The TRAF-associated protein TANK facilitates cross-talk within the IkappaB kinase family during Toll-like receptor signaling

Abstract

Toll-like receptor (TLR) ligands that signal via TIR-domain-containing adapter-inducing IFNβ (TRIF) activate the IκB kinase (IKK)-related kinases, TRAF associated NFκB activator (TANK)-binding kinase-1 (TBK1) and IKKε, which then phosphorylate IRF3 and induce the production of IFNβ. Here we show that TBK1 and IKKε are also activated by TLR ligands that signal via MyD88. Notably, the activation of IKKε is rapid, transient, and it precedes a more prolonged activation of TBK1. The MyD88- and TRIF-dependent signaling pathways activate the IKK-related kinases by two signaling pathways. One is mediated by the canonical IKKs, whereas the other culminates in the autoactivation of the IKK-related kinases. Once activated, TBK1/IKKε then phosphorylate and inhibit the canonical IKKs. The negative regulation of the canonical IKKs by the IKK-related kinases occurs in both the TRIF- and MyD88-dependent TLR pathways, whereas IRF3 phosphorylation is restricted to the TRIF-dependent signaling pathway. We have discovered that the activation of IKKε is abolished, the activation of TBK1 is reduced, and the interaction between the IKK-related kinases and the canonical IKKs is suppressed in TANK(-/-) macrophages, preventing the IKK-related kinases from negatively regulating the canonical IKKs. In contrast, IRF3 phosphorylation and IFNβ production was normal in TANK(-/-) macrophages. Our results demonstrate a key role for TANK in enabling the canonical IKKs and the IKK-related kinases to regulate each other, which is required to limit the strength of TLR signaling and ultimately, prevent autoimmunity.

Fig. 1.

Kinetics of activation of TBK1 and IKKε by different TLR agonists. BMDMs were stimulated for the times indicated with (A) 1 μg/mL Pam₃CSK₄, (B) 10 μg/mL poly(I:C), (C) 100 ng/mL LPS, or (D) 2 μM CpG. TBK1 and IKKε were immunoprecipitated (IP) and their catalytic activities were measured by incubating the immunoprecipitated kinases with GST-IRF3 and Mg[γ-³²P]-ATP. Reactions were terminated in SDS, the proteins resolved by SDS/PAGE, and the gel autoradiographed (A–D, Upper two panels). Kinase activity was quantified by phosphorimager analysis (mean ± SEM, n = 3–6). An aliquot of each immunoprecipitation was also immunoblotted for TBK1 and IKKε as a loading control (Lower). Cell extract (20 μg protein) was additionally immunoblotted with antibodies that recognize TBK1 phosphorylated at Ser172 (to monitor activation by a second independent method) and for the phosphorylation of IRF3 at Ser396 (A–D, Lower three panels). (E) BMDMs from the LPS-resistant mouse strain C3H/HeJ were stimulated for 60 min with 2 μg/mL LTA, 1 μg/mL Pam₃CSK₄, 10 μg/mL poly(I:C), 100 ng/mL LPS, 2 μg/mL R837 or 2 μM CpG. Cell extracts (20 μg protein) were immunoblotted with the same antibodies used in A–D.

Fig. 2.

Activation of TBK1 and IKKε in response to LPS occurs via both the MyD88- and the TRIF-dependent pathways, but only the latter leads to the phosphorylation of IRF3. (A) BMDMs from MyD88^−/− or TRIF^−/− mice or WT littermates were stimulated for the times indicated with 100 ng/mL LPS. The catalytic activities of TBK1 and IKKε (A, Upper two panels) were measured as described in Fig. 1 and Materials and Methods. Cell extracts (20 μg protein) were immunoblotted with the antibodies indicated (A, Lower four panels). (B) Quantitation of TBK1 and IKKε activities by phosphorimager analysis (mean ± SEM, n = 3).

Fig. 3.

Cross-talk between the canonical IKKs and the IKK-related kinases during MyD88 signaling in macrophages. (A) Activation of the IKK-related kinases. BMDMs were treated for 1 h with either 1 μM 5Z-7-oxozeaenol, 2 μM MRT67307, or both kinase inhibitors before stimulation for 10 min with 1 μg/mL Pam₃CSK₄. The catalytic activities of TBK1 and IKKε (Upper two panels) were measured as in Fig. 1 and Materials and Methods. In these experiments, TBK1 and IKKε were immunoprecipitated from the cell extracts and the immunoprecipitates washed to remove MRT67307 before assaying in the absence of this compound, because MRT67307 is a reversible inhibitor of the IKK-related kinases. Cell extracts (20 μg protein) were also immunoblotted with the antibodies indicated (Lower two panels). (B) The cell extracts in A were immunoblotted with the antibodies indicated. The phospho-specific antibody that recognizes IKKβ phosphorylated at Ser177 and Ser181 also recognizes IKKα phosphorylated at Ser176 and Ser180. However, the bands shown correspond to phosphorylated IKKβ only. Phosphorylated IKKα migrates more rapidly than IKKβ and is recognized very poorly by the antibody (figure S1A in ref. 9), which may be explained by lower levels of expression and/or activation of IKKα compared with IKKβ.

Fig. 4.

TANK is required for efficient activation of the IKK-related kinases by TLR agonists. (A–C) BMDMs from TANK^−/− mice or WT littermates were stimulated for the times indicated with (A) 1 μg/mL Pam₃CSK₄, (B) 100 ng/mL LPS, or (C) 2 μM CpG. The catalytic activities of TBK1 and IKKε (A–C, Upper two panels) were measured as described in Fig. 1 and Materials and Methods. Cell extracts (20 μg protein) were also immunoblotted with the antibodies indicated (A and C, Lower three panels and B, Lower four panels). NS in B denotes a nonspecific band detected by the TANK antibody. (D) BMDMs from TANK^−/− mice or WT littermates were stimulated for 6 h with 100 ng/mL LPS and the concentration of IFNβ in the culture supernatant measured with the ELISA kit from R&D Systems (mean ± SEM, n = 3).

Fig. 5.

Negative regulation of the canonical IKKs by the IKK-related kinases is impaired in TANK^−/− macrophages. BMDMs from TANK^−/− mice or WT littermates were stimulated for the times indicated with (A) 1 μg/mL Pam₃CSK₄, (B) 100 ng/mL LPS, or (C) 2 μM CpG. Cell extracts (20 μg protein) were immunoblotted with the antibodies indicated. (D) BMDMs from TANK^−/− mice or WT (TANK^+/+) littermates were treated for 1 h with 1 μM 5Z-7-oxozeaenol, and/or 2 μM MRT67307, before stimulation for 10 min with 100 ng/mL LPS. Cell extracts were immunoblotted as described above.

Fig. 6.

Association of the canonical IKKs and IKK-related kinases is dependent on TANK. (A) Lysates of RAW264.7 macrophages were incubated with antibodies from nonimmunized sheep (IgG) or antibodies against TBK1, NEMO, or IKKβ. The immunoprecipitates were washed, separated by SDS/PAGE, and immunoblotted with the antibodies indicated. (B) Cell extracts from TANK^+/+ and TANK^−/− macrophages were used to immunoprecipitate IKKβ and coimmunoprecipitating proteins were monitored by immunoblotting.

Fig. 7.

Model for the cross-talk between IKK family members in the MyD88 signaling pathway. K63-pUb chains formed by TRAF6 in response to IL-1 or TLR stimulation bind to TAB2/3–TAK1 and IKKα/β–NEMO–TANK–TBK1/IKKε complexes enabling the sequential activation of the canonical IKKs by TAK1 and then the activation of the IKK-related kinases by the canonical IKKs and by autoactivation, as discussed in the text. The IKK-related kinases then negatively regulate the canonical IKKs by phosphorylating sites that inhibit activity.