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SAPK Activation by TRAF2
Molecular Cell, Vol. 2, 389–395, September, 1998, Copyright 1998 by Cell Press
ASK1 Is Essential for JNK/SAPK Activation by TRAF2 Hideki Nishitoh,* † Masao Saitoh,*† Yoshiyuki Mochida,* † Kohsuke Takeda,*† Hiroyasu Nakano,‡ Mike Rothe,§ Kohei Miyazono,* and Hidenori Ichijo*† k * Department of Biochemistry The Cancer Institute, Tokyo Japanese Foundation for Cancer Research 1-37-1 Kami-Ikebukuro, Toshima-ku, Tokyo 170-8455 † Department of Biomaterials Science Faculty of Dentistry Tokyo Medical and Dental University 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8549 ‡ Department of Immunology Juntendo University School of Medicine 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-0033 Japan Core Research for Evolutional Science and Technology Japan Science and Technology Corporation 2-3 Surugadai, Kanada, Chiyoda-ku, Tokyo 101 Japan § Tularik Inc. 2 Corporate Drive South San Francisco, California 94080
Summary Tumor necrosis factor (TNF)-induced activation of the c-jun N-terminal kinase (JNK, also known as SAPK; stress-activated protein kinase) requires TNF receptor-associated factor 2 (TRAF2). The apoptosis signalregulating kinase 1 (ASK1) is activated by TNF and stimulates JNK activation. Here we show that ASK1 interacts with members of the TRAF family and is activated by TRAF2, TRAF5, and TRAF6 overexpression. A truncated derivative of TRAF2, which inhibits JNK activation by TNF, blocks TNF-induced ASK1 activation. A catalytically inactive mutant of ASK1 is a dominant-negative inhibitor of TNF- and TRAF2-induced JNK activation. In untransfected mammalian cells, ASK1 rapidly associates with TRAF2 in a TNF-dependent manner. Thus, ASK1 is a mediator of TRAF2induced JNK activation. Introduction TNF is a pleiotropic cytokine that plays a pivotal role in inflammation (Tracey and Cerami, 1993). TNF activates two transcription factors, activator protein 1 (AP-1) (Brenner et al., 1989) and nuclear factor-kB (NF-kB) (Rothe et al., 1995a; Baeuerle and Baltimore, 1996; Takeuchi et al., 1996). Both transcription factors are activated through the phosphorylation of JNK (Minden et al., 1994) and IkB kinases (DiDonato et al., 1997; Mercurio et al., 1997; Re´gnier et al., 1997; Woronicz et al., 1997; Zandi k To whom correspondence should be addressed at the Department
of Biomaterials Science at Tokyo Medical and Dental University (e-mail: [email protected]).
et al., 1997), respectively. These signals of TNF are mediated by two cell surface receptors, TNF receptor TNFR-1 and TNFR-2 (Tartaglia and Goeddel, 1992). TNF binding induces receptor aggregation, resulting in the recruitment of a number of cytoplasmic signaling proteins to the two distinct TNFR complexes (Rothe et al., 1994, 1995b; Hsu et al., 1995, 1996a, 1996b; Shu et al., 1996). One of these signaling proteins is TRAF2. TRAF2 interacts directly with TNFR-2 (Rothe et al., 1994), but it is recruited to TNFR-1 via its interaction with TNFR-1associated death domain protein (TRADD; Hsu et al., 1995, 1996b). TRAF2 is a member of the TRAF protein family. To date, six members of the TRAF family have been identified (Hu et al., 1994; Rothe et al., 1994; Cheng et al., 1995; Mosialos et al., 1995; Re´gnier et al., 1995; Cao et al., 1996; Nakano et al., 1996). They do not possess enzymatic activity, suggesting that they operate as adaptor proteins. All TRAF proteins contain a conserved C-terminal TRAF domain that is used for homoor heterooligomerization and for interaction with the cytoplasmic regions of the TNFR superfamily. Except for TRAF1, all TRAF proteins contain an N-terminal RING finger and several zinc finger structures that appear critical for their effector functions (Hu et al., 1994; Rothe et al., 1994; Cheng et al., 1995; Mosialos et al., 1995; Re´gnier et al., 1995; Cao et al., 1996; Nakano et al., 1996). Overexpression studies in mammalian cells have implicated TRAF2 (or other members of the TRAF protein family) in TNF-induced activation of both JNK and NF-kB (Rothe et al., 1995a; Hsu et al., 1996b; Liu et al., 1996; Takeuchi et al., 1996; Natoli et al., 1997; Reinhard et al., 1997). In vivo TNF signaling to JNK but not NF-kB was defective in TRAF2-deficient cells (Yeh et al., 1997). Recent studies have identified several serine/threonine protein kinases involved in TNF signal transduction. The NF-kB-inducing kinase (NIK) is a MAP kinase kinase kinase (MAPKKK)-related protein kinase that associates with TRAF2 and other members of the TRAF family and mediates activation of NF-kB (Malinin et al., 1997), but not JNK (Song et al., 1997). On the other hand, TRAF2-mediated activation of JNK was reported to be inhibited by catalytically inactive mutants of MAPK/extracellular signal-regulated kinase kinase 1 (MEKK1), another member of the MAPKKK family (Liu et al., 1996; Song et al., 1997), and germinal center kinase related (GCKR), which signals via MEKK1 to JNK (Shi and Kehrl, 1997). However, none of these kinases were shown to associate with TRAF2 (Shi and Kehrl, 1997; Song et al., 1997). These studies indicate that the JNK and NF-kB signaling pathways diverge at some point downstream of TRAF2, although a direct target of TRAF2 in the JNK signaling pathway is unknown. Apoptosis signal-regulating kinase 1 (ASK1) is a MAP KKK that activates the SEK1-JNK and MKK3/MKK6-p38 signaling cascades (Wang et al., 1996; Ichijo et al., 1997). Overexpression of ASK1 in epithelial cells under low serum conditions induced apoptosis, and a kinase-inactive mutant of ASK1 reduced TNF-induced apoptosis, suggesting that ASK1 is involved in the TNF signaling pathway leading to apoptosis (Ichijo et al., 1997).
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Through genetic screening for ASK1-binding proteins, thioredoxin (Trx) was recently identified as a physiological inhibitor of ASK1 (Saitoh et al., 1998). Only a reduced form of Trx associates with the N-terminal portion of ASK1 and keeps ASK1 kinase activity inactive. Upon treatment of cells with TNF or reactive oxygen species (ROS) such as hydrogen peroxide, Trx appears to be oxidized, and ASK1 is released from Trx and activated. Thus, Trx acts as a negative regulator in the TNF- and ROS-induced activation mechanism of ASK1. However, the molecular mechanism by which TNF activates ASK1 is not fully understood. Here we show that ASK1 interacts with members of the TRAF family and that ASK1 is a sufficient and necessary component in the TNF- and TRAF2-induced JNK activation mechanism. Results Activation of ASK1 by TRAF Family Members Overexpression of TRAF2, TRAF5, and TRAF6, but not TRAF1 or TRAF3, has been reported to activate JNK (Song et al., 1997). To investigate a potential role of ASK1 as a molecular target of TRAF2 in the TNF-induced JNK pathway, we tested whether TRAF family proteins can stimulate the kinase activity of ASK1. Hemagglutinin (HA) epitope-tagged ASK1 (HA-ASK1) was coexpressed with Flag epitope-tagged TRAF proteins (Flag-TRAF) in 293 cells and immunoprecipitated with anti-HA antibody. The immune complexes were subjected to an immune complex-coupled kinase assay using GSTMKK6 and GST-SAPK3/p38g as sequential substrates. Consistent with the ability to activate JNK, ASK1 was strongly activated by coexpression of TRAF2 and TRAF6 (Figure 1A); TRAF5 but not TRAF1 or TRAF3 also weakly activated ASK1. These results suggest that ASK1 is a downstream target of TRAF2, TRAF5, and TRAF6 in the JNK signaling pathways. Association of ASK1 with TRAF Family Members We next determined whether ASK1 can physically interact with TRAF proteins in a transfection-based coimmunoprecipitation assay. Flag-TRAFs were coexpressed with HA-ASK1 in 293 cells and immunoprecipitated with anti-Flag antibody. The immune complexes were subjected to immunoblotting with anti-HA antibody. ASK1 was found to associate with all TRAF proteins tested (Figure 1B). Interaction of ASK1 with TRAF2 (Figure 1C) and the other TRAF proteins (data not shown) was not detected by immunoprecipitation with an isotypematched control antibody. These findings suggest that ASK1 binds TRAF proteins within the conserved C-terminal homology region termed TRAF domain (Rothe et al., 1994). Consistently, a mutant TRAF2 protein lacking the N-terminal RING finger domain, TRAF2(87–501), as well as a TRAF2 derivative comprising only the TRAF domain, TRAF2(272–501), still associated with ASK1 (Figures 2A and 2B). However, in contrast to wild-type TRAF2, the N-terminally truncated TRAF2 proteins, which have been shown to be defective in JNK activation and NF-kB activation (Rothe et al., 1995a; Hsu et al., 1996b; Liu et al., 1996; Natoli et al., 1997; Reinhard et
Figure 1. Activation and Interaction of ASK1 with TRAF Family Proteins (A) Activation of ASK1 by TRAF proteins. HA-tagged ASK1 expression plasmid (pcDNA3-HA-ASK1) (1 mg; lanes 2–7) was transiently cotransfected with Flag-tagged TRAF expression plasmid (pRKFlag-TRAF1 and -TRAF2, and pCR3-Flag-TRAF3, -TRAF5, and -TRAF6) (1 mg; lanes 3–7) into 293 cells. After 12 hr, ASK1 was immunoprecipitated by anti-HA antibody. The immune complex was incubated with GST-MKK6, and then the kinase activity was measured with the substrate GST-p38gKN. Samples were analyzed by SDS-PAGE (8.5%) and an image analyzer. Top, in vitro kinase assay (IVK) for ASK1 activity. Bottom, immunoblotting (WB) of immunoprecipitated HA-ASK1 in the same sample. Kinase activity relative to the amount of ASK1 protein was calculated, and the activity is shown as fold increase relative to that of HA-ASK1 from TRAF-negative cells (lane 2). (B) Interaction of ASK1 with TRAF proteins. pcDNA3-HA-ASK1 (1 mg; lanes 2, 4, 6, 8, 10, and 12) was transiently cotransfected with each TRAF expression vector (1 mg; lanes 3–12) into 293 cells. After 36 hr, transfected cells were extracted with lysis buffer and immunoprecipitated (IP) with anti-Flag antibody, and immunoblotted (WB) with anti-HA antibody (top). The presence of Flag-TRAF (middle) and HA-ASK1 (bottom) in the same lysates is shown. Markers of molecular mass are shown on the left. (C) Specific interaction of ASK1 with TRAF2. 293 cells were transiently cotransfected with pcDNA3-HA-ASK1 (1 mg; lanes 1–4) and pRK-Flag-TRAF2 (1 mg; lanes 2 and 4). Lysates were divided and immunoprecipitated with anti-Flag antibody (lanes 1 and 2) or control antibody (lanes 3 and 4). The interaction was detected by immunoblotting with anti-HA antibody and anti-Flag antibody. The presence of HA-ASK1 and Flag-TRAF2 in the same lysates was verified by immunoblotting.
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Figure 2. Binding Sites between TRAF2 and ASK1 (A) Schematic representation of wild-type and mutant TRAF2 proteins. Their abilities to interact with and activate ASK1 when cotransfected into 293 cells are shown. (1), activation of or interaction with ASK1. (2), lack of such activities. (B) Interaction of ASK1 with wild-type and mutant TRAF2. pcDNA3-HA-ASK1 (1 mg; lanes 2, 4, 6, and 8) was transiently cotransfected into 293 cells with expression plasmids for Flag-tagged wild-type and mutant TRAF2 (1 mg; lanes 3–10). Lysates were immunoprecipitated and immunoblotted as described in Figure 1B. (C) Schematic representation of wild-type and mutant ASK1 proteins. The kinase domain is shown by the hatched boxes. ASK1KM represents a catalytically inactive mutant in which Lys-709 has been replaced by Met. Positive interaction with wild-type TRAF2 in 293 cells is shown by a (1). (D) Interaction of TRAF2 with wild-type and mutant ASK1. pRK-Flag-TRAF2 (1 mg; lanes 2, 4, 6, 8, and 10) was transiently cotransfected into 293 cells with HA-tagged wild-type and mutant ASK1 (1 mg; lanes 3–10). Lysates were immunoprecipitated and immunoblotted as described in Figure 1B. Markers of molecular mass are shown on the left.
al., 1997; Song et al., 1997), did not activate ASK1 kinase activity (Figure 2A; Figure 3A, lane 3). Thus, the RING finger domain of TRAF2 is required for activation of ASK1. To map the interaction domain of ASK1 with TRAF2, truncation mutants of ASK1 were analyzed for their interaction with TRAF2. HA-ASK1 deletion mutants were cotransfected with Flag-TRAF2 into 293 cells. In the coimmunoprecipitation assay, similar to wild-type ASK1 (ASK1WT), an N-terminally truncated ASK1 protein (ASK1DN) as well as a C-terminal fragment of ASK1 lacking the kinase domain (ASK1-CT), but not a C-terminally truncated ASK1 protein (ASK1-DC), coimmunoprecipitated with TRAF2 (Figure 2C and 2D), indicating that TRAF2 associates with the C-terminal noncatalytic region of ASK1. TNF-Induced JNK Activation Requires TRAF2 and ASK1 TRAF2(87–501) acts as a dominant-negative inhibitor of both TNF-induced JNK and NF-kB activation in mammalian cells (Hsu et al., 1996b; Liu et al., 1996; Natoli et al.,
1997; Reinhard et al., 1997; Song et al., 1997). To examine whether TRAF2 is required for activation of ASK1 by TNF, 293 cells were cotransfected with HA-ASK1 and Flag-TRAF2(87–501) expression plasmids, and ASK1 kinase activity was determined after TNF stimulation. In this assay, TRAF2(87–501) inhibited the TNF-induced activation of ASK1 in a dose-dependent manner (Figure 3A), suggesting that TRAF2 mediates ASK1 activation by TNF. We next examined whether ASK1 is required for TNFand TRAF2-induced JNK activation using a catalytically inactive form of ASK1 (ASK1KM; Figure 2C). 293 cells were transfected with expression vectors encoding HAtagged JNK (HA-JNK) and ASK1KM, stimulated with TNF, and JNK activity was measured by immune complex kinase assay. TNF-induced JNK activation was suppressed by ASK1KM (Figure 3B). When 293 cells were cotransfected with expression plasmids for FlagTRAF2, ASK1KM, and HA-JNK, TRAF2-induced JNK activation was markedly inhibited by ASK1KM in a dosedependent manner (Figure 3C). These results indicate that ASK1 acts as the downstream kinase of TRAF2 in
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the TNF-induced JNK activation pathway. In contrast, ASK1 did not induce NF-kB activity (Song et al., 1997; K. Tobiume, Y. M., and H. I., unpublished data), suggesting that the TNF signaling pathways leading to JNK and NFkB activation diverge upstream of ASK1.
Figure 3. Dominant-Negative Effects of TRAF2 and ASK1 Mutants in TNF-Induced JNK Pathway (A) Inhibition of the TNF-induced activation of ASK1 by a dominantnegative TRAF2 mutant. Indicated amounts of pRK-Flag-TRAF2(87– 501) were transiently cotransfected into 293 cells with pcDNA3-HAASK1 (1 mg). After 12 hr, the cells were incubated with (1) or without (2) TNF (200 ng/ml) for 15 min. ASK1 was immunoprecipitated with anti-HA antibody and assayed for kinase activity as described in Figure 1A. Top, phosphorylation of GST-p38gKN. Middle, immunoblotting of immunoprecipitated HA-ASK1 in the same sample. Bottom, immunoblotting of Flag-TRAF2(87–501) in the same lysate. Kinase activity relative to the amount of ASK1 protein is shown as fold increase relative to that of HA-ASK1 from TRAF2(87–501)- and TNF-negative cells (lane 2). (B) ASK1KM inhibits TNF-induced JNK activation. HA-tagged JNK expression plasmid (pcDNA3-HA-JNK; 1 mg; lane 2–6) was transiently cotransfected into 293 cells with the indicated amounts of ASK1KM expression plasmid (pcDNA3-ASK1KM; lane 3, 5, and 6). After 12 hr, cells were treated with TNF (200 ng/ml) for 15 min. Immunoprecipitated HA-JNK activity was measured by GST-cJUN phosphorylation as a direct substrate for JNK. Samples were analyzed by SDS-PAGE (10%) and an image analyzer. Expression of ASK1KM was verified by the immunoblotting with anti-ASK1 antiserum (DAV). Top, phosphorylation of GST-cJUN. Middle, immunoblotting of HA-JNK in the same sample. Bottom, expression of ASK1KM in the same lysate. Kinase activity relative to the amount of JNK protein is shown as fold increase relative to that of HA-JNK from ASK1KM- and TNF-negative cells (lane 2). (C) ASK1KM inhibits TRAF2-induced JNK activation. 293 cells were transiently cotransfected with pcDNA3-HA-JNK (2 mg; lane 2–7), pcDNA3-Flag-TRAF2 (0.25 mg; lane 4–7), and the indicated amounts of pcDNA3-ASK1KM (lane 3, 5–7). The relative kinase activity of JNK was determined by immune complex kinase assay as described in (B).
TNF-Dependent Endogenous Interaction between ASK1 and TRAF2 To evaluate the observed TRAF2–ASK1 interaction under more physiological conditions, we examined the association of endogenous ASK1 and TRAF2 in mouse L929 cells, which express a relatively large amount of endogenous ASK1 (Y. Sawada and H. I., unpublished data) and are sensitive to TNF-induced apoptosis (Saitoh et al., 1998). An anti-ASK1 polyclonal antibody (DAV) specifically immunoprecipitated and detected the endogenous ASK1 protein as a major doublet of bands with sizes of 160 and 170 kDa (Figure 4A). We determined whether ASK1 interacts with TRAF2 following TNF treatment. Lysates from TNF-treated cells were immunoprecipitated with nonimmune serum or anti-TRAF2 polyclonal antiserum, and the immunoprecipitates were analyzed by immunoblotting with anti-ASK1 antiserum. ASK1, mainly the 170 kDa component, rapidly associated with TRAF2 in a TNF-dependent manner (Figure 4B). The interaction was observed within 5 min after TNF treatment, peaked at 15–30 min, and decreased thereafter (Figure 4B), which correlates well with the time course of TNF-induced activation of ASK1 (Ichijo et al., 1997). We also tested whether the induced interaction between ASK1 and TRAF2 was specific for TNF stimulation. While treatment of L929 cells with IL-1 or H2O2 also activated endogenous ASK1 (Figure 4C, bottom panel), these stimuli did not induce the interaction between ASK1 and TRAF2 (Figure 4C, top panel). Thus, the recruitment of ASK1 to TRAF2 is signaled specifically and physiologically by TNF. Discussion The recent identification of several serine/threonine kinases has provided important insights into the mechanism of TNF-induced NF-kB activation (DiDonato et al., 1997; Mercurio et al., 1997; Re´gnier et al., 1997; Woronicz et al., 1997; Zandi et al., 1997, Nakano et al., 1998). Although TRAF2 is now known to be essential for JNK activation rather than NF-kB activation in the TNF signaling pathways (Lee et al., 1997; Yeh et al., 1997), a direct molecular target of TRAF2 to transduce signals to JNK has not been identified. We now report the identification of the MAPKKK ASK1 as a component of the TNF induced JNK activation pathway via TRAF2. ASK1 is a mediator of TRAF2-dependent JNK activation that associates with and links TRAF2 to downstream steps in this kinase cascade. Figure 5 represents a current model of TNF signaling pathways. TNF-induced activation of NFkB and JNK bifurcates at the level of TRAF2-associated MAPKKK family. One pathway is initiated by the interaction of TRAF2 with NIK, which triggers the NF-kB signaling cascade. Given that TNF signaling to JNK but not NF-kB was strongly reduced in TRAF2-deficient cells (Yeh et al., 1997), TNF might also utilize other TRAF
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Figure 5. Schematic Representation of the Role of ASK1 in TNF Signaling See text for details.
Figure 4. TNF-Dependent Endogenous Interaction between ASK1 and TRAF2 (A) Identification of endogenous ASK1 protein in mouse L929 cells. Lysate from L929 cells (2 3 10 8) was immunoprecipitated with the polyclonal anti-ASK1 antiserum (DAV) with (1) or without (2) blocking peptide (5 mg/ml) against the antiserum, and immunoblotted with DAV. Arrowheads indicate two major ASK1 proteins. Another specific band with an apparent size of 120 kDa may represent a degradation product of ASK1 or an unidentified ASK1-related molecule. Markers of molecular mass are shown on the left. (B) TNF-induced interaction of ASK1 with TRAF2. L929 cells (2 3 108 ) were treated with TNF (200 ng/ml) for the indicated time periods (lanes 1 and 3–6) or left untreated (lane 2). Cell lysates were immunoprecipitated with the nonimmune antiserum (lane 1) or the polyclonal anti-TRAF2 antiserum (lanes 2–6). Copurified ASK1 proteins were detected by immunoblotting with anti-ASK1 antiserum (top). Arrowheads indicate ASK1 proteins that specifically associated with endogenous TRAF2. Consistent expression of ASK1 (middle) and TRAF2 (bottom) after TNF treatment was confirmed by immunoblotting using similarly treated cell lysates. This experiment was performed four times with similar results. (C) TNF-specific interaction of ASK1 with TRAF2. L929 cells (2 3 108 ) were exposed for 15 min to TNF (200 ng/ml; lane 2), IL-1a (100 ng/ml; lane 3), or H2 O2 (1 mM; lane 4). Coimmunoprecipitated ASK1 (top) with TRAF2 was determined as described in Figure 5B. The presence of ASK1 and TRAF2 proteins in the same lysate was verified by the immunoblotting. The endogenous ASK1 from similarly treated cells was immunoprecipitated by DAV, and the kinase activity was measured (bottom) as described in Figure 1A.
proteins such as TRAF5 to activate NIK. In addition, the death domain kinase RIP is required for TNF-induced NF-kB activation (Ting et al., 1996; Kelliher et al., 1998). Another pathway is mediated by ASK1 that is recruited
to TRAF2 upon TNF-stimulation and activates the JNK but not NF-kB pathway. While GCKR and MEKK1 appears to constitute an ASK1-independent signaling pathway to JNK activation (Shi and Kehrl, 1997), these kinases might form a signaling complex with ASK1 as in the case for IkB kinase complex in the NF-kB signaling pathway (DiDonato et al., 1997; Mercurio et al., 1997; Re´ gnier et al., 1997; Woronicz et al., 1997; Zandi et al., 1997). Considering that GCKR belongs to a MAPKKK kinase family (Shi and Kehrl, 1997), it is also possible that GCKR or its close relative could be an upstream kinase of ASK1. Currently, we do not know how ASK1 is molecularly activated after binding TRAF2. Reactive oxygen species (ROS) such as superoxide, hydrogen peroxide, and hydroxyl radical are also known to function as important signaling intermediates in TNF signaling (Jacobson, 1996). Interestingly, N-acetyl-L-cystein (Nac), a thiolbased antioxidant, is able to block TNF- and TRAF2induced activation of JNK (Natoli et al., 1997), suggesting that JNK activation by TNF and TRAF2 may be dependent on thiols/disulfides exchanging reactions. In this respect, our recent finding of Trx, a redox-regulatory protein, as a cellular inhibitor of ASK1 could be of particular importance, in that Trx in its reduced form but not oxidized form binds to the N-terminal region of ASK1 and inhibits TNF-induced activation of ASK1 (Saitoh et al., 1998). Thus, the activation of ASK1 by TRAF2 might be regulated by the inactivation and subsequent dissociation of Trx from ASK1. Although ASK1 is an activator of the JNK pathway and is required for TNF-induced apoptosis in certain cells (Ichijo et al., 1997), JNK activity has been reported not to be involved in TNF-induced apoptosis (Liu et al., 1996). This potential discrepancy might be due to the difference of cell types analyzed. Alternatively, JNK activity might not be required for the ASK1-dependent apoptosis in the TNF signal transduction. Finally, targeted inactivation of ASK1 in mice will enable evaluation of the role of ASK1 in TNF-induced JNK activation as well as apoptosis in vivo.
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Experimental Procedures Cell Culture and Cytokines 293 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS), 4.5 mg/ml glucose, and 100 U/ml penicillin. L929 cells were cultured in DMEM containing 10% FBS and antibiotics. Human recombinant TNF-a and IL-1a were purchased from Pepro Tech EC Ltd. and Sigma, respectively. Expression Vectors and Transfections pcDNA3-HA-ASK1, pcDNA3-HA-ASK1DN, and pcDNA3-HA-ASK1 DC have been described (Saitoh et al., 1998). pRK-Flag-TRAF1 (Hu et al., 1994), pRK-Flag-TRAF2 (Rothe et al., 1995a), pRK-FlagTRAF2(87–501) (Rothe et al., 1995a), pRK-Flag-TRAF2(272–501) (Takeuchi et al., 1996), pCR3-Flag-TRAF3 (Kashiwada et al., 1998), pCR3-Flag-TRAF5 (Nakano et al., 1996), and pCR3-Flag-TRAF6 (Kashiwada et al., 1998) have been described. HA-JNK, HA-ASK1CT, and ASK1KM were constructed in pcDNA3. The plasmids of GSTMKK6 and GST-p38gKN for bacterial fusion protein were constructed in pGEX-4T-1 (Pharmacia) by PCR. Transfection was performed with Tfx-50 (Promega) according to the manufacturer’s instructions. Antiserum Rabbit polyclonal antisera to ASK1 (DAV) (Saitoh et al., 1998) and TRAF2 (Shu et al., 1996) were described. Immune Complex–Coupled Kinase Assay for ASK1 Immune complex–coupled kinase assay has been described (Ichijo et al., 1997; Saitoh et al., 1998). In brief, cells were lysed in the lysis buffer containing 20 mM Tris-HCl (pH 7.5), 12 mM b-glycerophosphate, 150 mM NaCl, 5 mM EGTA, 10 mM NaF, 1% Triton X-100, 1% deoxycholate, 1 mM DTT, 1 mM sodium orthovanadate, 1 mM PMSF, and 1.5% aprotinin. Cell extracts were clarified by centrifugation, and the supernatants were immunoprecipitated with anti-HA antibody (12CA5, Boehringer Mannheim) or antiserum to ASK1 (DAV) using protein A–Sepharose (Zymed). The beads were washed twice with the washing buffer containing 150 mM NaCl, 20 mM Tris-HCl (pH 7.5), 5 mM EGTA, and 1 mM DTT, and subjected to kinase assays. GST-MKK6 (0.2 mg) was first incubated with the immune complex for 10 min at 308C in a final volume of 10 ml of a solution containing 20 mM Tris-HCl (pH 7.5), 20 mM MgCl2 , and 100 mM ATP. Thereafter, the activated complex was incubated with 0.3 mCi of [g- 32P]ATP and 1 mg of GST-p38gKN in the same solution (final volume 20 ml) for 10 min at room temperature (RT). Kinase reactions were stopped by adding SDS sample buffer and subjected to SDSpolyacrylamide gel electrophoresis (PAGE) under reducing conditions. Phosphorylation of GST-p38gKN was analyzed by a Fuji BAS2000 image analyzer. To determine the amount of ASK1 protein in the same sample, the upper part of the gel (larger than 100 kDa) was cut out and immunoblotted with the rat anti-HA antibody (3F10, Boehringer Mannheim). The protein was detected with the ECL system (Amersham), in which less than 10 min exposure to X-ray film did not detect any 32P-radioactivity derived from autophosphorylation of ASK1. The amount of protein was quantified by densitometric analysis (Quantity One program: pdi, Inc.). Immune Complex Kinase Assay for JNK Transfected HA-JNK was immunoprecipitated with anti-HA antibody (12CA5) using protein A–Sepharose. The beads were washed twice with the washing buffer and subjected to kinase assays. One microgram of GST-cJUN(1–79) was incubated with the immune complex for 10 min at RT in a final volume of 20 ml of a solution containing 20 mM Tris-HCl (pH 7.5), 20 mM MgCl2 , and 0.3 mCi of [g-32P]ATP. Phosphorylation of GST-cJUN(1–79) was analyzed by SDS-PAGE. The amount of JNK protein in the same sample were determined by immunoblotting with rat anti-HA antibody (3F10). Coimmunoprecipitation Assay Transfected cells were lysed with the lysis buffer. Cell lysates were immunoprecipitated with anti-Flag antibody (M2, Kodak), or control mouse IgG1 monoclonal antibody (Biogenesis), using protein
G–Sepharose. For endogenous coimmunoprecipitations, 1 3 108 L929 cells were lysed in 2 ml of the lysis buffer. Cell lysates were immunoprecipitated with antiserum to TRAF2, using protein A-Sepharose. The beads were washed twice with the washing buffer, separated by SDS-PAGE, and immunoblotted with rat antiHA antibody (3F10), anti-Flag antibody (M2), or antiserum to ASK1 (DAV). The proteins were detected by the ECL system. Aliquots of whole-cell lysates were subjected to immunoblotting analysis by anti-HA antibody (3F10), anti-Flag antibody (M2), antiserum to ASK1 (DAV), or antiserum to TRAF2 to confirm appropriate expression of ASK1 and TRAF proteins. The proteins were detected with the ECL system.
Acknowledgments We thank D. V. Goeddel, K. Okumura, H. Yagita, and T. Kitagawa for discussion. We also thank M. Goedert, J. Han, R. J. Ulevitch, H. Chang, and D. Baltimore for plasmids. H. N. is a recipient of fellowships of the Japan Society for the Promotion of Science. This work was supported by Grants-in-Aid for scientific research from the Ministry of Education, Science, and Culture of Japan.
Received June 8, 1998; revised July 28, 1998.
References Baeuerle, P.A., and Baltimore, D. (1996). NF-kB: ten years after. Cell 87, 13–20. Brenner, D.A., O’Hara, M., Angel, P., Chojkier, M., and Karin, M. (1989). Prolonged activation of jun and collagenase genes by tumour necrosis factor-a. Nature 337, 661–663. Cao, Z., Xiong, J., Takeuchi, M., Kurama, T., and Goeddel, D.V. (1996). TRAF6 is a signal transducer for interleukin-1. Nature 383, 443–446. Cheng, G., Cleary, A.M., Ye, Z.S., Hong, D.I., Lederman, S., and Baltimore, D. (1995). Involvement of CRAF1, a relative of TRAF, in CD40 signaling. Science 267, 1494–1498. DiDonato, J.A., Hayakawa, M., Rothwarf, D.M., Zandi, E., and Karin, M. (1997). A cytokine-responsive IkB kinase that activates the transcription factor NF-kB. Nature 388, 548–554. Hsu, H., Xiong, J., and Goeddel, D.V. (1995). The TNF receptor 1–associated protein TRADD signals cell death and NF-kB activation. Cell 81, 495–504. Hsu, H., Huang, J., Shu, H.-B., Baichwal, V., and Goeddel, D.V. (1996a). TNF-dependent recruitment of the protein kinase RIP to the TNF receptor-1 signaling complex. Immunity 4, 387–396. Hsu, H., Shu, H.-B., Pan, M.-G., and Goeddel, D.V. (1996b). TRADD– TRAF2 and TRADD–FADD interactions define two distinct TNF receptor 1 signal transduction pathways. Cell 84, 299–308. Hu, H.M., O’Rourke, K., Boguski, M.S., and Dixit, V.M. (1994). A novel RING finger protein interacts with the cytoplasmic domain of CD40. J. Biol. Chem. 269, 30069–30072. Ichijo, H., Nishida, E., Irie, K., ten Dijke, P., Saitoh, M., Moriguchi, T., Takagi, M., Matsumoto, K., Miyazono, K., and Gotoh, Y. (1997). Induction of apoptosis by ASK1, a mammalian MAPKKK that activates SAPK/JNK and p38 signaling pathways. Science 275, 90–94. Jacobson, M.D. (1996). Reactive oxygen species and programmed cell death. Trends Biochem. Sci. 21, 83–86. Kashiwada, M., Shirakata, Y., Inoue, J.I., Nakano, H., Okazaki, K., Okumura, K., Yamamoto, T., Nagaoka, H., and Takemori, T. (1998). Tumor necrosis factor receptor-associated factor 6 (TRAF6) stimulates extracellular signal-regulated kinase (ERK) activity in CD40 signaling along a ras-independent pathway. J. Exp. Med. 187, 237–244. Kelliher, M.A., Grimm, S., Ishida, Y., Kuo, F., Stanger, B.Z., and Leder, P. (1998). The death domain kinase RIP mediates the TNFinduced NF-kB signal. Immunity 8, 297–303. Lee, S.Y., Reichlin, A., Santana, A., Sokol, K.A., Nussenzweig, M.C.,
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and Choi, Y. (1997). TRAF2 is essential for JNK but not NF-kB activation and regulates lymphocyte proliferation and survival. Immunity 7, 703–713. Liu, Z.-g., Hsu, H., Goeddel, D.V., and Karin, M. (1996). Dissection of TNF receptor 1 effector functions: JNK activation is not linked to apoptosis while NF-kB activation prevents cell death. Cell 87, 565–576. Malinin, N.L., Boldin, M.P., Kovalenko, A.V., and Wallach, D. (1997). MAP3K-related kinase involved in NF-kB induction by TNF, CD95 and IL-1. Nature 385, 540–544. Mercurio, F., Zhu, H., Murray, B.W., Shevchenko, A., Bennett, B.L., Li, J., Young, D.B., Barbosa, M., Mann, M., Manning, A., and Rao, A. (1997). IKK-1 and IKK-2: cytokine-activated IkB kinases essential for NF-kB activation. Science 278, 860–866. Minden, A., Lin, A., McMahon, M., Lange, C.C., Derijard, B., Davis, R.J., Johnson, G.L., and Karin, M. (1994). Differential activation of ERK and JNK mitogen-activated protein kinases by Raf-1 and MEKK. Science 266, 1719–1723. Mosialos, G., Birkenbach, M., Yalamanchili, R., VanArsdale, T., Ware, C., and Kieff, E. (1995). The Epstein-Barr virus transforming protein LMP1 engages signaling proteins for the tumor necrosis factor receptor family. Cell 80, 389–399. Nakano, H., Oshima, H., Chung, W., Williams, A.L., Ware, C.F., Yagita, H., and Okumura, K. (1996). TRAF5, an activator of NF-kB and putative signal transducer for the lymphotoxin-b receptor. J. Biol. Chem. 271, 14661–14664. Nakano, H., Shindo, M., Sakon, S., Nishinaka, S., Mihara, M., Yagita, H., and Okumura, K. (1998). Differential regulation of IkB kinase a and b by two upstream kinases, NF-kB-inducing kinase and mitogenactivated protein kinase/ERK kinase kinase-1. Proc. Natl. Acad. Sci. USA 95, 3537–3542. Natoli, G., Costanzo, A., Ianni, A., Templeton, D.J., Woodgett, J.R., Balsano, C., and Levrero, M. (1997). Activation of SAPK/JNK by TNF receptor 1 through a noncytotoxic TRAF2-dependent pathway. Science 275, 200–203. Re´gnier, C.H., Tomasetto, C., Moog-Lutz, C., Chenard, M.P., Wendling, C., Basset, P., and Rio, M.C. (1995). Presence of a new conserved domain in CART1, a novel member of the tumor necrosis factor receptor-associated protein family, which is expressed in breast carcinoma. J. Biol. Chem. 270, 25715–25721. Re´gnier, C.H., Song, H.Y., Gao, X., Goeddel, D.V., Cao, Z., and Rothe, M. (1997). Identification and characterization of an IkB kinase. Cell 90, 373–383. Reinhard, C., Shamoon, B., Shyamala, V., and Williams, L.T. (1997). Tumor necrosis factor a-induced activation of c-jun N-terminal kinase is mediated by TRAF2. EMBO J. 16, 1080–1092. Rothe, M., Wong, S.C., Henzel, W.J., and Goeddel, D.V. (1994). A novel family of putative signal transducers associated with the cytoplasmic domain of the 75 kDa tumor necrosis factor receptor. Cell 78, 681–692. Rothe, M., Sarma, V., Dixit, V.M., and Goeddel, D.V. (1995a). TRAF2mediated activation of NF-kB by TNF receptor 2 and CD40. Science 269, 1424–1427. Rothe, M., Pan, M.-G., Henzel, W.J., Ayres, T.M., and Goeddel, D.V. (1995b). The TNFR2–TRAF signaling complex contains two novel proteins related to baculoviral inhibitor of apoptosis proteins. Cell 83, 1243–1252. Saitoh, M., Nishitoh, H., Fujii, M., Takeda, K., Tobiume, K., Sawada, Y., Kawabata, M., Miyazono, K., and Ichijo, H. (1998). Mammalian thioredoxin is a direct inhibitor of apoptosis signal-regulating kinase (ASK) 1. EMBO J. 17, 2596–2606. Shi, C.S., and Kehrl, J.H. (1997). Activation of stress-activated protein kinase/c-Jun N-terminal kinase, but not NF-kB, by the tumor necrosis factor (TNF) receptor 1 through a TNF receptor-associated factor 2- and germinal center kinase related-dependent pathway. J. Biol. Chem. 272, 32102–32107. Shu, H.B., Takeuchi, M., and Goeddel, D.V. (1996). The tumor necrosis factor receptor 2 signal transducers TRAF2 and c-IAP1 are components of the tumor necrosis factor receptor 1 signaling complex. Proc. Natl. Acad. Sci. USA 93, 13973–13978.
Song, H.Y., Re´ gnier, C.H., Kirschning, C.J., Goeddel, D.V., and Rothe, M. (1997). Tumor necrosis factor (TNF)-mediated kinase cascades: bifurcation of nuclear factor-kB and c-jun N-terminal kinase (JNK/SAPK) pathways at TNF receptor-associated factor 2. Proc. Natl. Acad. Sci. USA 94, 9792–9796. Takeuchi, M., Rothe, M., and Goeddel, D.V. (1996). Anatomy of TRAF2. Distinct domains for nuclear factor-kB activation and association with tumor necrosis factor signaling proteins. J. Biol. Chem. 271, 19935–19942. Tartaglia, L.A., and Goeddel, D.V. (1992). Two TNF receptors. Immunol. Today 13, 151–153. Ting, A.T., Pimentel-Muinos, F.X., and Seed, B. (1996). RIP mediates tumor necrosis factor receptor 1 activation of NF-kB but not Fas/ APO-1-initiated apoptosis. EMBO J. 15, 6189–6196. Tracey, K.J., and Cerami, A. (1993). Tumor necrosis factor, other cytokines and disease. Annu. Rev. Cell Biol. 9, 317–343. Wang, X.S., Diener, K., Jannuzzi, D., Trollinger, D., Tan, T.H., Lichenstein, H., Zukowski, M., and Yao, Z. (1996). Molecular cloning and characterization of a novel protein kinase with a catalytic domain homologous to mitogen-activated protein kinase kinase kinase. J. Biol. Chem. 271, 31607–31611. Woronicz, J.D., Gao, X., Cao, Z., Rothe, M., and Goeddel, D.V. (1997). IkB kinase-b: NF-kB activation and complex formation with IkB kinase-a and NIK. Science 278, 866–869. Yeh, W.-C., Shahinian, A., Speiser, D., Kraunus, J., Billia, F., Wakeham, A., Pompa, J.L., Ferrick, D., Hum, B., Iscove, N., et al. (1997). Early lethality, functional NF-kB activation, and increased sensitivity to TNF-induced cell death in TRAF2-deficient mice. Immunity 7, 715–725. Zandi, E., Rothwarf, D.M., Delhase, M., Hayakawa, M., and Karin, M. (1997). The IkB kinase complex (IKK) contains two kinase subunits, IKKa and IKKb, necessary for IkB phosphorylation and NFkB activation. Cell 91, 243–252.
ASK1 Is Essential for JNK/SAPK Activation by TRAF2 Hideki Nishitoh,* † Masao Saitoh,*† Yoshiyuki Mochida,* † Kohsuke Takeda,*† Hiroyasu Nakano,‡ Mike Rothe,§ Kohei Miyazono,* and Hidenori Ichijo*† k * Department of Biochemistry The Cancer Institute, Tokyo Japanese Foundation for Cancer Research 1-37-1 Kami-Ikebukuro, Toshima-ku, Tokyo 170-8455 † Department of Biomaterials Science Faculty of Dentistry Tokyo Medical and Dental University 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8549 ‡ Department of Immunology Juntendo University School of Medicine 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-0033 Japan Core Research for Evolutional Science and Technology Japan Science and Technology Corporation 2-3 Surugadai, Kanada, Chiyoda-ku, Tokyo 101 Japan § Tularik Inc. 2 Corporate Drive South San Francisco, California 94080
Summary Tumor necrosis factor (TNF)-induced activation of the c-jun N-terminal kinase (JNK, also known as SAPK; stress-activated protein kinase) requires TNF receptor-associated factor 2 (TRAF2). The apoptosis signalregulating kinase 1 (ASK1) is activated by TNF and stimulates JNK activation. Here we show that ASK1 interacts with members of the TRAF family and is activated by TRAF2, TRAF5, and TRAF6 overexpression. A truncated derivative of TRAF2, which inhibits JNK activation by TNF, blocks TNF-induced ASK1 activation. A catalytically inactive mutant of ASK1 is a dominant-negative inhibitor of TNF- and TRAF2-induced JNK activation. In untransfected mammalian cells, ASK1 rapidly associates with TRAF2 in a TNF-dependent manner. Thus, ASK1 is a mediator of TRAF2induced JNK activation. Introduction TNF is a pleiotropic cytokine that plays a pivotal role in inflammation (Tracey and Cerami, 1993). TNF activates two transcription factors, activator protein 1 (AP-1) (Brenner et al., 1989) and nuclear factor-kB (NF-kB) (Rothe et al., 1995a; Baeuerle and Baltimore, 1996; Takeuchi et al., 1996). Both transcription factors are activated through the phosphorylation of JNK (Minden et al., 1994) and IkB kinases (DiDonato et al., 1997; Mercurio et al., 1997; Re´gnier et al., 1997; Woronicz et al., 1997; Zandi k To whom correspondence should be addressed at the Department
of Biomaterials Science at Tokyo Medical and Dental University (e-mail: [email protected]).
et al., 1997), respectively. These signals of TNF are mediated by two cell surface receptors, TNF receptor TNFR-1 and TNFR-2 (Tartaglia and Goeddel, 1992). TNF binding induces receptor aggregation, resulting in the recruitment of a number of cytoplasmic signaling proteins to the two distinct TNFR complexes (Rothe et al., 1994, 1995b; Hsu et al., 1995, 1996a, 1996b; Shu et al., 1996). One of these signaling proteins is TRAF2. TRAF2 interacts directly with TNFR-2 (Rothe et al., 1994), but it is recruited to TNFR-1 via its interaction with TNFR-1associated death domain protein (TRADD; Hsu et al., 1995, 1996b). TRAF2 is a member of the TRAF protein family. To date, six members of the TRAF family have been identified (Hu et al., 1994; Rothe et al., 1994; Cheng et al., 1995; Mosialos et al., 1995; Re´gnier et al., 1995; Cao et al., 1996; Nakano et al., 1996). They do not possess enzymatic activity, suggesting that they operate as adaptor proteins. All TRAF proteins contain a conserved C-terminal TRAF domain that is used for homoor heterooligomerization and for interaction with the cytoplasmic regions of the TNFR superfamily. Except for TRAF1, all TRAF proteins contain an N-terminal RING finger and several zinc finger structures that appear critical for their effector functions (Hu et al., 1994; Rothe et al., 1994; Cheng et al., 1995; Mosialos et al., 1995; Re´gnier et al., 1995; Cao et al., 1996; Nakano et al., 1996). Overexpression studies in mammalian cells have implicated TRAF2 (or other members of the TRAF protein family) in TNF-induced activation of both JNK and NF-kB (Rothe et al., 1995a; Hsu et al., 1996b; Liu et al., 1996; Takeuchi et al., 1996; Natoli et al., 1997; Reinhard et al., 1997). In vivo TNF signaling to JNK but not NF-kB was defective in TRAF2-deficient cells (Yeh et al., 1997). Recent studies have identified several serine/threonine protein kinases involved in TNF signal transduction. The NF-kB-inducing kinase (NIK) is a MAP kinase kinase kinase (MAPKKK)-related protein kinase that associates with TRAF2 and other members of the TRAF family and mediates activation of NF-kB (Malinin et al., 1997), but not JNK (Song et al., 1997). On the other hand, TRAF2-mediated activation of JNK was reported to be inhibited by catalytically inactive mutants of MAPK/extracellular signal-regulated kinase kinase 1 (MEKK1), another member of the MAPKKK family (Liu et al., 1996; Song et al., 1997), and germinal center kinase related (GCKR), which signals via MEKK1 to JNK (Shi and Kehrl, 1997). However, none of these kinases were shown to associate with TRAF2 (Shi and Kehrl, 1997; Song et al., 1997). These studies indicate that the JNK and NF-kB signaling pathways diverge at some point downstream of TRAF2, although a direct target of TRAF2 in the JNK signaling pathway is unknown. Apoptosis signal-regulating kinase 1 (ASK1) is a MAP KKK that activates the SEK1-JNK and MKK3/MKK6-p38 signaling cascades (Wang et al., 1996; Ichijo et al., 1997). Overexpression of ASK1 in epithelial cells under low serum conditions induced apoptosis, and a kinase-inactive mutant of ASK1 reduced TNF-induced apoptosis, suggesting that ASK1 is involved in the TNF signaling pathway leading to apoptosis (Ichijo et al., 1997).
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Through genetic screening for ASK1-binding proteins, thioredoxin (Trx) was recently identified as a physiological inhibitor of ASK1 (Saitoh et al., 1998). Only a reduced form of Trx associates with the N-terminal portion of ASK1 and keeps ASK1 kinase activity inactive. Upon treatment of cells with TNF or reactive oxygen species (ROS) such as hydrogen peroxide, Trx appears to be oxidized, and ASK1 is released from Trx and activated. Thus, Trx acts as a negative regulator in the TNF- and ROS-induced activation mechanism of ASK1. However, the molecular mechanism by which TNF activates ASK1 is not fully understood. Here we show that ASK1 interacts with members of the TRAF family and that ASK1 is a sufficient and necessary component in the TNF- and TRAF2-induced JNK activation mechanism. Results Activation of ASK1 by TRAF Family Members Overexpression of TRAF2, TRAF5, and TRAF6, but not TRAF1 or TRAF3, has been reported to activate JNK (Song et al., 1997). To investigate a potential role of ASK1 as a molecular target of TRAF2 in the TNF-induced JNK pathway, we tested whether TRAF family proteins can stimulate the kinase activity of ASK1. Hemagglutinin (HA) epitope-tagged ASK1 (HA-ASK1) was coexpressed with Flag epitope-tagged TRAF proteins (Flag-TRAF) in 293 cells and immunoprecipitated with anti-HA antibody. The immune complexes were subjected to an immune complex-coupled kinase assay using GSTMKK6 and GST-SAPK3/p38g as sequential substrates. Consistent with the ability to activate JNK, ASK1 was strongly activated by coexpression of TRAF2 and TRAF6 (Figure 1A); TRAF5 but not TRAF1 or TRAF3 also weakly activated ASK1. These results suggest that ASK1 is a downstream target of TRAF2, TRAF5, and TRAF6 in the JNK signaling pathways. Association of ASK1 with TRAF Family Members We next determined whether ASK1 can physically interact with TRAF proteins in a transfection-based coimmunoprecipitation assay. Flag-TRAFs were coexpressed with HA-ASK1 in 293 cells and immunoprecipitated with anti-Flag antibody. The immune complexes were subjected to immunoblotting with anti-HA antibody. ASK1 was found to associate with all TRAF proteins tested (Figure 1B). Interaction of ASK1 with TRAF2 (Figure 1C) and the other TRAF proteins (data not shown) was not detected by immunoprecipitation with an isotypematched control antibody. These findings suggest that ASK1 binds TRAF proteins within the conserved C-terminal homology region termed TRAF domain (Rothe et al., 1994). Consistently, a mutant TRAF2 protein lacking the N-terminal RING finger domain, TRAF2(87–501), as well as a TRAF2 derivative comprising only the TRAF domain, TRAF2(272–501), still associated with ASK1 (Figures 2A and 2B). However, in contrast to wild-type TRAF2, the N-terminally truncated TRAF2 proteins, which have been shown to be defective in JNK activation and NF-kB activation (Rothe et al., 1995a; Hsu et al., 1996b; Liu et al., 1996; Natoli et al., 1997; Reinhard et
Figure 1. Activation and Interaction of ASK1 with TRAF Family Proteins (A) Activation of ASK1 by TRAF proteins. HA-tagged ASK1 expression plasmid (pcDNA3-HA-ASK1) (1 mg; lanes 2–7) was transiently cotransfected with Flag-tagged TRAF expression plasmid (pRKFlag-TRAF1 and -TRAF2, and pCR3-Flag-TRAF3, -TRAF5, and -TRAF6) (1 mg; lanes 3–7) into 293 cells. After 12 hr, ASK1 was immunoprecipitated by anti-HA antibody. The immune complex was incubated with GST-MKK6, and then the kinase activity was measured with the substrate GST-p38gKN. Samples were analyzed by SDS-PAGE (8.5%) and an image analyzer. Top, in vitro kinase assay (IVK) for ASK1 activity. Bottom, immunoblotting (WB) of immunoprecipitated HA-ASK1 in the same sample. Kinase activity relative to the amount of ASK1 protein was calculated, and the activity is shown as fold increase relative to that of HA-ASK1 from TRAF-negative cells (lane 2). (B) Interaction of ASK1 with TRAF proteins. pcDNA3-HA-ASK1 (1 mg; lanes 2, 4, 6, 8, 10, and 12) was transiently cotransfected with each TRAF expression vector (1 mg; lanes 3–12) into 293 cells. After 36 hr, transfected cells were extracted with lysis buffer and immunoprecipitated (IP) with anti-Flag antibody, and immunoblotted (WB) with anti-HA antibody (top). The presence of Flag-TRAF (middle) and HA-ASK1 (bottom) in the same lysates is shown. Markers of molecular mass are shown on the left. (C) Specific interaction of ASK1 with TRAF2. 293 cells were transiently cotransfected with pcDNA3-HA-ASK1 (1 mg; lanes 1–4) and pRK-Flag-TRAF2 (1 mg; lanes 2 and 4). Lysates were divided and immunoprecipitated with anti-Flag antibody (lanes 1 and 2) or control antibody (lanes 3 and 4). The interaction was detected by immunoblotting with anti-HA antibody and anti-Flag antibody. The presence of HA-ASK1 and Flag-TRAF2 in the same lysates was verified by immunoblotting.
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Figure 2. Binding Sites between TRAF2 and ASK1 (A) Schematic representation of wild-type and mutant TRAF2 proteins. Their abilities to interact with and activate ASK1 when cotransfected into 293 cells are shown. (1), activation of or interaction with ASK1. (2), lack of such activities. (B) Interaction of ASK1 with wild-type and mutant TRAF2. pcDNA3-HA-ASK1 (1 mg; lanes 2, 4, 6, and 8) was transiently cotransfected into 293 cells with expression plasmids for Flag-tagged wild-type and mutant TRAF2 (1 mg; lanes 3–10). Lysates were immunoprecipitated and immunoblotted as described in Figure 1B. (C) Schematic representation of wild-type and mutant ASK1 proteins. The kinase domain is shown by the hatched boxes. ASK1KM represents a catalytically inactive mutant in which Lys-709 has been replaced by Met. Positive interaction with wild-type TRAF2 in 293 cells is shown by a (1). (D) Interaction of TRAF2 with wild-type and mutant ASK1. pRK-Flag-TRAF2 (1 mg; lanes 2, 4, 6, 8, and 10) was transiently cotransfected into 293 cells with HA-tagged wild-type and mutant ASK1 (1 mg; lanes 3–10). Lysates were immunoprecipitated and immunoblotted as described in Figure 1B. Markers of molecular mass are shown on the left.
al., 1997; Song et al., 1997), did not activate ASK1 kinase activity (Figure 2A; Figure 3A, lane 3). Thus, the RING finger domain of TRAF2 is required for activation of ASK1. To map the interaction domain of ASK1 with TRAF2, truncation mutants of ASK1 were analyzed for their interaction with TRAF2. HA-ASK1 deletion mutants were cotransfected with Flag-TRAF2 into 293 cells. In the coimmunoprecipitation assay, similar to wild-type ASK1 (ASK1WT), an N-terminally truncated ASK1 protein (ASK1DN) as well as a C-terminal fragment of ASK1 lacking the kinase domain (ASK1-CT), but not a C-terminally truncated ASK1 protein (ASK1-DC), coimmunoprecipitated with TRAF2 (Figure 2C and 2D), indicating that TRAF2 associates with the C-terminal noncatalytic region of ASK1. TNF-Induced JNK Activation Requires TRAF2 and ASK1 TRAF2(87–501) acts as a dominant-negative inhibitor of both TNF-induced JNK and NF-kB activation in mammalian cells (Hsu et al., 1996b; Liu et al., 1996; Natoli et al.,
1997; Reinhard et al., 1997; Song et al., 1997). To examine whether TRAF2 is required for activation of ASK1 by TNF, 293 cells were cotransfected with HA-ASK1 and Flag-TRAF2(87–501) expression plasmids, and ASK1 kinase activity was determined after TNF stimulation. In this assay, TRAF2(87–501) inhibited the TNF-induced activation of ASK1 in a dose-dependent manner (Figure 3A), suggesting that TRAF2 mediates ASK1 activation by TNF. We next examined whether ASK1 is required for TNFand TRAF2-induced JNK activation using a catalytically inactive form of ASK1 (ASK1KM; Figure 2C). 293 cells were transfected with expression vectors encoding HAtagged JNK (HA-JNK) and ASK1KM, stimulated with TNF, and JNK activity was measured by immune complex kinase assay. TNF-induced JNK activation was suppressed by ASK1KM (Figure 3B). When 293 cells were cotransfected with expression plasmids for FlagTRAF2, ASK1KM, and HA-JNK, TRAF2-induced JNK activation was markedly inhibited by ASK1KM in a dosedependent manner (Figure 3C). These results indicate that ASK1 acts as the downstream kinase of TRAF2 in
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the TNF-induced JNK activation pathway. In contrast, ASK1 did not induce NF-kB activity (Song et al., 1997; K. Tobiume, Y. M., and H. I., unpublished data), suggesting that the TNF signaling pathways leading to JNK and NFkB activation diverge upstream of ASK1.
Figure 3. Dominant-Negative Effects of TRAF2 and ASK1 Mutants in TNF-Induced JNK Pathway (A) Inhibition of the TNF-induced activation of ASK1 by a dominantnegative TRAF2 mutant. Indicated amounts of pRK-Flag-TRAF2(87– 501) were transiently cotransfected into 293 cells with pcDNA3-HAASK1 (1 mg). After 12 hr, the cells were incubated with (1) or without (2) TNF (200 ng/ml) for 15 min. ASK1 was immunoprecipitated with anti-HA antibody and assayed for kinase activity as described in Figure 1A. Top, phosphorylation of GST-p38gKN. Middle, immunoblotting of immunoprecipitated HA-ASK1 in the same sample. Bottom, immunoblotting of Flag-TRAF2(87–501) in the same lysate. Kinase activity relative to the amount of ASK1 protein is shown as fold increase relative to that of HA-ASK1 from TRAF2(87–501)- and TNF-negative cells (lane 2). (B) ASK1KM inhibits TNF-induced JNK activation. HA-tagged JNK expression plasmid (pcDNA3-HA-JNK; 1 mg; lane 2–6) was transiently cotransfected into 293 cells with the indicated amounts of ASK1KM expression plasmid (pcDNA3-ASK1KM; lane 3, 5, and 6). After 12 hr, cells were treated with TNF (200 ng/ml) for 15 min. Immunoprecipitated HA-JNK activity was measured by GST-cJUN phosphorylation as a direct substrate for JNK. Samples were analyzed by SDS-PAGE (10%) and an image analyzer. Expression of ASK1KM was verified by the immunoblotting with anti-ASK1 antiserum (DAV). Top, phosphorylation of GST-cJUN. Middle, immunoblotting of HA-JNK in the same sample. Bottom, expression of ASK1KM in the same lysate. Kinase activity relative to the amount of JNK protein is shown as fold increase relative to that of HA-JNK from ASK1KM- and TNF-negative cells (lane 2). (C) ASK1KM inhibits TRAF2-induced JNK activation. 293 cells were transiently cotransfected with pcDNA3-HA-JNK (2 mg; lane 2–7), pcDNA3-Flag-TRAF2 (0.25 mg; lane 4–7), and the indicated amounts of pcDNA3-ASK1KM (lane 3, 5–7). The relative kinase activity of JNK was determined by immune complex kinase assay as described in (B).
TNF-Dependent Endogenous Interaction between ASK1 and TRAF2 To evaluate the observed TRAF2–ASK1 interaction under more physiological conditions, we examined the association of endogenous ASK1 and TRAF2 in mouse L929 cells, which express a relatively large amount of endogenous ASK1 (Y. Sawada and H. I., unpublished data) and are sensitive to TNF-induced apoptosis (Saitoh et al., 1998). An anti-ASK1 polyclonal antibody (DAV) specifically immunoprecipitated and detected the endogenous ASK1 protein as a major doublet of bands with sizes of 160 and 170 kDa (Figure 4A). We determined whether ASK1 interacts with TRAF2 following TNF treatment. Lysates from TNF-treated cells were immunoprecipitated with nonimmune serum or anti-TRAF2 polyclonal antiserum, and the immunoprecipitates were analyzed by immunoblotting with anti-ASK1 antiserum. ASK1, mainly the 170 kDa component, rapidly associated with TRAF2 in a TNF-dependent manner (Figure 4B). The interaction was observed within 5 min after TNF treatment, peaked at 15–30 min, and decreased thereafter (Figure 4B), which correlates well with the time course of TNF-induced activation of ASK1 (Ichijo et al., 1997). We also tested whether the induced interaction between ASK1 and TRAF2 was specific for TNF stimulation. While treatment of L929 cells with IL-1 or H2O2 also activated endogenous ASK1 (Figure 4C, bottom panel), these stimuli did not induce the interaction between ASK1 and TRAF2 (Figure 4C, top panel). Thus, the recruitment of ASK1 to TRAF2 is signaled specifically and physiologically by TNF. Discussion The recent identification of several serine/threonine kinases has provided important insights into the mechanism of TNF-induced NF-kB activation (DiDonato et al., 1997; Mercurio et al., 1997; Re´gnier et al., 1997; Woronicz et al., 1997; Zandi et al., 1997, Nakano et al., 1998). Although TRAF2 is now known to be essential for JNK activation rather than NF-kB activation in the TNF signaling pathways (Lee et al., 1997; Yeh et al., 1997), a direct molecular target of TRAF2 to transduce signals to JNK has not been identified. We now report the identification of the MAPKKK ASK1 as a component of the TNF induced JNK activation pathway via TRAF2. ASK1 is a mediator of TRAF2-dependent JNK activation that associates with and links TRAF2 to downstream steps in this kinase cascade. Figure 5 represents a current model of TNF signaling pathways. TNF-induced activation of NFkB and JNK bifurcates at the level of TRAF2-associated MAPKKK family. One pathway is initiated by the interaction of TRAF2 with NIK, which triggers the NF-kB signaling cascade. Given that TNF signaling to JNK but not NF-kB was strongly reduced in TRAF2-deficient cells (Yeh et al., 1997), TNF might also utilize other TRAF
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Figure 5. Schematic Representation of the Role of ASK1 in TNF Signaling See text for details.
Figure 4. TNF-Dependent Endogenous Interaction between ASK1 and TRAF2 (A) Identification of endogenous ASK1 protein in mouse L929 cells. Lysate from L929 cells (2 3 10 8) was immunoprecipitated with the polyclonal anti-ASK1 antiserum (DAV) with (1) or without (2) blocking peptide (5 mg/ml) against the antiserum, and immunoblotted with DAV. Arrowheads indicate two major ASK1 proteins. Another specific band with an apparent size of 120 kDa may represent a degradation product of ASK1 or an unidentified ASK1-related molecule. Markers of molecular mass are shown on the left. (B) TNF-induced interaction of ASK1 with TRAF2. L929 cells (2 3 108 ) were treated with TNF (200 ng/ml) for the indicated time periods (lanes 1 and 3–6) or left untreated (lane 2). Cell lysates were immunoprecipitated with the nonimmune antiserum (lane 1) or the polyclonal anti-TRAF2 antiserum (lanes 2–6). Copurified ASK1 proteins were detected by immunoblotting with anti-ASK1 antiserum (top). Arrowheads indicate ASK1 proteins that specifically associated with endogenous TRAF2. Consistent expression of ASK1 (middle) and TRAF2 (bottom) after TNF treatment was confirmed by immunoblotting using similarly treated cell lysates. This experiment was performed four times with similar results. (C) TNF-specific interaction of ASK1 with TRAF2. L929 cells (2 3 108 ) were exposed for 15 min to TNF (200 ng/ml; lane 2), IL-1a (100 ng/ml; lane 3), or H2 O2 (1 mM; lane 4). Coimmunoprecipitated ASK1 (top) with TRAF2 was determined as described in Figure 5B. The presence of ASK1 and TRAF2 proteins in the same lysate was verified by the immunoblotting. The endogenous ASK1 from similarly treated cells was immunoprecipitated by DAV, and the kinase activity was measured (bottom) as described in Figure 1A.
proteins such as TRAF5 to activate NIK. In addition, the death domain kinase RIP is required for TNF-induced NF-kB activation (Ting et al., 1996; Kelliher et al., 1998). Another pathway is mediated by ASK1 that is recruited
to TRAF2 upon TNF-stimulation and activates the JNK but not NF-kB pathway. While GCKR and MEKK1 appears to constitute an ASK1-independent signaling pathway to JNK activation (Shi and Kehrl, 1997), these kinases might form a signaling complex with ASK1 as in the case for IkB kinase complex in the NF-kB signaling pathway (DiDonato et al., 1997; Mercurio et al., 1997; Re´ gnier et al., 1997; Woronicz et al., 1997; Zandi et al., 1997). Considering that GCKR belongs to a MAPKKK kinase family (Shi and Kehrl, 1997), it is also possible that GCKR or its close relative could be an upstream kinase of ASK1. Currently, we do not know how ASK1 is molecularly activated after binding TRAF2. Reactive oxygen species (ROS) such as superoxide, hydrogen peroxide, and hydroxyl radical are also known to function as important signaling intermediates in TNF signaling (Jacobson, 1996). Interestingly, N-acetyl-L-cystein (Nac), a thiolbased antioxidant, is able to block TNF- and TRAF2induced activation of JNK (Natoli et al., 1997), suggesting that JNK activation by TNF and TRAF2 may be dependent on thiols/disulfides exchanging reactions. In this respect, our recent finding of Trx, a redox-regulatory protein, as a cellular inhibitor of ASK1 could be of particular importance, in that Trx in its reduced form but not oxidized form binds to the N-terminal region of ASK1 and inhibits TNF-induced activation of ASK1 (Saitoh et al., 1998). Thus, the activation of ASK1 by TRAF2 might be regulated by the inactivation and subsequent dissociation of Trx from ASK1. Although ASK1 is an activator of the JNK pathway and is required for TNF-induced apoptosis in certain cells (Ichijo et al., 1997), JNK activity has been reported not to be involved in TNF-induced apoptosis (Liu et al., 1996). This potential discrepancy might be due to the difference of cell types analyzed. Alternatively, JNK activity might not be required for the ASK1-dependent apoptosis in the TNF signal transduction. Finally, targeted inactivation of ASK1 in mice will enable evaluation of the role of ASK1 in TNF-induced JNK activation as well as apoptosis in vivo.
Molecular Cell 394
Experimental Procedures Cell Culture and Cytokines 293 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS), 4.5 mg/ml glucose, and 100 U/ml penicillin. L929 cells were cultured in DMEM containing 10% FBS and antibiotics. Human recombinant TNF-a and IL-1a were purchased from Pepro Tech EC Ltd. and Sigma, respectively. Expression Vectors and Transfections pcDNA3-HA-ASK1, pcDNA3-HA-ASK1DN, and pcDNA3-HA-ASK1 DC have been described (Saitoh et al., 1998). pRK-Flag-TRAF1 (Hu et al., 1994), pRK-Flag-TRAF2 (Rothe et al., 1995a), pRK-FlagTRAF2(87–501) (Rothe et al., 1995a), pRK-Flag-TRAF2(272–501) (Takeuchi et al., 1996), pCR3-Flag-TRAF3 (Kashiwada et al., 1998), pCR3-Flag-TRAF5 (Nakano et al., 1996), and pCR3-Flag-TRAF6 (Kashiwada et al., 1998) have been described. HA-JNK, HA-ASK1CT, and ASK1KM were constructed in pcDNA3. The plasmids of GSTMKK6 and GST-p38gKN for bacterial fusion protein were constructed in pGEX-4T-1 (Pharmacia) by PCR. Transfection was performed with Tfx-50 (Promega) according to the manufacturer’s instructions. Antiserum Rabbit polyclonal antisera to ASK1 (DAV) (Saitoh et al., 1998) and TRAF2 (Shu et al., 1996) were described. Immune Complex–Coupled Kinase Assay for ASK1 Immune complex–coupled kinase assay has been described (Ichijo et al., 1997; Saitoh et al., 1998). In brief, cells were lysed in the lysis buffer containing 20 mM Tris-HCl (pH 7.5), 12 mM b-glycerophosphate, 150 mM NaCl, 5 mM EGTA, 10 mM NaF, 1% Triton X-100, 1% deoxycholate, 1 mM DTT, 1 mM sodium orthovanadate, 1 mM PMSF, and 1.5% aprotinin. Cell extracts were clarified by centrifugation, and the supernatants were immunoprecipitated with anti-HA antibody (12CA5, Boehringer Mannheim) or antiserum to ASK1 (DAV) using protein A–Sepharose (Zymed). The beads were washed twice with the washing buffer containing 150 mM NaCl, 20 mM Tris-HCl (pH 7.5), 5 mM EGTA, and 1 mM DTT, and subjected to kinase assays. GST-MKK6 (0.2 mg) was first incubated with the immune complex for 10 min at 308C in a final volume of 10 ml of a solution containing 20 mM Tris-HCl (pH 7.5), 20 mM MgCl2 , and 100 mM ATP. Thereafter, the activated complex was incubated with 0.3 mCi of [g- 32P]ATP and 1 mg of GST-p38gKN in the same solution (final volume 20 ml) for 10 min at room temperature (RT). Kinase reactions were stopped by adding SDS sample buffer and subjected to SDSpolyacrylamide gel electrophoresis (PAGE) under reducing conditions. Phosphorylation of GST-p38gKN was analyzed by a Fuji BAS2000 image analyzer. To determine the amount of ASK1 protein in the same sample, the upper part of the gel (larger than 100 kDa) was cut out and immunoblotted with the rat anti-HA antibody (3F10, Boehringer Mannheim). The protein was detected with the ECL system (Amersham), in which less than 10 min exposure to X-ray film did not detect any 32P-radioactivity derived from autophosphorylation of ASK1. The amount of protein was quantified by densitometric analysis (Quantity One program: pdi, Inc.). Immune Complex Kinase Assay for JNK Transfected HA-JNK was immunoprecipitated with anti-HA antibody (12CA5) using protein A–Sepharose. The beads were washed twice with the washing buffer and subjected to kinase assays. One microgram of GST-cJUN(1–79) was incubated with the immune complex for 10 min at RT in a final volume of 20 ml of a solution containing 20 mM Tris-HCl (pH 7.5), 20 mM MgCl2 , and 0.3 mCi of [g-32P]ATP. Phosphorylation of GST-cJUN(1–79) was analyzed by SDS-PAGE. The amount of JNK protein in the same sample were determined by immunoblotting with rat anti-HA antibody (3F10). Coimmunoprecipitation Assay Transfected cells were lysed with the lysis buffer. Cell lysates were immunoprecipitated with anti-Flag antibody (M2, Kodak), or control mouse IgG1 monoclonal antibody (Biogenesis), using protein
G–Sepharose. For endogenous coimmunoprecipitations, 1 3 108 L929 cells were lysed in 2 ml of the lysis buffer. Cell lysates were immunoprecipitated with antiserum to TRAF2, using protein A-Sepharose. The beads were washed twice with the washing buffer, separated by SDS-PAGE, and immunoblotted with rat antiHA antibody (3F10), anti-Flag antibody (M2), or antiserum to ASK1 (DAV). The proteins were detected by the ECL system. Aliquots of whole-cell lysates were subjected to immunoblotting analysis by anti-HA antibody (3F10), anti-Flag antibody (M2), antiserum to ASK1 (DAV), or antiserum to TRAF2 to confirm appropriate expression of ASK1 and TRAF proteins. The proteins were detected with the ECL system.
Acknowledgments We thank D. V. Goeddel, K. Okumura, H. Yagita, and T. Kitagawa for discussion. We also thank M. Goedert, J. Han, R. J. Ulevitch, H. Chang, and D. Baltimore for plasmids. H. N. is a recipient of fellowships of the Japan Society for the Promotion of Science. This work was supported by Grants-in-Aid for scientific research from the Ministry of Education, Science, and Culture of Japan.
Received June 8, 1998; revised July 28, 1998.
References Baeuerle, P.A., and Baltimore, D. (1996). NF-kB: ten years after. Cell 87, 13–20. Brenner, D.A., O’Hara, M., Angel, P., Chojkier, M., and Karin, M. (1989). Prolonged activation of jun and collagenase genes by tumour necrosis factor-a. Nature 337, 661–663. Cao, Z., Xiong, J., Takeuchi, M., Kurama, T., and Goeddel, D.V. (1996). TRAF6 is a signal transducer for interleukin-1. Nature 383, 443–446. Cheng, G., Cleary, A.M., Ye, Z.S., Hong, D.I., Lederman, S., and Baltimore, D. (1995). Involvement of CRAF1, a relative of TRAF, in CD40 signaling. Science 267, 1494–1498. DiDonato, J.A., Hayakawa, M., Rothwarf, D.M., Zandi, E., and Karin, M. (1997). A cytokine-responsive IkB kinase that activates the transcription factor NF-kB. Nature 388, 548–554. Hsu, H., Xiong, J., and Goeddel, D.V. (1995). The TNF receptor 1–associated protein TRADD signals cell death and NF-kB activation. Cell 81, 495–504. Hsu, H., Huang, J., Shu, H.-B., Baichwal, V., and Goeddel, D.V. (1996a). TNF-dependent recruitment of the protein kinase RIP to the TNF receptor-1 signaling complex. Immunity 4, 387–396. Hsu, H., Shu, H.-B., Pan, M.-G., and Goeddel, D.V. (1996b). TRADD– TRAF2 and TRADD–FADD interactions define two distinct TNF receptor 1 signal transduction pathways. Cell 84, 299–308. Hu, H.M., O’Rourke, K., Boguski, M.S., and Dixit, V.M. (1994). A novel RING finger protein interacts with the cytoplasmic domain of CD40. J. Biol. Chem. 269, 30069–30072. Ichijo, H., Nishida, E., Irie, K., ten Dijke, P., Saitoh, M., Moriguchi, T., Takagi, M., Matsumoto, K., Miyazono, K., and Gotoh, Y. (1997). Induction of apoptosis by ASK1, a mammalian MAPKKK that activates SAPK/JNK and p38 signaling pathways. Science 275, 90–94. Jacobson, M.D. (1996). Reactive oxygen species and programmed cell death. Trends Biochem. Sci. 21, 83–86. Kashiwada, M., Shirakata, Y., Inoue, J.I., Nakano, H., Okazaki, K., Okumura, K., Yamamoto, T., Nagaoka, H., and Takemori, T. (1998). Tumor necrosis factor receptor-associated factor 6 (TRAF6) stimulates extracellular signal-regulated kinase (ERK) activity in CD40 signaling along a ras-independent pathway. J. Exp. Med. 187, 237–244. Kelliher, M.A., Grimm, S., Ishida, Y., Kuo, F., Stanger, B.Z., and Leder, P. (1998). The death domain kinase RIP mediates the TNFinduced NF-kB signal. Immunity 8, 297–303. Lee, S.Y., Reichlin, A., Santana, A., Sokol, K.A., Nussenzweig, M.C.,
ASK1 Is Essential for JNK Activation by TRAF2 395
and Choi, Y. (1997). TRAF2 is essential for JNK but not NF-kB activation and regulates lymphocyte proliferation and survival. Immunity 7, 703–713. Liu, Z.-g., Hsu, H., Goeddel, D.V., and Karin, M. (1996). Dissection of TNF receptor 1 effector functions: JNK activation is not linked to apoptosis while NF-kB activation prevents cell death. Cell 87, 565–576. Malinin, N.L., Boldin, M.P., Kovalenko, A.V., and Wallach, D. (1997). MAP3K-related kinase involved in NF-kB induction by TNF, CD95 and IL-1. Nature 385, 540–544. Mercurio, F., Zhu, H., Murray, B.W., Shevchenko, A., Bennett, B.L., Li, J., Young, D.B., Barbosa, M., Mann, M., Manning, A., and Rao, A. (1997). IKK-1 and IKK-2: cytokine-activated IkB kinases essential for NF-kB activation. Science 278, 860–866. Minden, A., Lin, A., McMahon, M., Lange, C.C., Derijard, B., Davis, R.J., Johnson, G.L., and Karin, M. (1994). Differential activation of ERK and JNK mitogen-activated protein kinases by Raf-1 and MEKK. Science 266, 1719–1723. Mosialos, G., Birkenbach, M., Yalamanchili, R., VanArsdale, T., Ware, C., and Kieff, E. (1995). The Epstein-Barr virus transforming protein LMP1 engages signaling proteins for the tumor necrosis factor receptor family. Cell 80, 389–399. Nakano, H., Oshima, H., Chung, W., Williams, A.L., Ware, C.F., Yagita, H., and Okumura, K. (1996). TRAF5, an activator of NF-kB and putative signal transducer for the lymphotoxin-b receptor. J. Biol. Chem. 271, 14661–14664. Nakano, H., Shindo, M., Sakon, S., Nishinaka, S., Mihara, M., Yagita, H., and Okumura, K. (1998). Differential regulation of IkB kinase a and b by two upstream kinases, NF-kB-inducing kinase and mitogenactivated protein kinase/ERK kinase kinase-1. Proc. Natl. Acad. Sci. USA 95, 3537–3542. Natoli, G., Costanzo, A., Ianni, A., Templeton, D.J., Woodgett, J.R., Balsano, C., and Levrero, M. (1997). Activation of SAPK/JNK by TNF receptor 1 through a noncytotoxic TRAF2-dependent pathway. Science 275, 200–203. Re´gnier, C.H., Tomasetto, C., Moog-Lutz, C., Chenard, M.P., Wendling, C., Basset, P., and Rio, M.C. (1995). Presence of a new conserved domain in CART1, a novel member of the tumor necrosis factor receptor-associated protein family, which is expressed in breast carcinoma. J. Biol. Chem. 270, 25715–25721. Re´gnier, C.H., Song, H.Y., Gao, X., Goeddel, D.V., Cao, Z., and Rothe, M. (1997). Identification and characterization of an IkB kinase. Cell 90, 373–383. Reinhard, C., Shamoon, B., Shyamala, V., and Williams, L.T. (1997). Tumor necrosis factor a-induced activation of c-jun N-terminal kinase is mediated by TRAF2. EMBO J. 16, 1080–1092. Rothe, M., Wong, S.C., Henzel, W.J., and Goeddel, D.V. (1994). A novel family of putative signal transducers associated with the cytoplasmic domain of the 75 kDa tumor necrosis factor receptor. Cell 78, 681–692. Rothe, M., Sarma, V., Dixit, V.M., and Goeddel, D.V. (1995a). TRAF2mediated activation of NF-kB by TNF receptor 2 and CD40. Science 269, 1424–1427. Rothe, M., Pan, M.-G., Henzel, W.J., Ayres, T.M., and Goeddel, D.V. (1995b). The TNFR2–TRAF signaling complex contains two novel proteins related to baculoviral inhibitor of apoptosis proteins. Cell 83, 1243–1252. Saitoh, M., Nishitoh, H., Fujii, M., Takeda, K., Tobiume, K., Sawada, Y., Kawabata, M., Miyazono, K., and Ichijo, H. (1998). Mammalian thioredoxin is a direct inhibitor of apoptosis signal-regulating kinase (ASK) 1. EMBO J. 17, 2596–2606. Shi, C.S., and Kehrl, J.H. (1997). Activation of stress-activated protein kinase/c-Jun N-terminal kinase, but not NF-kB, by the tumor necrosis factor (TNF) receptor 1 through a TNF receptor-associated factor 2- and germinal center kinase related-dependent pathway. J. Biol. Chem. 272, 32102–32107. Shu, H.B., Takeuchi, M., and Goeddel, D.V. (1996). The tumor necrosis factor receptor 2 signal transducers TRAF2 and c-IAP1 are components of the tumor necrosis factor receptor 1 signaling complex. Proc. Natl. Acad. Sci. USA 93, 13973–13978.
Song, H.Y., Re´ gnier, C.H., Kirschning, C.J., Goeddel, D.V., and Rothe, M. (1997). Tumor necrosis factor (TNF)-mediated kinase cascades: bifurcation of nuclear factor-kB and c-jun N-terminal kinase (JNK/SAPK) pathways at TNF receptor-associated factor 2. Proc. Natl. Acad. Sci. USA 94, 9792–9796. Takeuchi, M., Rothe, M., and Goeddel, D.V. (1996). Anatomy of TRAF2. Distinct domains for nuclear factor-kB activation and association with tumor necrosis factor signaling proteins. J. Biol. Chem. 271, 19935–19942. Tartaglia, L.A., and Goeddel, D.V. (1992). Two TNF receptors. Immunol. Today 13, 151–153. Ting, A.T., Pimentel-Muinos, F.X., and Seed, B. (1996). RIP mediates tumor necrosis factor receptor 1 activation of NF-kB but not Fas/ APO-1-initiated apoptosis. EMBO J. 15, 6189–6196. Tracey, K.J., and Cerami, A. (1993). Tumor necrosis factor, other cytokines and disease. Annu. Rev. Cell Biol. 9, 317–343. Wang, X.S., Diener, K., Jannuzzi, D., Trollinger, D., Tan, T.H., Lichenstein, H., Zukowski, M., and Yao, Z. (1996). Molecular cloning and characterization of a novel protein kinase with a catalytic domain homologous to mitogen-activated protein kinase kinase kinase. J. Biol. Chem. 271, 31607–31611. Woronicz, J.D., Gao, X., Cao, Z., Rothe, M., and Goeddel, D.V. (1997). IkB kinase-b: NF-kB activation and complex formation with IkB kinase-a and NIK. Science 278, 866–869. Yeh, W.-C., Shahinian, A., Speiser, D., Kraunus, J., Billia, F., Wakeham, A., Pompa, J.L., Ferrick, D., Hum, B., Iscove, N., et al. (1997). Early lethality, functional NF-kB activation, and increased sensitivity to TNF-induced cell death in TRAF2-deficient mice. Immunity 7, 715–725. Zandi, E., Rothwarf, D.M., Delhase, M., Hayakawa, M., and Karin, M. (1997). The IkB kinase complex (IKK) contains two kinase subunits, IKKa and IKKb, necessary for IkB phosphorylation and NFkB activation. Cell 91, 243–252.