Activation mechanisms of ASK1 in response to various stresses and its significance in intracellular signaling

Advances in Biological Regulation xxx (2012) 1–10

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Activation mechanisms of ASK1 in response to various stresses and its significance in intracellular signaling Shigeru Shiizaki, Isao Naguro, Hidenori Ichijo* Laboratory of Cell signaling, Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan

a b s t r a c t Apoptosis signal-regulating kinase 1 (ASK1) is a member of the mitogen-activated protein kinase kinase kinase family. ASK1 activates c-jun N-terminal kinase (JNK) and p38 in response to various stimuli such as oxidative stress, endoplasmic reticulum stress, infection and calcium influx. Under these stress conditions, ASK1 plays important roles in intracellular signaling pathways and biological functions. Diverse proteins are known to interact with ASK1 and regulate the activity of ASK1. However, activation mechanisms of ASK1 and ASK1-binding proteins which regulate the activity of ASK1 have not been completely understood. In this review, we focus on the recent findings on ASK1 and update the regulatory mechanisms of ASK1 activity. Ó 2012 Published by Elsevier Ltd.

Introduction All living organisms are exposed to various types of stresses throughout their lifetime. Cells are imposed to respond to numerous stresses appropriately in order to maintain homeostasis. The mitogen-activated protein kinase (MAPK) pathways are evolutionally conserved intracellular signaling systems which regulate cellular stress response (Kyriakis and Avruch, 2012; Widmann et al., 1999). The MAPK pathways are essential for various cellular functions; however, the mechanisms by which cells sense stresses and transduce the information into cellular signals have not been fully understood. All MAPK pathways include a cascade of three classes of protein kinases: the MAPK kinase kinase (MAP3K), the MAPK kinase (MAP2K), and the MAPK. MAP3Ks are activated or inactivated by various

* Corresponding author. Tel.: þ81 3 5841 4858; fax: þ81 3 5841 4798. E-mail address: [email protected] (H. Ichijo). 2212-4926/$ – see front matter Ó 2012 Published by Elsevier Ltd. http://dx.doi.org/10.1016/j.jbior.2012.09.006

Please cite this article in press as: Shiizaki S, et al., Activation mechanisms of ASK1 in response to various stresses and its significance in intracellular signaling, Advances in Biological Regulation (2012), http:// dx.doi.org/10.1016/j.jbior.2012.09.006

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stimuli. The activated MAP3K phosphorylates and activates the MAP2K, which in turn phosphorylates and activates the MAPK. The activated MAPK phosphorylates other protein kinases and transcription factors, thus regulates various cellular responses. In mammals, three major MAPK family members have been well characterized: Extracellular signal-regulated kinase (ERK), c-jun NH2-terminal kinase (JNK) and p38 MAPK. Since the activation of MAPK pathways largely depends on the activation of MAP3Ks, it is important to understand the regulatory mechanisms of MAP3Ks. Apoptosis signal-regulating kinase 1 (ASK1) is a member of MAP3K family. ASK1 activates the MAP2K4/7 (MKK4/7)-JNK pathway and MAP2K3/6 (MKK3/6)-p38 pathway (Ichijo et al., 1997). It has been revealed that ASK1 is activated in response to various types of stresses including oxidative stress, endoplasmic reticulum (ER) stress and death receptor ligands to properly respond to these stresses (Hattori et al., 2009; Matsukawa et al., 2004; Takeda et al., 2003). Here, we summarize the recent findings on ASK1 and discuss the activation mechanisms of ASK1 and ASK1-mediated signaling pathways in various cellular responses. Minimal components of ASK1 signalosome Human and mouse ASK1 consist of 1,374 and 1,379 amino acids, respectively. Although whole tertiary structure of full-length ASK1 remains unclear, crystal structure of the human ASK1 catalytic domain in complex with staurosporine has been solved (Bunkoczi et al., 2007). ASK1 has a serine/ threonine kinase domain in the middle part of the molecule and two coiled-coil domains flanked by Nterminal coiled-coil domain (NCC) and C-terminal coiled-coil domain (CCC). Phosphorylation of a threonine residue within the kinase domain (Thr838 and Thr845 of human and mouse, respectively) is essential for the activation of ASK1. When Thr845 of human ASK1 was replaced by alanine, the kinase activity of the mutant ASK1 was abolished. Moreover, the phosphorylation state of Thr845 of ASK1 parallels the kinase activity of ASK1. The Thr845 phosphorylation of ASK1 has been shown to occur through the trans-autophosphorylation under oxidative stress (Tobiume et al., 2002). ASK1 forms homo-oligomer thorough the CCC. The C-terminal deletion mutant of ASK1 (ASK1 DCCC) lost its ability to homo-oligomerize. The phosphorylation level of Thr845 was reduced in case of ASK1 DCCC. Consistent with the result, in vitro kinase assay showed that ASK1 DCCC had lower basal kinase activity than wild-type ASK1. The activity and the phosphorylation level of ASK1 DCCC could be markedly enhanced by the compulsive homo-oligomerization induced by artificial system (Tobiume et al., 2002). These results suggest that homo-oligomerization through the ASK1 CCC is required for the full activation of ASK1. ASK1 forms a complex with not only ASK1 but also various proteins in the cell. The high molecular (1500–2000 kDa) mass complex is called ASK1 signalosome (Noguchi et al., 2005). ROS and tumor necrosis factor a (TNF-a) induced the formation of higher mass complex of ASK1 (>3000 kDa). In steady state, ASK1 signalosome includes thioredoxin (Trx), which negatively regulates ASK1 (see next chapter). Under hydrogen peroxide (H2O2) stimulation, tumor necrosis factor receptor-associated factor 2 (TRAF2) and TRAF6 are recruited to ASK1 signalosome and positively regulate ASK1 activity (Fig. 1). The expected molecular mass of monomeric ASK1, Trx and TRAF2 is w160 kDa, w12 kDa and w53 kDa respectively. Although ASK1 forms homo-oligomer, considering the high mass molecular weight of ASK1 signalosome, it is conceivable that ASK1 signalosome may contain further unknown components involved in regulation of ASK1. ASK1 in oxidative stress All aerobic organisms are exposed to reactive oxygen species (ROS) generated by aerobic metabolism. ROS are also generated by UV irradiation. The generation of ROS induces oxidative stress. It has been known that oxidative stress is associated with various diseases (Circu and Aw, 2010; Finkel, 2003). ASK1 is activated under ROS generation and plays a pivotal role in cellular responses to oxidative stress (Soga et al., 2012). Various ASK1-binding proteins regulate the activation of ASK1 in oxidative stress. Trx was identified as ASK1-binding protein by a yeast two-hybrid screening (Saitoh et al., 1998). When increasing amount of Trx was cotransfected with ASK1 to HEK293A cells, the kinase activity of ASK1 was inhibited in Please cite this article in press as: Shiizaki S, et al., Activation mechanisms of ASK1 in response to various stresses and its significance in intracellular signaling, Advances in Biological Regulation (2012), http:// dx.doi.org/10.1016/j.jbior.2012.09.006

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Fig. 1. The activation mechanism of ASK1 in response to ROS generation. ASK1 forms homo-oligomer thorough the CCC. In steady state, Trx and 14-3-3 proteins bind to ASK1 and negatively regulate the activation of ASK1. In response to ROS generation, Trx and 14-3-3 proteins dissociate from ASK1 while TRAF2 and TRAF6 are recruited to ASK1 and bind to ASK1, resulting in homophilic interaction of ASK1 through the NCC and activation of ASK1. USP9X counteracts proteasome-dependent degradation of activated ASK1 by deubiquitination.

a dose-dependent manner. It is reported that in reducing state, Trx binds to ASK1 through the Nterminal region of ASK1 and negatively regulates ASK1 activity (Fujino et al., 2007; Saitoh et al., 1998). In response to ROS stimulation, Trx is oxidized and dissociate from ASK1. This dissociation of Trx allows homophilic interaction of ASK1 through the NCC and ASK1 is activated (Fig. 1). Consistent with this notion, the N-terminus-deleted mutant of ASK1 (ASK1 DN), which lacks Trx binding region, had higher basal kinase activity and this activity was not inhibited by the coexpression with Trx (Saitoh et al., 1998). 14-3-3 proteins also negatively regulate ASK1. 14-3-3 proteins bind to ASK1 via the site including Ser966 when Ser966 of ASK1 is phosphorylated in steady state (Goldman et al., 2004). ROS induces dephosphorylation of Ser966 of ASK1, resulting in dissociation of 14-3-3 proteins from ASK1 and activation of ASK1 (Goldman et al., 2004; Zhang et al., 1999). In addition, it has been reported that oxidative stress activates the mammalian sterile 20 (Mst) family member SOK-1 and activated SOK-1 phosphorylates Ser58 of 14-3-3z. This phosphorylation of 14-3-3z induces the dissociation of ASK1 from 14-3-3z (Zhou et al., 2009). 14-3-3 proteins are also involved in TNF-a-induced activation of ASK1 (see next chapter). Not only negative regulators, but also positive regulators are involved in the activation of ASK1. Among TRAFs, TRAF2, TRAF5 and TRAF6 activated ASK1 when they were coexpressed with ASK1 (Nishitoh et al., 1998). Coexpression of ASK1 and TRAF2 enhanced ASK1 homo-oligomerization and activation. (Liu et al., 2000). Using deletion mutants of ASK1, TRAF2 and TRAF6 have been suggested to bind to amino-terminal region (amino acids 384–655) of ASK1 (Fujino et al., 2007). The association of TRAF2/TRAF6 with ASK1 is required for ROS-induced homophilic interaction of ASK1 through the NCC. In summary, upon ROS stimulation, Trx is dissociated from ASK1, while TRAF2/TRAF6 are recruited to ASK1 signalosome to facilitate the auto-phosphorylation of ASK1 (Noguchi et al., 2005) (Fig. 1). ASK1 is also regulated by ubiquitin-proteasome system under oxidative stress (Nagai et al., 2009). ASK1 is ubiquitinated and degraded upon oxidative stress. It has been reported that Ubiquitin-specific peptidase 9, X-linked (USP9X) binds to ASK1 and deubiquitinates ASK1 in response to oxidative stress, resulting in the stabilization of ASK1. The oxidative stress-induced activation of ASK1 was suppressed in USP9X knockdown cells. Thus, USP9X contributes to the sustained activation of ASK1 activity in oxidative stress (Nagai et al., 2009). Death receptor signaling and ASK1 The generation of ROS is also crucial for TNF-a-induced signaling pathway. TNF-a is a proinflammatory cytokine and plays a role in immune responses. Previous studies have demonstrated that ASK1 is important for TNF-a-induced activation of JNK. In ASK1-deficient (ASK1/) mouse embryonic fibroblasts (MEFs), the TNF-a-induced sustained activation of JNK was attenuated, compared to that in ASK1þ/þ MEFs. TNF-a-induced apoptosis was also attenuated in ASK1/ MEFs. Moreover, when ASK1þ/þ MEFs were treated with antioxidants such as N-acetyl-L-cysteine (NAC), TNF-a-induced Please cite this article in press as: Shiizaki S, et al., Activation mechanisms of ASK1 in response to various stresses and its significance in intracellular signaling, Advances in Biological Regulation (2012), http:// dx.doi.org/10.1016/j.jbior.2012.09.006

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apoptosis was inhibited (Tobiume et al., 2001). As described above, the generation of ROS induces the dissociation of Trx from ASK1 and recruitment of TRAF2/TRAF6 to ASK1. Thus, the TNF-a-induced activation of ASK1 is mediated by the generation of ROS. ASK1-interacting protein 1 (AIP1) is supposed to function as a scaffolding protein to recruit the TNFa signaling complex (TRADD-RIP1-TRAF2) to ASK1, resulting in the activation of ASK1 (Zhang et al., 2004). On the other hand, AIP1 is crucial for the TNF-a-induced association between ASK1 and PP2A, a member of serine/threonine phosphatase family (Min et al., 2008). Recruited PP2A dephosphorylates Ser966 of ASK1 and 14-3-3 proteins dissociate from ASK1, followed by the activation of ASK1. It has been reported that the TNF-a-induced activation of ASK1 and JNK enhances phosphorylation and polyubiquitination of Daxx, which was identified as a Fas death domain-interacting protein (Fukuyo et al., 2009; Kitamura et al., 2009). The activated ASK1 phosphorylated Daxx at Ser176 and Ser184 directly under TNF-a treatment. Then K-63 linked ubiquitination on Lys122 of Daxx occurred. The polyubquitinated Daxx in turn activated ASK1 in a positive feedback manner. When K63 ubiquitination-deficient Daxx mutant K122R was expressed, TNF-a-induced sustained JNK activation and apoptosis were attenuated. In addition, kinase-defective ASK1 failed to induce the Daxxdependent sustained activation of JNK. Thus, it has been suggested that Daxx-ASK1-JNK pathway is important for TNF-a-induced apoptosis. Fas is a member of the death receptor family. When Fas recognizes Fas ligand (FasL), two signaling pathways are activated. One involves Fas-associated death domain protein (FADD) and caspase 8 (Nagata, 1999). The other involves the activation of Fas- binding protein Daxx. The activated Daxx in turn activates ASK1 and then JNK and p38 (Chang et al., 1998). Indeed, Fas-induced activation of JNK and p38 was decreased in ASK1/ thymocytes. However, Fas-induced apoptosis was indistinguishable between ASK1þ/þ and ASK1/ thymocytes and MEFs (Tobiume et al., 2001). It has been suggested that the Daxx-ASK1-JNK/p38 axis is not crucial for Fas-induced apoptosis at least in these cells. ASK1 and immune response It has been indicated that ASK1 is involved in innate immunity. Lipopolysaccharide (LPS) is a component of the outer membrane of Gram-negative bacteria. When LPS is recognized by Toll-like receptor 4 (TLR4), the production of proinflammatory cytokines occurs (Lu et al., 2008). p38 is known to play a crucial role in the LPS-induced production of cytokine (Bode et al., 2012). It has been demonstrated that the binding of LPS to TLR4 induces the activation of TRAF6-ASK1-p38 signaling pathway (Matsuzawa et al., 2005). Pre-treatment with antioxidants suppressed the LPS-induced activation of ASK1 and p38, suggesting that ROS mediates the LPS-ASK1-p38 signaling pathway. Moreover, ASK1/ mice were more resistant to LPS-induced septic shock than ASK1þ/þ mice. The serum concentration of TNF-a and nitric oxide (NO), the principal factors responsible for septic shock, were also reduced in ASK1/ mice. TNF-a plays a protective role against infection through the production of inflammatory mediator (Bode et al., 2012). Therefore, the ASK1-p38 axis is important for the activation of mammalian innate immunity against bacterial infection (Matsuzawa et al., 2005). Cell volume regulation and ASK1 Previous studies have suggested that regulation of cell volume is implicated in apoptosis and necrosis (Barros et al., 2001; Bortner and Cidlowski, 2007). Hypertonic condition induces a decrease of cell volume. After the osmotic shrinkage, cells try to recover their volume via uptake of sodium and chloride ions and osmotically obliged water. This cellular response is called regulatory volume increase (RVI). However, cells undergoing apoptosis lack RVI. Even under isotonic condition, apoptotic cells undergo persistent cell shrinkage. This shrinkage is called apoptotic volume decrease (AVD). AVD is a morphological hallmark of an apoptotic process. Some studies have investigated whether lack of RVI plays a role in AVD (Algharabil et al., 2012; Numata et al., 2008). Akt (protein kinase B) is a serine/threonine protein kinase and known as an effector of phosphatidylinositol-3 kinase (PI3K) (Franke et al., 1995). It has been suggested that PI3K and Akt1 are activated under hypertonic stress and prevent cells from hyperosmolality-induced apoptosis in Please cite this article in press as: Shiizaki S, et al., Activation mechanisms of ASK1 in response to various stresses and its significance in intracellular signaling, Advances in Biological Regulation (2012), http:// dx.doi.org/10.1016/j.jbior.2012.09.006

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mIMCD3 cells and MDCK cells (Terada et al., 2001; Zhang et al., 2000). It has been reported that Akt1 and ASK1 are involved in apoptotic inhibition of hyperosmolality-induced RVI in HeLa cells (Subramanyam et al., 2010). According to this report, when Akt1 was depleted by siRNA, the induction of RVI under hyperosmotic conditions was suppressed. Treatment with apoptosis inducers inhibited the activation of Akt1 under hypertonic stress and RVI was also suppressed by apoptosis inducers under hypertonic stress. The inhibition of hyperosmolality-induced phosphorylation of Akt1 and RVI by apoptotic inducers was restored by treatment with ROS scavengers. On the other hand, the treatment of ROS scavengers under hypertonic condition attenuated the activation of ASK1 caused by apoptosis inducers. Moreover, the siRNA-mediated knockdown of ASK1 restored the hyperosmolalityinduced phosphorylation of Akt1 and RVI even under the treatment of apoptotic inducers. Taken together, it has been suggested that ROS-mediated activation of ASK1 under apoptotic situation inhibits the activation of Akt1 and thus suppresses RVI reaction, resulting in persistent cell shrinkage under hyperosmotic condition. Further studies are needed to reveal whether this mechanism can be applicable to AVD in isotonic situation. ASK1 in hypoxia and ischemia Hypoxia (low oxygen) and ischemia (low supply of blood, causing low levels of oxygen and glucose) are mainly caused by dysfunction of blood vessels. Shortage of oxygen or glucose generates ROS from mitochondria. Several reports have shown that ASK1 is activated under hypoxic or ischemic state (Bitto et al., 2010; Harding et al., 2010; Kwon et al., 2005). One study has suggested that ischemia causes dissociation of glutaredoxin and Trx from ASK1 by ROS generation, leading to the activation of ASK1p38 pathway. The study also has suggested that activated p38 triggers the phosphorylation and stabilization of hypoxia-inducible factor-1-a (HIF1-a), which regulates the expression of target genes encoding proteins required for adaption to hypoxia (Ke and Costa, 2006; Kwon et al., 2005) It has been reported that ASK1 translocates to punctate cytoplasmic structures, which are insoluble in Triton-X100, under ischemic stress in cardiac myocytes. ASK1 returned to the cytosol again by reperfusion, suggesting that the translocation is reversible (Browne and Dickens, 2011). So far, the precise character of these punctate structures is unclear. If the protein(s) which mediates the translocation of ASK1 is identified, the significance of ASK1 translocation would be explored further. ASK1 may interact with some components in the granules to activate downstream effecters required for cellular response to ischemia. ER stress response and ASK1 Endoplasmic reticulum (ER) stress is caused by accumulation of unfolded or misfolded proteins in ER lumen. Several cellular mechanisms handle these immature proteins in ER lumen. For example, in ER-associated protein degradation (ERAD) pathway, the immature proteins in ER lumen translocate into the cytosol and are degraded by the ubiquitin-proteasome system (Ron and Walter, 2007). Under ER stress, IRE1, which is localized on ER membrane, is activated and forms a complex with TRAF2 and ASK1. It has been suggested that the formation of IRE1-TRAF2-ASK1 complex activates the ASK1-JNK pathway, which induces apoptosis under excess ER stress (Nishitoh et al., 2002; Urano et al., 2000) Polyglutamine (PolyQ) sequences (CAG trinucleotide) are found in various proteins. Expanded PolyQ repeats are known to be a cause of several neurodegenerative disorders such as Huntington’s disease (Kakizuka, 1998; Paulson et al., 2000; Wetzel, 2012). Aggregation of PolyQ containing protein inhibits proteosomal activity and induces ER stress. Previous study has reported that primary neurons derived from ASK1/ mice were resistant to PolyQ-induced cell death, suggesting the involvement of the ER stress-induced IRE1-TRAF2-ASK1 pathway in PolyQ diseases (Nishitoh et al., 2002). The ER stress-induced activation of IRE1-TRAF2-ASK1 pathway is also involved in amyotrophic lateral sclerosis (ALS). Mutations of Cu/Zn-superoxide dismutase (SOD1) have been reported as a cause of familial ALS. Previous study has suggested that mutant SOD1 protein (SOD1mut) triggers ER stress and activates the IRE1-TRAF2-ASK1 pathway (Nishitoh et al., 2008). SOD1mut interacted with one of ERAD components, Derlin-1 (Fujisawa et al., in press; Nishitoh et al., 2008). SOD1mut attenuated the translocation of ERAD substrates and prevented the substrates from degradation. In addition, SOD1mutPlease cite this article in press as: Shiizaki S, et al., Activation mechanisms of ASK1 in response to various stresses and its significance in intracellular signaling, Advances in Biological Regulation (2012), http:// dx.doi.org/10.1016/j.jbior.2012.09.006

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induced cell death was significantly attenuated in ASK1/ neurons. These data suggest that the association of SOD1mut with Derin-1 causes the dysfunction of ERAD and activates the IRE1-TRAF2ASK1 signaling pathway via ER stress, leading to induction of motor neuron cell death in ALS. Several reports have discussed the relationship between autophagy and apoptosis under ER stress (Heath-Engel et al., 2008; Moretti et al., 2007). Although autophagy seems to be associated with ER stress and to act as a protective response to ER stress, the precise molecular mechanisms activating autophagy under ER stress have been unclear. When neuroblastoma SK-N-SH cells and MEFs were treated with ER stressors, tunicamycin and thapsigargin, the formation of autophagosomes was enhanced (Ogata et al., 2006). IRE1-TRAF2-JNK signaling pathway is supposed to be important for the formation of autophagosomes because the formation of autophagosomes under ER stress was attenuated in IRE1-deficient MEFs. In addition, the formation of autophagosomes was inhibited by the introduction of dominant-negative TRAF2 and the pretreatment with a JNK inhibitor (Ogata et al., 2006). As described above, since ASK1 is activated by ER stress via IRE1-TREAF2 axis and activates JNK, ASK1 might be involved in the ER stress-induced formation of autophagosomes. However, the involvement of ASK1 in autophagy under ER stress has not been investigated in detail. It has been reported that ASK1 overexpression induces autophagy in SH-SY5Y cells, but the requirement of ASK1 for autophagy or the mechanisms causing autophagy by ER stress were not clearly defined (NisoSantano et al., 2011). Further studies are needed to elucidate whether ASK1 plays a role in autophagy under ER stress. ASK1 and virus infection Influenza virus (IV) infection induces apoptosis in various cell types both in vitro and in vivo (Hinshaw et al., 1994; Mori et al., 1995; Takizawa et al., 1993). It has been suggested that ASK1 plays an important role in the IV infection-induced apoptosis (Maruoka et al., 2003; Sumbayev and Yasinska, 2006). IV infection induced the activation of ASK1 and downstream JNK and p38 in human bronchial epithelial cells (BEC). The phosphorylation of JNK and p38 was suppressed in ASK1/ MEFs and these ASK1/ MEFs were resistant to cell death under IV infection (Maruoka et al., 2003). Several possible mechanisms which can explain IV infection-induced ASK1 activation have been proposed. One possible mechanism is through ASK1-associated protein(s). Viral infection causes the generation of double-stranded RNA molecules, which activate double-stranded RNA-activated protein kinase (PKR). It has been reported that PKR binds to ASK1 and induces the activation of ASK1 (Takizawa et al., 2002). Another possible mechanism is ER stress-mediated signaling pathway. As described above, ER stress activates ASK1. BiP mRNA expression, known as a marker for ER stress, was up-regulated in IV-infected BEC, suggesting that IV infection causes ER stress (Maruoka et al., 2003). On the other hand, infection of influenza A virus activated PI3K-Akt pathway and Akt suppressed the activation of ASK1 in A549 cells (Lu et al., 2010). Further studies are needed to elucidate the precise regulatory mechanism of ASK1 against IV infection. It has been suggested that infection of the human immunodeficiency virus type 1 (HIV-1) leads to efficient apoptosis of T cells (Geleziunas et al., 2001; Masutani et al., 2005). HIV-1 Nef, which is known as a member of so-called “accessory” proteins, is expressed in HIV infected cells (Vermeire et al., 2011). HIV-1 Nef induces the expression of FasL on the surface of host cells. FasL induces apoptosis in the uninfected cells near the HIV infected cells. Simultaneously, HIV-1 Nef protects host cells from apoptosis induced by TNF-a and FasL. It has been suggested that HIV-1 Nef inhibits the dissociation of ASK1 from Trx, thus prevents the activation of ASK1 and ASK1-mediated apoptosis in host cells (Geleziunas et al., 2001; Masutani et al., 2005). ASK1 in calcium signaling Calcium ion (Ca2þ) is known as a second messenger and involved in various cellular processes including apoptosis (Zhivotovsky and Orrenius, 2011). The increase of Ca2þ in cytosol is achieved by the influx of extracellular Ca2þ or by the release from intracellular compartments such as ER. Various proteins interact with Ca2þ. Ca2þ/calmodulin-dependent protein kinase (CAMK) family proteins are downstream target of Ca2þ signaling. In Caenorhabditis elegans, it has been reported that nsy-1, which Please cite this article in press as: Shiizaki S, et al., Activation mechanisms of ASK1 in response to various stresses and its significance in intracellular signaling, Advances in Biological Regulation (2012), http:// dx.doi.org/10.1016/j.jbior.2012.09.006

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Fig. 2. Overview of upstream signaling of ASK1 in response to various stresses. ASK1 is activated by various stimuli. H2O2 treatment generates ROS and activates ASK1. TNF-a, LPS and ischemia also trigger the generation of ROS. ER stress induces the formation of IRE1-TRAF2-ASK1 complex and activates ASK1. Infection of IV has been suggested to induce ER stress. Ca2þ influx activates ASK1 through the regulation of Ca2þ-binding proteins, such as CAMKII.

encodes a homolog of mammalian ASK1, acts as a downstream of unc-43, which encodes a homolog of mammalian CAMKII (Sagasti et al., 2001). Several reports have suggested the interaction of CAMKII and ASK1 also in mammalian cells (Brnjic et al., 2010; Kashiwase et al., 2005; Takeda et al., 2004). In primary neurons derived from ASK1þ/þ mice, the influx of Ca2þ from extracellular space activated p38. This Ca2þ-induced activation of p38 was attenuated in primary neurons derived from ASK1/ mice. Treatment of CAMKII inhibitor KN-93 and transfection of siRNA targeting CAMKII to ASK1þ/þ primary neurons also impaired the activation of p38 induced by Ca2þ. In vitro kinase assay suggested that CAMKII directly phosphorylated ASK1, although the phosphorylation site was not at Thr845 of ASK1. Taken together, it has been proposed that Ca2þ influx activates CAMKII and CAMKII phosphorylates ASK1 at the sites other than Thr845. According to this phosphorylation, Thr845 is phosphorylated by an unidentified kinase or by ASK1 per se. Activated ASK1 induces the phosphorylation of p38 and regulates cellular functions. The phosphorylation sites and the activation mechanism of ASK1 by CAMKII remain to be elucidated. Calcium and integrin binding protein 1 (CIB1) is a Ca2þ-binding protein. It has been reported that CIB1 functions as negative regulator of ASK1 (Yoon et al., 2009). CIB1 bound to NH2-terminal domain of ASK1 and inhibited the activity of ASK1 probably by blocking the association of ASK1 and TRAF2. Conformational change of CIB1 induced CIB1 dissociation from ASK1 by the increase of intracellular Ca2þ. Protein serine/threonine phosphatase 5 (PP5) is a negative regulator of ASK1. PP5 directly binds to the activated form(s) of ASK1 and dephosphorylates ASK1. Therefore, PP5 inhibits H2O2-induced sustained activation of ASK1 by negative feedback (Morita et al., 2001). Recently, Ca2þ/S100 proteins, which consist of S100A1-16, S100B, S100P, S100Z and CALB3, have been reported to regulate the function of PP5 (Marenholz et al., 2004; Yamaguchi et al., 2012). S100A1 protein caused the dissociation of PP5 from ASK1 under treatment of ionomycin, which elevates the intracellular Ca2þ concentration. Moreover, overexpression of permanently active S100P mutant protein, which keeps Ca2þ binding form even in the absence of Ca2þ, constitutively inhibited the binding between ASK1 and PP5, resulting in the inhibition of the dephosphorylation of ASK1 by PP5. These findings suggest that S100 proteins activated by Ca2þ induce the dissociation of PP5 from ASK1, resulting in the activation of ASK1. Thus, it is possible that dissociation of ASK1 from PP5 by S100 protein may play a role in the ASK1 activation mechanism induced by Ca2þ signaling. Conclusions As described throughout this review, ASK1 is involved in a wide variety of stress signaling (Fig. 2). Recent studies have suggested that ASK1 possesses various biological functions besides the induction of apoptosis. Although various regulatory mechanisms of ASK1 have been elucidated, there are still Please cite this article in press as: Shiizaki S, et al., Activation mechanisms of ASK1 in response to various stresses and its significance in intracellular signaling, Advances in Biological Regulation (2012), http:// dx.doi.org/10.1016/j.jbior.2012.09.006

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unknown regulatory mechanisms as described in this review. Endogenous ASK1 forms the ASK1 signalosome (Noguchi et al., 2005). It is possible that the components of the ASK1 signalosome are different in response to stimulations. Search for the unknown ASK1-binding proteins may reveal novel regulatory mechanisms of ASK1 in response to various stimuli. The elucidation of precise functions of ASK1 in stress signaling may disclose promising strategies to overcome ASK1-related human diseases. Acknowledgments We are grateful to all the members of the Laboratory of Cell Signaling for meaningful discussions. References Algharabil J, Kintner DB, Wang Q, Begum G, Clark PA, Yang SS, et al. Inhibition of Na-K(þ)-2Cl() Cotransporter isoform 1 accelerates temozolomidemediated apoptosis in glioblastoma cancer cells. Cell Physiol Biochem 2012;30:33–48. Barros LF, Hermosilla T, Castro J. Necrotic volume increase and the early physiology of necrosis. 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