Domain-containing E3 Ubiquitin Ligase Ubiquitin ligases (E3s) confer specificity to ubiquitination by recognizing target substrates. However, the substrates of most E3s have not been extensively discovered, and new methods are needed to efficiently and comprehensively identify these substrates. Mostly, E3s specifically recognize substrates via their protein interaction domains. We developed a novel integrated strategy to identify substrates of E3s containing protein interaction domains on a proteomic scale. The binding properties of the protein interaction domains were characterized by screening a random peptide library using a yeast two-hybrid system. Artificial degrons, consisting of a preferential ubiquitination sequence and particular interaction domain-binding motifs, were tested as potential substrates by <i>in vitro</i> ubiquitination assays. Using this strategy, not only substrates but also nonsubstrate regulators can be discovered. The detailed substrate recognition mechanisms, which are useful for drug discovery, can also be characterized. We used the Ligand of Numb protein X (LNX) family of E3s, a group of PDZ domain-containing RING-type E3 ubiquitin ligases, to demonstrate the feasibility of this strategy. Many potential substrates of LNX E3s were identified. Eight of the nine selected candidates were ubiquitinated <i>in vitro</i>, and two novel endogenous substrates, PDZ-binding kinase (PBK) and breakpoint cluster region protein (BCR), were confirmed <i>in vivo</i>. We further revealed that the LNX1-mediated ubiquitination and degradation of PBK inhibited cell proliferation and enhanced sensitivity to doxorubicin-induced apoptosis. The substrate recognition mechanism of LNX E3s was also characterized; this process involves the recognition of substrates via their specific PDZ domains by binding to the C-termini of the target proteins. This strategy can potentially be extended to a variety of E3s that contain protein interaction domain(s), thereby serving as a powerful tool for the comprehensive identification of their substrates on a proteomic scale. Numb protein X;substrate recognition mechanisms;PBK;E 3s;breakpoint cluster region protein;BCR;LNX E 3s;PDZ;proteomic scale;substrate recognition mechanism;protein interaction domains 2012-10-05
    https://acs.figshare.com/articles/dataset/Proteomics_Strategy_to_Identify_Substrates_of_LNX_a_PDZ_Domain_containing_E3_Ubiquitin_Ligase/2481076
10.1021/pr300674c.s002