Proteomics Strategy to Identify Substrates of LNX, a PDZ Domain-containing E3 Ubiquitin Ligase

Abstract

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.

Decreased Anxiety-Related Behaviour but Apparently Unperturbed NUMB Function in Ligand of NUMB Protein-X (LNX) 1/2 Double Knockout Mice

“…Nevertheless, given the absence of obvious NUMBrelated abnormalities in DKO mice, the possibility that LNX functions in the CNS are mediated by interacting proteins other than NUMB needs to be considered. Many LNX1 interacting proteins have been identified [ 13,14 ]. Most of these are PDZ domain ligands, with the second PDZ domain mediating a large proportion of these interactions.…”

“…While many LNXinteracting proteins are known, most were found by yeast twohybrid assays and protein/peptide arrays, and only a minority of these have been confirmed in mammalian cells using full length proteins. Noting that a large proportion of previously reported LNX1 and LNX2 interactions involve their second PDZ domain [ 13,14 ], we reasoned that analysis of PDZ2 may thus be sufficient to capture a significant fraction of all LNX1 and LNX2interacting proteins. To compare the range of ligands that bind LNX1 and LNX2 PDZ2 in a neural context, we purified recombinant GST tagged PDZ2 domains and used these proteins to 'pull down' interacting proteins from mouse brain lysates.…”

“…PDZ domains function as protein-protein interaction modules, most commonly binding to the carboxyltermini of other proteins. Wolting et al [ 13 ] catalogued around 220 LNXinteracting proteins both from their own work and the published literature, while a subsequent study by Guo et al [ 14 ] added approximately 30 additional proteins to this list. Most of these interactions are PDZ domainmediated and were identified using either yeast twohybrid assays or arrays of PDZ domains and PDZbinding motifs.…”

“…To date, only a small number of the described LNXinteracting proteins have been shown to be substrates for ubiquitination by LNX. For example, ubiquitination of cSrc and PDZbinding kinase (PBK) by LNX1 targets them for proteasomal degradation [ 8,14 ], while LNXmediated ubiquitination of CLAUDINS and CD8α appears to cause their internalization from the cell surface, via endocytic pathways [ 15,16 ]. Nevertheless, these examples indicate that the ubiquitin ligase activity of LNX proteins can be targeted to specific substrates via PDZ mediated interactions.…”

Rapid development of proteomics in China: from the perspective of the Human Liver Proteome Project and technology development

“…However, the substrates of most E3s have not been extensively explored. Gao YouHe and his colleagues [54] developed a proteomics tool to identify E3-specific substrates by affinity purification with protein interaction domains recognizable by E3. They also verified the ubiquitination features using an in vitro assay.…”