Fig 1.
Characterisation of LNX1 interacting proteins.
(A) Schematic diagram of the domain structure of LNX1p80 and LNX2 showing the RING and four PDZ domains. N represents the NUMB-binding NPAY/NPAF motif. (B) The ability of the indicated proteins to interact with transfected GFP-tagged LNX constructs was assessed in HEK 293 cells. For each interacting protein, top panels show western blots of cell lysates (Lys), while the bottom panels show the output of a GFP “pull down” assay (PD). In the panels on the left, the specificity of interactions for LNX1 versus LNX2 was assessed, while on the right the interaction site on LNX1 was mapped to individual protein domains. Binding of endogenous proteins to LNX was assessed for liprinα-1, KIF7 and NUMB. For the other proteins, interactions of transfected HA or GST epitope-tagged proteins were assessed. For AKAP13, the mapping to LNX domains was performed in two separate experiments. Successful expression and pull down of GFP-tagged LNX proteins was verified in all assays and representative “pull down” blots probed for GFP are shown. n = 2–3.
Table 1.
Proteomic analysis of GFP-LNX1 interacting proteins purified from HEK293 cells.
The top 30 proteins identified, as ranked by Mascot score, are shown. The full table is available as supplementary material. Previously known interactions are underlined, as are carboxyl-terminal cysteine residues.
Fig 2.
Representative fluorescence immunocytochemistry images examining localisation of the LNX1 and interacting proteins following transient expression in MCF-7 cells.
(A) LNX1p80 transfected alone (B)-(F) LNX1p80 transfected in combination with the indicated interacting proteins that had been tagged with either HA or FLAG epitope tags. Anti-FLAG (green), anti-HA (green) and anti-LNX (red) were used to visualize the proteins of interest. Nuclei were stained with DAPI (blue). The different wavelengths were scanned individually and digitally merged (overlay). The regions highlighted by the small dashed boxes in B and D are shown enlarged in the bottom right corners of these images. Scale bar indicates 20 μm.
Fig 3.
Liprin-α1 interacts with LNX1 via its C-terminus and is a substrate for LNX-mediated ubiquitination.
(A) GFP pull-down assays performed on HEK 293T cells transiently transfected with either wild-type FLAG-liprin-α1 or a similar liprin-α1 construct with a carboxyl-terminal YSC* to DSE* mutation (FLAG-liprin-α1-C-mut) and either GFP-LNX1, GFP-LNX1-PDZ2 or GFP. Cell lysates and purified proteins were subjected to western blotting (WB) with the indicated antibodies. n = 2. (B) Ubiquitination of liprin-α1 assessed in a cell based assay. HEK 293T cells were co-transfected with the indicated LNX and liprin-α1 expression constructs as well with a construct encoding HA epitope-tagged ubiquitin. Ubiquitinated proteins were immunoprecipitated from cell lysates using an anti-HA antibody. Western blotting of immunoprecipitates for liprin- α1 revealed its ubiquitination as a high molecular weight smear. Western blot of whole cell lysates confirmed expression of all constructs. Levels of ubiquitinated liprin-α1 were quantified by densitometry and normalised for liprin-α1 expression in whole cell lysates. Data in the upper graph are expressed as mean ± SEM. n = 4. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; one-way ANOVA followed by Bonferroni post-hoc test. Data from a separate experiment are shown in the lower graph as mean ± SEM. n = 3. (C) Ubiquitination of wild-type FLAG-liprin-α1 or FLAG-liprin-α1-C-mut in the presence of the indicated LNX1 constructs was assessed as in (B) above. An obvious high molecular weight smear corresponding to ubiquitinated wild-type liprin-α1, but not liprin-α1-C-mut with a carboxyl-terminal mutation, was detected for both wild-type LNX1p80 and the catalytically inactive LNXp80-C48A mutant. (D) Liprin-α1 protein levels assessed in the presence of either wild-type LNX1p80 or the LNXp80-C48A mutant. Whole cell lysates were analysed by Western blot using anti-FLAG, anti-LNX1/2-PDZ3/4 and anti-β-actin antibodies. Liprin-α1 protein levels were quantified, normalised to β-actin levels and subjected to one-way ANOVA followed by Bonferroni post-hoc test. Data are expressed as mean ± SEM. n = 4. *p < 0.05.
Fig 4.
LNX1 mediated ubiquitination of KLHL11, KIF7 and ERC2.
HEK 293T cells were transiently transfected with the indicated constructs and ubiquitinated proteins detected as in Fig 3B above. The ubiquitination status of (A) KLHL11, (B) KIF7, (C) ERC2 and (D) SRGAP2 was then revealed by Western blot analysis using the indicated antibodies. Western blot of whole cell lysates confirmed expression of all constructs. n = 2.
Fig 5.
Interactions of LNX1 with the TRIM E3 ubiquitin ligase family.
A, Schematic diagram of the domain structure of MID2 showing the RING and B-BOX type zinc finger domains, the coiled-coil dimerization domain (COIL), the microtubule-binding COS (C-terminal subgroup one signature) domain, the fibronectin type III (FN3) domain and the SPRY (in splA kinase and ryanodine receptor) domain. MID1 shares a very similar domain organization, whereas TRIM27 lacks the COS and FN3 domains [24]. B, Mapping of the LNX1 binding site on MID2. The ability of the indicated FLAG epitope tagged MID2 constructs to interact with GFP-tagged LNX1p80 was assessed following transfection into in HEK 293 cells by GFP “pull down” assays. Successful expression of constructs was verified by western blotting of cell lysates and interactions detected in pull down samples. n = 2. C, Analysis of LNX1 binding to MID1. The ability of FLAG epitope-tagged MID1 to interact with GFP-tagged LNX1p80 was assessed in the presence or absence of HA epitope-tagged MID2. n = 2 D, Confirmation of TRIM27 interaction with LNX1 in a GFP “pull down” assay. n = 2. E, Investigation of the ability of MID2, LNX1p70 and liprin-α1 to form a trimolecular complex. Interaction of untagged LNX1p70 and FLAG-tagged liprin-α1 with GFP-tagged MID2 was assessed in GFP “pull down” assays. n = 3.