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The ASK1–MAP kinase pathways in immune and stress responses
Microbes and Infection 8 (2006) 1098e1107 www.elsevier.com/locate/micinf
Review
The ASK1eMAP kinase pathways in immune and stress responses Teruyuki Hayakawa, Atsushi Matsuzawa, Takuya Noguchi, Kohsuke Takeda, Hidenori Ichijo* Laboratory of Cell Signaling, Graduate School of Pharmaceutical Sciences, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan Available online 19 January 2006
Abstract Apoptosis signal-regulating kinase 1 (ASK1) is a mitogen-activated protein kinase kinase kinase which plays pivotal roles in stress and immune responses. This review is focused on three major subjects on ASK1: the regulatory mechanisms of the kinase activity, the pathophysiological roles in stress response and innate immunity, and the evolutionary perspectives. Ó 2005 Elsevier SAS. All rights reserved. Keywords: MAP kinase cascades; Apoptosis signal-regulating kinase 1; Innate immunity; Inflammation; Oxidative stresses
1. Introduction Living organisms are continuously exposed to various environmental stresses, such as oxidative stress, ultraviolet radiation and infection. In such circumstances, reactive oxygen species (ROS) appear to be commonly utilized by cells and tissues as a signaling intermediate that can be generated from the extracellular environment or intracellular organelles, such as mitochondria and phagosomes. ROS are also thought to be one of the main causes of cell senescence and dysfunction of biochemical systems. Therefore, cells must respond appropriately to such environmental stresses by using their intracellular signaling pathways. The immune system is essential for host defense against a wide range of pathogens. To clear pathogens, the host must distinguish ‘‘non-self’’ from ‘‘self’’ and then trigger the immune responses. Recognition of the molecular patterns differentially expressed among pathogens, so-called pathogenassociated molecular patterns (PAMPs), is the first step to discriminate ‘‘non-self’’ from ‘‘self’’. Toll-like receptors (TLRs) have been identified as receptors for the PAMPs in the last decade (reviewed in [1]); for example, TLR4, TLR1/2/6, TLR3 and TLR9 recognize lipopolysaccharide (LPS), peptidoglycan * Corresponding author. Tel.: þ81 3 5841 4859; fax: þ81 3 5841 4778. E-mail address: [email protected] (H. Ichijo). 1286-4579/$ - see front matter Ó 2005 Elsevier SAS. All rights reserved. doi:10.1016/j.micinf.2005.12.001
(PG), dsRNA and CpG-DNA, respectively. After recognition, TLRs elicit the intracellular signals (finally to the nucleus and other organelles) that are indispensable for triggering the immune responses. These intracellular signals include phosphorylatione dephosphorylation relays via protein kinases/phosphatases and formation of signaling complexes (signalosomes), eventually leading to the induction of cytokines that are required for the activation of innate and adaptive immune systems. In this review, recent progress in our knowledge on stress responses will be summarized by focusing on a serine/threonine kinase: apoptosis signal-regulating kinase 1 (ASK1). ASK1 is activated in response to various stimuli including LPS, ROS, ER stress, influx of calcium ions and various cytokines such as tumor necrosis factor and Fas ligand. We have recently demonstrated that the TRAF6eASK1ep38 axis is essential for inflammatory response on LPS stimulation. It is important that ROS-dependent ASK1 activation is specific, unlike other MAPKKKs, to LPSeTLR4 signaling [2]. 2. Roles of ASK1 in MAP kinase cascades: a stress-responsive kinase 2.1. Overview of the MAP kinase cascades In response to various extracellular and intracellular stimuli, mitogen-activated protein kinases (MAPKs) are activated or
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inactivated. MAPKs regulate a wide variety of cellular responses, such as cell growth, differentiation, and apoptosis. In the MAPK signaling cascades, MAP kinase kinase kinases (MAPKKKs) phosphorylate and activate MAP kinase kinases (MAPKKs), and MAPKKs subsequently phosphorylate and activate MAPKs. In all MAPK cascades, MAPKs are activated by MAPKK-catalyzed phosphorylation of Tyr and Thr residues at T-X-Y motif in the activation loop of the kinase domain. MAPKKs are also activated by MAPKKKs-catalyzed phosphorylation of Ser/Thr residues in the kinase domain (Fig. 1). Through these sequential biochemical events, the signals are amplified and diversified in a step-by-step manner. All eukaryotic cells possess multiple MAPK pathways. Among them, roles of mammalian MAPKs such as extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and p38 MAPK pathways have been defined to some extent (Fig. 1). ERK pathway is activated by various growth factors and closely linked to the regulation of cell cycle. JNK and p38 are preferentially activated by various environmental factors such as osmotic shock, oxidative stress and pro-inflammatory cytokines, and linked to various stress responses. JNK and p38 are thus also called stress-activated protein kinases (SAPKs). MAPK cascades are evolutionarily conserved in eukaryotic cells. In Fig. 1, stress-responsive MAP kinase cascades in mammals, Drosophila melanogaster, and Caenorhabditis elegans
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are shown (details in each organism are also reviewed in [3e5]). All of these organisms possess JNK and p38 pathways, although biological output can differ among them. Most of the MAPKKKs shown in Fig. 1 are capable of activating JNK and p38 pathways through MAPKKs, such as SEK1(MKK4)/ MKK7 and MKK3/MKK6, respectively. MAPKKKs in the JNK and p38 pathways are highly divergent in number (at least eleven MAPKKKs have been identified upstream of JNK) and structure. This diversity is seemingly of little use for activating only two MAPKs JNK and p38, but its diversity and complexity are rather consistent with the variety of stimuli that activate MAPK pathways. MAPKKKs thus appear to play pivotal roles in sensing and signaling of cellular and environmental stress [3]. The following sections will focus on ASK1, one of the well-studied MAPKKKs. ASK1 was originally identified as an apoptosis-inducing MAPKKK that activates both p38 and JNK pathways [6]. Recent studies revealed that ASK1 contributes not only to regulation of cell death but also to cytokine responses, cell differentiation and immune regulation [7]. 2.2. ASK1 activation in stress response In this section we will focus on molecular features of ASK1. Although ASK1 is activated by various stresses, common mechanisms appear to operate to activate ASK1 in response to various stresses. ASK1 has two important structural
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Fig. 1. Components of stress-responsive MAP kinase cascades are evolutionarily conserved. JNK and p38 pathways, two major stress-responsive MAP kinase cascades in mammals, Drosophila melanogaster and Caenorhabditis elegans are shown. There are two other major cascades (ERK1 and ERK5), which are not illustrated here. In all MAPK cascades, MAPKs are activated by MAPKK-catalyzed phosphorylation of Tyr and Thr residues at T-X-Y motif in the activation loop of the kinase domain. MAPKKs are also activated by MAPKKKs-catalyzed phosphorylation of Ser/Thr residues in the kinase domain. It is uncertain whether common activation mechanisms for MAPKKKs exist; however autophosphorylation of Ser/Thr residues in the kinase domain appears to be required for many of the known MAPKKKs.
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2.2.2. ASK1 signalosome: a node for signal transduction Following the dissociation of Trx, it has recently been reported that recruitment of tumor necrosis factor receptorassociated factor (TRAF) family proteins is also required for oxidative stress-induced ASK1 activation [16]. Endogenous ASK1 constitutively forms a high molecular weight complex (approximately 1500e2000 kDa) which was designated ‘‘ASK1 signalosome’’. Upon H2O2 stimulation, the ASK1 signalosome forms higher molecular weight complex (approximately >3000 kDa) due at least to the recruitment of TRAF2 and TRAF6, which have previously been reported to activate ASK1 (discussed in Section 2.3.2). The ASK1 signalosome contains not only Trx or TRAFs, but also other unidentified factors, suggesting the multiple roles of the ASK1 signalosome as the ‘‘node’’ for signal transduction. A large number of ASK1-interacting proteins (both positive and negative regulators) have been reported, most of which may be contained in the ASK1 signalosome. Elucidation of the roles of these components may shed light on the precise mechanisms of the ASK1-dependent signaling cascade which links various stresses to the biological events.
features: N-terminal thioredoxin binding site and C-terminal homophilic interaction site. 2.2.1. Molecular structure and general activation mechanism of ASK1 Human and mouse ASK1 consist of 1374 and 1380 amino acids, respectively, and possess a serine/threonine kinase domain in the middle of the molecule [6] (Fig. 2A). When wild-type ASK1 or its constitutively active mutant are overexpressed in mammalian cells, they directly phosphorylate and activate MKK3/MKK6 and MKK4/MKK7, and induce apoptosis in various cells [8]. However, the downstream events of ASK1 are variable depending on the cell types and cellular context, including cell death [6,9e11], differentiation [12,13], neurite outgrowth [14] and induction of cytokines [2]. ASK1 has two coiled-coil domains at its N- and C-terminals, of which C-terminal coiled-coil domain is essential for forming ‘‘ASK1 signalosome’’ (see below) and activating ASK1 itself [15,16]. ASK1 in its resting state forms a static homo-oligomer through its C-terminal coiled-coil domain. A ubiquitously expressed reduction/oxidation (redox) protein thioredoxin (Trx) binds to the N-terminal of ASK1 and inhibits its activity in the absence of oxidative stress (Fig. 2B). Upon oxidative stress stimulation such as hydrogen peroxide (H2O2), intramolecular disulfide bonding is formed between two Cys residues in Trx. Oxidized form of Trx dissociates from ASK1, which leads to the autophosphorylation of threonine residue (Thr 838 in human and Thr 845 in mouse) in the activation loop of ASK1. The kinase activity of ASK1 is fully elevated by this phosphorylation. It is thus proposed that following ROS production (either from exogenous or endogenous sources), dissociation of Trx and tight homo-oligomerization of ASK1 are the sequential events that are essential for ASK1 activation [15,17] (Fig. 2B). The phospho-specific antibody that recognizes phospho-Thr in the activation loop has been found to be a useful indicator of the ASK1 activity [15].
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2.3. Stimuli and stresses that activate ASK1 ASK1 is activated by cytokines and various environmental and cellular stresses. In the following subsections we discuss some of these stresses that activate ASK1. Note that ASK1e Trx system is widely used as a regulatory mechanism of ASK1 (Fig. 3). 2.3.1. Oxidative stress: ASK1eTrx system ROS is not only a cellular stress that is produced at the electron transport chain or under pathological conditions, but is also used as a second messenger in signal transduction: for example, it is required for cell proliferation induced by various growth factors [18]. An appropriate amount of ROS is
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Fig. 2. General features of ASK1. (A) Domain structure of ASK1. Human ASK1 consists of 1374 amino acids and contains a serine/threonine kinase domain in the middle of the molecule. ASK1 also has two coiled-coil domains at the N- and C-terminals. (B) ASK1eTrx system. ASK1 in the resting state forms a homooligomer via C-terminal coiled-coil domain and binds to reduced Trx at the N-terminus. Upon ROS generation, intramolecular disulfide bonding is formed in Trx and subsequently Trx dissociates from ASK1, which leads to the autophosphorylation of the Thr 845 residue in its activation loop. ROS are the key to cancel the ‘‘safety lock’’ of the ‘‘bomb’’ ASK1.
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Fig. 3. Molecular mechanisms of ASK1 activation in response to various stimuli. Signaling mechanisms of representative stimuli that activate ASK1 are illustrated. (Left) LPS-induced ASK1 activation requires generation of ROS and recruitment of the adaptor protein TRAF6. Only p38 but not JNK and IKK pathways were impaired in ASK1/ cells. (Center) TNF binds to TNR receptor (TNFR1), which activates ASK1ep38/JNK pathways. This signal is mediated by TRAF2 and dependent on ROS generation. (Right) ER stress-induced ASK1 activation is mediated by IRE1, one of the ER transmembrane proteins. ASK1 interacts with IRE1eTRAF2 complex and is activated by ER stress, leading to the activation of JNK pathway. It is currently unknown whether the dissociation of Trx is required for activation of ASK1 by ER stress.
necessary for physiological cell signaling and normal cellular response, whereas excessive production of ROS behaves as cellular stress and eventually induces necrosis or apoptosis. ASK1 is strongly activated when cells are treated with hydrogen peroxide (H2O2) or other ROS-generating stimuli. In response to the oxidative stress, a common ROS-sensory mechanism, ASK1eTrx system, appears to operate. Trx was initially identified in a screening for ASK1-interacting proteins [17]. Reduced form of Trx, which possesses two sulfhydryl groups at the Cys-32 and Cys-35 residues, specifically binds to the N-terminal region of ASK1 and inhibits ASK1’s kinase activity. In contrast, oxidized form of Trx, which possesses intramolecular disulfide bonding between the two cysteine residues, does not bind to ASK1. Upon H2O2 stimulation, Trx dissociates from ASK1, and the freed ASK1 appears to be tightly oligomerized through N- and C-terminal coiled-coil domains, leading to the autophosphorylation of the Thr residue at the activation loop ([15,17]; G. Fujino and H. Ichijo, unpublished observation). ASK1eTrx system is thus thought to be a redox sensor which functions as a signal converter of intracellular redox changes into the signaling of kinase cascades (Fig. 2B). ASK1-null mice have been generated to investigate the roles of ASK1 in oxidative stress-induced activation of JNK/ p38 cascades and apoptosis [11]. ASK1/ mice were viable and histologically indistinguishable from wild-type littermates. However, in mouse embryonic fibroblasts (MEFs) isolated from ASK1/, H2O2- and tumor necrosis factor a-induced sustained but not transient activation of JNK and p38 was suppressed. H2O2-induced apoptosis was also reduced in ASK1/ mice MEFs, suggesting that ASK1 is required for oxidative stress-induced apoptosis.
2.3.2. Tumor necrosis factor (TNF) Whether cells survive or undergo apoptosis is regulated by both intracellular conditions and extracellular factors such as death ligands. Receptors that respond to these ligands are called death receptors and are among members of the TNFreceptor family, all of which have extracellular cysteine-rich repeats and a cytoplasmic death domain (DD) [19]. Tumor necrosis factor alpha (TNFa) is a pleiotropic cytokine that plays important roles in inflammation, immune responses and apoptosis. TNFa activates two major transcription factors: activator ptotein-1 (AP-1) and nuclear factor-kB (NF-kB). Both transcription factors are activated through the activation of JNK/p38 and IkB kinases, respectively [20]. Upon TNFa treatment, ASK1 is activated and subsequently activates JNK and p38 cascades (Fig. 3). This activation is mediated by TNF receptor-associated factor 2 (TRAF2), an adaptor protein that possesses E3 ubiquitin ligase activity essential for TNFa-mediated signaling pathways [21]. Interestingly, this TRAF2-mediated activation of ASK1 appears to be ROSdependent. TNF stimulation or overexpression of TRAF2 generates intracellular ROS, and activation of ASK1 is largely dependent on ROS (i.e. suppressed by treatment with antioxidants) [22,23]. It is consistent with the ASK1eTrx system mentioned earlier; ROS-mediated dissociation of Trx from ASK1 is necessary for TNF-induced activation of ASK1. Requirement of ASK1 in TNFa signaling has been demonstrated by using MEFs derived from ASK1/ mice [11]. ASK1/ MEFs were resistant to TNFa-induced apoptosis, and sustained but not transient activation of JNK and p38 were diminished in ASK1/ MEFs. It has been reported that the TRAF2e GCKseMEKK1 pathway is also involved in TRAF2-mediated transient JNK activation. Two MAPKKKs, ASK1 and MEKK1,
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are thought to act in TNFa and TRAF2-mediated JNK activation, although whether they act redundantly or complementarily remains to be studied. 2.3.3. ER stress and neurodegenerative conditions In living cells newly synthesized proteins often fail to fold correctly or fail to find their correct partner in a larger protein complex. Such a misfolded protein is not only useless to cells but toxic, and therefore is normally digested in proteasome to be detoxified. Accumulation of unfolded and misfolded proteins in endoplasmic reticulum (ER) lumen induces ER stress. ER stress has recently been reported to be a potential cause of neurodegenerative disorders such as Alzheimer’s disease [24], Parkinson’s disease [25], polyglutamine diseases [9] and diabetes mellitus [26]. Cells try to adapt themselves against ER stress by stressresponsive pathways called unfolded protein response (UPR). IRE1, ATF6, OASIS and PERK are the ER transmembrane proteins that mediate UPR by reducing influx of proteins into ER lumen. If this adaptation is insufficient for the clearance of ER stress, cells undergo apoptosis. ASK1 is activated by ER stress in the IRE1eTRAF2eASK1eJNK axis [9]. ASK1 interacts with TRAF2 and is activated upon ER stress (Fig. 3). Moreover, in primary neurons derived from ASK1/ mice, ER stress- and polyglutamine-induced JNK activation and apoptosis were reduced. These data demonstrate the essential role of ASK1 in ER stress-induced cell death. It was recently shown that ASK1 is also required for amyloid b (Ab)-induced neuronal cell death [27]. Upon Ab treatment, the ASK1eJNK pathway was activated in a ROS-dependent manner, but not through ER stress. ASK1/ neurons were resistant to Ab-induced cell death. These two reports on the roles of ASK1 in neuronal cell death suggest the importance of ASK1 in the pathogenesis of neurodegenerative diseases. ASK1 may be a potential therapeutic target for prevention and treatment of various neurodegenerative diseases. 2.3.4. Other stimuli that activate ASK1 G-protein coupled receptor (GPCR)-mediated signals also activate ASK1. For example angiotensin II (Ang II) treatment induces the activation of ASK1 in cardiomyocytes. A recent study demonstrated that Ang II-mediated ASK1 activation was mediated by generation of ROS and was closely related to cardiac hypertrophy and remodeling, although the intermediate molecule between the AngII receptor and ASK1 is elusive [28]. It is also reported that b-Arrestin 2 acts as an scaffold protein for ASK1eMKK4eJNK3 and is involved in JNK3 activation induced by Ang II [29]. ASK1 was also reported to be involved in Fas signaling through an adaptor protein Daxx [30]. Daxx interacts both with intracellular DD of Fas and N-terminus of ASK1, and subsequently activates JNK. However, the sensitivity to Fas-induced apoptosis was indistinguishable between in wildtype and ASK1/ thymocytes, suggesting the role of Daxxe ASK1eJNK pathway in other physiological events unrelated to Fas-induced apoptosis [11].
Ca2þ influx and LPS stimulation also activate ASK1 [2,31], which will be discussed in the following sections (Fig. 3). 3. Emerging roles of ASK1: lessons from Drosophila and C. elegans Recently the orthologues corresponding to mammalian ASK1 have been reported in two model organisms, the fruit fly Drosophila melanogaster and the nematode Caenorhabditis elegans. The merit of using these organisms includes phenotype-based genetic screening. Both Drosophila and C. elegans ASK1 have been identified in forward genetic screening, and the information from these studies is also useful for studies in mammals. 3.1. Drosophila ASK1 Drosophila ASK1 (DASK1) was found to be a structural and functional orthologue of ASK1. Overexpression of Reaper (Rpr) using an eye-specific promoter gives rise to caspasedependent apoptosis of the eye cells, resulting in small eyes. Kuranaga et al. identified DASK1 and Drosophila TRAF1 (DTRAF1, most similar to human TRAF4) as the dominant modifiers of Rpr, in which suppression of Rpr-induced small-eye phenotype was used as a criteria for the genetic screening [32]. Rpr-mediated cell death of eyes was also suppressed by co-expression of the kinase mutant of DASK1 (DASK1K618M). Furthermore, in Drosophila S2 cells, expression of DTRAF1 activates DASK1 and activated DASK1 then activates Drosophila JNK (DJNK, also called Basket) (Fig. 1). DASK1 is thought to possess molecular function as MAPKKK. Since little is known about the biological functions of DASK1 except for its apoptosis-inducing activity, future availability of DASK1 mutant fly will be a powerful tool to understand the in vivo roles of ASK1. 3.2. NSY-1: ASK1 in C. elegans The nematode Caenorhabditis elegans is also an excellent model organism used to perform large-scale genetic screenings. Until now, NSY-1, an orthologue of mammalian ASK1, has been identified in two independent genetic screenings: Nsy (neuronal symmetry) and Esp (enhanced susceptibility) (Fig. 4). 3.2.1. Neuronal symmetry C. elegans detects odorants with the bilaterally symmetric pair of neurons: AWC neurons [33]. The candidate odorant receptor gene str-2 is expressed in only one neuron, either the left or the right neuron, and its asymmetric expression is required for odor discrimination [34]. A genetic screen for mutants defective in lefteright asymmetry was performed and the identified mutants were named neuronal symmetry (nsy) genes [35]. Sagasti et al. reported that nsy-1 encodes an orthologue of the mammalian ASK1, and it acts downstream of the Ca2þ/calmodulin-dependent protein kinase II (CaMKII) UNC-43 [36]. Tanaka-Hino et al. subsequently reported that SEK-1 MAPKK (an orthologue of mammalian MKK3 and
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Fig. 4. Physiological roles of NSY-1 and ASK1 in C. elegans and mammals. In C. elegans AWC neurons, the NSY-1eSEK-1eMAPK cascade is activated by Ca2þ signaling through TIR-1 and UNC-43 (CaMKII), and determines the asymmetric cell fate. In the C. elegans intestine, NSY-1 is activated by the pathogens through TIR-1 but not through UNC-43, and it activates the downstream SEK-1ePMK-1 pathway, leading to the innate immune response. In the mammalian neuron, ASK1 is activated by Ca2þ influx through the direct phosphorylation by CaMKII. Ca2þ-activated ASK1 then activates p38. Combined with the fact that constitutively active ASK1 induces neurite outgrowth in PC12 cells, ASK1 might play critical roles in synaptic plasticity. In the immune cells such as macrophage and dendritic cell, ASK1 is activated by LPS treatment and is required for LPS-induced p38 activation and innate immune responses such as inflammation.
MKK6) is also required for asymmetric expression in AWC neurons and that SEK-1 acts in a pathway downstream of UNC-43 and NSY-1 [37]. Thus, the CaMKIIeNSY-1eSEK-1 axis is essential for asymmetric expression of str-2 and, as a result, neuronal differentiation. These elegant analyses prompted us to question whether this pathway is conserved in humans. Using ASK1-deficient mice, Takeda et al. have demonstrated the essential role of ASK1 in Ca2þ-induced p38 MAPK activation in neuronal cells [31]. In PC12 cells and primary neurons, ASK1 was activated in response to influx of Ca2þ ions evoked by membrane depolarization, which was inhibited by the treatment with L-type Ca2þ channel blocker (nifedipine) and CaMK inhibitor (KN-93). CaMKII directly phosphorylates (other than activation loop) and somehow activates ASK1. Consistent with the results from C. elegans, these data demonstrate the important role of CaMKII as an upstream activator of ASK1. Detailed biochemical analysis of phosphorylation-dependent activation mechanisms of ASK1 will be an important issue to be addressed. 3.2.2. Enhanced susceptibility C. elegans is also a useful genetic tool for screening mutants of immune responses because it is relatively easy to isolate mutants with higher susceptibility to pathogens compared to the screening with other higher organisms such as the fruit fly or mouse [38]. Kim and colleagues isolated 10 Esp mutants that were more susceptible to infection by Pseudomonas aeruginosa strain PA14 [39]. esp-2 and esp-8 were the two most susceptible mutants and were shown to correspond to two
known genes, sek-1 and nsy-1, respectively. It was strongly suggested that NSY-1eSEK-1ePMK-1 pathway is the essential pathway to resist PA14. This was strengthened by the RNA interference (RNAi) experiment of PMK-1; inhibition of PMK-1 activity by knock-down gave rise to a strong Esp phenotype. Moreover, PA14-induced p38 activity was almost completely suppressed in esp-2/sek-1 and esp-8/nsy-1 mutants compared to wild-type N2 worms. Interestingly, unc-43 (mammalian CaMKII) mutant, which shows a null Nsy phenotype, had almost no Esp phenotype. It has therefore been suggested that another molecule other than UNC-43 may exist upstream of the NSY-1 in the Esp pathway; TIR-1 was recently identified to act as an upstream activator of NSY-1 (see below). 4. ASK1 in TLR signaling Recently it was demonstrated that TRAF6eASK1ep38 pathway plays important roles in LPS-mediated inflammatory signaling. ASK1 is specifically activated by LPS, but not other TLR ligands tested, and is required for the activation of p38, but not JNK or NF-kB pathways [2]. 4.1. Overview of TLR signaling In the innate immune responses, certain pathogens are first sensed by cell surface receptors or intracellular components that recognize PAMPs. The recognition is then converted to the intracellular signal pathways that culminate in immune responses such as inflammation and cytokine production.
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4.1.1. Pathogen recognition by TLRs TLRs are the major components for the recognition of a broad range of pathogenic microorganisms (reviewed in [1]). There are at least ten TLRs in human (TLR1e10) and thirteen in mice (TLR1e13), and the cognate ligands for most of them have been identified. They are roughly divided in two groups: TLRs that recognize bacterial components (TLR1/2/6, TLR4 and TLR5) or viral components (TLR3, 7 and 8). TLR9 recognizes CpG DNA motif derived from both bacteria and virus. The TLRs are type I integral membrane glycoproteins. Those extracellular regions contain leucine-rich repeat (LRR) motifs, which bind and recognize pathogens. On the other hand, the cytoplasmic tails of TLRs contain Toll/interleukin-1 receptor (TIR) domains that bind to intracellular signaling molecules. TIR domain is named so because it is conserved in cytoplasmic tails of all TLRs and interleukin-1 receptors (IL-1Rs).
molecule 1), and TRIF-related adaptor molecule (TRAM; also known as TICAM2, TIR domain-containing molecule 2) [40]. Knockout mice of these genes were generated and analyzed, and they were found to be deficient in transducing specific pathogen’s stimuli to intracellular events. Upon stimulation, TLRs recruit at least one TIR adaptor protein, and downstream events occur in a TIR adaptor-dependent manner. These pathways are often classified in two groups: MyD88-dependent and TRIF-dependent pathways. Activation of MyD88-dependent pathway leads to the production of pro-inflammatory cytokines such as interleukin (IL)-6 and TNF-a whereas activation of TRIF-dependent pathway leads to production of type-I interferon (IFNa/b). Downstream molecules of such TIR adaptor proteins include IL-1R-associated kinases (IRAKs), transforming growth factor-b (TGF-b)-activated kinase (TAK1), TAK1-binding protein 1 (TAB1), TAB2, and TNF receptor-associated factor 6 (TRAF6). Upon LPS stimulation, TLR4 activates both MyD88- and TRIF-dependent pathways, which eventually induce transcription of pro-inflammatory cytokines and type-I interferon, respectively. In MyD88-dependent pathway, the IRAK4/ IRAK1eTRAF6 complex is recruited to the cytoplasmic TIR domain of TLR4 by TIRAPeMyD88 adaptor proteins. TRAF6 then activates cytoplasmic kinases, including TAK1 and MEKK3 [41e43] (Fig. 5). Other reports demonstrate the essential roles of Tpl2/Cot proto-oncogene [44] and MEKK1 [45] in LPS-induced activation of MAPK cascades. To what extent TRAF6 contributes to the activation of Tpl2/ Cot and MEKK1 is yet to be studied. ASK1 is also involved in TLR4 signaling, which we will discuss in the next section.
4.1.2. Intracellular signaling downstream of TLRs After pathogens bind to extracellular regions of TLRs, TLRs undergo conformational changes and their cytoplasmic TIR domains then activate common signaling pathways, which include the activation of transcription factor nuclear factor-kB (NF-kB) and stress-activated protein kinases including p38 MAPK and JNK. TIR domains of cytoplasmic tails of TLRs interact with cytoplasmic TIR domain-containing adaptor proteins, which include myeloid differentiation primary-response protein 88 (MyD88), TIR domain-containing adaptor protein (TIRAP; also known as Mal, MyD88-adaptor-like protein), TIR domain-containing adaptor protein inducing interferon (IFN)-b (TRIF; also known as TICAM1, TIR domain-containing
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Fig. 5. Roles of ASK1 and other MAPKKKs in TLR4 signaling. LPS is recognized by TLR4eMD2 complex and the signal is transduced to the intracellular kinase cascades. ASK1 is activated by TLR4 but not by other TLRs tested. ROS generation and direct interaction of ASK1 with TRAF6 are required for its activation. The activation of the downstream kinase p38 was diminished in ASK1/ cells, whereas JNK and IKK seemed to be almost normal. TAK1 and MEKK3 are also activated by interaction with TRAF6 and activate the p38, JNK, and NF-kB pathways. Tpl2/Cot is essential in LPS-mediated ERK activation. MEKK1 has also been reported to be involved in LPS-mediated JNK activation (not shown).
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4.2. TRAF6eASK1ep38 axis in TLR4 signaling In this decade, a number of signaling molecules have been identified in the innate immunity: Toll-like receptors, adaptor proteins that determine specificity of downstream signals, and transcription factors for cytokine production. However, the roles of MAPK cascades in mammalian innate immunity have been largely unknown. As described earlier, the role of ASK1ep38 pathway in immune responses has been reported from the studies of C. elegans [39]. Matsuzawa et al. have recently demonstrated the importance of ASK1 in the mammalian innate immunity system using ASK1/ mice [2] (Fig. 5). In ASK1/ splenocytes and bone marrow-derived dendritic cells (BMDCs), LPS-induced activation of p38 MAPK was specifically diminished, whereas activation of JNK and NF-kB was unaffected. Production of inflammatory cytokines such as TNF, IL-6 and IL-1b was also diminished in ASK1/ splenocytes and BMDCs, indicating that the ASK1ep38 axis is required for inflammation in response to LPS. Interestingly, ASK1 was shown to be selectively required for TLR4 signaling. LPS-induced activation of ASK1 and downstream p38 was specifically abolished in ASK1/ cells, whereas PGN, Poly(I:C) and CpG DNA-induced responses (correspond to TLR2, TLR3 and TLR9, respectively) were not. As a candidate for generation of the specificity for TLR4e ASK1 pathway, two factors, ROS and TRAF6, have been studied in detail. In RAW264.7 macrophage cell line, LPS-induced activation of ASK1ep38 pathway and cytokine production were strikingly suppressed by various anti-oxidants including propyl gallate and N-acetyl-L-cysteine. Moreover, LPSinduced interaction between ASK1 and TRAF6 was disrupted by the presence of anti-oxidant. These data suggest the possible mechanism for LPS-induced ASK1 activation as follows: (1) after LPS stimulation TLR4 somehow generates ROS, which triggers the dissociation of the inhibitor Trx from ASK1; (2) freed ASK1 effectively interacts with TRAF6 (and probably with other unidentified components) and forms the active signalosome of ASK1, which facilitates ASK1 activation and selective p38 activation. In vivo study also supports the role of ASK1 in innate immunity. When wild-type and ASK1/ mice were injected with LPS, ASK1/ mice were resistant to LPS-induced septic shock, whereas most wild-type mice died within 40 h. The levels of two principal factors responsible for septic shock, TNF and nitric oxide,
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were also reduced in serum of ASK1/ mice. In conclusion, these data demonstrate that the ROS-dependent TRAF6e ASK1ep38 axis is crucial for TLR4-mediated mammalian innate immunity. 5. Perspectives and conclusion As we have seen earlier, NSY-1ePMK-1/ASK1ep38 signaling pathway is conserved in C. elegans and mammals and plays pivotal roles in pathogen resistance and inflammation. However, there are some discrepancies between these organisms. We will wind up this review by discussing unsolved important issues on ASK1 such as evolutionary conservation and roles of ROS and ASK1 in the stress and immune responses. 5.1. To what extent innate immunity is evolutionarily conserved? There are some discrepancies between C. elegans and other organisms such as Drosophila and mice. It goes without saying that in mammals and Drosophila NF-kB/Relish and TLRs/Toll are the key players in innate immunity and inflammation. However, these components do not seem to be evolutionarily conserved in C. elegans [46]. C. elegans possesses two TIR domain-containing proteins, TOL-1 and TIR-1. TOL-1 is most similar to the members of the TLR family: TOL-1 extracellular domain contains LRRs, followed by a putative transmembrane domain and an intracellular TIR domain. TIR-1 is the intracellular TIR-containing protein with two sterile a-motif domains at the center and HEAT/armadillo repeats at the N-terminus [47,48]. Mutations in tol-1 have been reported to show no phenotype on pathogen susceptibility [46]. In contrast, tir-1 RNAi-inactivated worms were more sensitive to the pathogens: the Gram-negative bacterial pathogens P. aeruginosa and Serratia marcescens, and the fungal pathogen Drechmeria coniospora [47,48]. Moreover, in tir-1 RNAi-inactivated worms, the activity of PMK-1 was diminished [47]. These results suggest that TIR-1 acts upstream of the NSY-1eSEK-1ePMK-1 pathway and that the TIR-1e NSY-1eSEK-1ePMK-1 pathway may be evolutionarily the most ancient pathway for innate immunity. The upstream components of NSY-1 and ASK1 are still unknown. As described earlier, TIR-1 does not seem to participate in TLR signaling, although mammalian ASK1ep38 pathway plays an important role at least in TLR4 signaling.
Table 1 Molecular profiles of stimuli that activate ASK1 Stimuli
Involvement of ROS in ASK1 activation
Activator
Contribution of ASK1 p38
JNK
Oxidative stress TNFa ER stress LPS Amyloid b Calcium influx
Yes Yes ? Yes Yes (by NADPH oxidase?) No
TRAF2, 6 TRAF2, (6?) TRAF2, (6?) TRAF6, (2?) ? CaMKII
YesL* YesL* ? Yes ? YesE*
YesL* YesL* Yes No Yes ?
*Contribution only at: E, early phase; L, late phase.
Cell type analyzed
MEF MEF MEF DC, splenocyte Cortical neuron Neuron
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It is therefore possible that another conserved immune signaling pathway exists in mammals, in which the mammalian orthologue of TIR-1 may play a pivotal role. One possibility is that TIR-1 might be coupled to non-TLR-type receptors; or alternatively, TIR-1 itself might be the pathogen receptor. The orthologue of TIR-1 in mammals, known as sterile a motif and armadillo motif (SARM), has been cloned by Mink et al. [49], although its cellular function remains to be elucidated.
plasticity. Moreover, TRAF6eASK1ep38 pathway plays an essential role in inflammatory and innate immune responses. ASK1 forms a mega-complex and rearranges its components in response to various stimuli. It is conceivable that ASK1 functions as a signaling node in stress response. Further investigation of regulatory mechanisms of ASK1 and its relatives will provide the clues for understanding immune and stress responses. Acknowledgements
5.2. Molecular mechanisms of ASK1 activation, again Let us go back again to the molecular mechanisms of ASK1 activation. As we have seen, ROS generated by various stimuli appear to be the common ‘‘key’’ to open the ‘‘safety lock’’ Trx (Fig. 3, Table 1). ROS is thus considered to be the common second messenger for ASK1 activation. The source for ROS is still largely unknown, but NAD(P)H oxidase is one of the likely candidates. Indeed, after LPS stimulation, ROS is known to be generated from NAD(P)H oxidase complex including NAD(P)H oxidase 4 isozyme, which directly interacts with TLR4 [50]. However, the relationship between NAD(P)H oxidase and ASK1 in LPS signaling is yet to be investigated. Another candidate is mitochondria, the main source of oxygen radicals in eukaryotic metabolism. It has been established that ROS is generated by TNFa in many cell types [51]. Complex formation with activators and homo-oligomerization are also the indispensable steps for ASK1 activation. Among the components of ASK1 complex, TRAF family proteins may be the common adaptor proteins required for ASK1 activation. Further studies are required to uncover the unidentified factors, which may shed light on the precise mechanism of ROS-dependent ASK1 activation. 5.3. Roles of ASK1 in stress and immune response As described throughout this review, ASK1 is activated by various environmental stresses. These include oxidative stress, TNF as a pro-inflammatory cytokine, LPS as a TLR agonist. ASK1-deficient mice were born without apparent phenotype and grew normally, suggesting that ASK1 is not essential for cell growth and differentiation. On the other hand, ASK1/ MEFs were more resistance to oxidative stress-, TNF-, and ER stress-induced cell death. These two phenomena suggest that ASK1 activity is tightly regulated under unstressed conditions. This is consistent with the ASK1eTrx system, in which ASK1 in the steady state is tightly inactivated by Trx. ASK1 is able not only to induce apoptosis, but also to mediate a wide range of cellular responses. ASK1 induces apoptosis through ASK1ep38/JNK cascades in response to proapoptotic stresses (e.g. oxidative stress and TNF) and pathogenic stresses (e.g. ER stress, GPCR- and Ab-induced ROS production). On the other hand, constitutively active ASK1 induces neurite outgrowth in PC12 cells. ASK1 is activated by CaMKII, which activates ASK1ep38 pathway in neurons, suggesting that ASK1 might play critical roles in synaptic
We thank all the members of the Laboratory of Cell Signaling for valuable discussion and comments. References [1] S. Akira, K. Takeda, Toll-like receptor signalling, Nat. Rev. Immunol. 4 (2004) 499e511. [2] A. Matsuzawa, K. Saegusa, T. Noguchi, C. Sadamitsu, H. Nishitoh, S. Nagai, S. Koyasu, K. Matsumoto, K. Takeda, H. Ichijo, ROS-dependent activation of the TRAF6-ASK1-p38 pathway is selectively required for TLR4-mediated innate immunity, Nat. Immunol. 6 (2005) 587e592. [3] J.M. Kyriakis, J. Avruch, Mammalian mitogen-activated protein kinase signal transduction pathways activated by stress and inflammation, Physiol. Rev. 81 (2001) 807e869. [4] B. Stronach, Dissecting JNK signaling, one KKKinase at a time, Dev. Dyn. 232 (2005) 575e584. [5] A. Sakaguchi, K. Matsumoto, N. Hisamoto, Roles of MAP kinase cascades in Caenorhabditis elegans, J. Biochem. (Tokyo) 136 (2004) 7e11. [6] H. Ichijo, E. Nishida, K. Irie, P. Ten Dijke, M. Saitoh, T. Moriguchi, M. Takagi, K. Matsumoto, K. Miyazono, Y. Gotoh, Induction of apoptosis by ASK1, a mammalian MAPKKK that activates SAPK/JNK and p38 signaling pathways, Science 275 (1997) 90e94. [7] J. Matsukawa, A. Matsuzawa, K. Takeda, H. Ichijo, The ASK1eMAP kinase cascades in mammalian stress response, J. Biochem. (Tokyo) 136 (2004) 261e265. [8] K. Takeda, A. Matsuzawa, H. Nishitoh, H. Ichijo, Roles of MAPKKK ASK1 in stress-induced cell death, Cell Struct. Funct. 28 (2003) 23e29. [9] H. Nishitoh, A. Matsuzawa, K. Tobiume, K. Saegusa, K. Takeda, K. Inoue, S. Hori, A. Kakizuka, H. Ichijo, ASK1 is essential for endoplasmic reticulum stress-induced neuronal cell death triggered by expanded polyglutamine repeats, Genes Dev. 16 (2002) 1345e1355. [10] T. Hatai, A. Matsuzawa, S. Inoshita, Y. Mochida, T. Kuroda, K. Sakamaki, K. Kuida, S. Yonehara, H. Ichijo, K. Takeda, Execution of apoptosis signal-regulating kinase 1 (ASK1)-induced apoptosis by the mitochondria-dependent caspase activation, J. Biol. Chem. 275 (2000) 26576e26581. [11] K. Tobiume, A. Matsuzawa, T. Takahashi, H. Nishitoh, K. Morita, K. Takeda, O. Minowa, K. Miyazono, T. Noda, H. Ichijo, ASK1 is required for sustained activations of JNK/p38 MAP kinases and apoptosis, EMBO Rep. 2 (2001) 222e228. [12] K. Sayama, H. Komatsuzawa, K. Yamasaki, Y. Shirakata, Y. Hanakawa, K. Ouhara, S. Tokumaru, X. Dai, M. Tohyama, P. Ten Dijke, M. Sugai, H. Ichijo, K. Hashimoto, New mechanisms of skin innate immunity: ASK1-mediated keratinocyte differentiation regulates the expression of beta-defensins, LL37, and TLR2, Eur. J. Immunol. 35 (2005) 1886e1895. [13] K. Sayama, Y. Hanakawa, Y. Shirakata, K. Yamasaki, Y. Sawada, L. Sun, K. Yamanishi, H. Ichijo, K. Hashimoto, Apoptosis signal-regulating kinase 1 (ASK1) is an intracellular inducer of keratinocyte differentiation, J. Biol. Chem. 276 (2001) 999e1004. [14] K. Takeda, T. Hatai, T.S. Hamazaki, H. Nishitoh, M. Saitoh, H. Ichijo, Apoptosis signal-regulating kinase 1 (ASK1) induces neuronal differentiation and survival of PC12 cells, J. Biol. Chem. 275 (2000) 9805e9813.
T. Hayakawa et al. / Microbes and Infection 8 (2006) 1098e1107 [15] K. Tobiume, M. Saitoh, H. Ichijo, Activation of apoptosis signal-regulating kinase 1 by the stress-induced activating phosphorylation of preformed oligomer, J. Cell. Physiol. 191 (2002) 95e104. [16] T. Noguchi, K. Takeda, A. Matsuzawa, K. Saegusa, H. Nakano, J. Gohda, J.I. Inoue, H. Ichijo, Recruitment of TRAF family proteins to the ASK1 signalosome is essential for oxidative stress-induced cell death, J. Biol. Chem. 280 (2005) 37033e37040. [17] M. Saitoh, H. Nishitoh, M. Fujii, K. Takeda, K. Tobiume, Y. Sawada, M. Kawabata, K. Miyazono, H. Ichijo, Mammalian thioredoxin is a direct inhibitor of apoptosis signal-regulating kinase (ASK) 1, EMBO J. 17 (1998) 2596e2606. [18] P.G. Winyard, C.J. Moody, C. Jacob, Oxidative activation of antioxidant defence, Trends Biochem. Sci. 30 (2005) 453e461. [19] R.M. Locksley, N. Killeen, M.J. Lenardo, The TNF, TNF receptor superfamilies: integrating mammalian biology, Cell 104 (2001) 487e501. [20] H. Wajant, K. Pfizenmaier, P. Scheurich, Tumor necrosis factor signaling, Cell Death Differ. 10 (2003) 45e65. [21] H. Nishitoh, M. Saitoh, Y. Mochida, K. Takeda, H. Nakano, M. Rothe, K. Miyazono, H. Ichijo, ASK1 is essential for JNK/SAPK activation by TRAF2, Mol. Cell 2 (1998) 389e395. [22] H. Liu, H. Nishitoh, H. Ichijo, J.M. Kyriakis, Activation of apoptosis signal-regulating kinase 1 (ASK1) by tumor necrosis factor receptorassociated factor 2 requires prior dissociation of the ASK1 inhibitor thioredoxin, Mol. Cell. Biol. 20 (2000) 2198e2208. [23] Y. Gotoh, J.A. Cooper, Reactive oxygen species- and dimerizationinduced activation of apoptosis signal-regulating kinase 1 in tumor necrosis factor-alpha signal transduction, J. Biol. Chem. 273 (1998) 17477e 17482. [24] T. Nakagawa, H. Zhu, N. Morishima, E. Li, J. Xu, B.A. Yankner, J. Yuan, Caspase-12 mediates endoplasmic-reticulum-specific apoptosis and cytotoxicity by amyloid-beta, Nature 403 (2000) 98e103. [25] Y. Imai, M. Soda, H. Inoue, N. Hattori, Y. Mizuno, R. Takahashi, An unfolded putative transmembrane polypeptide, which can lead to endoplasmic reticulum stress, is a substrate of Parkin, Cell 105 (2001) 891e902. [26] H.P. Harding, H. Zeng, Y. Zhang, R. Jungries, P. Chung, H. Plesken, D.D. Sabatini, D. Ron, Diabetes mellitus and exocrine pancreatic dysfunction in perk/ mice reveals a role for translational control in secretory cell survival, Mol Cell 7 (2001) 1153e1163. [27] H. Kadowaki, H. Nishitoh, F. Urano, C. Sadamitsu, A. Matsuzawa, K. Takeda, H. Masutani, J. Yodoi, Y. Urano, T. Nagano, H. Ichijo, Amyloid beta induces neuronal cell death through ROS-mediated ASK1 activation, Cell Death Differ. 12 (2005) 19e24. [28] Y. Izumiya, S. Kim, Y. Izumi, K. Yoshida, M. Yoshiyama, A. Matsuzawa, H. Ichijo, H. Iwao, Apoptosis signal-regulating kinase 1 plays a pivotal role in angiotensin II-induced cardiac hypertrophy and remodeling, Circ. Res. 93 (2003) 874e883. [29] P.H. McDonald, C.W. Chow, W.E. Miller, S.A. Laporte, M.E. Field, F.T. Lin, R.J. Davis, R.J. Lefkowitz, Beta-arrestin 2: a receptor-regulated MAPK scaffold for the activation of JNK3, Science 290 (2000) 1574e1577. [30] H.Y. Chang, H. Nishitoh, X. Yang, H. Ichijo, D. Baltimore, Activation of apoptosis signal-regulating kinase 1 (ASK1) by the adapter protein Daxx, Science 281 (1998) 1860e1863. [31] K. Takeda, A. Matsuzawa, H. Nishitoh, K. Tobiume, S. Kishida, J. Ninomiya-Tsuji, K. Matsumoto, H. Ichijo, Involvement of ASK1 in Ca2þ-induced p38 MAP kinase activation, EMBO Rep. 5 (2004) 161e166. [32] E. Kuranaga, H. Kanuka, T. Igaki, K. Sawamoto, H. Ichijo, H. Okano, M. Miura, Reaper-mediated inhibition of DIAP1-induced DTRAF1 degradation results in activation of JNK in Drosophila, Nat. Cell Biol. 4 (2002) 705e710. [33] C.I. Bargmann, E. Hartwieg, H.R. Horvitz, Odorant-selective genes and neurons mediate olfaction in C. elegans, Cell 74 (1993) 515e527.
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[34] P.D. Wes, C.I. Bargmann, C. elegans odour discrimination requires asymmetric diversity in olfactory neurons, Nature 410 (2001) 698e701. [35] E.R. Troemel, A. Sagasti, C.I. Bargmann, Lateral signaling mediated by axon contact and calcium entry regulates asymmetric odorant receptor expression in C. elegans, Cell 99 (1999) 387e398. [36] A. Sagasti, N. Hisamoto, J. Hyodo, M. Tanaka-Hino, K. Matsumoto, C.I. Bargmann, The CaMKII UNC-43 activates the MAPKKK NSY-1 to execute a lateral signaling decision required for asymmetric olfactory neuron fates, Cell 105 (2001) 221e232. [37] M. Tanaka-Hino, A. Sagasti, N. Hisamoto, M. Kawasaki, S. Nakano, J. Ninomiya-Tsuji, C.I. Bargmann, K. Matsumoto, SEK-1 MAPKK mediates Ca2þ signaling to determine neuronal asymmetric development in Caenorhabditis elegans, EMBO Rep. 3 (2002) 56e62. [38] C.L. Kurz, J.J. Ewbank, Caenorhabditis elegans: an emerging genetic model for the study of innate immunity, Nat. Rev. Genet. 4 (2003) 380e390. [39] D.H. Kim, R. Feinbaum, G. Alloing, F.E. Emerson, D.A. Garsin, H. Inoue, M. Tanaka-Hino, N. Hisamoto, K. Matsumoto, M.W. Tan, F.M. Ausubel, A conserved p38 MAP kinase pathway in Caenorhabditis elegans innate immunity, Science 297 (2002) 623e626. [40] L.A. O’Neill, K.A. Fitzgerald, A.G. Bowie, The Toll-IL-1 receptor adaptor family grows to five members, Trends Immunol. 24 (2003) 286e290. [41] C. Wang, L. Deng, M. Hong, G.R. Akkaraju, J. Inoue, Z.J. Chen, TAK1 is a ubiquitin-dependent kinase of MKK and IKK, Nature 412 (2001) 346e351. [42] S. Sato, H. Sanjo, K. Takeda, J. Ninomiya-Tsuji, M. Yamamoto, T. Kawai, K. Matsumoto, O. Takeuchi, S. Akira, Essential function for the kinase TAK1 in innate and adaptive immune responses, Nat. Immunol. in press. [43] Q. Huang, J. Yang, Y. Lin, C. Walker, J. Cheng, Z.G. Liu, B. Su, Differential regulation of interleukin 1 receptor and Toll-like receptor signaling by MEKK3, Nat. Immunol. 5 (2004) 98e103. [44] C.D. Dumitru, J.D. Ceci, C. Tsatsanis, D. Kontoyiannis, K. Stamatakis, J.H. Lin, C. Patriotis, N.A. Jenkins, N.G. Copeland, G. Kollias, P.N. Tsichlis, TNF-alpha induction by LPS is regulated posttranscriptionally via a Tpl2/ERK-dependent pathway, Cell 103 (2000) 1071e1083. [45] Y. Xia, C. Makris, B. Su, E. Li, J. Yang, G.R. Nemerow, M. Karin, MEK kinase 1 is critically required for c-Jun N-terminal kinase activation by proinflammatory stimuli and growth factor-induced cell migration, Proc. Natl. Acad. Sci. U.S.A. 97 (2000) 5243e5248. [46] N. Pujol, E.M. Link, L.X. Liu, C.L. Kurz, G. Alloing, M.W. Tan, K.P. Ray, R. Solari, C.D. Johnson, J.J. Ewbank, A reverse genetic analysis of components of the Toll signaling pathway in Caenorhabditis elegans, Curr Biol. 11 (2001) 809e821. [47] N.T. Liberati, K.A. Fitzgerald, D.H. Kim, R. Feinbaum, D.T. Golenbock, F.M. Ausubel, Requirement for a conserved Toll/interleukin-1 resistance domain protein in the Caenorhabditis elegans immune response, Proc. Natl. Acad. Sci. U.S.A. 101 (2004) 6593e6598. [48] C. Couillault, N. Pujol, J. Reboul, L. Sabatier, J.F. Guichou, Y. Kohara, J.J. Ewbank, TLR-independent control of innate immunity in Caenorhabditis elegans by the TIR domain adaptor protein TIR-1, an ortholog of human SARM, Nat. Immunol. 5 (2004) 488e494. [49] M. Mink, B. Fogelgren, K. Olszewski, P. Maroy, K. Csiszar, A novel human gene (SARM) at chromosome 17q11 encodes a protein with a SAM motif and structural similarity to Armadillo/beta-catenin that is conserved in mouse, Drosophila, and Caenorhabditis elegans, Genomics 74 (2001) 234e244. [50] H.S. Park, H.Y. Jung, E.Y. Park, J. Kim, W.J. Lee, Y.S. Bae, Cutting edge: direct interaction of TLR4 with NAD(P)H oxidase 4 isozyme is essential for lipopolysaccharide-induced production of reactive oxygen species and activation of NF-kappa B, J. Immunol. 173 (2004) 3589e3593. [51] J.F. Curtin, M. Donovan, T.G. Cotter, Regulation and measurement of oxidative stress in apoptosis, J. Immunol. Methods 265 (2002) 49e72.
Review
The ASK1eMAP kinase pathways in immune and stress responses Teruyuki Hayakawa, Atsushi Matsuzawa, Takuya Noguchi, Kohsuke Takeda, Hidenori Ichijo* Laboratory of Cell Signaling, Graduate School of Pharmaceutical Sciences, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan Available online 19 January 2006
Abstract Apoptosis signal-regulating kinase 1 (ASK1) is a mitogen-activated protein kinase kinase kinase which plays pivotal roles in stress and immune responses. This review is focused on three major subjects on ASK1: the regulatory mechanisms of the kinase activity, the pathophysiological roles in stress response and innate immunity, and the evolutionary perspectives. Ó 2005 Elsevier SAS. All rights reserved. Keywords: MAP kinase cascades; Apoptosis signal-regulating kinase 1; Innate immunity; Inflammation; Oxidative stresses
1. Introduction Living organisms are continuously exposed to various environmental stresses, such as oxidative stress, ultraviolet radiation and infection. In such circumstances, reactive oxygen species (ROS) appear to be commonly utilized by cells and tissues as a signaling intermediate that can be generated from the extracellular environment or intracellular organelles, such as mitochondria and phagosomes. ROS are also thought to be one of the main causes of cell senescence and dysfunction of biochemical systems. Therefore, cells must respond appropriately to such environmental stresses by using their intracellular signaling pathways. The immune system is essential for host defense against a wide range of pathogens. To clear pathogens, the host must distinguish ‘‘non-self’’ from ‘‘self’’ and then trigger the immune responses. Recognition of the molecular patterns differentially expressed among pathogens, so-called pathogenassociated molecular patterns (PAMPs), is the first step to discriminate ‘‘non-self’’ from ‘‘self’’. Toll-like receptors (TLRs) have been identified as receptors for the PAMPs in the last decade (reviewed in [1]); for example, TLR4, TLR1/2/6, TLR3 and TLR9 recognize lipopolysaccharide (LPS), peptidoglycan * Corresponding author. Tel.: þ81 3 5841 4859; fax: þ81 3 5841 4778. E-mail address: [email protected] (H. Ichijo). 1286-4579/$ - see front matter Ó 2005 Elsevier SAS. All rights reserved. doi:10.1016/j.micinf.2005.12.001
(PG), dsRNA and CpG-DNA, respectively. After recognition, TLRs elicit the intracellular signals (finally to the nucleus and other organelles) that are indispensable for triggering the immune responses. These intracellular signals include phosphorylatione dephosphorylation relays via protein kinases/phosphatases and formation of signaling complexes (signalosomes), eventually leading to the induction of cytokines that are required for the activation of innate and adaptive immune systems. In this review, recent progress in our knowledge on stress responses will be summarized by focusing on a serine/threonine kinase: apoptosis signal-regulating kinase 1 (ASK1). ASK1 is activated in response to various stimuli including LPS, ROS, ER stress, influx of calcium ions and various cytokines such as tumor necrosis factor and Fas ligand. We have recently demonstrated that the TRAF6eASK1ep38 axis is essential for inflammatory response on LPS stimulation. It is important that ROS-dependent ASK1 activation is specific, unlike other MAPKKKs, to LPSeTLR4 signaling [2]. 2. Roles of ASK1 in MAP kinase cascades: a stress-responsive kinase 2.1. Overview of the MAP kinase cascades In response to various extracellular and intracellular stimuli, mitogen-activated protein kinases (MAPKs) are activated or
T. Hayakawa et al. / Microbes and Infection 8 (2006) 1098e1107
inactivated. MAPKs regulate a wide variety of cellular responses, such as cell growth, differentiation, and apoptosis. In the MAPK signaling cascades, MAP kinase kinase kinases (MAPKKKs) phosphorylate and activate MAP kinase kinases (MAPKKs), and MAPKKs subsequently phosphorylate and activate MAPKs. In all MAPK cascades, MAPKs are activated by MAPKK-catalyzed phosphorylation of Tyr and Thr residues at T-X-Y motif in the activation loop of the kinase domain. MAPKKs are also activated by MAPKKKs-catalyzed phosphorylation of Ser/Thr residues in the kinase domain (Fig. 1). Through these sequential biochemical events, the signals are amplified and diversified in a step-by-step manner. All eukaryotic cells possess multiple MAPK pathways. Among them, roles of mammalian MAPKs such as extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and p38 MAPK pathways have been defined to some extent (Fig. 1). ERK pathway is activated by various growth factors and closely linked to the regulation of cell cycle. JNK and p38 are preferentially activated by various environmental factors such as osmotic shock, oxidative stress and pro-inflammatory cytokines, and linked to various stress responses. JNK and p38 are thus also called stress-activated protein kinases (SAPKs). MAPK cascades are evolutionarily conserved in eukaryotic cells. In Fig. 1, stress-responsive MAP kinase cascades in mammals, Drosophila melanogaster, and Caenorhabditis elegans
Mammal
P S/T MAPKKK
P
P
S
S/T
MAPKK
P
P
T
Y
MAPK
MLK2/3 DLK TAO1/2
are shown (details in each organism are also reviewed in [3e5]). All of these organisms possess JNK and p38 pathways, although biological output can differ among them. Most of the MAPKKKs shown in Fig. 1 are capable of activating JNK and p38 pathways through MAPKKs, such as SEK1(MKK4)/ MKK7 and MKK3/MKK6, respectively. MAPKKKs in the JNK and p38 pathways are highly divergent in number (at least eleven MAPKKKs have been identified upstream of JNK) and structure. This diversity is seemingly of little use for activating only two MAPKs JNK and p38, but its diversity and complexity are rather consistent with the variety of stimuli that activate MAPK pathways. MAPKKKs thus appear to play pivotal roles in sensing and signaling of cellular and environmental stress [3]. The following sections will focus on ASK1, one of the well-studied MAPKKKs. ASK1 was originally identified as an apoptosis-inducing MAPKKK that activates both p38 and JNK pathways [6]. Recent studies revealed that ASK1 contributes not only to regulation of cell death but also to cytokine responses, cell differentiation and immune regulation [7]. 2.2. ASK1 activation in stress response In this section we will focus on molecular features of ASK1. Although ASK1 is activated by various stresses, common mechanisms appear to operate to activate ASK1 in response to various stresses. ASK1 has two important structural
Caenorhabditis elegans
Drosophila melanogaster MEKK1/2/3 MTK1 ASK1 TAK1 Cot
MKK4(SEK1) MKK3 MKK6 MKK7
JNK1/2/3
p38α/β/γ/δ
Apoptosis, Differentiation, Stress response
1099
Slpr(MLK)
dMEKK1
DASK1 DLK
dTAK1
MLK-1 DLK-1 KIN-18
MTK-1 NSY-1 MOM-4 ZAK-1
MKK4 Hep(MKK7)
Lic (dMKK3)
MKK-4 JKK-1 MEK-1
Bsk(JNK)
D-p38a/b
JNK-1 KGB-1/2 PMK-1/2/3
SEK-1
Dorsal closure, Developmental Resistance to Resistance to heavy metals infections, Tissue polarity processes? and starvation Cell-fate decision
Fig. 1. Components of stress-responsive MAP kinase cascades are evolutionarily conserved. JNK and p38 pathways, two major stress-responsive MAP kinase cascades in mammals, Drosophila melanogaster and Caenorhabditis elegans are shown. There are two other major cascades (ERK1 and ERK5), which are not illustrated here. In all MAPK cascades, MAPKs are activated by MAPKK-catalyzed phosphorylation of Tyr and Thr residues at T-X-Y motif in the activation loop of the kinase domain. MAPKKs are also activated by MAPKKKs-catalyzed phosphorylation of Ser/Thr residues in the kinase domain. It is uncertain whether common activation mechanisms for MAPKKKs exist; however autophosphorylation of Ser/Thr residues in the kinase domain appears to be required for many of the known MAPKKKs.
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2.2.2. ASK1 signalosome: a node for signal transduction Following the dissociation of Trx, it has recently been reported that recruitment of tumor necrosis factor receptorassociated factor (TRAF) family proteins is also required for oxidative stress-induced ASK1 activation [16]. Endogenous ASK1 constitutively forms a high molecular weight complex (approximately 1500e2000 kDa) which was designated ‘‘ASK1 signalosome’’. Upon H2O2 stimulation, the ASK1 signalosome forms higher molecular weight complex (approximately >3000 kDa) due at least to the recruitment of TRAF2 and TRAF6, which have previously been reported to activate ASK1 (discussed in Section 2.3.2). The ASK1 signalosome contains not only Trx or TRAFs, but also other unidentified factors, suggesting the multiple roles of the ASK1 signalosome as the ‘‘node’’ for signal transduction. A large number of ASK1-interacting proteins (both positive and negative regulators) have been reported, most of which may be contained in the ASK1 signalosome. Elucidation of the roles of these components may shed light on the precise mechanisms of the ASK1-dependent signaling cascade which links various stresses to the biological events.
features: N-terminal thioredoxin binding site and C-terminal homophilic interaction site. 2.2.1. Molecular structure and general activation mechanism of ASK1 Human and mouse ASK1 consist of 1374 and 1380 amino acids, respectively, and possess a serine/threonine kinase domain in the middle of the molecule [6] (Fig. 2A). When wild-type ASK1 or its constitutively active mutant are overexpressed in mammalian cells, they directly phosphorylate and activate MKK3/MKK6 and MKK4/MKK7, and induce apoptosis in various cells [8]. However, the downstream events of ASK1 are variable depending on the cell types and cellular context, including cell death [6,9e11], differentiation [12,13], neurite outgrowth [14] and induction of cytokines [2]. ASK1 has two coiled-coil domains at its N- and C-terminals, of which C-terminal coiled-coil domain is essential for forming ‘‘ASK1 signalosome’’ (see below) and activating ASK1 itself [15,16]. ASK1 in its resting state forms a static homo-oligomer through its C-terminal coiled-coil domain. A ubiquitously expressed reduction/oxidation (redox) protein thioredoxin (Trx) binds to the N-terminal of ASK1 and inhibits its activity in the absence of oxidative stress (Fig. 2B). Upon oxidative stress stimulation such as hydrogen peroxide (H2O2), intramolecular disulfide bonding is formed between two Cys residues in Trx. Oxidized form of Trx dissociates from ASK1, which leads to the autophosphorylation of threonine residue (Thr 838 in human and Thr 845 in mouse) in the activation loop of ASK1. The kinase activity of ASK1 is fully elevated by this phosphorylation. It is thus proposed that following ROS production (either from exogenous or endogenous sources), dissociation of Trx and tight homo-oligomerization of ASK1 are the sequential events that are essential for ASK1 activation [15,17] (Fig. 2B). The phospho-specific antibody that recognizes phospho-Thr in the activation loop has been found to be a useful indicator of the ASK1 activity [15].
A
1
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2.3. Stimuli and stresses that activate ASK1 ASK1 is activated by cytokines and various environmental and cellular stresses. In the following subsections we discuss some of these stresses that activate ASK1. Note that ASK1e Trx system is widely used as a regulatory mechanism of ASK1 (Fig. 3). 2.3.1. Oxidative stress: ASK1eTrx system ROS is not only a cellular stress that is produced at the electron transport chain or under pathological conditions, but is also used as a second messenger in signal transduction: for example, it is required for cell proliferation induced by various growth factors [18]. An appropriate amount of ROS is
649
940
1236 1293 1394(aa)
NH2-
-COOH coiled-coil Trx binding
kinase domain
coiled-coil
B autophosphorylation HS ASK HS - Trx 1
ROS
P ASK1
HS - - Trx 1 ASK HS
Steady State
ASK1 P Trx S-S
Trx
Active State
S-S
Fig. 2. General features of ASK1. (A) Domain structure of ASK1. Human ASK1 consists of 1374 amino acids and contains a serine/threonine kinase domain in the middle of the molecule. ASK1 also has two coiled-coil domains at the N- and C-terminals. (B) ASK1eTrx system. ASK1 in the resting state forms a homooligomer via C-terminal coiled-coil domain and binds to reduced Trx at the N-terminus. Upon ROS generation, intramolecular disulfide bonding is formed in Trx and subsequently Trx dissociates from ASK1, which leads to the autophosphorylation of the Thr 845 residue in its activation loop. ROS are the key to cancel the ‘‘safety lock’’ of the ‘‘bomb’’ ASK1.
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TNF LPS CD14
TLR4 TNFR1
MD2
plasma membrane
TIRAP
MyD88
TRADD (ER stress)
TRAF2
IRAK4/1
TRAF6 ROS
ROS TRAF2
Trx
ASK1
IRE1
Trx
ASK1
ASK1
TRAF6
p38
ASK1
ASK1
TRAF2
p38 JNK
JNK
Fig. 3. Molecular mechanisms of ASK1 activation in response to various stimuli. Signaling mechanisms of representative stimuli that activate ASK1 are illustrated. (Left) LPS-induced ASK1 activation requires generation of ROS and recruitment of the adaptor protein TRAF6. Only p38 but not JNK and IKK pathways were impaired in ASK1/ cells. (Center) TNF binds to TNR receptor (TNFR1), which activates ASK1ep38/JNK pathways. This signal is mediated by TRAF2 and dependent on ROS generation. (Right) ER stress-induced ASK1 activation is mediated by IRE1, one of the ER transmembrane proteins. ASK1 interacts with IRE1eTRAF2 complex and is activated by ER stress, leading to the activation of JNK pathway. It is currently unknown whether the dissociation of Trx is required for activation of ASK1 by ER stress.
necessary for physiological cell signaling and normal cellular response, whereas excessive production of ROS behaves as cellular stress and eventually induces necrosis or apoptosis. ASK1 is strongly activated when cells are treated with hydrogen peroxide (H2O2) or other ROS-generating stimuli. In response to the oxidative stress, a common ROS-sensory mechanism, ASK1eTrx system, appears to operate. Trx was initially identified in a screening for ASK1-interacting proteins [17]. Reduced form of Trx, which possesses two sulfhydryl groups at the Cys-32 and Cys-35 residues, specifically binds to the N-terminal region of ASK1 and inhibits ASK1’s kinase activity. In contrast, oxidized form of Trx, which possesses intramolecular disulfide bonding between the two cysteine residues, does not bind to ASK1. Upon H2O2 stimulation, Trx dissociates from ASK1, and the freed ASK1 appears to be tightly oligomerized through N- and C-terminal coiled-coil domains, leading to the autophosphorylation of the Thr residue at the activation loop ([15,17]; G. Fujino and H. Ichijo, unpublished observation). ASK1eTrx system is thus thought to be a redox sensor which functions as a signal converter of intracellular redox changes into the signaling of kinase cascades (Fig. 2B). ASK1-null mice have been generated to investigate the roles of ASK1 in oxidative stress-induced activation of JNK/ p38 cascades and apoptosis [11]. ASK1/ mice were viable and histologically indistinguishable from wild-type littermates. However, in mouse embryonic fibroblasts (MEFs) isolated from ASK1/, H2O2- and tumor necrosis factor a-induced sustained but not transient activation of JNK and p38 was suppressed. H2O2-induced apoptosis was also reduced in ASK1/ mice MEFs, suggesting that ASK1 is required for oxidative stress-induced apoptosis.
2.3.2. Tumor necrosis factor (TNF) Whether cells survive or undergo apoptosis is regulated by both intracellular conditions and extracellular factors such as death ligands. Receptors that respond to these ligands are called death receptors and are among members of the TNFreceptor family, all of which have extracellular cysteine-rich repeats and a cytoplasmic death domain (DD) [19]. Tumor necrosis factor alpha (TNFa) is a pleiotropic cytokine that plays important roles in inflammation, immune responses and apoptosis. TNFa activates two major transcription factors: activator ptotein-1 (AP-1) and nuclear factor-kB (NF-kB). Both transcription factors are activated through the activation of JNK/p38 and IkB kinases, respectively [20]. Upon TNFa treatment, ASK1 is activated and subsequently activates JNK and p38 cascades (Fig. 3). This activation is mediated by TNF receptor-associated factor 2 (TRAF2), an adaptor protein that possesses E3 ubiquitin ligase activity essential for TNFa-mediated signaling pathways [21]. Interestingly, this TRAF2-mediated activation of ASK1 appears to be ROSdependent. TNF stimulation or overexpression of TRAF2 generates intracellular ROS, and activation of ASK1 is largely dependent on ROS (i.e. suppressed by treatment with antioxidants) [22,23]. It is consistent with the ASK1eTrx system mentioned earlier; ROS-mediated dissociation of Trx from ASK1 is necessary for TNF-induced activation of ASK1. Requirement of ASK1 in TNFa signaling has been demonstrated by using MEFs derived from ASK1/ mice [11]. ASK1/ MEFs were resistant to TNFa-induced apoptosis, and sustained but not transient activation of JNK and p38 were diminished in ASK1/ MEFs. It has been reported that the TRAF2e GCKseMEKK1 pathway is also involved in TRAF2-mediated transient JNK activation. Two MAPKKKs, ASK1 and MEKK1,
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are thought to act in TNFa and TRAF2-mediated JNK activation, although whether they act redundantly or complementarily remains to be studied. 2.3.3. ER stress and neurodegenerative conditions In living cells newly synthesized proteins often fail to fold correctly or fail to find their correct partner in a larger protein complex. Such a misfolded protein is not only useless to cells but toxic, and therefore is normally digested in proteasome to be detoxified. Accumulation of unfolded and misfolded proteins in endoplasmic reticulum (ER) lumen induces ER stress. ER stress has recently been reported to be a potential cause of neurodegenerative disorders such as Alzheimer’s disease [24], Parkinson’s disease [25], polyglutamine diseases [9] and diabetes mellitus [26]. Cells try to adapt themselves against ER stress by stressresponsive pathways called unfolded protein response (UPR). IRE1, ATF6, OASIS and PERK are the ER transmembrane proteins that mediate UPR by reducing influx of proteins into ER lumen. If this adaptation is insufficient for the clearance of ER stress, cells undergo apoptosis. ASK1 is activated by ER stress in the IRE1eTRAF2eASK1eJNK axis [9]. ASK1 interacts with TRAF2 and is activated upon ER stress (Fig. 3). Moreover, in primary neurons derived from ASK1/ mice, ER stress- and polyglutamine-induced JNK activation and apoptosis were reduced. These data demonstrate the essential role of ASK1 in ER stress-induced cell death. It was recently shown that ASK1 is also required for amyloid b (Ab)-induced neuronal cell death [27]. Upon Ab treatment, the ASK1eJNK pathway was activated in a ROS-dependent manner, but not through ER stress. ASK1/ neurons were resistant to Ab-induced cell death. These two reports on the roles of ASK1 in neuronal cell death suggest the importance of ASK1 in the pathogenesis of neurodegenerative diseases. ASK1 may be a potential therapeutic target for prevention and treatment of various neurodegenerative diseases. 2.3.4. Other stimuli that activate ASK1 G-protein coupled receptor (GPCR)-mediated signals also activate ASK1. For example angiotensin II (Ang II) treatment induces the activation of ASK1 in cardiomyocytes. A recent study demonstrated that Ang II-mediated ASK1 activation was mediated by generation of ROS and was closely related to cardiac hypertrophy and remodeling, although the intermediate molecule between the AngII receptor and ASK1 is elusive [28]. It is also reported that b-Arrestin 2 acts as an scaffold protein for ASK1eMKK4eJNK3 and is involved in JNK3 activation induced by Ang II [29]. ASK1 was also reported to be involved in Fas signaling through an adaptor protein Daxx [30]. Daxx interacts both with intracellular DD of Fas and N-terminus of ASK1, and subsequently activates JNK. However, the sensitivity to Fas-induced apoptosis was indistinguishable between in wildtype and ASK1/ thymocytes, suggesting the role of Daxxe ASK1eJNK pathway in other physiological events unrelated to Fas-induced apoptosis [11].
Ca2þ influx and LPS stimulation also activate ASK1 [2,31], which will be discussed in the following sections (Fig. 3). 3. Emerging roles of ASK1: lessons from Drosophila and C. elegans Recently the orthologues corresponding to mammalian ASK1 have been reported in two model organisms, the fruit fly Drosophila melanogaster and the nematode Caenorhabditis elegans. The merit of using these organisms includes phenotype-based genetic screening. Both Drosophila and C. elegans ASK1 have been identified in forward genetic screening, and the information from these studies is also useful for studies in mammals. 3.1. Drosophila ASK1 Drosophila ASK1 (DASK1) was found to be a structural and functional orthologue of ASK1. Overexpression of Reaper (Rpr) using an eye-specific promoter gives rise to caspasedependent apoptosis of the eye cells, resulting in small eyes. Kuranaga et al. identified DASK1 and Drosophila TRAF1 (DTRAF1, most similar to human TRAF4) as the dominant modifiers of Rpr, in which suppression of Rpr-induced small-eye phenotype was used as a criteria for the genetic screening [32]. Rpr-mediated cell death of eyes was also suppressed by co-expression of the kinase mutant of DASK1 (DASK1K618M). Furthermore, in Drosophila S2 cells, expression of DTRAF1 activates DASK1 and activated DASK1 then activates Drosophila JNK (DJNK, also called Basket) (Fig. 1). DASK1 is thought to possess molecular function as MAPKKK. Since little is known about the biological functions of DASK1 except for its apoptosis-inducing activity, future availability of DASK1 mutant fly will be a powerful tool to understand the in vivo roles of ASK1. 3.2. NSY-1: ASK1 in C. elegans The nematode Caenorhabditis elegans is also an excellent model organism used to perform large-scale genetic screenings. Until now, NSY-1, an orthologue of mammalian ASK1, has been identified in two independent genetic screenings: Nsy (neuronal symmetry) and Esp (enhanced susceptibility) (Fig. 4). 3.2.1. Neuronal symmetry C. elegans detects odorants with the bilaterally symmetric pair of neurons: AWC neurons [33]. The candidate odorant receptor gene str-2 is expressed in only one neuron, either the left or the right neuron, and its asymmetric expression is required for odor discrimination [34]. A genetic screen for mutants defective in lefteright asymmetry was performed and the identified mutants were named neuronal symmetry (nsy) genes [35]. Sagasti et al. reported that nsy-1 encodes an orthologue of the mammalian ASK1, and it acts downstream of the Ca2þ/calmodulin-dependent protein kinase II (CaMKII) UNC-43 [36]. Tanaka-Hino et al. subsequently reported that SEK-1 MAPKK (an orthologue of mammalian MKK3 and
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UNC-2/36 (Ca2+ channel) UNC-43 (CaMKII)
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LPS
bacterial infection
Ca2+ channel
TLR4
? Adaptor proteins
CaMKII TRAF6
TIR-1
TIR-1
NSY-1
NSY-1
MAPKKK
ASK1
ASK1
SEK-1
SEK-1
MAPKK
MKK3/6
MKK3/6
MAPK
PMK-1
MAPK
p38
p38
asymmetric str-2 expression
Immune response (ex. anti-microbial peptides)
synaptic plasticity?
inflammation, septic shock
Fig. 4. Physiological roles of NSY-1 and ASK1 in C. elegans and mammals. In C. elegans AWC neurons, the NSY-1eSEK-1eMAPK cascade is activated by Ca2þ signaling through TIR-1 and UNC-43 (CaMKII), and determines the asymmetric cell fate. In the C. elegans intestine, NSY-1 is activated by the pathogens through TIR-1 but not through UNC-43, and it activates the downstream SEK-1ePMK-1 pathway, leading to the innate immune response. In the mammalian neuron, ASK1 is activated by Ca2þ influx through the direct phosphorylation by CaMKII. Ca2þ-activated ASK1 then activates p38. Combined with the fact that constitutively active ASK1 induces neurite outgrowth in PC12 cells, ASK1 might play critical roles in synaptic plasticity. In the immune cells such as macrophage and dendritic cell, ASK1 is activated by LPS treatment and is required for LPS-induced p38 activation and innate immune responses such as inflammation.
MKK6) is also required for asymmetric expression in AWC neurons and that SEK-1 acts in a pathway downstream of UNC-43 and NSY-1 [37]. Thus, the CaMKIIeNSY-1eSEK-1 axis is essential for asymmetric expression of str-2 and, as a result, neuronal differentiation. These elegant analyses prompted us to question whether this pathway is conserved in humans. Using ASK1-deficient mice, Takeda et al. have demonstrated the essential role of ASK1 in Ca2þ-induced p38 MAPK activation in neuronal cells [31]. In PC12 cells and primary neurons, ASK1 was activated in response to influx of Ca2þ ions evoked by membrane depolarization, which was inhibited by the treatment with L-type Ca2þ channel blocker (nifedipine) and CaMK inhibitor (KN-93). CaMKII directly phosphorylates (other than activation loop) and somehow activates ASK1. Consistent with the results from C. elegans, these data demonstrate the important role of CaMKII as an upstream activator of ASK1. Detailed biochemical analysis of phosphorylation-dependent activation mechanisms of ASK1 will be an important issue to be addressed. 3.2.2. Enhanced susceptibility C. elegans is also a useful genetic tool for screening mutants of immune responses because it is relatively easy to isolate mutants with higher susceptibility to pathogens compared to the screening with other higher organisms such as the fruit fly or mouse [38]. Kim and colleagues isolated 10 Esp mutants that were more susceptible to infection by Pseudomonas aeruginosa strain PA14 [39]. esp-2 and esp-8 were the two most susceptible mutants and were shown to correspond to two
known genes, sek-1 and nsy-1, respectively. It was strongly suggested that NSY-1eSEK-1ePMK-1 pathway is the essential pathway to resist PA14. This was strengthened by the RNA interference (RNAi) experiment of PMK-1; inhibition of PMK-1 activity by knock-down gave rise to a strong Esp phenotype. Moreover, PA14-induced p38 activity was almost completely suppressed in esp-2/sek-1 and esp-8/nsy-1 mutants compared to wild-type N2 worms. Interestingly, unc-43 (mammalian CaMKII) mutant, which shows a null Nsy phenotype, had almost no Esp phenotype. It has therefore been suggested that another molecule other than UNC-43 may exist upstream of the NSY-1 in the Esp pathway; TIR-1 was recently identified to act as an upstream activator of NSY-1 (see below). 4. ASK1 in TLR signaling Recently it was demonstrated that TRAF6eASK1ep38 pathway plays important roles in LPS-mediated inflammatory signaling. ASK1 is specifically activated by LPS, but not other TLR ligands tested, and is required for the activation of p38, but not JNK or NF-kB pathways [2]. 4.1. Overview of TLR signaling In the innate immune responses, certain pathogens are first sensed by cell surface receptors or intracellular components that recognize PAMPs. The recognition is then converted to the intracellular signal pathways that culminate in immune responses such as inflammation and cytokine production.
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4.1.1. Pathogen recognition by TLRs TLRs are the major components for the recognition of a broad range of pathogenic microorganisms (reviewed in [1]). There are at least ten TLRs in human (TLR1e10) and thirteen in mice (TLR1e13), and the cognate ligands for most of them have been identified. They are roughly divided in two groups: TLRs that recognize bacterial components (TLR1/2/6, TLR4 and TLR5) or viral components (TLR3, 7 and 8). TLR9 recognizes CpG DNA motif derived from both bacteria and virus. The TLRs are type I integral membrane glycoproteins. Those extracellular regions contain leucine-rich repeat (LRR) motifs, which bind and recognize pathogens. On the other hand, the cytoplasmic tails of TLRs contain Toll/interleukin-1 receptor (TIR) domains that bind to intracellular signaling molecules. TIR domain is named so because it is conserved in cytoplasmic tails of all TLRs and interleukin-1 receptors (IL-1Rs).
molecule 1), and TRIF-related adaptor molecule (TRAM; also known as TICAM2, TIR domain-containing molecule 2) [40]. Knockout mice of these genes were generated and analyzed, and they were found to be deficient in transducing specific pathogen’s stimuli to intracellular events. Upon stimulation, TLRs recruit at least one TIR adaptor protein, and downstream events occur in a TIR adaptor-dependent manner. These pathways are often classified in two groups: MyD88-dependent and TRIF-dependent pathways. Activation of MyD88-dependent pathway leads to the production of pro-inflammatory cytokines such as interleukin (IL)-6 and TNF-a whereas activation of TRIF-dependent pathway leads to production of type-I interferon (IFNa/b). Downstream molecules of such TIR adaptor proteins include IL-1R-associated kinases (IRAKs), transforming growth factor-b (TGF-b)-activated kinase (TAK1), TAK1-binding protein 1 (TAB1), TAB2, and TNF receptor-associated factor 6 (TRAF6). Upon LPS stimulation, TLR4 activates both MyD88- and TRIF-dependent pathways, which eventually induce transcription of pro-inflammatory cytokines and type-I interferon, respectively. In MyD88-dependent pathway, the IRAK4/ IRAK1eTRAF6 complex is recruited to the cytoplasmic TIR domain of TLR4 by TIRAPeMyD88 adaptor proteins. TRAF6 then activates cytoplasmic kinases, including TAK1 and MEKK3 [41e43] (Fig. 5). Other reports demonstrate the essential roles of Tpl2/Cot proto-oncogene [44] and MEKK1 [45] in LPS-induced activation of MAPK cascades. To what extent TRAF6 contributes to the activation of Tpl2/ Cot and MEKK1 is yet to be studied. ASK1 is also involved in TLR4 signaling, which we will discuss in the next section.
4.1.2. Intracellular signaling downstream of TLRs After pathogens bind to extracellular regions of TLRs, TLRs undergo conformational changes and their cytoplasmic TIR domains then activate common signaling pathways, which include the activation of transcription factor nuclear factor-kB (NF-kB) and stress-activated protein kinases including p38 MAPK and JNK. TIR domains of cytoplasmic tails of TLRs interact with cytoplasmic TIR domain-containing adaptor proteins, which include myeloid differentiation primary-response protein 88 (MyD88), TIR domain-containing adaptor protein (TIRAP; also known as Mal, MyD88-adaptor-like protein), TIR domain-containing adaptor protein inducing interferon (IFN)-b (TRIF; also known as TICAM1, TIR domain-containing
Other TLRs TLR4 LPS MD2 CD14 TRIF TRAM
TIRAP MyD88
TIR adaptors
IRAK4/1 TRAF6 TRIF-dependent pathway TRAF6
TRAF6
TRAF6
MAPKKK
ASK1
MEKK3
TAK1
Cot/Tpl2
MAPK
p38
JNK
NF-κB
ERK
ROS
Fig. 5. Roles of ASK1 and other MAPKKKs in TLR4 signaling. LPS is recognized by TLR4eMD2 complex and the signal is transduced to the intracellular kinase cascades. ASK1 is activated by TLR4 but not by other TLRs tested. ROS generation and direct interaction of ASK1 with TRAF6 are required for its activation. The activation of the downstream kinase p38 was diminished in ASK1/ cells, whereas JNK and IKK seemed to be almost normal. TAK1 and MEKK3 are also activated by interaction with TRAF6 and activate the p38, JNK, and NF-kB pathways. Tpl2/Cot is essential in LPS-mediated ERK activation. MEKK1 has also been reported to be involved in LPS-mediated JNK activation (not shown).
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4.2. TRAF6eASK1ep38 axis in TLR4 signaling In this decade, a number of signaling molecules have been identified in the innate immunity: Toll-like receptors, adaptor proteins that determine specificity of downstream signals, and transcription factors for cytokine production. However, the roles of MAPK cascades in mammalian innate immunity have been largely unknown. As described earlier, the role of ASK1ep38 pathway in immune responses has been reported from the studies of C. elegans [39]. Matsuzawa et al. have recently demonstrated the importance of ASK1 in the mammalian innate immunity system using ASK1/ mice [2] (Fig. 5). In ASK1/ splenocytes and bone marrow-derived dendritic cells (BMDCs), LPS-induced activation of p38 MAPK was specifically diminished, whereas activation of JNK and NF-kB was unaffected. Production of inflammatory cytokines such as TNF, IL-6 and IL-1b was also diminished in ASK1/ splenocytes and BMDCs, indicating that the ASK1ep38 axis is required for inflammation in response to LPS. Interestingly, ASK1 was shown to be selectively required for TLR4 signaling. LPS-induced activation of ASK1 and downstream p38 was specifically abolished in ASK1/ cells, whereas PGN, Poly(I:C) and CpG DNA-induced responses (correspond to TLR2, TLR3 and TLR9, respectively) were not. As a candidate for generation of the specificity for TLR4e ASK1 pathway, two factors, ROS and TRAF6, have been studied in detail. In RAW264.7 macrophage cell line, LPS-induced activation of ASK1ep38 pathway and cytokine production were strikingly suppressed by various anti-oxidants including propyl gallate and N-acetyl-L-cysteine. Moreover, LPSinduced interaction between ASK1 and TRAF6 was disrupted by the presence of anti-oxidant. These data suggest the possible mechanism for LPS-induced ASK1 activation as follows: (1) after LPS stimulation TLR4 somehow generates ROS, which triggers the dissociation of the inhibitor Trx from ASK1; (2) freed ASK1 effectively interacts with TRAF6 (and probably with other unidentified components) and forms the active signalosome of ASK1, which facilitates ASK1 activation and selective p38 activation. In vivo study also supports the role of ASK1 in innate immunity. When wild-type and ASK1/ mice were injected with LPS, ASK1/ mice were resistant to LPS-induced septic shock, whereas most wild-type mice died within 40 h. The levels of two principal factors responsible for septic shock, TNF and nitric oxide,
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were also reduced in serum of ASK1/ mice. In conclusion, these data demonstrate that the ROS-dependent TRAF6e ASK1ep38 axis is crucial for TLR4-mediated mammalian innate immunity. 5. Perspectives and conclusion As we have seen earlier, NSY-1ePMK-1/ASK1ep38 signaling pathway is conserved in C. elegans and mammals and plays pivotal roles in pathogen resistance and inflammation. However, there are some discrepancies between these organisms. We will wind up this review by discussing unsolved important issues on ASK1 such as evolutionary conservation and roles of ROS and ASK1 in the stress and immune responses. 5.1. To what extent innate immunity is evolutionarily conserved? There are some discrepancies between C. elegans and other organisms such as Drosophila and mice. It goes without saying that in mammals and Drosophila NF-kB/Relish and TLRs/Toll are the key players in innate immunity and inflammation. However, these components do not seem to be evolutionarily conserved in C. elegans [46]. C. elegans possesses two TIR domain-containing proteins, TOL-1 and TIR-1. TOL-1 is most similar to the members of the TLR family: TOL-1 extracellular domain contains LRRs, followed by a putative transmembrane domain and an intracellular TIR domain. TIR-1 is the intracellular TIR-containing protein with two sterile a-motif domains at the center and HEAT/armadillo repeats at the N-terminus [47,48]. Mutations in tol-1 have been reported to show no phenotype on pathogen susceptibility [46]. In contrast, tir-1 RNAi-inactivated worms were more sensitive to the pathogens: the Gram-negative bacterial pathogens P. aeruginosa and Serratia marcescens, and the fungal pathogen Drechmeria coniospora [47,48]. Moreover, in tir-1 RNAi-inactivated worms, the activity of PMK-1 was diminished [47]. These results suggest that TIR-1 acts upstream of the NSY-1eSEK-1ePMK-1 pathway and that the TIR-1e NSY-1eSEK-1ePMK-1 pathway may be evolutionarily the most ancient pathway for innate immunity. The upstream components of NSY-1 and ASK1 are still unknown. As described earlier, TIR-1 does not seem to participate in TLR signaling, although mammalian ASK1ep38 pathway plays an important role at least in TLR4 signaling.
Table 1 Molecular profiles of stimuli that activate ASK1 Stimuli
Involvement of ROS in ASK1 activation
Activator
Contribution of ASK1 p38
JNK
Oxidative stress TNFa ER stress LPS Amyloid b Calcium influx
Yes Yes ? Yes Yes (by NADPH oxidase?) No
TRAF2, 6 TRAF2, (6?) TRAF2, (6?) TRAF6, (2?) ? CaMKII
YesL* YesL* ? Yes ? YesE*
YesL* YesL* Yes No Yes ?
*Contribution only at: E, early phase; L, late phase.
Cell type analyzed
MEF MEF MEF DC, splenocyte Cortical neuron Neuron
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It is therefore possible that another conserved immune signaling pathway exists in mammals, in which the mammalian orthologue of TIR-1 may play a pivotal role. One possibility is that TIR-1 might be coupled to non-TLR-type receptors; or alternatively, TIR-1 itself might be the pathogen receptor. The orthologue of TIR-1 in mammals, known as sterile a motif and armadillo motif (SARM), has been cloned by Mink et al. [49], although its cellular function remains to be elucidated.
plasticity. Moreover, TRAF6eASK1ep38 pathway plays an essential role in inflammatory and innate immune responses. ASK1 forms a mega-complex and rearranges its components in response to various stimuli. It is conceivable that ASK1 functions as a signaling node in stress response. Further investigation of regulatory mechanisms of ASK1 and its relatives will provide the clues for understanding immune and stress responses. Acknowledgements
5.2. Molecular mechanisms of ASK1 activation, again Let us go back again to the molecular mechanisms of ASK1 activation. As we have seen, ROS generated by various stimuli appear to be the common ‘‘key’’ to open the ‘‘safety lock’’ Trx (Fig. 3, Table 1). ROS is thus considered to be the common second messenger for ASK1 activation. The source for ROS is still largely unknown, but NAD(P)H oxidase is one of the likely candidates. Indeed, after LPS stimulation, ROS is known to be generated from NAD(P)H oxidase complex including NAD(P)H oxidase 4 isozyme, which directly interacts with TLR4 [50]. However, the relationship between NAD(P)H oxidase and ASK1 in LPS signaling is yet to be investigated. Another candidate is mitochondria, the main source of oxygen radicals in eukaryotic metabolism. It has been established that ROS is generated by TNFa in many cell types [51]. Complex formation with activators and homo-oligomerization are also the indispensable steps for ASK1 activation. Among the components of ASK1 complex, TRAF family proteins may be the common adaptor proteins required for ASK1 activation. Further studies are required to uncover the unidentified factors, which may shed light on the precise mechanism of ROS-dependent ASK1 activation. 5.3. Roles of ASK1 in stress and immune response As described throughout this review, ASK1 is activated by various environmental stresses. These include oxidative stress, TNF as a pro-inflammatory cytokine, LPS as a TLR agonist. ASK1-deficient mice were born without apparent phenotype and grew normally, suggesting that ASK1 is not essential for cell growth and differentiation. On the other hand, ASK1/ MEFs were more resistance to oxidative stress-, TNF-, and ER stress-induced cell death. These two phenomena suggest that ASK1 activity is tightly regulated under unstressed conditions. This is consistent with the ASK1eTrx system, in which ASK1 in the steady state is tightly inactivated by Trx. ASK1 is able not only to induce apoptosis, but also to mediate a wide range of cellular responses. ASK1 induces apoptosis through ASK1ep38/JNK cascades in response to proapoptotic stresses (e.g. oxidative stress and TNF) and pathogenic stresses (e.g. ER stress, GPCR- and Ab-induced ROS production). On the other hand, constitutively active ASK1 induces neurite outgrowth in PC12 cells. ASK1 is activated by CaMKII, which activates ASK1ep38 pathway in neurons, suggesting that ASK1 might play critical roles in synaptic
We thank all the members of the Laboratory of Cell Signaling for valuable discussion and comments. References [1] S. Akira, K. Takeda, Toll-like receptor signalling, Nat. Rev. Immunol. 4 (2004) 499e511. [2] A. Matsuzawa, K. Saegusa, T. Noguchi, C. Sadamitsu, H. Nishitoh, S. Nagai, S. Koyasu, K. Matsumoto, K. Takeda, H. Ichijo, ROS-dependent activation of the TRAF6-ASK1-p38 pathway is selectively required for TLR4-mediated innate immunity, Nat. Immunol. 6 (2005) 587e592. [3] J.M. Kyriakis, J. Avruch, Mammalian mitogen-activated protein kinase signal transduction pathways activated by stress and inflammation, Physiol. Rev. 81 (2001) 807e869. [4] B. Stronach, Dissecting JNK signaling, one KKKinase at a time, Dev. Dyn. 232 (2005) 575e584. [5] A. Sakaguchi, K. Matsumoto, N. Hisamoto, Roles of MAP kinase cascades in Caenorhabditis elegans, J. Biochem. (Tokyo) 136 (2004) 7e11. [6] H. Ichijo, E. Nishida, K. Irie, P. Ten Dijke, M. Saitoh, T. Moriguchi, M. Takagi, K. Matsumoto, K. Miyazono, Y. Gotoh, Induction of apoptosis by ASK1, a mammalian MAPKKK that activates SAPK/JNK and p38 signaling pathways, Science 275 (1997) 90e94. [7] J. Matsukawa, A. Matsuzawa, K. Takeda, H. Ichijo, The ASK1eMAP kinase cascades in mammalian stress response, J. Biochem. (Tokyo) 136 (2004) 261e265. [8] K. Takeda, A. Matsuzawa, H. Nishitoh, H. Ichijo, Roles of MAPKKK ASK1 in stress-induced cell death, Cell Struct. Funct. 28 (2003) 23e29. [9] H. Nishitoh, A. Matsuzawa, K. Tobiume, K. Saegusa, K. Takeda, K. Inoue, S. Hori, A. Kakizuka, H. Ichijo, ASK1 is essential for endoplasmic reticulum stress-induced neuronal cell death triggered by expanded polyglutamine repeats, Genes Dev. 16 (2002) 1345e1355. [10] T. Hatai, A. Matsuzawa, S. Inoshita, Y. Mochida, T. Kuroda, K. Sakamaki, K. Kuida, S. Yonehara, H. Ichijo, K. Takeda, Execution of apoptosis signal-regulating kinase 1 (ASK1)-induced apoptosis by the mitochondria-dependent caspase activation, J. Biol. Chem. 275 (2000) 26576e26581. [11] K. Tobiume, A. Matsuzawa, T. Takahashi, H. Nishitoh, K. Morita, K. Takeda, O. Minowa, K. Miyazono, T. Noda, H. Ichijo, ASK1 is required for sustained activations of JNK/p38 MAP kinases and apoptosis, EMBO Rep. 2 (2001) 222e228. [12] K. Sayama, H. Komatsuzawa, K. Yamasaki, Y. Shirakata, Y. Hanakawa, K. Ouhara, S. Tokumaru, X. Dai, M. Tohyama, P. Ten Dijke, M. Sugai, H. Ichijo, K. Hashimoto, New mechanisms of skin innate immunity: ASK1-mediated keratinocyte differentiation regulates the expression of beta-defensins, LL37, and TLR2, Eur. J. Immunol. 35 (2005) 1886e1895. [13] K. Sayama, Y. Hanakawa, Y. Shirakata, K. Yamasaki, Y. Sawada, L. Sun, K. Yamanishi, H. Ichijo, K. Hashimoto, Apoptosis signal-regulating kinase 1 (ASK1) is an intracellular inducer of keratinocyte differentiation, J. Biol. Chem. 276 (2001) 999e1004. [14] K. Takeda, T. Hatai, T.S. Hamazaki, H. Nishitoh, M. Saitoh, H. Ichijo, Apoptosis signal-regulating kinase 1 (ASK1) induces neuronal differentiation and survival of PC12 cells, J. Biol. Chem. 275 (2000) 9805e9813.
T. Hayakawa et al. / Microbes and Infection 8 (2006) 1098e1107 [15] K. Tobiume, M. Saitoh, H. Ichijo, Activation of apoptosis signal-regulating kinase 1 by the stress-induced activating phosphorylation of preformed oligomer, J. Cell. Physiol. 191 (2002) 95e104. [16] T. Noguchi, K. Takeda, A. Matsuzawa, K. Saegusa, H. Nakano, J. Gohda, J.I. Inoue, H. Ichijo, Recruitment of TRAF family proteins to the ASK1 signalosome is essential for oxidative stress-induced cell death, J. Biol. Chem. 280 (2005) 37033e37040. [17] M. Saitoh, H. Nishitoh, M. Fujii, K. Takeda, K. Tobiume, Y. Sawada, M. Kawabata, K. Miyazono, H. Ichijo, Mammalian thioredoxin is a direct inhibitor of apoptosis signal-regulating kinase (ASK) 1, EMBO J. 17 (1998) 2596e2606. [18] P.G. Winyard, C.J. Moody, C. Jacob, Oxidative activation of antioxidant defence, Trends Biochem. Sci. 30 (2005) 453e461. [19] R.M. Locksley, N. Killeen, M.J. Lenardo, The TNF, TNF receptor superfamilies: integrating mammalian biology, Cell 104 (2001) 487e501. [20] H. Wajant, K. Pfizenmaier, P. Scheurich, Tumor necrosis factor signaling, Cell Death Differ. 10 (2003) 45e65. [21] H. Nishitoh, M. Saitoh, Y. Mochida, K. Takeda, H. Nakano, M. Rothe, K. Miyazono, H. Ichijo, ASK1 is essential for JNK/SAPK activation by TRAF2, Mol. Cell 2 (1998) 389e395. [22] H. Liu, H. Nishitoh, H. Ichijo, J.M. Kyriakis, Activation of apoptosis signal-regulating kinase 1 (ASK1) by tumor necrosis factor receptorassociated factor 2 requires prior dissociation of the ASK1 inhibitor thioredoxin, Mol. Cell. Biol. 20 (2000) 2198e2208. [23] Y. Gotoh, J.A. Cooper, Reactive oxygen species- and dimerizationinduced activation of apoptosis signal-regulating kinase 1 in tumor necrosis factor-alpha signal transduction, J. Biol. Chem. 273 (1998) 17477e 17482. [24] T. Nakagawa, H. Zhu, N. Morishima, E. Li, J. Xu, B.A. Yankner, J. Yuan, Caspase-12 mediates endoplasmic-reticulum-specific apoptosis and cytotoxicity by amyloid-beta, Nature 403 (2000) 98e103. [25] Y. Imai, M. Soda, H. Inoue, N. Hattori, Y. Mizuno, R. Takahashi, An unfolded putative transmembrane polypeptide, which can lead to endoplasmic reticulum stress, is a substrate of Parkin, Cell 105 (2001) 891e902. [26] H.P. Harding, H. Zeng, Y. Zhang, R. Jungries, P. Chung, H. Plesken, D.D. Sabatini, D. Ron, Diabetes mellitus and exocrine pancreatic dysfunction in perk/ mice reveals a role for translational control in secretory cell survival, Mol Cell 7 (2001) 1153e1163. [27] H. Kadowaki, H. Nishitoh, F. Urano, C. Sadamitsu, A. Matsuzawa, K. Takeda, H. Masutani, J. Yodoi, Y. Urano, T. Nagano, H. Ichijo, Amyloid beta induces neuronal cell death through ROS-mediated ASK1 activation, Cell Death Differ. 12 (2005) 19e24. [28] Y. Izumiya, S. Kim, Y. Izumi, K. Yoshida, M. Yoshiyama, A. Matsuzawa, H. Ichijo, H. Iwao, Apoptosis signal-regulating kinase 1 plays a pivotal role in angiotensin II-induced cardiac hypertrophy and remodeling, Circ. Res. 93 (2003) 874e883. [29] P.H. McDonald, C.W. Chow, W.E. Miller, S.A. Laporte, M.E. Field, F.T. Lin, R.J. Davis, R.J. Lefkowitz, Beta-arrestin 2: a receptor-regulated MAPK scaffold for the activation of JNK3, Science 290 (2000) 1574e1577. [30] H.Y. Chang, H. Nishitoh, X. Yang, H. Ichijo, D. Baltimore, Activation of apoptosis signal-regulating kinase 1 (ASK1) by the adapter protein Daxx, Science 281 (1998) 1860e1863. [31] K. Takeda, A. Matsuzawa, H. Nishitoh, K. Tobiume, S. Kishida, J. Ninomiya-Tsuji, K. Matsumoto, H. Ichijo, Involvement of ASK1 in Ca2þ-induced p38 MAP kinase activation, EMBO Rep. 5 (2004) 161e166. [32] E. Kuranaga, H. Kanuka, T. Igaki, K. Sawamoto, H. Ichijo, H. Okano, M. Miura, Reaper-mediated inhibition of DIAP1-induced DTRAF1 degradation results in activation of JNK in Drosophila, Nat. Cell Biol. 4 (2002) 705e710. [33] C.I. Bargmann, E. Hartwieg, H.R. Horvitz, Odorant-selective genes and neurons mediate olfaction in C. elegans, Cell 74 (1993) 515e527.
1107
[34] P.D. Wes, C.I. Bargmann, C. elegans odour discrimination requires asymmetric diversity in olfactory neurons, Nature 410 (2001) 698e701. [35] E.R. Troemel, A. Sagasti, C.I. Bargmann, Lateral signaling mediated by axon contact and calcium entry regulates asymmetric odorant receptor expression in C. elegans, Cell 99 (1999) 387e398. [36] A. Sagasti, N. Hisamoto, J. Hyodo, M. Tanaka-Hino, K. Matsumoto, C.I. Bargmann, The CaMKII UNC-43 activates the MAPKKK NSY-1 to execute a lateral signaling decision required for asymmetric olfactory neuron fates, Cell 105 (2001) 221e232. [37] M. Tanaka-Hino, A. Sagasti, N. Hisamoto, M. Kawasaki, S. Nakano, J. Ninomiya-Tsuji, C.I. Bargmann, K. Matsumoto, SEK-1 MAPKK mediates Ca2þ signaling to determine neuronal asymmetric development in Caenorhabditis elegans, EMBO Rep. 3 (2002) 56e62. [38] C.L. Kurz, J.J. Ewbank, Caenorhabditis elegans: an emerging genetic model for the study of innate immunity, Nat. Rev. Genet. 4 (2003) 380e390. [39] D.H. Kim, R. Feinbaum, G. Alloing, F.E. Emerson, D.A. Garsin, H. Inoue, M. Tanaka-Hino, N. Hisamoto, K. Matsumoto, M.W. Tan, F.M. Ausubel, A conserved p38 MAP kinase pathway in Caenorhabditis elegans innate immunity, Science 297 (2002) 623e626. [40] L.A. O’Neill, K.A. Fitzgerald, A.G. Bowie, The Toll-IL-1 receptor adaptor family grows to five members, Trends Immunol. 24 (2003) 286e290. [41] C. Wang, L. Deng, M. Hong, G.R. Akkaraju, J. Inoue, Z.J. Chen, TAK1 is a ubiquitin-dependent kinase of MKK and IKK, Nature 412 (2001) 346e351. [42] S. Sato, H. Sanjo, K. Takeda, J. Ninomiya-Tsuji, M. Yamamoto, T. Kawai, K. Matsumoto, O. Takeuchi, S. Akira, Essential function for the kinase TAK1 in innate and adaptive immune responses, Nat. Immunol. in press. [43] Q. Huang, J. Yang, Y. Lin, C. Walker, J. Cheng, Z.G. Liu, B. Su, Differential regulation of interleukin 1 receptor and Toll-like receptor signaling by MEKK3, Nat. Immunol. 5 (2004) 98e103. [44] C.D. Dumitru, J.D. Ceci, C. Tsatsanis, D. Kontoyiannis, K. Stamatakis, J.H. Lin, C. Patriotis, N.A. Jenkins, N.G. Copeland, G. Kollias, P.N. Tsichlis, TNF-alpha induction by LPS is regulated posttranscriptionally via a Tpl2/ERK-dependent pathway, Cell 103 (2000) 1071e1083. [45] Y. Xia, C. Makris, B. Su, E. Li, J. Yang, G.R. Nemerow, M. Karin, MEK kinase 1 is critically required for c-Jun N-terminal kinase activation by proinflammatory stimuli and growth factor-induced cell migration, Proc. Natl. Acad. Sci. U.S.A. 97 (2000) 5243e5248. [46] N. Pujol, E.M. Link, L.X. Liu, C.L. Kurz, G. Alloing, M.W. Tan, K.P. Ray, R. Solari, C.D. Johnson, J.J. Ewbank, A reverse genetic analysis of components of the Toll signaling pathway in Caenorhabditis elegans, Curr Biol. 11 (2001) 809e821. [47] N.T. Liberati, K.A. Fitzgerald, D.H. Kim, R. Feinbaum, D.T. Golenbock, F.M. Ausubel, Requirement for a conserved Toll/interleukin-1 resistance domain protein in the Caenorhabditis elegans immune response, Proc. Natl. Acad. Sci. U.S.A. 101 (2004) 6593e6598. [48] C. Couillault, N. Pujol, J. Reboul, L. Sabatier, J.F. Guichou, Y. Kohara, J.J. Ewbank, TLR-independent control of innate immunity in Caenorhabditis elegans by the TIR domain adaptor protein TIR-1, an ortholog of human SARM, Nat. Immunol. 5 (2004) 488e494. [49] M. Mink, B. Fogelgren, K. Olszewski, P. Maroy, K. Csiszar, A novel human gene (SARM) at chromosome 17q11 encodes a protein with a SAM motif and structural similarity to Armadillo/beta-catenin that is conserved in mouse, Drosophila, and Caenorhabditis elegans, Genomics 74 (2001) 234e244. [50] H.S. Park, H.Y. Jung, E.Y. Park, J. Kim, W.J. Lee, Y.S. Bae, Cutting edge: direct interaction of TLR4 with NAD(P)H oxidase 4 isozyme is essential for lipopolysaccharide-induced production of reactive oxygen species and activation of NF-kappa B, J. Immunol. 173 (2004) 3589e3593. [51] J.F. Curtin, M. Donovan, T.G. Cotter, Regulation and measurement of oxidative stress in apoptosis, J. Immunol. Methods 265 (2002) 49e72.