Use of the pharmacological inhibitor BX795 to study the regulation and physiological roles of TBK1 and IkappaB kinase epsilon: a distinct upstream kinase mediates Ser-172 phosphorylation and activation

Abstract

TANK-binding kinase 1 (TBK1) and IkappaB kinase epsilon (IKKepsilon) regulate the production of Type 1 interferons during bacterial and viral infection, but the lack of useful pharmacological inhibitors has hampered progress in identifying additional physiological roles of these protein kinases and how they are regulated. Here we demonstrate that BX795, a potent and relatively specific inhibitor of TBK1 and IKKepsilon, blocked the phosphorylation, nuclear translocation, and transcriptional activity of interferon regulatory factor 3 and, hence, the production of interferon-beta in macrophages stimulated with poly(I:C) or lipopolysaccharide (LPS). In contrast, BX795 had no effect on the canonical NFkappaB signaling pathway. Although BX795 blocked the autophosphorylation of overexpressed TBK1 and IKKepsilon at Ser-172 and, hence, the autoactivation of these protein kinases, it did not inhibit the phosphorylation of endogenous TBK1 and IKKepsilon at Ser-172 in response to LPS, poly(I:C), interleukin-1alpha (IL-1alpha), or tumor necrosis factor alpha and actually enhanced the LPS, poly(I:C), and IL-1alpha-stimulated phosphorylation of this residue. These results demonstrate that the phosphorylation of Ser-172 and the activation of TBK1 and IKKepsilon are catalyzed by a distinct protein kinase(s) in vivo and that TBK1 and IKKepsilon control a feedback loop that limits their activation by LPS, poly(I:C) and IL-1alpha (but not tumor necrosis factor alpha) to prevent the hyperactivation of these enzymes.

FIGURE 1.

BX795 blocks the phosphorylation, nuclear translocation, and transcriptional activity of IRF3 and production of interferon β in response to TLR3 and TLR4 agonists. A, HEK293-TLR3 cells were incubated for 60 min without (–) or with (+)1 μm BX795 and subsequently stimulated with 50 μg/ml poly(I:C) for the times indicated. Cell extract protein (40 μg) was subjected to SDS-PAGE and immunoblotted for IRF3. B, HEK293-TLR3 cells were stimulated for 2 h with poly(I:C) as in A. The cells were fixed, stained for IRF3, and visualized by confocal microscopy. C, HEK293-TLR3 cells were co-transfected with DNA encoding an IRF3 luciferase reporter construct and pTK-Renilla luciferase plasmid DNA. 24 h post-transfection cells were incubated for 1 h with varying concentrations of BX795 before stimulation for 6 h with 50μg/ml poly(I:C). Luciferase activity was measured and normalized to Renilla luciferase activity (mean ± S.E., n = 4). D, RAW264.7 cells were incubated for 1 h at various concentrations of BX795 and then stimulated for 6 h with 100 ng/ml LPS. The concentration of IFN-β released into the culture medium was measured by enzyme-linked immunosorbent assay. (mean ± S.E., n = 4). E, the experiment was carried out as in D except that cells were incubated without (white bars) or with (black bars)1 μm BX795 and stimulated with either 100 ng/ml LPS or 10 μg/ml of poly(I:C). (mean ± S.E., n = 3; *, p < 0.01). F, the effects of BX795 on TLR signaling do not result from inhibition of PDK1. RAW264.7 macrophages were treated for 30 min without (control) or with 1 μm BX795 then stimulated for 45 min without (–) or with (+) 100 ng/ml LPS. Cell extracts (40 μg protein) were immunoblotted with antibodies recognizing IRF3, IRF3 phosphorylated at Ser-396, and S6 kinase 1 (S6K1) phosphorylated at Thr-229.

FIGURE 2.

BX795 selectively blocks IRF3 but not NFκB signaling. A, RAW264.7 cells were incubated without (–) or with (+)1 μm BX795 and then either left unstimulated (–) or stimulated (+) for 30 min with 100 ng/ml LPS. Cell extracts (40 μg protein) were then immunoblotted with the antibodies used in Fig. 1 and with antibodies that recognize TBK1, RelA phosphorylated at Ser-468 or Ser-536, IκBα phosphorylated at Ser-32 and Ser-36, or p105 phosphorylated at Ser-933. B, same as A except that the RAW264.7 cells were stimulated with 10 μg/ml poly(I:C) for 60 min, and immunoblotting for IκBα was carried out using an antibody recognizing all forms of IκBα instead of the antibody recognizing phosphorylated IκBα. C, HEK293-TLR3 cells were co-transfected with DNA encoding an IRF3 or a NFκB luciferase reporter construct and pTK-Renilla luciferase plasmid DNA. 24 h post-transfection cells were treated without (white bars) or with (black bars)1 μm BX795 and then stimulated for 6 h with 50μg/ml poly(I:C). Luciferase activity was measured and normalized to Renilla luciferase activity (mean ± S.E., n = 4; *, p < 0.001). D and E, MEFs were serum-starved overnight and incubated without (–) or with (+)1 μm BX795 for 60 min before stimulation with 10 ng/ml IL-1α (D) or TNF-α (E). Cell extracts were immunoblotted as described in A and B.

FIGURE 3.

Phosphorylation of TBK1 and IKKε at Ser-172 correlates with catalytic activity. A, RAW264.7 macrophages were stimulated with 100 ng/ml LPS for the times indicated. Catalytic activity was measured by immunoprecipitating TBK1 and incubating the immunocomplexes with GST-IRF3 in presence of Mg[γ-³²P]ATP. Proteins were resolved by SDS-PAGE and stained with Coomassie Blue, and phosphorylated IRF3 detected by autoradiography (top panel). Phosphorylation of Ser-172 on TBK1 was monitored by immunoprecipitating the phosphorylated TBK1 with the anti-IKKε Ser(P)-172 antibody and immunoblotting with an antibody recognizing all forms of TBK1 (middle panel). A further aliquot of cell extract was subjected to SDS-PAGE and immunoblotted with the same TBK1 antibody. B, same as A, except that HEK293-TLR3 cells were stimulated for the times indicated with 50 μg/ml poly(I:C). C, RAW264.7 macrophages were left unstimulated (–) or stimulated (+) for 45 min with 100 ng/ml LPS. The Ser-172-phosphorylated forms of TBK1 and IKKε were immunoprecipitated from cell extracts using the anti-Ser(P)-172 IKKε antibody, and the presence of TBK1 and/or IKKε was revealed by immunoblotting using antibodies recognizing all forms of TBK1 (top panel) or IKKε (second panel from top) or a mixture of the two antibodies (third panel from the top). In the bottom panel, 40 μg of extract protein was immunoblotted without immunoprecipitation using a mixture of the antibodies recognizing all forms of TBK1 and IKKε.

FIGURE 4.

The overexpression of TBK1 and IKKε leads to autophosphorylation and transphosphorylation of Ser-172. A, wild type (WT) and two different catalytically-inactive FLAG-tagged mutants of TBK1 (TBK1-(K38A) and TBK1[D157A]) were expressed in HEK293 cells. Cell extract (40 μg protein) was then subjected to SDS-PAGE and immunoblotted using anti-Ser(P)-172 TBK1 and anti-FLAG. B, FLAG-WT-TBK1 was transfected into HEK293 cells. 24 h later the cells were treated without (–) or with (+)1 μm BX795 for the times indicated. Cell extracts (40 μg protein) were then immunoblotted using anti-FLAG and anti-Ser(P)-172 TBK1 as in A. C, a vector encoding FLAG WT-TBK1 was transfected into HEK293 cells, then incubated for 1 h without (–) or with (+)1 μm BX795. TBK1 was then immunoprecipitated from 0.1 mg of cell extract protein with anti-FLAG (washed) and assayed for activity using the IκBα peptide substrate (mean ± S.E., n = 4; *, p < 0.001), D, a vector encoding FLAG-tagged TBK1-(K38A) was co-expressed in HEK293 cells with an empty vector (–), a vector encoding GST (+), or a vector encoding GST-WT-TBK1 (TBK1) GST-WT-IKKε (IKKε). The cells were lysed, and 40 μg of cell extract protein was immunoblotted (WB) using anti-Ser(P)-172 TBK1/IKKε or anti-FLAG as in A (upper two panels). A further 0.5 mg of cell extract protein was incubated for 1 h at 4 °C with 0.01 ml of glutathione-Sepharose beads with continuous mixing. The beads were collected by centrifugation and washed three times in lysis buffer, and bound proteins were released with SDS and immunoblotted with anti-GST or anti-FLAG antibodies (lower two panels). E, wild type and catalytically-inactive mutants of FLAG-TBK1 and FLAG-IKKε were expressed in HEK293 cells, and the lysates were immunoblotted using anti-FLAG as in A. F, WT-FLAG-IKKε was expressed in HEK293 cells. 24 h later the cells were treated with 1 μm BX795 for the times indicated. Cell extracts (40 μg protein) were then immunoblotted using anti-FLAG as in A. Anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control.

FIGURE 5.

Endogenous IKKε protein expression is unaffected by treatment with BX795 and does not form a complex with TBK1. A, RAW264.7 cells were treated for 1 h without (–) or with (+)1 μm BX795 before stimulation for 16 h without (–) or with (+) 100 ng/ml LPS. Cell extracts were immunoblotted with antibodies raised against IKKε, TBK1, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). B, endogenous TBK1 and IKKε were immunoprecipitated (IP) from cell extracts of RAW264.7 cells. IgG isolated from a non-immunized sheep served as a negative control. Proteins present in the immunoprecipitates were detected by immunoblotting with antibodies that recognize IKKε, TBK1, and TANK.

FIGURE 6.

BX795 increases the phosphorylation of Ser-172 and the catalytic activity of TBK1 and IKKε in response to LPS and poly(I:C). A, RAW264.7 cells were treated for 1 h without (–) or with (+)1 μm BX795 before stimulation for 45 min without (–) or with (+) 100 ng/ml LPS. TBK1 was immunoprecipitated from 1 mg of cell extract protein, and its catalytic activity was measured by incubation for 30 min at 30 °C with GST-IRF3 and Mg[γ-³²P]ATP. Reactions were terminated in SDS, proteins were resolved by SDS-PAGE, and the gel was autoradiographed (top panel). The gel was subjected to phosphorimaging analysis, and the catalytic activity of TBK1 was quantitated and normalized to that observed in the unstimulated cells (mean ± S.E., n = 3; *, p < 0.05). B, an aliquot of the cell extract in A was used to analyze the phosphorylation of TBK1 and IKKε at Ser-172 as in Fig. 3C (top panel). As a loading control, cell extract (40 μg of protein) was also immunoblotted with antibodies recognizing all forms of TBK1 and IKKε (lower panel). This antibody also recognizes a nonspecific band migrating slower than TBK1 and IKKε. C, RAW264.7 cells were incubated for 1 h with the indicated concentrations of BX795 and then either left unstimulated (–) or stimulated (+) for 45 min with 100 ng/ml LPS. The phosphorylation of TBK1 and IKKε at Ser-172 was then examined by immunoprecipitation/immunoblotting as in Fig. 3C. D, bone marrow-derived macrophages were incubated for 1 h without (–) or with (+)1 μm BX795 followed by stimulation for 30 or 60 min with 100 ng/ml LPS. Cell extract (20 μg protein) was then immunoblotted with the antibodies indicated. E, the experiment was performed as in D, but the cells were stimulated for 60 min with 10 μg/ml poly(I:C). F, RAW264.7 cells were stimulated for 45 min with 100 ng/ml LPS, TBK1 was immunoprecipitated from 0.5 mg of cell extract protein, and its ability to phosphorylate GST-IRF3 (2 μm) was measured at varying concentrations of BX795 using Mg[γ-³²P]ATP. Reactions were terminated in SDS, proteins were resolved by SDS-PAGE, and the gel was autoradiographed (top panel). The gel was subjected to phosphorimaging analysis, and the catalytic activity of TBK1 was quantitated and normalized to that measured in the absence of inhibitor (mean ± S.E., n = 4).

FIGURE 7.

Activation and phosphorylation of TBK1 and IKKε by IL-1α and TNFα. A and B, kinetics of activation of TBK1 and IKKε. MEFs were serum-starved overnight and stimulated for the indicated times using 10 ng/ml IL-1α (A) or TNFα (B). Catalytic activity was measured by immunoprecipitating TBK1 and incubating the immune complexes with GST-IRF3 in the presence of Mg[γ-³²P]ATP. Proteins were resolved by SDS-PAGE, and phosphorylated proteins were detected by autoradiography (top panel). Phosphorylation of Ser-172 on TBK1 was monitored by immunoprecipitating the phosphorylated TBK1 and IKKε with the anti-IKKε Ser(P)-172 antibody (S051C) and immunoblotting with an antibody recognizing all forms of TBK1 and IKKε (middle panel). C and D, effect of BX795 on the activation of TBK1 and IKKε. MEFs were incubated for 1 h without (–) or with (+)1μm BX795 before stimulation for 10 min with 10 ng/ml IL-1α (C) or TNFα (D). Catalytic activity and phosphorylation of TBK1 and IKKε were measured as in A and B.

FIGURE 8.

TNFα, but not IL-1α, induced phosphorylation of TBK1 and IKKε is reduced in TAK1-deficient MEFs. Wild type (WT) MEFs and MEFs expressing a truncated inactive form of TAK1 (26) were stimulated with 10 ng/ml IL-1α (A) or TNFα (B) for 10 min. The phosphorylation of TBK1 and IKKε was monitored using the immunoprecipitation/immunoblotting assay described in Fig. 3C. The phosphorylation of p105 as well as total TAK1 and TBK1/IKKε was detected in total protein extracts (40 μg) by immunoblotting with the respective antibodies. TAK1Δ is an inactive form of TAK1, which lacks 37 amino acid residues in a region critical for ATP binding. Similar results were obtained in two independent experiments. KO, knockout.

FIGURE 9.

Effect of BX795 on the activation of MAP kinases. A and B, RAW264.7 cells were treated without (–) or with (+)1 μm BX795 and either left unstimulated (–) or stimulated (+) with 100 ng/ml LPS (A) or 10 μg/ml poly(I:C) (B). The cells were lysed, and aliquots of the lysates were subjected to SDS-PAGE and immunoblotting with antibodies that recognize the phosphorylated forms of ERK1/2, p38α MAPK, and JNK1/2. As a loading control, cell extracts were immunoblotted with an antibody recognizing all forms of p38α MAP kinase. C and D, same as A and B, except that MEFs were deprived of serum overnight and then incubated without (–) or with (+)1 μm BX795 for 60 min before stimulation with 10 ng/ml IL-1α (C) or TNFα (D).

FIGURE 10.

Model for the activation and feedback control of TBK1 and IKKε by proinflammatory stimuli. The binding of IL-1α, LPS, and poly(I:C) to their respective receptors induces the activation of an unidentified protein kinase (PKX). PKX subsequently phosphorylates TBK1 and IKKε at Ser-172, thereby triggering their activation. TBK1 and IKKε can then phosphorylate substrates such as the transcription factor IRF3. As shown in this study, TBK1 and IKKε also exert a negative feedback control on their activation by IL-1α, LPS, and poly(I:C) presumably by phosphorylation and inhibition of an upstream component of the pathway and/or by activating a Ser-172 protein phosphatase (not illustrated). The TNFα-stimulated activation of TBK1 and IKKε appears to require a separate or additional protein kinase that is at least partially dependent on TAK1 activity, and this arm of the pathway is not subject to feedback control.