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 in vitro 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 in vitro, and two novel endogenous substrates, PDZ-binding kinase (PBK) and breakpoint cluster region protein (BCR), were confirmed in vivo. 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.
National Key Laboratory of Medical Molecular Biology, Department of Physiology and Pathophysiology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences/School of Basic Medicine, Peking Union Medical College, Beijing 100005, China.
Zhengguang
Z
Guo
Eli
E
Song
Sucan
S
Ma
Xiaorong
X
Wang
Shijuan
S
Gao
Chen
C
Shao
Siqi
S
Hu
Lulu
L
Jia
Rui
R
Tian
Tao
T
Xu
Youhe
Y
Gao
D000595:Amino Acid Sequence; D000903:Antibiotics, Antineoplastic / Q000494:pharmacology; D049109:Cell Proliferation; D002470:Cell Survival / Q000187:drug effects; D016384:Consensus Sequence; D004317:Doxorubicin / Q000494:pharmacology; D055785:Gene Knockdown Techniques; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D061986:MCF-7 Cells; D020929:Mitogen-Activated Protein Kinase Kinases / Q000378:metabolism; D058977:Molecular Sequence Annotation; D010446:Peptide Fragments / Q000737:chemistry; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D011499:Protein Processing, Post-Translational; D040901:Proteomics; D034622:RNA Interference; D020798:Two-Hybrid System Techniques; D044767:Ubiquitin-Protein Ligases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D054875:Ubiquitination
D000903:Antibiotics, Antineoplastic; D010446:Peptide Fragments; D004317:Doxorubicin; C484733:LNX1 protein, human; D044767:Ubiquitin-Protein Ligases; D020929:Mitogen-Activated Protein Kinase Kinases; C409022:PDZ-binding kinase