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An antiapoptotic protein, c-FLIPL, directly binds to MKK7 and inhibits the JNK pathway

2006; Springer Nature; Volume: 25; Issue: 23 Linguagem: Inglês

10.1038/sj.emboj.7601423

1460-2075

Akihito Nakajima, Sachiko Komazawa‐Sakon, Mutsuhiro Takekawa, Tomonari Sasazuki, Wen‐Chen Yeh, Hideo Yagita∥, Ko Okumura, Hiroyasu Nakano,

Immune Cell Function and Interaction

Article16 November 2006free access An antiapoptotic protein, c-FLIPL, directly binds to MKK7 and inhibits the JNK pathway Akihito Nakajima Akihito Nakajima Department of Immunology, Juntendo University School of Medicine, Tokyo, Japan Search for more papers by this author Sachiko Komazawa-Sakon Sachiko Komazawa-Sakon Department of Immunology, Juntendo University School of Medicine, Tokyo, Japan Search for more papers by this author Mutsuhiro Takekawa Mutsuhiro Takekawa Division of Molecular Cell Signaling, Institute of Medical Science, The University of Tokyo, Tokyo, Japan Search for more papers by this author Tomonari Sasazuki Tomonari Sasazuki Department of Immunology, Juntendo University School of Medicine, Tokyo, Japan Search for more papers by this author Wen-Chen Yeh Wen-Chen Yeh Campbell Family Institute for Breast Cancer Research, University Health Network, Department of Medical Biophysics, University of Toronto, Toronto, Ontario, Canada Search for more papers by this author Hideo Yagita Hideo Yagita Department of Immunology, Juntendo University School of Medicine, Tokyo, Japan Search for more papers by this author Ko Okumura Ko Okumura Department of Immunology, Juntendo University School of Medicine, Tokyo, Japan Search for more papers by this author Hiroyasu Nakano Corresponding Author Hiroyasu Nakano Department of Immunology, Juntendo University School of Medicine, Tokyo, Japan Search for more papers by this author Akihito Nakajima Akihito Nakajima Department of Immunology, Juntendo University School of Medicine, Tokyo, Japan Search for more papers by this author Sachiko Komazawa-Sakon Sachiko Komazawa-Sakon Department of Immunology, Juntendo University School of Medicine, Tokyo, Japan Search for more papers by this author Mutsuhiro Takekawa Mutsuhiro Takekawa Division of Molecular Cell Signaling, Institute of Medical Science, The University of Tokyo, Tokyo, Japan Search for more papers by this author Tomonari Sasazuki Tomonari Sasazuki Department of Immunology, Juntendo University School of Medicine, Tokyo, Japan Search for more papers by this author Wen-Chen Yeh Wen-Chen Yeh Campbell Family Institute for Breast Cancer Research, University Health Network, Department of Medical Biophysics, University of Toronto, Toronto, Ontario, Canada Search for more papers by this author Hideo Yagita Hideo Yagita Department of Immunology, Juntendo University School of Medicine, Tokyo, Japan Search for more papers by this author Ko Okumura Ko Okumura Department of Immunology, Juntendo University School of Medicine, Tokyo, Japan Search for more papers by this author Hiroyasu Nakano Corresponding Author Hiroyasu Nakano Department of Immunology, Juntendo University School of Medicine, Tokyo, Japan Search for more papers by this author Author Information Akihito Nakajima1, Sachiko Komazawa-Sakon1, Mutsuhiro Takekawa2, Tomonari Sasazuki1, Wen-Chen Yeh3, Hideo Yagita1, Ko Okumura1 and Hiroyasu Nakano 1 1Department of Immunology, Juntendo University School of Medicine, Tokyo, Japan 2Division of Molecular Cell Signaling, Institute of Medical Science, The University of Tokyo, Tokyo, Japan 3Campbell Family Institute for Breast Cancer Research, University Health Network, Department of Medical Biophysics, University of Toronto, Toronto, Ontario, Canada *Corresponding author. Department of Immunology, Juntendo University School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421, Japan. Tel.: +81 3 5802 1045; Fax: +81 3 3813 0421; E-mail: [email protected] The EMBO Journal (2006)25:5549-5559https://doi.org/10.1038/sj.emboj.7601423 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Inhibition of NF-κB activation increases susceptibility to tumor necrosis factor (TNF)α-induced cell death, concurrent with caspases and prolonged c-Jun N-terminal kinase (JNK) activation, and reactive oxygen species (ROS) accumulation. However, the detailed mechanisms are unclear. Here we show that cellular FLICE-inhibitory protein (c-FLIP) is rapidly lost in NF-κB activation-deficient, but not wild-type fibroblasts upon TNFα stimulation, indicating that NF-κB normally maintains the cellular levels of c-FLIP. The ectopic expression of the long form of c-FLIP (c-FLIPL) inhibits TNFα-induced prolonged JNK activation and ROS accumulation in NF-κB activation-deficient fibroblasts. Conversely, TNFα induces prolonged JNK activation and ROS accumulation in c-Flip−/− fibroblasts. Moreover, c-FLIPL directly interacts with a JNK activator, MAP kinase kinase (MKK)7, in a TNFα-dependent manner and inhibits the interactions of MKK7 with MAP/ERK kinase kinase 1, apoptosis-signal-regulating kinase 1, and TGFβ-activated kinase 1. This stimuli-dependent interaction of c-FLIPL with MKK7 might selectively suppress the prolonged phase of JNK activation. Taken that ROS promote JNK activation and activation of the JNK pathway may promote ROS accumulation, c-FLIPL might block this positive feedback loop, thereby suppressing ROS accumulation. Introduction NF-κB is a collective term of dimeric transcriptional factors that belong to the Rel family of proteins, and regulates expression of various inflammatory cytokines, chemokines, and adhesion molecules (Ghosh and Karin, 2002). NF-κB is activated by various inflammatory cytokines and cellular stress including tumor necrosis factor (TNF)α, interleukin-1 (IL-1), UV, and γ-irradiation. Moreover, NF-κB, especially the RelA-containing complex, inhibits cell death induced by TNFα, Fas ligand, TRAIL, and genotoxic stress. Currently, the antiapoptotic functions of NF-κB are supported to be mainly mediated by the upregulation of antiapoptotic genes such as cellular FLICE-inhibitory protein (c-Flip), the members of the Bcl-2 family, and X-chromosome-linked inhibitor of apoptosis (Xiap) (Karin and Lin, 2002). However, the detailed molecular mechanisms are not completely understood. The regulation of cell death and survival is also controlled in part by another signaling cascade activated by the mitogen-activated protein kinase (MAPK) following cellular stress or cytokine signaling (Davis, 2000; Kyriakis and Avruch, 2001). In mammals, the MAPK cascades are composed of three distinct signaling modules, the c-Jun N-terminal kinase (JNK), the p38MAPK, and the extracellular signal-regulated kinase (ERK) cascades. Each MAPK is activated by sequential protein phosphorylation through a MAPK module. In the case of the JNK cascade, the MAP kinase kinase kinases (MAPKKKs) include apoptosis-signal-regulating kinase (ASK)1, MAP/ERK kinase kinases (MEKKs), MTK1 (also known as MEKK4), and TGFβ-activated kinase (TAK)1. These MAPKKKs activate MAP kinase kinase (MKK)4 and/or MKK7, which in turn activate JNKs (Davis, 2000; Kyriakis and Avruch, 2001). Cytokines and growth factors including TNFα and IL-1 induce rapid (within 10 min) yet transient MAPK activation, cellular stress, such as UV- or γ-irradiation, induces prolonged MAPK activation. Several lines of evidence suggest that transient MAPK activation is associated with gene expression, proliferation, and differentiation, whereas prolonged MAPK activation promotes cell death in a cell-type- and stimuli-dependent manner (Chen et al, 1996; Guo et al, 1998). In the past, the contributions of the NF-κB and JNK pathways have been discussed independently. However, two recent studies have revealed that inhibition of NF-κB activation induces prolonged JNK activation that promotes TNFα-induced cell death (De Smaele et al, 2001; Tang et al, 2001). Later studies have shown that TNFα-induced accumulation of reactive oxygen species (ROS) is enhanced in NF-κB activation-deficient cells including RelA−/−, IκB kinase β−/−, and TNF receptor-associated factor (traf)2−/−traf5−/− murine embryonic fibroblasts (MEFs) (Sakon et al, 2003; Pham et al, 2004; Ventura et al, 2004; Kamata et al, 2005). Notably, this TNFα-induced ROS accumulation and prolonged JNK activation are completely suppressed by antioxidants, but not caspase inhibitors, indicating that TNFα-induced ROS accumulation is caspase-independent in NF-κB activation-deficient MEFs. Furthermore, expression of antioxidant enzyme genes including manganese-dependent superoxide dismutase (Mnsod) and ferritin heavy chain (fhc) is upregulated by TNFα in an NF-κB-dependent manner, and the ectopic expression of these genes inhibits TNFα-induced ROS accumulation in NF-κB-deficient MEFs (Pham et al, 2004; Kamata et al, 2005). Although these studies have revealed that the caspase-independent pathway plays a crucial role in ROS accumulation, a recent study has shown that activated caspase-3 cleaves the p75 subunit of complex I of the mitochondrial electron transport chain, which would impair the function of complex I, resulting in ROS accumulation (Ricci et al, 2004). In addition, Giorgio et al (2005) have shown that proapoptotic signals induce the release of p66Shc from a putative inhibitory complex and the released p66Shc then oxidizes reduced cytochrome c, thereby generating ROS. Therefore, the mechanisms whereby TNFα induces ROS accumulation are still controversial (Papa et al, 2004b; Luo et al, 2005; Nakano et al, 2006). c-FLIP, also designated as CASH or Casper, was first identified as a molecule that interacts with FADD or is structurally related to procaspase-8 (Goltsev et al, 1997; Irmler et al, 1997; Shu et al, 1997). c-Flip encodes two splicing variants, long form of c-FLIP (c-FLIPL) and short form of c-FLIP (c-FLIPs). While c-FLIPS only contains two N-terminal death effector domains (DEDs), c-FLIPL consists of the N-terminal DEDs and a C-terminal caspase-like domain that does not possess enzymatic activity. Many studies have shown that both c-FLIPL and c-FLIPS inhibit death receptor-induced apoptosis by binding to and inhibiting caspase-8 activation via DED–DED interaction (Thome and Tschopp, 2001). Consistently, c-Flip−/− cells showed an increased susceptibility to TNFα- and Fas ligand-induced cell death (Yeh et al, 2000). Moreover, recent studies have shown that c-FLIP and viral FLIP are also involved in the Wnt signaling pathways (Naito et al, 2004; Nakagiri et al, 2005). In the present study, we have shown that c-FLIPL is rapidly degraded after TNFα stimulation, and the ectopic expression of c-FLIPL inhibits the TNFα-induced ROS accumulation and prolonged JNK activation in NF-κB activation-deficient MEFs. Conversely, TNFα induces prolonged JNK and ERK activation, and ROS accumulation in c-Flip−/− cells. We have also shown that c-FLIPL binds to MKK7 and MEK1 in a TNFα-dependent manner, resulting in suppression of the MKK7–JNK and MEK1–ERK pathways. Taken that ROS promote JNK activation and activation of the JNK pathway may induce ROS accumulation, c-FLIPL might block this positive feedback loop, thereby suppressing ROS accumulation. Collectively, c-FLIPL is essential for suppression of TNFα-induced prolonged JNK activation and ROS accumulation. Results NF-κB protects signal-dependent degradation of c-FLIPL To explore signaling intermediate(s) that link the TNF receptor to ROS accumulation, we first compared protein expression levels of downstream signaling molecules of the TNF receptor in wild-type (WT), RelA−/−, and traf2−/−traf5−/− MEFs before and after TNFα stimulation. Among various signaling molecules investigated, we found that TNFα stimulation induced a rapid and progressive loss of c-FLIPL in both RelA−/− and traf2−/−traf5−/− MEFs, concurrent with the appearance of a degradation fragment of 43 kDa (Figure 1A). Notably, MEFs appeared to express 55 kDa c-FLIPL, but not 26 kDa c-FLIPs. A similar degradation fragment was also observed in WT MEFs after TNFα stimulation, while the levels of c-FLIPL were not apparently reduced. When pretreated with a protein synthesis inhibitor, cycloheximide (CHX), c-FLIPL was progressively lost also in WT MEFs (Figure 1B). These results indicate that TNFα induces rapid degradation of c-FLIP in both WT and NF-κB activation-deficient MEFs, but the expression levels of c-FLIPL are maintained in WT MEFs by NF-κB-dependent de novo synthesis. It has been shown that the processing of c-FLIPL to the p43 fragment is mediated by caspase-8 (Shu et al, 1997; Kataoka and Tschopp, 2004), and c-FLIP levels are also controlled in part by the ubiquitin/proteasome pathway (Kreuz et al, 2001; Micheau et al, 2001). Consistently, the TNFα-induced degradation of c-FLIPL at early time points was partially inhibited by z-VAD-fmk or a proteasome inhibitor, MG132, in RelA−/− and traf2−/−traf5−/− MEFs (Supplementary Figure S1). Therefore, the TNFα-induced rapid degradation of c-FLIPL may be at least partly mediated by caspases and the ubiquitin/proteasome pathway. Figure 1.TNFα induces rapid degradation of c-FLIPL and ectopic expression of c-FLIPL inhibits TNFα-induced accumulation of ROS, prolonged JNK activation, and cell death in RelA−/− and traf2−/−traf5−/− MEFs. (A) WT, RelA−/−, and traf2−/−traf5−/− MEFs were stimulated with TNFα (10 ng/ml) for the indicated times, then the lysates were analyzed by immunoblotting with anti-c-FLIP (upper panel) and anti-tubulin (lower panel) antibodies. p55 and p43 indicate full and degraded form of c-FLIPL, respectively. (B) WT MEFs were stimulated with TNFα (10 ng/ml) in the presence of CHX (10 μg/ml) for the indicated times. Immunoblotting were performed as in (A). The asterisks indicate nonspecific bands. (C) Transfection of c-FLIPL inhibits TNFα-induced ROS accumulation in RelA−/− and traf2−/−traf5−/− MEFs. Total cell lysates from mock (M) or respective transfectants (T) were immunoblotted with anti-Flag antibody. The transfectants were unstimulated (thin line) or stimulated (bold line) with TNFα (10 ng/ml) for 4 h, and then the cells were labeled with CM-H2DCFDA (1 μM) and analyzed by flow cytometry. (D) c-FLIPL inhibits prolonged, but not early JNK activation in RelA−/− and traf2−/−traf5−/− MEFs. Mock- or c-FLIPL-transfected RelA−/− and traf2−/−traf5−/− MEFs were stimulated with TNFα (10 ng/ml) for the indicated times, and then the lysates were immunoblotted with anti-phospho-JNK (upper panel) and anti-total JNK (lower panel) antibodies. (E) c-FLIPL inhibits TNFα-induced cell death in RelA−/− and traf2−/−traf5−/− MEFs. Mock- or c-FLIPL-transfected RelA−/− and traf2−/−traf5−/− MEFs were stimulated with the indicated amounts of TNFα for 16 h. Cell viability was determined by WST assay. Results are presented as the mean of triplicate samples and represent three independent experiments with similar results. Standard errors are within 5%. *P<0.05 compared to mock transfectant. Download figure Download PowerPoint c-FLIPL inhibits TNFα-induced ROS accumulation and prolonged JNK activation Consistent with our previous study (Sakon et al, 2003), TNFα-induced ROS accumulation and prolonged JNK activation are suppressed by an antioxidant, butylated hydroxyanisole (BHA), but not a broad caspase inhibitor, z-VAD-fmk in RelA−/− and traf2−/−traf5−/− MEFs (Supplementary Figure S2A). This indicates that ROS accumulation and prolonged JNK activation are induced in a caspase-independent manner in these cells. To test directly whether the degradation of c-FLIPL is responsible for the TNFα-induced ROS accumulation and prolonged JNK activation, we ectopically expressed c-FLIPL in RelA−/− and traf2−/−traf5−/− MEFs. Expression of c-FLIPL almost completely inhibited TNFα-induced ROS accumulation in RelA−/− and traf2−/−traf5−/− MEFs (Figure 1C), suggesting that c-FLIPL might also suppress the caspase-independent pathway leading to ROS accumulation. TNFα-induced JNK activation at 10 min was not different between WT and RelA−/− MEFs, but this early phase JNK activation was severely impaired in traf2−/−traf5−/− MEFs (Supplementary Figure S2B). Intriguingly, c-FLIPL specifically inhibited the sustained (at 120 min), but not early (at 10 min), phase of JNK activation in RelA−/− and traf2−/−traf5−/− MEFs (Figure 1D). Moreover, expression of c-FLIPL substantially increased cell viability after TNFα stimulation in RelA−/− and traf2−/−traf5−/− MEFs (Figure 1E). Taken that TNFα induces caspase-dependent and -independent cell death in NF-κB activation-deficient cells (Sakon et al, 2003), these results indicate that ectopic expression of c-FLIPL is sufficient for suppression of TNFα-induced ROS accumulation, prolonged JNK activation, and cell death through suppression of the caspase-dependent and -independent pathways in NF-κB activation-deficient cells. TNFα induces ROS accumulation and prolonged JNK activation in c-Flip−/− MEFs To test directly whether c-FLIPL is essential for suppression of JNK activation and ROS accumulation, we first investigated the time course of caspase-3 activities after TNFα stimulation in c-Flip−/− MEFs. TNFα did not induce activation of caspase-3 in WT MEFs, unless the cells were stimulated with TNFα in the presence of CHX (Figure 2A). In sharp contrast, TNFα stimulation induced a robust activation of caspase-3 at 1 h and then continued at 2 h, which was completely suppressed in the presence of z-VAD-fmk or z-VAD-fmk plus BHA, but not BHA in c-Flip−/− MEFs (Figure 2A). We next tested whether JNK activation is prolonged in c-Flip−/− MEFs. While TNFα stimulation only induced transient JNK activation in WT MEFs, the sustained phase of JNK activation gradually appeared at 1 h after stimulation in c-Flip−/− MEFs, which was completely suppressed by z-VAD-fmk (Figure 2B). However, JNK activation continued at 2 h even in the presence of z-VAD-fmk, and this activation was substantially inhibited by the further addition of BHA. These results suggest that the caspase-dependent pathway plays a dominant role in JNK activation at 1 h. This is then followed by the caspase-independent ROS-inducing pathway leading to JNK activation at 2 h. Similarly, TNFα-induced ERK activation was prolonged in c-Flip−/− MEFs. Phosphorylation of JNK and ERK in TNFα plus BHA-treated cells at 2 h appeared to decrease compared to TNFα plus zVAD-fmk-treated cells. Taken that BHA alone inhibited neither caspase activation nor ROS accumulation at 2 h (Figure 2A and C), these decreases might not be due to direct inhibitory effect of BHA on JNK and ERK activation, but rather due to massive cell death of TNFα plus BHA-treated c-Flip−/− MEFs (data not shown). Figure 2.TNFα induces ROS accumulation and prolonged JNK activation in c-Flip−/− MEFs. (A) WT MEFs and c-Flip−/− MEFs were stimulated with TNFα (10 ng/ml) in the absence or presence of CHX (10 μg/ml), z-VAD-fmk (zVAD) (50 μM), BHA (100 μM), or z-VAD-fmk (50 μM) plus BHA (100 μM) for the indicated times. Caspase-3 activities were measured by using fluorogenic substrates. Results are presented as mean±s.e.'s of triplicate samples. Caspase activities were not detected in z-VAD-fmk- or z-VAD-fmk plus BHA-treated cells. (B) WT and c-Flip−/− MEFs were stimulated as in (A), and then the lysates were analyzed as in Figure 1D. zB indicates z-VAD-fmk (50 μM) plus BHA (100 μM) treatment. (C) WT and c-Flip−/− MEFs were unstimulated (thin line) or stimulated (bold line) with TNFα (10 ng/ml) as in (A) for the indicated times, accumulated ROS were analyzed as in Figure 1C. NE (not examined) indicates that ROS accumulation was not analyzed due to massive cell death. (D) c-Flip−/− MEFs were stimulated with the indicated amounts of TNFα in the absence or presence of z-VAD-fmk (zVAD) (50 μM), BHA (100 μM), or z-VAD-fmk (50 μM) plus BHA (100 μM) for 14 h. Cell viability was determined by WST assay as in Figure 1E. *P<0.05 compared to TNFα plus z-VAD-fmk-treated cells. Download figure Download PowerPoint We tested whether TNFα induces ROS accumulation in c-Flip−/− MEFs. ROS slightly accumulated at 1 h, and then progressively accumulated at 2 h after stimulation in c-Flip−/− MEFs (Figure 2C). In contrast to NF-κB activation-deficient cells (Supplementary Figure S2A), TNFα-induced ROS accumulation was significantly suppressed by z-VAD-fmk at 2 h after simulation. However, a substantial amount of ROS still accumulated in c-Flip−/− MEFs at 2 and 4 h even in the presence of z-VAD-fmk (Figure 2C). We could not evaluate ROS accumulation in TNFα- or TNFα plus BHA-stimulated c-Flip−/− MEFs at 4 h due to cell death. Consistent with an inhibitory effect on TNFα-induced JNK activation, BHA plus z-VAD-fmk almost completely suppressed TNFα-induced ROS accumulation at 2 and 4 h after stimulation. Together, these results suggest that both caspase-dependent and -independent pathways may play crucial roles in TNFα-induced prolonged JNK activation and ROS accumulation in c-Flip−/− MEFs. The reason why BHA could not inhibit caspase-dependent ROS accumulation is currently unknown. Similarly, another antioxidant, N-acetyl cystein, could not inhibit TNFα-induced ROS accumulation in c-Flip−/− MEFs (data not shown). Consistent with these results, z-VAD-fmk plus BHA treatment more efficiently increased cell viability of c-Flip−/− MEFs than z-VAD-fmk alone (Figure 2D). Collectively, c-FLIP is essential for suppression of TNFα-induced ROS accumulation, prolonged JNK activation, and cell death. We also tested whether deficiency of c-FLIP could affect the duration of the JNK pathway induced by other stimuli. Consistent with a previous study (Chen et al, 1996), anisomycin and UV induced prolonged JNK activation even in WT MEFs (Supplementary Figure S3A and B). Similarly, these stimuli induced prolonged JNK activation in c-Flip−/− MEFs. In contrast, IL-1 induced transient JNK activation in both WT and c-Flip−/− MEFs (Supplementary Figure S3C), suggesting that c-FLIP-dependent suppression of the JNK pathway is specific to TNFα-induced JNK activation. TNFα-induced NF-κB activation and expression of fhc and Mnsod genes are not different between WT and c-Flip−/− MEFs Previous studies have shown that NF-κB-dependent upregulation of antioxidant enzyme genes including fhc and Mnsod are responsible for elimination of ROS (Pham et al, 2004; Kamata et al, 2005). Therefore, TNFα induces ROS accumulation in NF-κB activation-deficient cells. On the other hand, several studies have shown that c-FLIPL activates NF-κB under particular conditions (Kataoka et al, 2000; Golks et al, 2006). To test the possibility that accumulation of ROS is due to impaired upregulation of fhc and/or Mnsod genes in c-Flip−/− MEFs, we first investigated whether TNFα-induced IκBα degradation is impaired in c-Flip−/− MEFs. Consistent with a previous study (Yeh et al, 2000), TNFα-induced degradation of IκBα was not impaired in c-Flip−/− MEFs (Figure 3A). Moreover, TNFα-induced upregulation of fhc and Mnsod mRNAs were not significantly different up to 120 min after TNFα stimulation in WT and c-Flip−/− MEFs (Figure 3B). We could not investigate the induction of mRNAs of these genes at later time points after 120 min, as almost all c-Flip−/− MEFs died. Figure 3.TNFα-induced NF-κB activation and expression of fhc and Mnsod mRNAs are not different between WT and c-Flip−/− MEFs. (A) TNFα induces degradation of IκBα in WT and c-Flip−/− MEFs. WT and c-Flip−/− MEFs were stimulated with TNFα (10 ng/ml) for the indicated times, then the lysates were analyzed by immunoblotting with anti-IκBα (upper panel) and anti-tubulin (lower panel) antibodies. (B) Induction of fhc and Mnsod mRNAs upon TNFα stimulation. WT or c-Flip−/− MEFs were stimulated with TNFα as in (A), and then total RNAs were extracted. Expression of the indicated genes was analyzed by real-time PCR. mRNA levels of the indicated genes were compared as a ratio to the unstimulated time points. The data are shown as mean±s.e.'s of triplicates samples. (C) TNFα induces prolonged MEK1 activation in c-Flip−/− MEFs. MEFs were stimulated as in (A), and the lysates were analyzed by immunoblotting with anti-phospho MEK1 (upper panel) and anti-total MEK1 (lower panel) antibodies. The asterisks indicate the degraded form of MEK1. Download figure Download PowerPoint To further explore the mechanism underlying c-FLIP-dependent suppression of the ERK and JNK pathways, we examined whether TNFα-induced MEK1 phosphorylation is prolonged in c-Flip−/− MEFs. Phosphorylation of endogenous MEK1 was detected at 10 min upon TNFα stimulation in both WT and c-Flip−/− MEFs. Notably, phosphorylation of MEK1 reappeared at 60 min and persisted up to 120 min in c-Flip−/− MEFs (Figure 3C), whereas phosphorylation of MEK1 disappeared at 60 min in WT MEFs. The decrease in the size of phosphorylated MEK1 at 120 min in c-Flip−/− MEFs might be due to a degradation of MEK1. However, at least under our experimental conditions, we could not detect phosphorylation of endogenous MKK7 upon TNFαstimulation possibly due to a poor sensitivity of the antibody to recognize phosphorylated MKK7 (data not shown). Collectively, these results indicate that TNFα induces prolonged ERK and JNK activation through activation of MEK1 and possibly MKK7 in c-Flip−/− MEFs, which are not suppressed by NF-κB-inducible genes other than c-Flip. c-FLIPL inhibits JNK and ERK activation Taken that TNFα-induced ROS accumulation is abolished in Jnk1−/−Jnk2−/− (Jnk−/−) MEFs (Ventura et al, 2004) and the above results together, we surmised that c-FLIPL might directly inhibit the JNK pathway. Thus, we first tested whether c-FLIPL inhibits phosphorylation of MAPKs triggered by MAPKKKs including MEKK1, ASK1, and TAK1, which have been implicated in TNFα-induced MAPK activation (Nishitoh et al, 1998; Yuasa et al, 1998; Sato et al, 2005). We transfected HEK293 cells with HA-tagged MAPKs along with a constitutively active form of MEKK1 (MEKK1ΔN) or ASK1 in the presence or absence of c-FLIPL. Expression of c-FLIPL substantially inhibited the MEKK1ΔN-induced JNK and ERK phosphorylation (Figure 4A and B). c-FLIPL also inhibited the ASK1-induced JNK, but not p38 phosphorylation (Figure 4C and D). Collectively, c-FLIPL preferentially inhibits the JNK and ERK, but not p38 pathways. As we could not detect the interaction of c-FLIPL with JNK, ERK, or p38 in co-immunoprecipitation experiments (data not shown), we speculated that c-FLIPL inhibits the JNK and ERK pathways by suppressing their activators such as MAPKKs or MAPKKKs. Therefore, we next tested whether c-FLIPL inhibits MEKK1ΔN- and ASK1-triggered phosphorylation of MAPKKs in their activation loop motifs. c-FLIPL substantially inhibited the MEKK1ΔN- and ASK1-induced phosphorylation of MKK7 (Figure 4E and F). Similarly, c-FLIPL moderately inhibited the MEKK1ΔN-induced MEK1 and MKK4 phosphorylation (Figure 4G and H). In contrast, c-FLIPL did not inhibit the ASK1-induced MKK6 phosphorylation (Figure 4I). Moreover, c-FLIPL inhibited the TAK1-induced MKK7, MKK4, and JNK, but inhibitory effect of c-FLIPL on p38 phosphorylation was marginal. Similarly, c-FLIPL inhibited Tpl-2-induced JNK and ERK activation (Supplementary Figure S4). These results indicate that c-FLIPL selectively inhibits the MKK7–JNK, MKK4–JNK, and MEK1–ERK, but not MKK6–p38 pathways. Figure 4.c-FLIPL inhibits the MKK7–JNK, MKK4–JNK, and MEK1–ERK pathways. (A–I) HEK293 cells were transfected with the indicated expression vectors. After immunoprecipitation (IP) with anti-HA antibody, the phosphorylation of MAPKs or MAPKKs was analyzed by immunoblotting with the indicated phospho-specific antibodies (top panels). Expression levels of the transfected proteins in the immunoprecipitates (IP) (second panels) and the total lysates (TL) (third and bottom panels) were analyzed by immunoblotting with anti-HA, anti-Myc, and anti-Flag antibodies. Download figure Download PowerPoint c-FLIPL binds to MKK7 and MEK1 in a TNFα-dependent manner, and disrupts their interactions with MEKK1, ASK1, and TAK1 The inhibition of the MEKK1-induced MEK1, MKK4, and MKK7 phosphorylation by c-FLIPL prompted us to investigate whether c-FLIPL interacts with MAPKKs. Intriguingly, c-FLIPL interacted with MEK1 and MKK7, and their interactions were significantly enhanced in the presence of MEKK1ΔN (Figure 5A and D). These results suggest that the MEKK1-induced phosphorylation and/or conformational changes of MEK1 and MKK7 might enhance their binding to c-FLIPL. Similarly, MKK4 weakly interacted with c-FLIPL, but MKK6 did not (Figure 5B and C). We tested whether c-FLIPL could interact with MKK7 and MEK1 under more physiological conditions. We first used RelA−/− MEFs stably expressing Flag-c-FLIPL. Interactions of c-FLIPL with endogenous MKK7 and MEK1 were undetectable in unstimulated cells, but their interactions were weakly induced at 5 min and increased up to 2 h after TNFα stimulation (Figure 5E). Notably, the interactions of MKK7 or MEK1 with Flag-c-FLIPL were specific, as anti-Flag, but not control antibody, specifically co-immunoprecipitated MKK7 and MEK1. More importantly, the interaction of endogenous c-FLIPL with endogenous MKK7 was induced upon TNFα stimulation in HEK293 cells (Figure 5F). Together, c-FLIPL physically interacts with MKK7 and MEK1 in a TNFα-dependent manner. Figure 5.Interactions of c-FLIPL with MEK1 and MKK7 are induced in a TNFα-dependent, and c-FLIPL disrupts their interactions with MEKK1. (A–D) HEK293 cells were transfected with the indicated expression vectors. After immunoprecipitation (IP) with anti-Flag antibody, co-immunoprecipitated MAPKKs were detected by immunoblotting with anti-HA antibody (top panels). Expression levels of the transfected proteins in the immunoprecipitates (IP) (second panels) and the total lysates (TL) (third and bottom panels) were analyzed by immunoblotting with anti-Flag, anti-HA, and anti-Myc antibodies. (E) RelA−/− MEFs stably expressing Flag-c-FLIPL were stimulated with TNFα (10 ng/ml) for the indicated times. After immunoprecipitation with control (C) or anti-F