FAF1 Suppresses IκB Kinase (IKK) Activation by Disrupting the IKK Complex Assembly

Abstract

This study presents a molecular inhibitory mechanism by Fas-associated factor 1 (FAF1) on IκB kinase (IKK) activation, where divergent NF-κB-activating stimuli converge. FAF1 interacts with IKKβ in response to proinflammatory stimuli (such as tumor necrosis factor-α, interleukin-1β, and lipopolysaccharide) and suppresses IKK activation. Interaction of the leucine-zipper domain of IKKβ with FAF1 affected the IKK heterocomplex (IKKα/β) and homocomplex (IKKα/α, IKKβ/β) formations and attenuated IKKγ recruitment to IKKβ. Overexpression of FAF1 reduced the level of IKKβ activity, whereas FAF1 depletion increased the activity. These results indicate that FAF1 inhibits IKK activation and its downstream signaling by interrupting the IKK complex assembly through physical interaction with IKKβ. Taken together, FAF1 robustly suppresses NF-κB activation through the inhibition of IKK activation in combination with previously reported cytoplasmic retention of NF-κB p65 (Park, M. Y., Jang, H. D., Lee, S. Y., Lee, K. J., and Kim, E. (2004) J. Biol. Chem. 279, 2544–2549). Such redundant suppression would prevent inadvertent activation of the NF-κB pathway. This study presents a molecular inhibitory mechanism by Fas-associated factor 1 (FAF1) on IκB kinase (IKK) activation, where divergent NF-κB-activating stimuli converge. FAF1 interacts with IKKβ in response to proinflammatory stimuli (such as tumor necrosis factor-α, interleukin-1β, and lipopolysaccharide) and suppresses IKK activation. Interaction of the leucine-zipper domain of IKKβ with FAF1 affected the IKK heterocomplex (IKKα/β) and homocomplex (IKKα/α, IKKβ/β) formations and attenuated IKKγ recruitment to IKKβ. Overexpression of FAF1 reduced the level of IKKβ activity, whereas FAF1 depletion increased the activity. These results indicate that FAF1 inhibits IKK activation and its downstream signaling by interrupting the IKK complex assembly through physical interaction with IKKβ. Taken together, FAF1 robustly suppresses NF-κB activation through the inhibition of IKK activation in combination with previously reported cytoplasmic retention of NF-κB p65 (Park, M. Y., Jang, H. D., Lee, S. Y., Lee, K. J., and Kim, E. (2004) J. Biol. Chem. 279, 2544–2549). Such redundant suppression would prevent inadvertent activation of the NF-κB pathway. Fas-associated factor 1 (FAF1) 3The abbreviations used are: FAF1Fas-associated factor 1siFAF1FAF1-specific siRNAsiRNAsmall interfering RNANF-κBnuclear factor-κBImdimmune deficiencyIKKIκB kinaseLZleucine-zipperHLHhelix-loop-helixTNFtumor necrosis factorIL-1interleukin-1LPSlipopolysaccharideChIPchromatin immunoprecipitationFIDFas-interacting domainDEDIDdeath effector domain-interacting domainEGSethylene glycolbis-succinimidyl succinateGSTglutathione S-transferaseGFPgreen fluorescent protein.3The abbreviations used are: FAF1Fas-associated factor 1siFAF1FAF1-specific siRNAsiRNAsmall interfering RNANF-κBnuclear factor-κBImdimmune deficiencyIKKIκB kinaseLZleucine-zipperHLHhelix-loop-helixTNFtumor necrosis factorIL-1interleukin-1LPSlipopolysaccharideChIPchromatin immunoprecipitationFIDFas-interacting domainDEDIDdeath effector domain-interacting domainEGSethylene glycolbis-succinimidyl succinateGSTglutathione S-transferaseGFPgreen fluorescent protein. is evolutionarily conserved from flies to mammals (1Chu K. Niu X. Williams L.T. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 11894-11898Crossref PubMed Scopus (169) Google Scholar, 2Fröhlich T. Risau W. Flamme I. J. Cell Sci. 1998; 111: 2353-2363Crossref PubMed Google Scholar, 3Ryu S.W. Chae S.K. Lee K.J. Kim E. Biochem. Biophys. Res. Commun. 1999; 262: 388-394Crossref PubMed Scopus (43) Google Scholar, 4Kim M. Lee J.H. Lee S.Y. Kim E. Chung J. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 16358-16363Crossref PubMed Scopus (103) Google Scholar) and is involved in various key biological processes. FAF1 potentiates the Fas pathway as a member of the Fas death-inducing signaling complex and mediates chemotherapeutic-induced cell death (5Ryu S.W. Lee S.J. Park M.Y. Jun J.I. Jung Y.K. Kim E. J. Biol. Chem. 2003; 278: 24003-24010Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, 6Park M.Y. Ryu S.W. Kim K.D. Lim J.S. Lee Z.W. Kim E. Int. J. Cancer. 2005; 115: 412-418Crossref PubMed Scopus (25) Google Scholar). FAF1 also functions as an integral regulatory component of the transient receptor potential vanilloid type 1 (TRPV1) signaling pathway (7Kim S. Kang C. Shin C.Y. Hwang S.W. Yang Y.D. Shim W.S. Park M.Y. Kim E. Kim M. Kim B.M. Cho H. Shin Y. Oh U. J. Neurosci. 2006; 26: 2403-2412Crossref PubMed Scopus (52) Google Scholar). FAF1 is involved in the ubiquitination pathway and interacts with ubiquitin and valosin-containing protein, which is a multiubiquitin chain-targeting factor (8Song E.J. Yim S.H. Kim E. Kim N.S. Lee K.J. Mol. Cell. Biol. 2005; 25: 2511-2524Crossref PubMed Scopus (96) Google Scholar). In addition, FAF1 inhibits the chaperone activities of the heat-shock proteins Hsc70 and Hsp70 (9Kim H.J. Song E.J. Lee Y.S. Kim E. Lee K.J. J. Biol. Chem. 2005; 280: 8125-8133Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). Fas-associated factor 1 FAF1-specific siRNA small interfering RNA nuclear factor-κB immune deficiency IκB kinase leucine-zipper helix-loop-helix tumor necrosis factor interleukin-1 lipopolysaccharide chromatin immunoprecipitation Fas-interacting domain death effector domain-interacting domain ethylene glycolbis-succinimidyl succinate glutathione S-transferase green fluorescent protein. Fas-associated factor 1 FAF1-specific siRNA small interfering RNA nuclear factor-κB immune deficiency IκB kinase leucine-zipper helix-loop-helix tumor necrosis factor interleukin-1 lipopolysaccharide chromatin immunoprecipitation Fas-interacting domain death effector domain-interacting domain ethylene glycolbis-succinimidyl succinate glutathione S-transferase green fluorescent protein. FAF1 is also involved in the nuclear factor-κB (NF-κB) signaling pathway. FAF1 inhibits NF-κB activation in HEK 293 cells by binding to NF-κB p65 (10Park M.Y. Jang H.D. Lee S.Y. Lee K.J. Kim E. J. Biol. Chem. 2004; 279: 2544-2549Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). NF-κB inhibition by FAF1 has also been reported in Drosophila (4Kim M. Lee J.H. Lee S.Y. Kim E. Chung J. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 16358-16363Crossref PubMed Scopus (103) Google Scholar). Caspar, a fly homolog of human FAF1, selectively suppresses the immune deficiency (Imd) pathway. Loss-of-function caspar mutants constitutively expressed antibacterial genes in the absence of bacterial infections, indicating that caspar is an endogenous suppressor of the Imd pathway. Selective involvement of IκB kinase (IKK) complex in the fly Imd pathway led us to examine the regulatory mechanism, if any, between FAF1 and the IKK complex. The IKK complex is mainly composed of two catalytic subunits, IKKα/IKK1 and IKKβ/IKK2, and a regulatory subunit, IKKγ/NF-κB essential modulator (NEMO)/IKKAP1. Knockout mouse studies have demonstrated that IKKβ has a dominant role in NF-κB activation induced by proinflammatory cytokines, whereas IKKα is essential for morphogenic signaling (11Scheidereit C. Oncogene. 2006; 25: 6685-6705Crossref PubMed Scopus (541) Google Scholar). Although IKKγ lacks the catalytic function, IKKγ is essential for activation of the IKK complex. Both IKKα and IKKβ contain an N-terminal kinase domain, a central leucine-zipper (LZ) domain, and a C-terminal helix-loop-helix (HLH) domain (12Zandi E. Rothwarf D.M. Delhase M. Hayakawa M. Karin M. Cell. 1997; 91: 243-252Abstract Full Text Full Text PDF PubMed Scopus (1582) Google Scholar, 13Kwak Y.T. Guo J. Shen J. Gaynor R.B. J. Biol. Chem. 2000; 275: 14752-14759Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar, 14Delhase M. Hayakawa M. Chen Y. Karin M. Science. 1999; 284: 309-313Crossref PubMed Scopus (749) Google Scholar). Homo- and hetero-oligomerizations between IKKα and IKKβ occur through their LZ domains, and the HLH domains mediate recruitment of IKKγ to the IKK complex. This study reveals a novel function of FAF1: as an endogenous suppressor of IKK activation. FAF1 disrupts IKK complex assembly through physical interaction with the LZ domain of IKKβ. Association between FAF1 and IKKβ was induced by proinflammatory stimuli. Such an induced interaction indicates that FAF1 is an NF-κB pathway suppressor with a unique mode of action. Reagents and Constructs-Recombinant tumor necrosis factor-α (TNFα) and interleukin-1β (IL-1β) (R&D Systems Inc., Minneapolis, MN), lipopolysaccharide (LPS), anti-FLAG and anti-β-tubulin (Sigma-Aldrich), anti-IKKα, anti-IKKβ, anti-IKKγ, anti-IκBα, anti-p65, and anti-GFP (Santa Cruz Biotechnology, Santa Cruz, CA), anti-phospho-IKKα/β (Cell Signaling Technology, Beverly, MA), anti-glyceraldehyde-3-phosphate dehydrogenase (Lab Frontier, Seoul, Korea), and anti-S1 (Aprogen, Daejeon, Korea) antibodies were purchased from the indicated manufacturers. Constructions of FAF1, its deletion mutants, and pRK5-FLAG-IKKβ have been described previously (10Park M.Y. Jang H.D. Lee S.Y. Lee K.J. Kim E. J. Biol. Chem. 2004; 279: 2544-2549Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar, 15Ryu S.W. Kim E. Biochem. Biophys. Res. Commun. 2001; 286: 1027-1032Crossref PubMed Scopus (39) Google Scholar). We cloned IKKα, IKKβ, and IKKβ deletion constructs into the pMS1–2-S1 (Abcam, Cambridge, MA) and pGex4T-1 (Amersham Biosciences, Buckinghamshire, UK) vectors. The anti-FAF1 antibody and pCNS-IKKγ were provided by Dr. Jong-Seok Lim (Sookmyung Women's University, Seoul, Korea) and the Genome Research Center (Korea Research Institute of Bioscience and Biotechnology, Daejeon, Korea), respectively. Transfection and RNA Interference-Cell transfection experiments were performed using the calcium phosphate precipitation method with 1 μg of plasmid (15Ryu S.W. Kim E. Biochem. Biophys. Res. Commun. 2001; 286: 1027-1032Crossref PubMed Scopus (39) Google Scholar). Duplex siRNAs with two thymidine residues (dTdT) at the 3′-end of the sequence were provided by Qiagen Inc. (Valencia, CA) and transfected according to the manufacturer's instructions (Biontex, Munich, Germany). The target sequences were: IKKα (sense, 5′-GCAGGCUCUUUCAGGGACA-3′) (16Takaesu G. Surabhi R.M. Park K.J. Ninomiya-Tsuji J. Matsumoto K. Gaynor R.B. J. Mol. Biol. 2003; 326: 105-115Crossref PubMed Scopus (319) Google Scholar) and FAF1 (sense, 5′-CCACCUUCAUCAUCUAGUC-3′). The siRNA-resistant FAF1 and FAF1-FID (Fas-interacting domain) plasmids were constructed using site-directed mutagenesis of GFP-FAF1 and GFP-FAF1-FID using the following oligonucleotide: 5′-CCGCCGUCGUCGUCUAGUC-3′ (changed nucleotides are underlined). Immunoprecipitation-The HEK 293 cells were lysed in lysis buffer (10Park M.Y. Jang H.D. Lee S.Y. Lee K.J. Kim E. J. Biol. Chem. 2004; 279: 2544-2549Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar), and the lysates were subject to centrifugation. A total of 200 μg of protein from the supernatant was incubated with corresponding antibodies for 2 h at 4°C. Immunoprecipitates were washed and immunoblotted with the indicated antibodies. Luciferase Assay, GST Pulldown Assay, and in Vitro Kinase Assay-Luciferase assay (10Park M.Y. Jang H.D. Lee S.Y. Lee K.J. Kim E. J. Biol. Chem. 2004; 279: 2544-2549Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar, 17Hong S.Y. Yoon W.H. Park J.H. Kang S.G. Ahn J.H. Lee T.H. J. Biol. Chem. 2000; 1275: 18022-18028Abstract Full Text Full Text PDF Scopus (107) Google Scholar), GST pulldown assays (3Ryu S.W. Chae S.K. Lee K.J. Kim E. Biochem. Biophys. Res. Commun. 1999; 262: 388-394Crossref PubMed Scopus (43) Google Scholar, 10Park M.Y. Jang H.D. Lee S.Y. Lee K.J. Kim E. J. Biol. Chem. 2004; 279: 2544-2549Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar), and in vitro kinase assays (18DiDonato J.A. Hayakawa M. Rothwarf D.M. Zandi E. Karin M. Nature. 1997; 388: 548-554Crossref PubMed Scopus (1910) Google Scholar) were performed as described previously. Chromatin Immunoprecipitation (ChIP)-The HEK 293 cells were lysed in SDS lysis buffer (19Tian B. Nowak D.E. Jamaluddin M. Wang S. Brasier A.R. J. Biol. Chem. 2005; 280: 17435-17448Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar), and equal amounts of DNA were immunoprecipitated with 1 μg of anti-p65 antibody. Immunoprecipitated DNA was eluted and PCR-amplified. PCR products were separated by agarose gel electrophoresis and stained with ethidium bromide. ChIP primers for the IκBα gene (accession number NM_020529) were: IκBα promoter, sense, 5′-GACGACCCCAATTCAAATCG-3′, and antisense, 5′TCAGGCTCGGGGAATTTCC-3′. Interaction between FAF1 and IKKβ Is Induced by Proinflammatory Stimuli-Endogenous association of FAF1 and IKKβ was induced in response to proinflammatory stimuli such as TNFα, IL-1β, and LPS (Fig. 1A). GST pulldown assays using various GST fusion proteins of the truncated FAF1 showed that the N-terminal half of FAF1 (amino acids 1–381) interact with IKKβ (Fig. 1B). Similarly, GST pulldown assays between various domains of IKKβ and FAF1 demonstrated that the LZ domain of IKKβ strongly binds to FAF1 (Fig. 1C). The kinase domain of IKKβ also showed weak binding to FAF1. FAF1 Inhibits IKK Complex Formation-Considering that the LZ domain of IKKβ is responsible for interaction with FAF1, we examined oligomerization of IKKα and IKKβ leading to IKK complex assembly (20Tang E.D. Inohara N. Wang C.Y. Nunez G. Guan K.L. J. Biol. Chem. 2003; 278: 38566-38570Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). Levels of endogenous IKK complex (Fig. 2A) and homo-(IKKα/α, IKKβ/β) and hetero-complex (IKKα/β) (supplemental Fig. S1A) formations were increased when FAF1 was depleted by FAF1-specific siRNA (siFAF1) expression. IKK complex formations were decreased when FAF1 expression was rescued by the transfection of siRNA-resistant FAF1 construct, FAF1-res (Fig. 2A and supplemental Fig. S1C), indicating that FAF1 suppress IKK complex formation. We then examined which FAF1 domain had the potential to suppress IKK complex formation. Transfection of FAF1-FID reduced IKK complex formation, but those of FAF1-DEDID (death effector domain-interacting domain) and FAF1-ΔFID ·DEDID did not (supplemental Fig. S1B). Expression of siRNA-resistant FAF1-FID reduced the endogenous IKKα/β/γ oligomerization (Fig. 2A). This indicates that FAF1-FID has the potential for IKK complex disruption. We asked whether FAF1 affected IKKβ recruitment to the tetrameric IKKγ, which is the essential regulatory component that facilitates the trans-autophosphorylation of IKKβ necessary for IKK complex activation (21Tegethoff S. Behlke J. Scheidereit C. Mol. Cell. Biol. 2003; 23: 2029-2041Crossref PubMed Scopus (111) Google Scholar). In vitro-translated 35S-labeled IKKγ proteins were treated with ethylene glycol-bissuccinimidyl succinate (EGS), a cross-linking agent, to induce IKKγ tetramerization. Residual amounts of monomeric IKKγ were still detected after EGS treatment, as has been previously reported. The addition of FAF1 reduced IKKβ recruitment to the tetrameric and monomeric forms of IKKγ in a dose-dependent manner (Fig. 2B), whereas unrelated proteins, such as β-tubulin and glyceraldehyde-3-phosphate dehydrogenase, did not (supplemental Fig. S2). Consistent with the above results, binding between the IKKβ-HLH domain and IKKγ was strongly inhibited by the addition of FAF1 protein (Fig. 2C). Furthermore, IKK phosphorylation was blocked by the addition of FAF1 in a dose-dependent manner, whereas the total amount of exogenous IKKβ remained unchanged (Fig. 2D). These results demonstrate that FAF1 selectively inhibits IKK activation by disrupting assembly of the fully competent IKK complex. FAF1 Inhibits the Catalytic Activity of IKKβ-Next, the catalytic activity of IKKβ in the disrupted IKK complex by FAF1 was measured. FAF1 overexpression reduced the TNFα-induced catalytic activities of IKKβ in a dose-dependent manner (Fig. 3A, upper right panel). However, introduction of FAF1-specific siRNA increased the level of IKKβ activity (Fig. 3A, upper left panel). Similarly, FAF1 overexpression attenuated IL-1β- or LPS-induced IKKβ activations (Fig. 3A, lower panels). Consistent with these in vivo results, the addition of GST-FAF1 also inhibited the IKK catalytic activity in vitro in a dose-dependent manner (Fig. 3B, right panel). To further confirm the inhibitory function of FAF1, the level of IκBα, which is an endogenous marker of IKK activity, was examined. After TNFα treatment, the level of IκBα in mock-transfected cells was more rapidly reduced than the level in FAF1-transfected cells, indicating that FAF1 inhibits IκBα phosphorylation (Fig. 3C). The level of IKKβ activity was inversely correlated with the FAF1 expression level, indicating that FAF1 negatively regulates the IKKβ activity. When IKKα-specific siRNA was transfected, FAF1 inhibited IKKβ activation (supplemental Fig. S3). This indicates that FAF1-mediated IKKβ activity suppression occurred independently of IKKα. Considering that fly homologs of mammalian IKKβ and IKKγ, but not IKKα, participate in the fly Imd pathway (22Tanji T. Ip Y.T. Trends Immunol. 2005; 26: 193-198Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar), inhibition of the fly Imd pathway by FAF1 correlates well with the IKKα-independent IKKβ suppression in mammalian cells shown here. FAF1-FID Is Crucial for Suppression of IKKβ Activation-Both the FID and the DEDID of FAF1 interact with IKKβ (Fig. 1B); therefore, we investigated which domain is responsible for suppression of IKKβ activation. Transfection of fulllength FAF1 and FAF1-FID led to significant suppression of IKKβ catalytic activity, whereas FAF1-DEDID and FAF1-ΔFID·DEDID did not (Fig. 4A, left panel). Consistent with these cellular results, GST-FAF1-FID successfully inhibited the activity of IKKβ kinase to its substrate GST-IκBα in in vitro kinase assays (Fig. 4A, right panel). These data indicate that FAF1-FID is responsible and sufficient for the suppression of IKKβ activation. This result provides a molecular explanation of the suppression of IKK complex formation by FAF1-FID (Fig. 2A). We then tested whether FAF1-FID inhibits NF-κB activation. FAF1-FID overexpression strongly inhibited NF-κB activation by proinflammatory stimuli in HEK 293 cells (Fig. 4B, upper panel). The FAF1-FID-mediated suppression of IKK activation was also shown by reduced transcription of the NF-κB target gene cIAP2 (Fig. 4B, lower panel). ChIP results also showed that FAF1-FID significantly inhibited, whereas siFAF1 increased, recruitment of NF-κB to the IκBα promoter region (Fig. 4C). This study demonstrates that FAF1 has a novel function as a suppressor of IKK complex activation. 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However, FAF1 differs from the suppressors that bind to IKKs in quiescent cells. By contrast, interaction of FAF1 with IKKβ is induced by IKK-activating signals. Such a binding pattern would be useful to prevent over-activation of a pathway that requires tight regulation. The detailed mechanism underlying induced binding of FAF1-IKKβ is yet to be determined. Phosphorylation of FAF1 has been reported (31Olsen B.B. Jessen V. Hojrup P. Issinger O.G. Boldyreff B. FEBS Lett. 2003; 546: 218-222Crossref PubMed Scopus (20) Google Scholar); however, the phosphorylation status of FAF1 remained unchanged despite stimulation (data not shown). In addition, in vitro binding studies have shown that phosphorylation is not a prerequisite for the binding, indicating that phosphorylation of FAF1 might not be the determining factor. Other types of modification in FAF1 or in FAF1-interacting partners that occur in response to stimuli might facilitate the FAF1-IKKβ interaction. Proteomic analysis of FAF1-interacting proteins in response to NF-κB-activating stimuli would help us to investigate this issue. Download with