ALS-associated missense and nonsense TBK1 mutations can both cause loss of kinase function

Abstract

Mutations in TANK binding kinase 1 (TBK1) have been linked to amyotrophic lateral sclerosis. Some TBK1 variants are nonsense and are predicted to cause disease through haploinsufficiency; however, many other mutations are missense with unknown functional effects. We exome sequenced 699 familial amyotrophic lateral sclerosis patients and identified 16 TBK1 novel or extremely rare protein-changing variants. We characterized a subset of these: p.G217R, p.R357X, and p.C471Y. Here, we show that the p.R357X and p.G217R both abolish the ability of TBK1 to phosphorylate 2 of its kinase targets, IRF3 and optineurin, and to undergo phosphorylation. They both inhibit binding to optineurin and the p.G217R, within the TBK1 kinase domain, reduces homodimerization, essential for TBK1 activation and function. Finally, we show that the proportion of TBK1 that is active (phosphorylated) is reduced in 5 lymphoblastoid cell lines derived from patients harboring heterozygous missense or in-frame deletion TBK1 mutations. We conclude that missense mutations in functional domains of TBK1 impair the binding and phosphorylation of its normal targets, implicating a common loss of function mechanism, analogous to truncation mutations.

Keywords: ALS; FTD; Familial ALS; TBK1; WES.

Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

Figures

Fig. 1

TBK1 mutations identified to date and their location in TBK1 structure. (A, B) Schematic representation of TBK1 protein structure (Tu et al., 2013) showing a map of nonsense (A) and missense (B) variants found in the literature and in our cohort. For more details on the variants found in this study see Table 1. (C) TBK1 homodimer crystal structure (PDB 4IM0) mapping the mutations found in our study excluding premature stop codons and frameshift deletions. Abbreviations: CTD, C-terminal domain (light blue); KD, kinase domain (green); SDD, scaffold dimerization domain (pink); TBK1, TANK binding kinase 1; ULD, ubiquitin-like domain (purple). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 2

TBK1 p.G217R and p.R357X impair IRF3 phosphorylation as well as TBK1 binding with OPTN and its phosphorylation. (A) Western blot analysis of IRF3 (left) and pIRF3 (right). (B left) Quantitative analysis of blot in (A) left showing a similar expression level of endogenous IRF3 in cells (n = 3). (B right) Quantitative analysis of blot in (A) right showing a significant decrease of expression of endogenous pIRF3 in cells transfected with TBK1-p.G217R and p.R357X (n = 3, analyzed by one-way ANOVA followed by Dunnett's post test p < 0.0001). (C) Qualitative immunocytochemistry of HEK293T transfected with TBK1-WT, p.G217R, p.R357X, and probed for p-IRF3 confirming the result obtained by Western blot analysis (scale bar = 50 μm). (D) Co-IP with HA-tag pull down (TBK1) showing no binding of OPTN in any of the mutated samples with the exception of p.C471Y (n = 3). (E) HEK293T were transiently cotransfected with Flag-OPTN WT and HA-TBK1 WT, p.G217R, p.R357X, or p.C471Y, treated with alkaline phosphatase and analyzed by Western blot showing lack of OPTN phosphorylation in all mutated samples a part from p.C471Y. Abbreviations: IRF3, interferon regulatory factor 3; OPTN, optineurin; TBK1, TANK binding kinase 1; WT, wild type. **** p ≤ 0.0001.

Fig. 3

TBK1 p.G217R and p.R357X impair TBK1 phosphorylation and autophosphorylation and might reduce TBK1 homodimerization. (A) Western blot analysis of TBK1 expression levels (left) and pTBK1 (right). (B left) Quantitative analysis of blot in (A) left showing a similar expression level of TBK1 in cells (n = 4). (B right) Quantitative analysis of blot in (A) right showing a significant decrease of expression of pTBK1 in p.G217R and p.R357X (n = 4 analyzed by one-Way ANOVA followed by Dunnett's post test p < 0.0001). (C) Immunocytochemistry of HEK293T transfected with TBK1-WT, p.G217R, p.R357X, and probed for p-TBK1 confirming the result obtained by Western blot analysis (Fig. 3B) (Scale bar = 10 μm). (D) Native gel showing the dimer and monomer (indicated by black arrows) in TBK1-WT and TBK1-C471Y, the weaker dimer and monomer in the p.G217R sample, and no dimer or monomer in the R357X sample. (E) Quantitative analysis of gel in (D) showing significant reduction in dimer formation for p.G217R (positive control on the right TBK1 WT treated with DTT, n = 3, analyzed by one-way ANOVA followed by Dunnett's post test p < 0.05). Abbreviations: TBK1, tank binding kinase 1; WT, wild type. * p = 0.0306; *** p = 0.0006; **** p ≤ 0.0001.

Fig. 4

Patient-derived LCLs harboring TBK1 variants show a reduced level of TBK1 phosphorylation. (A top) Western blot of control- and patient-derived LCLs, harboring 5 different TBK1 variants, showing the level of total TBK1. (A bottom) Western blot showing phospho-TBK1 expression in patient- and control-derived lymphoblasts. (B) Dot plot showing a significant difference in the ratio of phospho-TBK1 and total TBK1 between control- and patient-derived LCLs (analyzed by unpaired t-test, 2 tailed, p = 0.0229). Abbreviations: LCL, lymphoblastoid cell line; TBK1, tank binding kinase 1. * p = 0.0229.

Supplementary figure 1

TBK1 variant c.992+1G>A produces the in-framedeletion of exon 8. Agarose gel on cDNA extracted from WT and c.992+1GA compared to the sample carrying both WT alleles (WT/C992+1G>A WTallele: 2190 bp, WT/C992+1G>A mutated allele: 1990bp, WT/WT: 2190bp). The boxes above and below show the sequencing of the WT/WT sample and the sequencing of the WT/c.992+1G>A sample, respectively.