Use of the Pharmacological Inhibitor BX795 to Study the Regulation and Physiological Roles of TBK1 and IκB Kinase ϵ

TANK-binding kinase 1 (TBK1) and IκB kinase ϵ (IKKϵ) 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 IKKϵ, blocked the phosphorylation, nuclear translocation, and transcriptional activity of interferon regulatory factor 3 and, hence, the production of interferon-β in macrophages stimulated with poly(I:C) or lipopolysaccharide (LPS). In contrast, BX795 had no effect on the canonical NFκB signaling pathway. Although BX795 blocked the autophosphorylation of overexpressed TBK1 and IKKϵ at Ser-172 and, hence, the autoactivation of these protein kinases, it did not inhibit the phosphorylation of endogenous TBK1 and IKKϵ at Ser-172 in response to LPS, poly(I:C), interleukin-1α (IL-1α), or tumor necrosis factor α and actually enhanced the LPS, poly(I:C), and IL-1α-stimulated phosphorylation of this residue. These results demonstrate that the phosphorylation of Ser-172 and the activation of TBK1 and IKKϵ are catalyzed by a distinct protein kinase(s) in vivo and that TBK1 and IKKϵ control a feedback loop that limits their activation by LPS, poly(I:C) and IL-1α (but not tumor necrosis factor α) to prevent the hyperactivation of these enzymes. TANK-binding kinase 1 (TBK1) and IκB kinase ϵ (IKKϵ) 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 IKKϵ, blocked the phosphorylation, nuclear translocation, and transcriptional activity of interferon regulatory factor 3 and, hence, the production of interferon-β in macrophages stimulated with poly(I:C) or lipopolysaccharide (LPS). In contrast, BX795 had no effect on the canonical NFκB signaling pathway. Although BX795 blocked the autophosphorylation of overexpressed TBK1 and IKKϵ at Ser-172 and, hence, the autoactivation of these protein kinases, it did not inhibit the phosphorylation of endogenous TBK1 and IKKϵ at Ser-172 in response to LPS, poly(I:C), interleukin-1α (IL-1α), or tumor necrosis factor α and actually enhanced the LPS, poly(I:C), and IL-1α-stimulated phosphorylation of this residue. These results demonstrate that the phosphorylation of Ser-172 and the activation of TBK1 and IKKϵ are catalyzed by a distinct protein kinase(s) in vivo and that TBK1 and IKKϵ control a feedback loop that limits their activation by LPS, poly(I:C) and IL-1α (but not tumor necrosis factor α) to prevent the hyperactivation of these enzymes. IntroductionInvading bacteria and viruses are sensed by the host pattern recognition receptors, which bind components of these organisms, called pathogen-associated molecular patterns. The binding of pathogen-associated molecular patterns to pattern recognition receptors activates signaling cascades that culminate in the production of proinflammatory cytokines, chemokines, and interferons, which are released from immune cells into the circulation, where they mount responses to combat the invading pathogen (1Akira S. Uematsu S. Takeuchi O. Cell. 2006; 124: 783-801Abstract Full Text Full Text PDF PubMed Scopus (8550) Google Scholar). The interaction between pathogen-associated molecular patterns and pattern recognition receptors leads invariably to the activation of the mitogen-activated protein (MAP) 3The abbreviations used are: MAP, mitogen-activated protein; MAPK, MAP kinase; MKK, MAPK kinase; MAP3K, MAPK kinase kinase; ASK1, apoptosis-stimulating kinase-1; ERK, extracellular signal-regulated kinase; IFN, interferon; IKK, IκB kinase; IRF3, interferon regulatory factor 3; JNK1/2, c-Jun N-terminal kinase; LPS, lipopolysaccharide; MLK, mixed lineage kinase; PDK1, 3-phosphoinositide-dependent protein kinase 1; TAK1, transforming growth factor β-activated kinase 1; TBK1, TANK-binding kinase 1; TLR, Toll-like receptor; TNF, tumor necrosis factor; IL, interleukin; MEF, mouse embryonic fibroblast; GST, glutathione S-transferase. kinases, termed p38 MAP kinases and c-Jun N-terminal kinases 1 and 2 (JNK1/2) and the IκB kinase (IKK) complex. The latter contains the protein kinases IKKα and IKKβ, which switch on the transcription factor NFκB and, hence, NFκB-dependent gene transcription, by phosphorylating IκBα and other IκB isoforms (2Hayden M.S. Ghosh S. Cell. 2008; 132: 344-362Abstract Full Text Full Text PDF PubMed Scopus (3442) Google Scholar). IKKβ also activates the protein kinase Tpl2 by phosphorylating its p105 regulatory subunit, leading to the activation of two other MAP kinases, termed extracellular signal-regulated kinase 1 (ERK1) and ERK2 (3Beinke S. Robinson M.J. Hugunin M. Ley S.C. Mol. Cell. Biol. 2004; 24: 9658-9667Crossref PubMed Scopus (170) Google Scholar, 4Waterfield M. Jin W. Reiley W. Zhang M. Sun S.C. Mol. Cell. Biol. 2004; 24: 6040-6048Crossref PubMed Scopus (116) Google Scholar). Together, the MAP kinases and NFκB regulate the production of many proinflammatory cytokines and chemokines.A subset of pattern recognition receptors, namely Toll-like receptors 3 and 4 (TLR3, TLR4) and the cytosolic receptors RIG-I (retinoic acid-inducible gene I) and MDA-5 (melanoma differentiation-associated gene 5), also activate a distinct signaling pathway requiring the IKK-related kinases, IKKϵ and TANK-binding kinase 1 (TBK1) (5Kawai T. Akira S. Nat. Immunol. 2006; 7: 131-137Crossref PubMed Scopus (1397) Google Scholar, 6Takeuchi O. Akira S. Curr. Opin. Immunol. 2008; 20: 17-22Crossref PubMed Scopus (450) Google Scholar). Early studies, largely based on overexpression experiments, suggested that a major role of TBK1 and IKKϵ was to activate NFκB and NFκB-dependent gene transcription, and for this reason, TBK1 has also been called NFκB-activating kinase (7Tojima Y. Fujimoto A. Delhase M. Chen Y. Hatakeyama S. Nakayama K. Kaneko Y. Nimura Y. Motoyama N. Ikeda K. Karin M. Nakanishi M. Nature. 2000; 404: 778-782Crossref PubMed Scopus (312) Google Scholar, 8Pomerantz J.L. Baltimore D. EMBO J. 1999; 18: 6694-6704Crossref PubMed Google Scholar, 9Shimada T. Kawai T. Takeda K. Matsumoto M. Inoue J. Tatsumi Y. Kanamaru A. Akira S. Int. Immunol. 1999; 11: 1357-1362Crossref PubMed Scopus (307) Google Scholar). However, later studies using cells from mice that do not express TBK1 and/or IKKϵ failed to support this conclusion (10Hemmi H. Takeuchi O. Sato S. Yamamoto M. Kaisho T. Sanjo H. Kawai T. Hoshino K. Takeda K. Akira S. J. Exp. Med. 2004; 199: 1641-1650Crossref PubMed Scopus (460) Google Scholar, 11Perry A.K. Chow E.K. Goodnough J.B. Yeh W.C. Cheng G. J. Exp. Med. 2004; 199: 1651-1658Crossref PubMed Scopus (307) Google Scholar). Instead, they indicated that these protein kinases play an essential role in regulating the production of type I interferons (IFNs) by phosphorylating the transcription factor, termed interferon regulatory factor 3 (IRF3) (10Hemmi H. Takeuchi O. Sato S. Yamamoto M. Kaisho T. Sanjo H. Kawai T. Hoshino K. Takeda K. Akira S. J. Exp. Med. 2004; 199: 1641-1650Crossref PubMed Scopus (460) Google Scholar, 11Perry A.K. Chow E.K. Goodnough J.B. Yeh W.C. Cheng G. J. Exp. Med. 2004; 199: 1651-1658Crossref PubMed Scopus (307) Google Scholar). Under basal conditions IRF3 is cytosolic, but after the TBK1/IKKϵ-mediated phosphorylation of its C terminus, IRF3 dimerizes and translocates to the nucleus, where it activates a gene transcription program leading to the production of IFN-β (12McWhirter S.M. Fitzgerald K.A. Rosains J. Rowe D.C. Golenbock D.T. Maniatis T. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 233-238Crossref PubMed Scopus (445) Google Scholar, 13Fitzgerald K.A. McWhirter S.M. Faia K.L. Rowe D.C. Latz E. Golenbock D.T. Coyle A.J. Liao S.M. Maniatis T. Nat. Immunol. 2003; 4: 491-496Crossref PubMed Scopus (2037) Google Scholar). The production of IFN-β may additionally require the TBK1/IKKϵ-catalyzed phosphorylation of other proteins, such as the Dead-box RNA-helicase DDX3 (14Schroder M. Baran M. Bowie A.G. EMBO J. 2008; 27: 2147-2157Crossref PubMed Scopus (275) Google Scholar, 15Soulat D. Burckstummer T. Westermayer S. Goncalves A. Bauch A. Stefanovic A. Hantschel O. Bennett K.L. Decker T. Superti-Furga G. EMBO J. 2008; 27: 2135-2146Crossref PubMed Scopus (217) Google Scholar) and MITA (16Zhong B. Yang Y. Li S. Wang Y.Y. Li Y. Diao F. Lei C. He X. Zhang L. Tien P. Shu H.B. Immunity. 2008; 29: 538-550Abstract Full Text Full Text PDF PubMed Scopus (968) Google Scholar). IKKϵ has also been implicated in the phosphorylation of the STAT1 transcription factor at Ser-708 in a pathway that protects cells against infection by influenza A virus (17Tenoever B.R. Ng S.L. Chua M.A. McWhirter S.M. Garcia-Sastre A. Maniatis T. Science. 2007; 315: 1274-1278Crossref PubMed Scopus (268) Google Scholar).However, mouse knock-out studies are not always definitive because the complete loss of a protein kinase(s) may be compensated for by other protein kinases, whereas the prolonged absence of a protein kinase may result in long term changes in gene transcription programs so that the effects observed may be indirect. The embryonic lethality of the TBK1 knock-out mouse also limits its use in understanding the physiological roles of this protein kinase. Moreover, papers continue to be published proposing roles for TBK1 and IKKϵ in phosphorylating defined sites on the RelA and c-Rel components of the NFκB transcription complex that are thought to control the expression of a subset of NFκB-dependent genes (18Buss H. Dorrie A. Schmitz M.L. Hoffmann E. Resch K. Kracht M. J. Biol. Chem. 2004; 279: 55633-55643Abstract Full Text Full Text PDF PubMed Scopus (301) Google Scholar, 19Mattioli I. Geng H. Sebald A. Hodel M. Bucher C. Kracht M. Schmitz M.L. J. Biol. Chem. 2006; 281: 6175-6183Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar, 20Harris J. Oliere S. Sharma S. Sun Q. Lin R. Hiscott J. Grandvaux N. J. Immunol. 2006; 177: 2527-2535Crossref PubMed Scopus (87) Google Scholar). Finally, there is considerable evidence that TBK1 and IKKϵ play additional roles in cells. For instance, TBK1 is activated by TNF, and TBK1 knock-out mice die just before birth because the fetal hepatocytes undergo TNFα-induced apoptosis (21Bonnard M. Mirtsos C. Suzuki S. Graham K. Huang J. Ng M. Itie A. Wakeham A. Shahinian A. Henzel W.J. Elia A.J. Shillinglaw W. Mak T.W. Cao Z. Yeh W.C. EMBO J. 2000; 19: 4976-4985Crossref PubMed Google Scholar). These observations imply that TBK1 plays a key role in preventing apoptosis in the fetal hepatocytes of wild type mice. TBK1 is also reported to be activated by hypoxia and to control the production of angiogenic factors, such as vasoendothelial growth factor (22Korherr C. Gille H. Schafer R. Koenig-Hoffmann K. Dixelius J. Egland K.A. Pastan I. Brinkmann U. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 4240-4245Crossref PubMed Scopus (85) Google Scholar), whereas the overexpression of IKKϵ in breast cancer lines is reported to contribute a survival signal to the transformed cells (23Boehm J.S. Zhao J.J. Yao J. Kim S.Y. Firestein R. Dunn I.F. Sjostrom S.K. Garraway L.A. Weremowicz S. Richardson A.L. Greulich H. Stewart C.J. Mulvey L.A. Shen R.R. Ambrogio L. Hirozane-Kishikawa T. Hill D.E. Vidal M. Meyerson M. Grenier J.K. Hinkle G. Root D.E. Roberts T.M. Lander E.S. Polyak K. Hahn W.C. Cell. 2007; 129: 1065-1079Abstract Full Text Full Text PDF PubMed Scopus (476) Google Scholar). The direct substrates of TBK1 and IKKϵ or molecular pathways underlying any of these responses and the possible roles of these protein kinases in the pathogenesis of human cancer are unknown.The identification of the physiological substrates and biological roles of protein kinases has been greatly aided by the use of relatively specific, small cell-permeable inhibitors of these enzymes. These compounds can be used simply and rapidly and provide a complementary approach to the use of mouse knockouts or RNA interference technology, avoiding the potential drawbacks in ablating the expression of a protein kinase that were mentioned above. The compound BX795 was originally developed as a small molecule inhibitor of 3-phosphoinositide-dependent protein kinase 1 (PDK1) (24Feldman R.I. Wu J.M. Polokoff M.A. Kochanny M.J. Dinter H. Zhu D. Biroc S.L. Alicke B. Bryant J. Yuan S. Buckman B.O. Lentz D. Ferrer M. Whitlow M. Adler M. Finster S. Chang Z. Arnaiz D.O. J. Biol. Chem. 2005; 280: 19867-19874Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar), but we recently found that it also inhibited TBK1 and IKKϵ at low nanomolar concentrations in vitro (25Bain J. Plater L. Elliott M. Shpiro N. Hastie C.J. McLauchlan H. Klevernic I. Arthur J.S. Alessi D.R. Cohen P. Biochem. J. 2007; 408: 297-315Crossref PubMed Scopus (2088) Google Scholar). Moreover, BX795 only inhibited a few other protein kinases significantly out of 70 tested and, importantly, did not inhibit IKKβ (25Bain J. Plater L. Elliott M. Shpiro N. Hastie C.J. McLauchlan H. Klevernic I. Arthur J.S. Alessi D.R. Cohen P. Biochem. J. 2007; 408: 297-315Crossref PubMed Scopus (2088) Google Scholar). These findings suggested that BX795 might be the first pharmacological inhibitor suitable for studying the regulation and roles of TBK1 and IKKϵ in cells. In this paper we demonstrate the utility of this compound and exploit it to show that, unexpectedly, the activation of TBK1 and IKKϵ is not an autophosphorylation event but is mediated by a distinct “upstream” protein kinase.EXPERIMENTAL PROCEDURESMaterials—BX795 was synthesized as described (25Bain J. Plater L. Elliott M. Shpiro N. Hastie C.J. McLauchlan H. Klevernic I. Arthur J.S. Alessi D.R. Cohen P. Biochem. J. 2007; 408: 297-315Crossref PubMed Scopus (2088) Google Scholar), dissolved in DMSO, and stored as a 10 mm solution at –20 °C. Poly(I:C) and LPS were from InvivoGen, and mouse IL-1α and TNFα were from Sigma.DNA Constructs—TBK1 (NCBI NP_037386.1) was amplified from IMAGE EST 5492519 (Geneservice) using KOD Hot Start DNA Polymerase (Novagen). The PCR product was cloned into pSC-b (Stratagene) and sequenced to completion. The insert was excised using BamHI and NotI and inserted into pCMV-FLAG-1 or pEBG6P to generate FLAG-TBK1 and GST-TBK1, respectively. IKKϵ (NCBI NP_054721.1) was cloned in a similar manner using IMAGE EST 3062062. Point mutations were created using the QuikChange mutagenesis kit (Stratagene) but using KOD Hot Start DNA Polymerase. IRF3 (GenBank™ CAA91227.1) was amplified by PCR using IMAGE EST 5494536, ligated with pSC-b, and sequenced. The insert was subcloned into NotI sites of pGEX6P-2. PRDII (NFκB) and PRDIII-I (IRF3) elements from the IFN-β promoter cloned into the pLuc-MCS vector were a kind gift from Katherine Fitzgerald (University of Massachusetts). pTK-RL was obtained from Stratagene.Cell Culture—HEK293 cells stably expressing TLR3-FLAG (termed HEK293-TLR3 cells) were provided by Katherine Fitzgerald (University of Massachusetts), immortalized mouse embryonic fibroblasts (MEFs) were from wild type mice, and mice expressing a truncated, inactive form of TAK1 (26Sato S. Sanjo H. Takeda K. Ninomiya-Tsuji J. Yamamoto M. Kawai T. Matsumoto K. Takeuchi O. Akira S. Nat. Immunol. 2005; 6: 1087-1095Crossref PubMed Scopus (740) Google Scholar) were provided by Professor Shizuo Akira (Osaka University, Japan). HEK293-TLR3, RAW264.7 cells (hereafter termed RAW cells) and MEFS were maintained in Dulbecco's modified Eagle's medium supplemented with 2 mm glutamine, 10% fetal calf serum, and the antibiotics penicillin and streptomycin. Bone-marrow derived macrophages were generated from mice as described (27Ananieva O. Darragh J. Johansen C. Carr J.M. McIlrath J. Park J.M. Wingate A. Monk C.E. Toth R. Santos S.G. Iversen L. Arthur J.S. Nat. Immunol. 2008; 9: 1028-1036Crossref PubMed Scopus (238) Google Scholar). Cells were transfected using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions.Antibodies—Antibodies were raised in sheep against the human TBK1 protein expressed in insect Sf21 cells (Sheep S041C, bleed 2) (25Bain J. Plater L. Elliott M. Shpiro N. Hastie C.J. McLauchlan H. Klevernic I. Arthur J.S. Alessi D.R. Cohen P. Biochem. J. 2007; 408: 297-315Crossref PubMed Scopus (2088) Google Scholar) and the C-terminal peptide of mouse IKKϵ (NRLIERLHRVPSAPDV) (Sheep S277C, bleed 2) and used for immunoprecipitation. The phosphopeptide CEKFVS*VYGTE (where S* indicates phosphoserine) corresponding to the sequence surrounding Ser-172 of TBK1 and IKKϵ was used to generate an antibody that immunoprecipitated the phosphorylated forms of these protein kinases (Sheep S051C, bleed 2). The phosphopeptide was coupled separately to keyhole limpet hemocyanin and bovine serum albumin, then mixed and injected into sheep at Diagnostics Scotland (Edinburgh, UK). The antisera were purified by affinity chromatography on immobilized TBK1 or the peptide antigen, respectively, in the Division of Signal Transduction Therapy (University of Dundee. The phospho-specific antibody was incubated with the unphosphorylated form of the peptide immunogen (10 μg of peptide per μg of antibody) before use to neutralize any antibodies recognizing the unphosphorylated forms of TBK1 and IKKϵ. The following antibodies were used for immunoblotting: anti-glyceraldehyde-3-phosphate dehydrogenase (Research Diagnostics Inc.), anti-GST (Division of Signal Transduction Therapy, University of Dundee), horseradish peroxidase-conjugated secondary antibodies (Pierce), anti-IRF3, anti-TAK1 (Santa Cruz), anti-FLAG, anti-IKKϵ (Sigma), anti-TBK1, anti-TANK and anti-IκBα (Cell Signaling Technology). Antibodies recognizing phosphorylated Ser-933 (Ser(P)-933) of p105 (NFκB1), Ser(P)-32/Ser(P)-36 of IκBα, Ser(P)-468 of RelA, Ser(P)-536 of RelA, Ser(P)-396 of IRF3, the Thr(P)-Glu-Tyr(P) sequence of ERK1 and ERK2, the Thr(P)-Gly-Tyr(P) sequence of p38 MAP kinases and a pan-PDK1 phosphorylation site antibody which recognizes the phosphorylated activation loop (Thr(P)-229) of S6 kinase 1 (28Collins B.J. Deak M. Murray-Tait V. Storey K.G. Alessi D.R. J. Cell Sci. 2005; 118: 5023-5034Crossref PubMed Scopus (39) Google Scholar) were also from Cell Signaling Technology. The antibody recognizing the Thr(P)-Pro-Tyr(P) sequence of JNK1/2 was from BIOSOURCE, whereas that recognizing Ser(P)-172 of TBK1 was from BD Biosciences.Immunoprecipitation and Immunoblotting—Pharmacological inhibitors dissolved in DMSO or an equivalent volume of DMSO for control incubations were added to the culture medium of cells grown as monolayers. After 1 h at 37 °C, the cells were stimulated with LPS, poly(I:C), IL-1α, or TNFα as described in all the figure legends. Thereafter, the cells were rinsed in ice-cold phosphate-buffered saline and extracted in lysis buffer (50 mm Tris/HCl, pH 7.4, 1 mm EDTA, 1 mm EGTA, 50 mm NaF, 5 mm sodium pyrophosphate, 10 mm sodium β-glycerol 1-phosphate, 1 mm dithiothreitol, 1 mm sodium orthovanadate, 0.27 m sucrose, 1% (v/v) Triton X-100, 1 mg/ml aprotinin, 1 mg/ml leupeptin, 1 mm phenylmethylsulfonyl fluoride). Cell extracts were clarified by centrifugation at 14,000 × g for 10 min at 4 °C, and protein concentration was determined using the Bradford assay. Proteins were immunoprecipitated by incubating 1 mg of cell extract protein with 10 μg of antibody for 90 min at 4 °C followed by the addition of Protein G-Sepharose. After mixing for 15 min at 4 °C and brief centrifugation, the immunocomplexes were washed 3 times in lysis buffer, denatured in SDS, and subjected to SDS-PAGE. To detect proteins in cell lysates, 40 μg of protein extract was separated by SDS-PAGE. After transfer to polyvinylidene difluoride membranes, proteins were detected by immunoblotting and visualized by treating the blots with ECL (Amersham Biosciences) followed by autoradiography.Protein Kinase Assays—IRF3 was expressed in Escherichia coli as a GST fusion protein and purified by affinity chromatography on glutathione-Sepharose. Endogenous TBK1 was immunoprecipitated from 1 mg of cell lysate protein using the sheep anti-TBK1 (sheep S041C, bleed 2) antibody and overexpressed FLAG-TBK1 with an anti-FLAG M2-agarose (Sigma). Immunocomplexes were washed 3 times in lysis buffer and twice in 50 mm Tris-HCl, pH 7.5, 0.1% (v/v) 2-mercaptoethanol, 0.1 mm EGTA, 10 mm magnesium acetate and then resuspended in the same buffer containing 2 μm GST-IRF3 (endogenous TBK1) or the peptide KKKKERLLDDRHDSGLDSMKDEE (0.3 mm), corresponding to the sequence surrounding Ser-32 and Ser-36 of IκBα, plus four lysine residues at the N terminus to facilitate binding to phosphocellulose paper (termed IκBα peptide substrate). Assays were initiated by adding [γ-32P]ATP (1000 cpm/pmol) to a final concentration of 0.1 mm. When GST-IRF3 was used as the substrate, the reactions were terminated after 30 min at 30 °C by the addition of SDS containing 40 mm EDTA, pH 7.0, heated for 5 min at 100 °C and separated by SDS-PAGE, and phosphorylated proteins were detected by autoradiography. Quantification was performed by phosphorimaging analysis. For assays with the IκBα substrate peptide, reactions were terminated after 10 min at 30 °C by spotting an aliquot of the reaction on to a 2 × 2-cm2 piece of phosphocellulose P81 paper (Whatman) followed by immersion in 75 mm phosphoric acid. After washing six times in phosphoric acid and once in acetone, the papers were dried and counted. One unit of TBK1 activity was defined as that amount of enzyme catalyzing the incorporation of 1 nmol of phosphate into substrate in 1 min.Luciferase Assays—Cells were co-transfected with PRD(II)2 or PRD(III-I)3-pLuc-MCS and pTK-RL plasmid DNA. 24 h later cells were stimulated and extracted in Passive Lysis Buffer (Promega). Luciferase activity was measured with a dual-luciferase assay system (Promega) according to the manufacturer's instructions.Measurement of IFN-β Production—Cells were treated for 1 h with or without inhibitors then stimulated for 6 h with 100 ng/ml LPS or 10 μg/ml poly(I:C). The cell culture medium was removed and clarified by centrifugation for 10 min at 14,000 × g, and the concentration of mouse IFN-β was measured using an enzyme-linked immunosorbent assay kit (R&D Systems) according to manufacturer's protocol.Microscopy—Cells seeded on glass coverslips were serum-starved overnight before stimulation with poly(I:C). The cells were fixed, permeabilized, and stained as described (29Clark K. Langeslag M. van Leeuwen B. Ran L. Ryazanov A.G. Figdor C.G. Moolenaar W.H. Jalink K. van Leeuwen F.N. EMBO J. 2006; 25: 290-301Crossref PubMed Scopus (289) Google Scholar) using anti-IRF3 (1:100; Santa Cruz) followed by incubation with Alexa546-conjugated anti-rabbit IgG (1:500; Molecular Probes). The cells were then visualized using a Zeiss-LSM 510-meta microscope fitted with an alpha Plan-Fluar 100x/1.45 oil objective.Statistical Analysis—Quantitative data are presented as the mean ± S.E. Statistical significance of differences between experimental groups was assessed with Student's t test. Differences in means were considered significant if p < 0.05.RESULTSSpecificity of BX795 in Vitro—We reported previously that six protein kinases were inhibited by >90% in vitro in the presence of 0.1 μm BX795, namely TBK1, IKKϵ, PDK1, Aurora B, ERK8, and MARK3, but 60 other protein kinases tested were unaffected or only slightly inhibited at this concentration (25Bain J. Plater L. Elliott M. Shpiro N. Hastie C.J. McLauchlan H. Klevernic I. Arthur J.S. Alessi D.R. Cohen P. Biochem. J. 2007; 408: 297-315Crossref PubMed Scopus (2088) Google Scholar). The IC50 values for these six protein kinases are shown in Table 1. MARK3 is a member of the subgroup of protein kinases that include the AMP-activated protein kinase. In the present study we found that the other MARK isoforms (MARK1, MARK2, and MARK4) and another AMP-activated protein kinase-related kinase (NUAK1) were inhibited with similar potency to MARK3 (Table 1). We also studied the effect of BX795 on a number of additional kinases not examined previously. These experiments showed that BX795 did not inhibit the following protein-tyrosine kinases at 1 μm: ephrin receptors A2 and B3, Syk, Bruton's tyrosine kinase, and fibroblast growth factor receptor 1. However, the vascular endothelial growth factor receptor was inhibited, albeit much less potently than TBK1 (Table 1). We reported previously that BX795 did not inhibit IKKβ (25Bain J. Plater L. Elliott M. Shpiro N. Hastie C.J. McLauchlan H. Klevernic I. Arthur J.S. Alessi D.R. Cohen P. Biochem. J. 2007; 408: 297-315Crossref PubMed Scopus (2088) Google Scholar), and in the present study we found that the IKKα isoform is also unaffected at 1 μm in vitro (results not shown). For reasons discussed later, we also examined the effect of BX795 on a number of MAP kinase kinases (MKKs) and MAP kinase kinase kinases (termed MAP3Ks). At 1 μm, BX795 did not inhibit MKK1, MKK3, MKK4, MKK6, and MKK7, or the MAP3Ks Tpl2 and c-Raf (results not shown) but did potently inhibit the mixed lineage kinases, termed MLK1 (MAP3K9), MLK2 (MAP3K10), and MLK3 (MAP3K11)) (Table 1). MAP3K7, also called transforming growth factor β-activated kinase-1 (TAK1), and MAP3K5, also called apoptosis-stimulating kinase-1 (ASK1), were inhibited >100-fold less potently than TBK1 (Table 1). The ability of BX795 to inhibit the TBK1-catalyzed phosphorylation of IRF3 at Ser-396 declined as the ATP concentration in the assay was increased (supplemental Fig. S1), indicating that BX795 is an ATP competitive inhibitor of TBK1 as is the case for PDK1 (24Feldman R.I. Wu J.M. Polokoff M.A. Kochanny M.J. Dinter H. Zhu D. Biroc S.L. Alicke B. Bryant J. Yuan S. Buckman B.O. Lentz D. Ferrer M. Whitlow M. Adler M. Finster S. Chang Z. Arnaiz D.O. J. Biol. Chem. 2005; 280: 19867-19874Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar).TABLE 1I50 values for the inhibition of selected protein kinases by BX795KinaseIC50ATPμmμmTBK10.006 ± 0.001100IKKϵ0.041 ± 0.001100PDK10.111 ± 0.013100Aurora B0.031 ± 0.002100ERK80.140 ± 0.027100MARK30.081 ± 0.008100MARK10.055 ± 0.004100MARK20.053 ± 0.005100MARK40.019 ± 0.001100NUAK10.005 ± 0.001100VEGFR0.157 ± 0.011100MLK10.050 ± 0.003100MLK20.046 ± 0.011100MLK30.042 ± 0.005100TAK10.70 ± 0.14100ASK10.620 ± 0.004100 Open table in a new tab BX795 Blocks TBK1- and IKKϵ-mediated Activation of IRF3 and Production of IFN-β—To examine whether BX795 inhibited TBK1 and IKKϵ when added to mammalian cells in culture, we used HEK293 cells that stably overexpress TLR3 (termed HEK293-TLR3 cells) for initial studies. IRF3 migrated as a doublet in unstimulated cells. Stimulation with poly(I:C), a synthetic TLR3 agonist, led to the appearance of a more slowly migrating species whose level became maximal after 2 h (Fig. 1A). The appearance of this species, which has been shown by others to result from phosphorylation (30Lin R. Heylbroeck C. Pitha P.M. Hiscott J. Mol. Cell. Biol. 1998; 18: 2986-2996Crossref PubMed Scopus (747) Google Scholar), was prevented by prior incubation with BX795 (Fig. 1A). We also observed that IRF3 accumulated in the nucleus on a similar time scale after poly(I:C) treatment, which was blocked by BX795 (Fig. 1B). The next step in this pathway is IRF3-stimulated gene transcription. As expected from the results presented above, BX795 inhibited IRF3-dependent gene transcription (Fig. 1C). Finally, BX795 blocked the secretion of IFN-β from macrophages whether stimulated by LPS, a TLR4 agonist (Fig. 1, D and E), or poly(I:C) (Fig. 1E).BX795 was originally developed as an inhibitor the protein kinase PDK1, but BX795 had no effect on the LPS-stimulated phosphorylation of p70 ribosomal S6 kinase 1 at Thr-229, the site that is targeted by PDK1 (31Pullen N. Dennis P.B. Andjelkovic M. Dufner A. Kozma S.C. Hemmings B.A. Thomas G. Science. 1998; 279: 707-710Crossref PubMed Scopus (723) Google Scholar), under conditions where it completely blocked the LPS-stimulated phosphorylation of IRF3 at Ser-396 (Fig. 1F). This experiment demonstrated that TBK1/IKKϵ was inhibited much more potently than PDK1 by BX795 in cells and excluded the possibility that BX795 suppressed the activation of IRF3 and production of IFN-β by inhibiting PDK1.BX795 Does Not Affect Activation of the IKKα/β Complex or NFκB-dependent Gene Transcription by LPS, poly(I:C), IL-1α, or TNFα—The question of whether TBK1 and IKKϵ play a role in regulating NFκB-dependent gene transcription is an issue that is still not fully resolved (see the Introduction). We, therefore, studied the effect of BX795 on the activation of NFκB and NFκB-dependent transcriptional activity. Under conditions where BX795 completely blocked the LPS (Fig. 2A)- or poly(I:C)-stimulated (Fig. 2B) phosphorylation of IRF3 at Ser-396, this compound did not affect the phosphorylation at Ser-32 and Ser-36 or degradation of the IκBα inhibitory component of the NFκB transcription complex or the phosphorylation at Ser-933 of the p105 (NFκB1) regulatory subunit of the protein kinase Tpl2 (Fig. 2, A and B), which are established physiological substrates of the IKKα/β complex. These results demonstrated that BX795 did not inhibit any of the steps involved in the LPS- or poly(I:C)-stimulated activation of the IKKα/β complex or the ability of IKKα/β to phosphorylate downstream substrates. Nor did BX795 inhibit the LPS- or poly(I:C)-stimulated phosphorylation of the RelA component of the NFκB transcription factor at Ser-468 and Ser-536 (Fig. 2, A and B), amino acid residues reported to become phosphorylated when TBK1 and/or IKKϵ were overexpressed in cells (18