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.
Highlights
PDZ domains are protein interaction domains often found in molecules involved in signal transduction, cell polarity and synaptic transmission
The strongest similarity as measured by percent sequence identity for each of the LNX1 PDZ domains was with the corresponding PDZ domain in LNX2
We examined the sequence identity in the 17 residues identified as the binding site sequence between each LNX1 PDZ domain and the respective top three similar PDZ domains
Summary
PDZ domains are protein interaction domains often found in molecules involved in signal transduction, cell polarity and synaptic transmission. PDZ domains were first found to recognize the extreme carboxy-terminal tail of target molecules by recognizing the last few amino acids as well as the terminal carboxylate residue [1]. Proteins that contain PDZ domains often have multiple copies with distinct binding specificities and function as scaffolds in multiprotein complexes [5,6]. LNX1 and LNX2 are RING finger domain-containing E3 ubiquitin ligases that each contain four PDZ domains [7]. Murine Lnx mRNA is expressed in brain, heart, lung, skeletal muscle and kidney [8]. Murine Lnx is more widely expressed and mRNA is detected in liver, spleen, lung, heart, kidney, thymus, skeletal muscle and brain [10]. In a pair-wise yeast two-hybrid assay, LNX1 has been shown to be able to interact with itself and LNX2 [10]
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