Identification of TBK1 complexes required for the phosphorylation of IRF3 and the production of interferon β

Abstract

The double-stranded RNA mimetic poly(I:C) and lipopolysaccharide (LPS) activate Toll-like receptors 3 (TLR3) and TLR4, respectively, triggering the activation of TANK (TRAF family member-associated NF-κB activator)-binding kinase 1 (TBK1) complexes, the phosphorylation of interferon regulatory factor 3 (IRF3) and transcription of the interferon β (IFNβ) gene. Here, we demonstrate that the TANK-TBK1 and optineurin (OPTN)-TBK1 complexes control this pathway. The poly(I:C)- or LPS-stimulated phosphorylation of IRF3 at Ser396 and production of IFNβ were greatly reduced in bone marrow-derived macrophages (BMDMs) from TANK knockout (KO) mice crossed to knockin mice expressing the ubiquitin-binding-defective OPTN[D477N] mutant. In contrast, IRF3 phosphorylation and IFNβ production were not reduced significantly in BMDM from OPTN[D477N] knockin mice and only reduced partially in TANK KO BMDM. The TLR3/TLR4-dependent phosphorylation of IRF3 and IFNβ gene transcription were not decreased in macrophages from OPTN[D477N] crossed to mice deficient in IκB kinase ε, a TANK-binding kinase related to TBK1. In contrast with the OPTN-TBK1 complex, TBK1 associated with OPTN[D477N] did not undergo phosphorylation at Ser172 in response to poly(I:C) or LPS, indicating that the interaction of ubiquitin chains with OPTN is required to activate OPTN-TBK1 in BMDM. The phosphorylation of IRF3 and IFNβ production induced by Sendai virus infection were unimpaired in BMDM from TANK KO × OPTN[D477N] mice, suggesting that other/additional TBK1 complexes control the RIG-I-like receptor-dependent production of IFNβ. Finally, we present evidence that, in human HACAT cells, the poly(I:C)-dependent phosphorylation of TBK1 at Ser172 involves a novel TBK1-activating kinase(s).

Keywords: IRF3; LPS; TBK1; TLR3; interferon; ubiquitin.

Figure 1.. IFNβ production is suppressed in BMDM from TANK KO × OPTN[D477N] mice.

BMDM from wild-type (WT) mice (closed bars), TANK KO mice (open bars), OPTN[D477N] mice (striped bars) and TANK KO × OPTN[D477N] mice (hatched bars) were stimulated with 10 µg/ml poly(I:C) (A and C) or 100 ng/ml LPS (B and D) for the times indicated. (A and B) The IFNβ mRNA produced was measured relative to 18S ribosomal mRNA and normalized to the level of wild-type IFNβ mRNA (100%) measured after 2 h stimulation with poly(I:C) (A) or 1 h stimulation with LPS (B). (C and D) The amount of IFNβ in the cell culture medium was measured by ELISA after 8 h stimulation with poly(I:C) (C) or 4 h stimulation with LPS (D). (A–D) The results are presented as arithmetic mean (±SEM for four independent experiments carried out on BMDM from nine different mice).

Figure 2.. Poly(I:C)- or LPS-induced phosphorylation of TBK1, IRF3 and MAP kinases in BMDM from different mouse lines.

(A–D) BMDM from WT mice and TANK KO mice (A and B), IKKε KO mice (C and D), OPTN[D477N] mice and either OPTN[D/N] × TANK KO mice (A and B) or OPTN[D/N] × IKKε KO mice (C and D) were stimulated with 10 µg/ml poly(I:C) or 100 ng/ml LPS for the times indicated. Aliquots of the cell extracts (20 µg protein) were subjected to SDS–PAGE and immunoblotted with antibodies that recognize TBK1 or IKKε phosphorylated at Ser172 (pTBK1 and pIKKε, respectively), IRF3 phosphorylated at Ser396 (pIRF3), JNK1 and JNK2 phosphorylated at their Thr-Pro-Tyr motifs (p-JNK1/2) and p38α phosphorylated at its Thr-Gly-Tyr motif (p-p38α), and with antibodies that recognize all forms of TBK1, IKKε, IRF3, TANK and OPTN. Antibodies to GAPDH were used as a loading control.

Figure 3.. IFNβ production is not suppressed in BMDM from IKKε × OPTN[D477N] mice.

BMDM from WT mice (closed bars), IKKε KO mice (open bars), OPTN[D477N] mice (striped bars) and IKKε KO × OPTN[D477N] mice (hatched bars) were stimulated with 10 µg/ml poly(I:C) (A and C) or 100 ng/ml LPS (C and D) for the times indicated. (A and B) The IFNβ mRNA produced was measured relative to 18S ribosomal mRNA and normalized to the level of wild-type IFNβ mRNA (100%) measured after 2 h stimulation with poly(I:C) (A) or 1 h stimulation with LPS (B). (C and D) The amount of IFNβ in the cell culture medium was measured by ELISA after stimulation for 8 h with poly(I:C) (C) or 4 h with LPS (D). (A–D) The results are presented as arithmetic mean (±SEM for three independent experiments carried out on BMDM from eight different mice).

Figure 4.. Poly(I:C)- or LPS-dependent activation of the individual TANK–TBK1 and OPTN–TBK1 complexes in BMDM.

(A and B) WT BMDMs were stimulated with 10 µg/ml poly(I:C) (A) or 100 ng/ml LPS (B) for the times indicated in Figure 1. TANK was immunoprecipitated from the extracts and phosphorylation of the TBK1 in the immunoprecipitates (pTBK1) was analyzed by immunoblotting as described in Materials and Methods. The membranes were also immunoblotted with antibodies that recognize all forms of TBK1 and TANK. (C and D) As in A, B except that OPTN was immunoprecipitated from the extracts of TANK KO and TANK KO x OPTN[D477N] mice and the presence of phospho-TBK1, total TBK1 and OPTN in the immunoprecipitates was analyzed by immunoblotting.

Figure 5.. Poly(I:C)-dependent phosphorylation of TBK1 and IFNβ gene transcription in TRIF KO and TAK1 KO HACAT cells.

(A) Poly(I:C)-dependent phosphorylation of TBK1 and IRF3 is abolished in TRIF KO HACAT cells. Cells were stimulated with 10 µg/ml poly(I:C) for the times indicated as in Figure 1, and the cell extracts (20 µg of protein) were subjected to SDS–PAGE and immunoblotting with antibodies that recognize TBK1 phosphorylated at Ser172 (pTBK1) or IRF3 phosphorylated at Ser396 (pIRF3), and with antibodies that recognize all forms of TBK1, IRF3 and TRIF. Antibodies to GAPDH were used as a loading control. (B) TAK1 KO and WT HACAT cells were incubated for 1 h with 2.0 µM MRT67307 and then stimulated with 10 µg/ml poly(I:C) for the times indicated. The cell extracts were processed as in A and immunoblotted with antibodies that recognize the phosphorylated (p), activated forms of IKKα/β, JNK1/2 and p38α MAP kinase (p38α) and all forms of TAK1, IKKβ, JNK and p38α. (C) The TAK1 KO and WT cells were incubated for 1 h with or without 2 µM MRT67307 prior to stimulation with poly(I:C). Other details are as in A,B. (D) WT HACAT cells (open bars) and TAK1 KO HACAT cells (closed bars) were stimulated with poly(I:C). IFNβ mRNA production was then measured relative to hypoxanthine-guanine phosphoribosyltransferase 1 (HPRT1) mRNA and normalized to the level of IFNβ mRNA measured in WT cells (100%) after stimulation for 4 h with poly(I:C). The results are presented as arithmetic mean (±SEM for two independent experiments each performed in triplicate).