Biochemical and Computational Analysis Of LNX1 Interacting Proteins

Abstract

PDZ (Post-synaptic density, 95 kDa, Discs large, Zona Occludens-1) domains are protein interaction domains that bind to the carboxy-terminal amino acids of binding partners, heterodimerize with other PDZ domains, and also bind phosphoinositides. PDZ domain containing proteins are frequently involved in the assembly of multi-protein complexes and clustering of transmembrane proteins. LNX1 (Ligand of Numb, protein X 1) is a RING (Really Interesting New Gene) domain-containing E3 ubiquitin ligase that also includes four PDZ domains suggesting it functions as a scaffold for a multi-protein complex. Here we use a human protein array to identify direct LNX1 PDZ domain binding partners. Screening of 8,000 human proteins with isolated PDZ domains identified 53 potential LNX1 binding partners. We combined this set with LNX1 interacting proteins identified by other methods to assemble a list of 220 LNX1 interacting proteins. Bioinformatic analysis of this protein list was used to select interactions of interest for future studies. Using this approach we identify and confirm six novel LNX1 binding partners: KCNA4, PAK6, PLEKHG5, PKC-alpha1, TYK2 and PBK, and suggest that LNX1 functions as a signalling scaffold.

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

“…These proteomic results provide confirmation of just four of the approximately 250 previously reported LNX interactions (BCR, CTNND2, ERC2, KRT15) [ 13,14 ]. This partly reflects our focus on just the second PDZ domain and the fact that previously reported proteins may not be expressed in P16 mouse brain tissue.…”

“…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.…”

Lnx2b, an E3 ubiquitin ligase, in dorsal forerunner cells and Kupffer's vesicle is required for specification of zebrafish left–right laterality

“…Through high-throughput screening, several putative interacting partners of LNX were identified including Nkd2, an Nkd1 homologue (Wolting et al 2011;Guo et al 2012). Nkd2, much like Nkd1, inhibits Wnt/beta-catenin signaling by binding to and destabilizing Disheveled (Dvl), a highly conserved scaffold protein family governing cell fate and cell polarity (Wharton et al 2001;Yan et al 2001;Schneider et al 2010).…”