%0 Generic
%A Guo, Zhengguang
%A Song, Eli
%A Ma, Sucan
%A Wang, Xiaorong
%A Gao, Shijuan
%A Shao, Chen
%A Hu, Siqi
%A Jia, Lulu
%A Tian, Rui
%A Xu, Tao
%A Gao, Youhe
%D 2012
%T Proteomics Strategy to
Identify Substrates of LNX,
a PDZ Domain-containing E3 Ubiquitin Ligase
%U https://acs.figshare.com/articles/dataset/Proteomics_Strategy_to_Identify_Substrates_of_LNX_a_PDZ_Domain_containing_E3_Ubiquitin_Ligase/2481076
%R 10.1021/pr300674c.s002
%2 https://acs.figshare.com/ndownloader/files/4123765
%K Numb protein X
%K substrate recognition mechanisms
%K PBK
%K E 3s
%K breakpoint cluster region protein
%K BCR
%K LNX E 3s
%K PDZ
%K proteomic scale
%K substrate recognition mechanism
%K protein interaction domains
%X 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.
%I ACS Publications