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.

Figure 1. Schematic diagram of protein domains and carboxy terminal residues of LNX1 and LNX2.

Percentages represent amino acid sequence identity between corresponding PDZ domains.

Figure 2. Two Invitrogen ProtoArrays® v4 were incubated with a mixture of the following Alexa647-fluorescently labelled proteins: 0.33 ug/mL LNX1 PDZ2, 0.5 ug/mL LNX1 PDZ3 and 0.2 ug/mL LNX1 PDZ4Δ3C.

(A,B) A sample grid (grid #43) is shown from each of the two arrays. Reference spots for grid identification are outlined in thin white rectangles. Thick yellow rectangles indicate LNX1 interactors identified on both protein arrays: BC004233.1 28AA in row 6 columns 3 and 4 and NM_172160.1 KCNAB1 in row 14 columns 3 and 4. (B) Thick white rectangles indicate LNX1 interactors identified on only one protein array: NM_020646.1 ASCL3 in row 5 columns 15 and 16, NM_020548.4 DBI in row 11 columns 1 and 2, NM_153752.1 untranslated mRNA in row 14 columns 1 and 2, and NM_016096.1 ZNF706 in row 14 columns 7 and 8.

Figure 3. Clustering by GO annotation similarity.

220 LNX1 interacting proteins were clustered according to the graph similarity of their GO annotation. Nodes represent proteins; lines between nodes represent the similarity of GO annotation between two proteins with thick dark lines representing high similarity and thin light lines representing lower similarity scores. Node colours represent GO annotation and were randomly assigned by Cytoscape. Proteins are grouped together in circles according to their clusters, i.e. circle 1 = cluster 1. (A) Proteins clustered according to the similarity of their GO Biological Process annotation, clusters BP1–BP9. (B) Proteins clustered according to the similarity of their GO Molecular Function annotation, clusters MF1–MF9. (C) Proteins clustered according to the similarity of their GO Cellular Component annotation, clusters CC1–CC9.

Figure 4. Confirmation of novel LNX1 and LNX2 binding partners by co-immunoprecipitation.

Flag-tagged LNX1, LNX1 C45A (inactive ubiquitin ligase), LNX2 or LNX2 C51A (inactive ubiquitin ligase) were cotransfected with Myc-tagged candidates into 293T cells. Cell lysates were immunoprecipitated with anti-Myc antibody and blotted with anti-Flag to identify novel interactions.

Figure 5. Interaction of individual LNX1 PDZ domains with novel binding partners as tested by fusion protein binding experiments.

Each PDZ domain of LNX1 was fused to GST. Purified fusion proteins were incubated with 293T cell lysate transfected with the myc-tagged protein indicated at left. Interactions were detected by immunoblotting with anti-myc antibody.

Figure 6. LNX1-lipid binding.

(A) Binding of purified LNX1 or GST-LNX1 PDZ1, 2, 3 or 4 to phosphatidylinositol phosphate lipids immobilized on membrane strips (Echelon Biosciences) was assessed by anti-LNX1 or anti-GST immunoblot. Exposure times of individual membrane strips were: LNX1, 1 seconds.; PDZ1, 30 min.; PDZ2, 1 min.; PDZ3, 30 min., PDZ4, 15 seconds. (B) Sequence comparisons of LNX1 PDZ 2 and 4 and the lipid membrane-binding PDZ 2 of Par3. Residue positions shown to be critical for forming a positive charge cluster (▴) or the binding pocket for the phosphatidylinositol head group (*) in Par3 PDZ 2 are indicated.