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. 2022 Dec 27;222(2):e202108144. doi: 10.1083/jcb.202108144

Figure 7.

Figure 7.

Poly(I:C) stimulation induces TBK1-dependent, specific degradation of TNIP1 by autophagy. (A) Immunofluorescence images showing colocalization between TNIP1 and pTBK1 upon poly(I:C) treatment. Cells were either left untreated or treated with 5 µg/ml poly(I:C) for 4 h, and subsequently stained for endogenous TNIP1 and pTBK1. Colocalization between TNIP1 and pTBK1 is indicated by arrowheads. Quantification of TNIP1 dots colocalizing with pTBK1 was done using Volocity software (PerkinElmer). Around 160–220 cells were counted for each condition in each independent experiment (n = 3). * = P < 0.05, unpaired two-sided t test. Error bars indicate SD. Scale bar in overview image is 10 µm, and scale bar in insert is 2 µm. (B) Time-course effect of poly(I:C) treatment on TBK1 activation and TNIP1. Representative blot and the corresponding quantification of the relative pTBK1 over total TBK1 levels are shown. (C and D) TBK1 phopshorylates TNIP1 N-terminal of LIR2. (C) In vivo phosphoproteomics using Elastase or ProAlanase as proteolytic enzymes identified indicated phosphopeptides. The single phosphorylation site could not be unambiguously localized to one of the three amino acid residues highlighted in red. Inhibition of TBK1 blocked the respective phosphorylation event (n ≥ 3). (D) In vitro kinase assay using purified TBK1 and TNIP1 coupled to phosphoproteomics indicates that TBK1 directly phosphorylates TNIP1 on one of the amino acid residues highlighted in red. * = P < 0.05, unpaired two-sided t test. Error bars indicate SEM. (E) In vitro GST-pulldown assay using 35S-labeled myc-TNIP1 and myc-TNIP1-S122E/S123E against recombinant GST and GST-tagged human ATG8s. Bound myc-TNIP1 WT and S122E/S123E was detected using autoradiography (AR). Quantification and fold change of n = 3, * = P < 0.05, ** = P < 0.01, *** = P < 0.001; unpaired two-sided t test. Error bars indicate SD. (F) The interaction between TNIP1 and LC3B is regulated by TBK1. GFP-LC3B is purified using GFP trap beads. Bound TNIP1 is deteced by Western blot. Inhibition of TBK1 by MRT67307 negatively regulates the poly(I:C)-dependent interaction of TNIP1 with LC3. KO1 cells were used for reconstitution. (G) Inhibition of TBK1 negatively interferes with poly(I:C)-dependent degradation of TNIP1. Western blots of whole cell lysate indicate TNIP1 stabilization by TBK1 inhibition. Actin was used as loading control (n = 3). Error bars indicate SEM. ** = P < 0.01, unpaired, two-sided t test. (H) TBK1 KO negatively interferes with poly(I:C)-dependent degradation of TNIP1. Western blots of whole cell lysate indicate TNIP1 stabilization by TBK1 KO in two independent cell lines. Tubulin was used as loading control (n = 3). Error bars indicate SD. ** = P < 0.01, unpaired, two-sided t test. In A, C, D, E, G, and H, data distribution was assumed to be normal, but this was not formally tested. Source data are available for this figure: SourceData F7.