SUMO-1 represses apoptosis signal-regulating kinase 1 activation through physical interaction and not through covalent modification

This study examined whether small ubiquitin-related modifier-1 (SUMO-1) regulates apoptosis signal-regulating kinase 1 (ASK 1). ASK 1 interacted with SUMO-1 in vitro as well as in BOSC 23 cells. Endogenous ASK 1-SUMO-1 interaction was disrupted following H(2)O(2) signal. SUMO-1 overexpression suppressed the self-oligomerization, kinase activity and apoptotic potential of ASK 1, whereas SUMO-1 depletion potentiated such activities. SUMO-1(Delta C 6), a sumoylation-incompetent mutant lacking carboxy-terminal six amino acids, suppressed AS 1 activation, implying that the suppressive effect of SUMO-1 on ASK 1 is independent of sumoylation. ASK 1(3M), an ASK 1 mutant in which all three lysines in the psiKXE motif were substituted with alanines, still retained the kinase activity and activated the Jun amino-terminal kinase pathway. However, SUMO-1 failed to interact with ASK 1(3M) and to suppress ASK 1(3M) activation, indicating that the three lysines are important for regulation by SUMO-1. This study shows that SUMO-1 exerts a negative regulatory effect on ASK 1 activation through physical interaction and not through covalent modification.     

Role of the ASK1-SEK1-JNK1-HIPK1 signal in Daxx trafficking and ASK1 oligomerization

Overexpression of JNK binding domain inhibited glucose deprivation-induced JNK1 activation, relocalization of Daxx from the nucleus to the cytoplasm, and apoptosis signal-regulating kinase 1 (ASK1) oligomerization in human prostate adenocarcinoma DU-145 cells. However, SB203580, a p38 inhibitor, did not prevent relocalization of Daxx and oligomerization of ASK1 during glucose deprivation. Studies from in vivo labeling and immune complex kinase assay demonstrated that phosphorylation of Daxx occurred during glucose deprivation, and its phosphorylation was mediated through the ASK1-SEK1-JNK1-HIPK1 signal transduction pathway. Data from immunofluorescence staining and protein interaction assay suggest that phosphorylated Daxx may be translocated to the cytoplasm, bind to ASK1, and subsequently lead to ASK1 oligomerization. Mutation of Daxx Ser667 to Ala results in suppression of Daxx relocalization during glucose deprivation, suggesting that Ser667 residue plays an important role in the relocalization of Daxx. Unlike wild-type Daxx, a Daxx deletion mutant (amino acids 501-625) mainly localized to the cytoplasm, where it associated with ASK1, activated JNK1, and induced ASK1 oligomerization without glucose deprivation. Taken together, these results show that glucose deprivation activates the ASK1-SEK1-JNK1-HIPK1 pathway, and the activated HIPK1 is probably involved in the relocalization of Daxx from the nucleus to the cytoplasm. The relocalized Daxx may play an important role in glucose deprivation-induced ASK1 oligomerization.     

ASK1 regulates influenza virus infection-induced apoptotic cell death

Apoptosis occurs in influenza virus (IV)-infected cells. There are a number of mechanisms for the regulation of apoptosis. However, the molecular mechanism of IV infection-induced apoptosis is still controversial. Apoptosis signal-regulating kinase1 (ASK1) is a ubiquitously expressed mitogen-activated protein kinase kinase kinase (MAPKKK) that activates the SEK1-c-Jun N-terminal kinase (JNK) and MKK3/MKK6-p38 MAPK signaling cascades. ASK1 has been implicated in cytokine- and stress-induced apoptosis. Here, we show the following: (1) IV infection activated ASK1 and concomitantly phosphorylated JNK and p38 MAPK in human bronchial epithelial cells; (2) the activation of JNK and p38 MAPK but not extracellular-regulated kinase (ERK) in embryonic fibroblasts (MEFs) derived from ASK1 knockout mice (ASK1(-/-) MEFs) was depressed compared to MEFs derived from wild type mice (ASK1(+/+) MEFs); and (3) ASK1(-/-) MEFs were defective in IV infection-induced caspase-3 activation and cell death. These results indicate that apoptosis in IV-infected BEC is mediated through ASK1-dependent cascades.     

Critical role of ASK1 in the 6-hydroxydopamine-induced apoptosis in human neuroblastoma SH-SY5Y cells

6-hydroxydopamine (6-OHDA)-induced apoptosis in dopaminergic neuronal cells is a common cell model of Parkinson's disease (PD). The role of apoptosis signal-regulating kinase 1 (ASK1) in this model has not been well studied. We observed significant activation of ASK1, p38 and JNK, as well as apoptosis in human dopaminergic neuroblastoma SH-SY5Y cells exposed to 6-OHDA. Over-expressing kinase-dead mutant ASK1(K709M) or knock-down of endogenous ASK1 by its small interfering RNA (siRNA) greatly suppressed activation of these kinases and apoptosis in the cells. It was found that the activation of p38 and JNK was suppressed to almost the same extent as that of ASK1 in the ASK1-knock-down cells, suggesting that activated ASK1 is almost totally responsible for activation of p38/JNK. It was also observed that the 6-OHDA-induced cell apoptosis could be effectively prevented by over-expressing the dominant-negative mutant of p38 or p38 inhibitor SB203580, demonstrating that activation of p38/JNK signalling is required for initiating the programmed cell death. Furthermore, suppression of the 6-OHDA-generated reactive oxygen species (ROS) by pre-incubation of cells with N-acetyl-L-cysteine effectively inhibited the 6-OHDA-induced activation of ASK1, p38 and JNK, and protected the cells from apoptosis. This study clearly shows the route from ROS generation by 6-OHDA to initiation of p38/JNK signalling via activation of ASK1 in the studied PD model.     

Mammalian thioredoxin is a direct inhibitor of apoptosis signal-regulating kinase (ASK) 1.

Apoptosis signal-regulating kinase (ASK) 1 was recently identified as a mitogen-activated protein (MAP) kinase kinase kinase which activates the c-Jun N-terminal kinase (JNK) and p38 MAP kinase pathways and is required for tumor necrosis factor (TNF)-alpha-induced apoptosis; however, the mechanism regulating ASK1 activity is unknown. Through genetic screening for ASK1-binding proteins, thioredoxin (Trx), a reduction/oxidation (redox)-regulatory protein thought to have anti-apoptotic effects, was identified as an interacting partner of ASK1. Trx associated with the N-terminal portion of ASK1 in vitro and in vivo. Expression of Trx inhibited ASK1 kinase activity and the subsequent ASK1-dependent apoptosis. Treatment of cells with N-acetyl-L-cysteine also inhibited serum withdrawal-, TNF-alpha- and hydrogen peroxide-induced activation of ASK1 as well as apoptosis. The interaction between Trx and ASK1 was found to be highly dependent on the redox status of Trx. Moreover, inhibition of Trx resulted in activation of endogenous ASK1 activity, suggesting that Trx is a physiological inhibitor of ASK1. The evidence that Trx is a negative regulator of ASK1 suggests possible mechanisms for redox regulation of the apoptosis signal transduction pathway as well as the effects of antioxidants against cytokine- and stress-induced apoptosis.     

Negative regulation of ASK1 by p21Cip1 involves a small domain that includes Serine 98 that is phosphorylated by ASK1 in vivo

The cyclin-dependent kinase inhibitor p21(Cip1) regulates multiple cellular functions and protects cells from genotoxic and other cellular stresses. Activation of apoptosis signal-regulating kinase 1 (ASK1) induced by inhibition of mTOR signaling leads to sustained phospho-c-Jun that is suppressed in cells with functional p53 or by forced expression of p21(Cip1). Here we show that small deletions of p21(Cip1) around S98 abrogate its association with ASK1 but do not affect binding to Cdk1, hence distinguishing between the cell cycle-regulating functions of p21(Cip1) and its ability to suppress activation of the ASK1/Jun N-terminal protein kinase (JNK) pathway. p21(Cip1) is phosphorylated in vitro by both ASK1 and JNK1 at S98. In vivo phosphorylation of p21(Cip1), predominantly carried out by ASK1, is associated with binding to ASK1 and inactivation of ASK1 kinase function. Binding of p21(Cip1) to ASK1 requires ASK1 kinase function and may involve phosphorylation of S98.     

Roles of MAPKKK ASK1 in stress-induced cell death

Apoptosis signal-regulating kinase 1 (ASK1) is a ubiquitously expressed mitogen-activated protein (MAP) kinase kinase kinase that activates the c-Jun N-terminal kinase (JNK) and p38 MAP kinase signaling cascades. Recent findings from analyses of ASK1-deficient mice have revealed that ASK1 is required for apoptosis induced by oxidative stress, TNF and endoplasmic reticulum (ER) stress. In addition, several lines of evidence have suggested that ASK1 has diverse functions in the decision of cell fate beyond its pro-apoptotic activity. Thus, ASK1 appears to be a pivotal component not only in stress-induced cell death but also in a broad range of biological activities in order for cells to adapt to or oppose various stresses.     

Superoxide-mediated early oxidation and activation of ASK1 are important for initiating methylglyoxal-induced apoptosis process

Methylglyoxal (MG) is a physiological metabolite, but it is known to be toxic, inducing stress and causing apoptosis. Our previous studies demonstrated that MG induced apoptosis in Jurkat cells by activating the c-Jun N-terminal kinase (JNK) signal transduction pathway, which induced an obvious decrease in mitochondrial membrane potential, followed by caspase-3 activation. Here, we observed that MG-induced apoptosis was associated with both rapid production of superoxide anion (O(2)(-)) followed by a marked increase in ROS and striking and temporal activation of ASK1. Overexpression of wild-type ASK1 could enhance the rate of apoptosis induced by MG, whereas the expression of the kinase-inactive form of ASK1 notably prevented cells from MG-induced death. NAC and PDTC blocked the activation of ASK1 and MG-induced apoptosis completely. Moreover, nonthiol antioxidants SOD-mimic MnTBAP and catalase together obviously inhibited MG-induced ASK1 activation and apoptosis induction. Correspondingly, MG-mediated ASK1 activation was enhanced by diethyldithiocarbamate (DDC). Addition of antioxidant into the culture of cells at a later stage (4-8 h after the initial MG treatment) failed to prevent their death. These results suggest that activating ASK1 at the early stage linking to production of O(2)(-) is crucial for subsequent progression of apoptosis in MG-treated Jurkat cells.     

ASK1-p38 MAPK/JNK signaling cascade mediates anandamide-induced PC12 cell death

Anandamide is a neuroimmunoregulatory molecule that triggers apoptosis in a number of cell types including PC12 cells. Here, we investigated the molecular mechanisms underlying anandamide-induced cell death in PC12 cells. Anandamide treatment resulted in the activation of p38 mitogen-activated protein kinase (MAPK), c-Jun N-terminal kinase (JNK), and p44/42 MAPK in apoptosing cells. A selective p38 MAPK inhibitor, SB203580, or dn-JNK, JNK1(A-F) or SAPKbeta(K-R), blocked anandamide-induced cell death, whereas a specific inhibitor of MEK-1/2, U0126, had no effect, indicating that activation of p38 MAPK and JNK is critical in anandamide-induced cell death. An important role for apoptosis signal-regulating kinase 1 (ASK1) in this event was also demonstrated by the inhibition of p38 MAPK/JNK activation and death in cells overexpressing dn-ASK1, ASK1 (K709M). Conversely, the constitutively active ASK1, ASK1DeltaN, caused prolonged p38 MAPK/JNK activation and increased cell death. These indicate that ASK1 mediates anandamide-induced cell death via p38 MAPK and JNK activation. Here, we also found that activation of p38 MAPK/JNK is accompanied by cytochrome c release from the mitochondria and caspase activation (which can be inhibited by SB203580), suggesting that anandamide triggers a mitochondrial dependent apoptotic pathway. The caspase inhibitor, zVAD, and the mitochondrial pore opening inhibitor, cyclosporine A, blocked anandamide-induced cell death but not p38 MAPK/JNK activation, suggesting that activation of these kinases may occur upstream of mitochondrial associated events.     

ASK1 is required for sustained activations of JNK/p38 MAP kinases and apoptosis

Apoptosis signal-regulating kinase (ASK) 1 is activated in response to various cytotoxic stresses including TNF, Fas and reactive oxygen species (ROS) such as H₂O₂, and activates c-Jun NH₂-terminal kinase (JNK) and p38. However, the roles of JNK and p38 signaling pathways during apoptosis have been controversial. Here we show that by deleting ASK1 in mice, TNF- and H₂O₂-induced sustained activations of JNK and p38 are lost in ASK1^–/– embryonic fibroblasts, and that ASK1^–/– cells are resistant to TNF- and H₂O₂-induced apoptosis. TNF- but not Fas-induced apoptosis requires ROS-dependent activation of ASK1–JNK/p38 pathways. Thus, ASK1 is selectively required for TNF- and oxidative stress-induced sustained activations of JNK/p38 and apoptosis.     

Positive regulation of ASK1-mediated c-Jun NH(2)-terminal kinase signaling pathway by the WD-repeat protein Gemin5

Gemin5 is a 170-kDa WD-repeat-containing protein that was initially identified as a component of the survival of motor neurons (SMN) complex. We now show that Gemin5 facilitates the activation of apoptosis signal-regulating kinase 1 (ASK1) and downstream signaling. Gemin5 physically interacted with ASK1 as well as with the downstream kinases SEK1 and c-Jun NH(2)-terminal kinase (JNK1), and it potentiated the H(2)O(2)-induced activation of each of these kinases in intact cells. Moreover, Gemin5 promoted the binding of ASK1 to SEK1 and to JNK1, as well as the ASK1-induced activation of JNK1. In comparison, Gemin5 did not physically associate with MKK7, MKK3, MKK6, or p38. Furthermore, depletion of endogenous Gemin5 by RNA interference (RNAi) revealed that Gemin5 contributes to the activation of ASK1 and JNK1, and to apoptosis induced by H(2)O(2) and tumor necrosis factor-alpha (TNFalpha) in HeLa cells. Together, our results suggest that Gemin5 functions as a scaffold protein for the ASK1-JNK1 signaling module and thereby potentiates ASK1-mediated signaling events.     

Amyloid beta induces neuronal cell death through ROS-mediated ASK1 activation

Amyloid beta (Abeta) is a main component of senile plaques in Alzheimer's disease and induces neuronal cell death. Reactive oxygen species (ROS), nitric oxide and endoplasmic reticulum (ER) stress have been implicated in Abeta-induced neurotoxicity. We have reported that apoptosis signal-regulating kinase 1 (ASK1) is required for ROS- and ER stress-induced JNK activation and apoptosis. Here we show the involvement of ASK1 in Abeta-induced neuronal cell death. Abeta activated ASK1 mainly through production of ROS but not through ER stress in cultured neuronal cells. Importantly, ASK1-/- neurons were defective in Abeta-induced JNK activation and cell death. These results indicate that ROS-mediated ASK1 activation is a key mechanism for Abeta-induced neurotoxicity, which plays a central role in Alzheimer's disease.     

A novel function of peroxiredoxin 1 (Prx-1) in apoptosis signal-regulating kinase 1 (ASK1)-mediated signaling pathway

We report a novel function of peroxiredoxin-1 (Prx-1) in the ASK1-mediated signaling pathway. Prx-1 interacts with ASK1 via the thioredoxin-binding domain of ASK1 and this interaction is highly inducible by H2O2. However, catalytic mutants of Prx1, C52A, C173A, and C52A/C173A, could not undergo H2O2 inducible interactions, indicating that the redox-sensitive catalytic activity of Prx-1 is required for the interaction with ASK1. Prx-1 overexpression inhibited the activation of ASK1, and resulted in the inhibition of downstream signaling cascades such as the MKK3/6 and p38 pathway. In Prx-1 knockdown cells, ASK1, p38, and JNK were quickly activated, leading to apoptosis in response to H2O2. These findings suggest a negative role of Prx-1 in ASK1-induced apoptosis.     

ASK1 resistant neuroblastoma is deficient in activation of p38 kinase

Apoptosis Signal-regulating Kinase 1 (ASK1) is known to either induce apoptosis or differentiation in various cell lines of neuronal origin. We analyzed the effect of the constitutively active mutant of ASK1 (ASK1-Delta N) in an adenoviral vector in four neuroblastoma cell lines, two murine, C1300 and NXS2, and two human, SH-SY5Y and IMR-32. Already after 24 h upon infection, C1300 and SH-SY5Y cells arrested in growth when judged by [(3)H]thymidine incorporation, and the majority of the cells demonstrated apoptotic appearance, which was confirmed by DNA-laddering in gel electrophoresis. In contrast, NXS2 and IMR-32 cell lines remained unaffected. Immunoblotting revealed strongly phosphorylated p38 MAPK accompanied by weakly phosphorylated JNK in C1300 and SH-SY5Y, whereas none of these kinases were activated by adenoviruses expressing the kinase negative ASK1 mutant or beta-galactosidase. There was no expression of phosphorylated kinases in IMR-32 cells, but NXS2 showed a faint band of phosphorylated p38 MAPK. Addition of the p38 MAPK specific inhibitor, SB203580, protected C1300 and SH-SY5Y cells from apoptosis induced by ASK1-Delta N. The anti-neoplastic agent, paclitaxel, activates ASK1 and JNK, and promotes the in vitro assembly of stable microtubules. Addition of 10 nM paclitaxel sensitised the NXS2 cell line to ASK1-induced cell death. Our results indicate that ASK1 induces apoptosis in neuroblastoma cells mainly via the p38 MAPK pathway, and resistant neuroblastoma cells can be sensitised to ASK1 by paclitaxel.     

Apoptosis signal-regulating kinase (ASK) 2 functions as a mitogen-activated protein kinase kinase kinase in a heteromeric complex with ASK1

Apoptosis signal-regulating kinase (ASK) 1 is a mitogen-activated protein kinase kinase kinase (MAP3K) in the c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase pathways that play multiple important roles in cytokine and stress responses. Here we show that ASK2, a highly related serine/threonine kinase to ASK1, also functions as a MAP3K only in a heteromeric complex with ASK1. We found that endogenous ASK2 was constitutively degraded in ASK1-deficient cells, suggesting that ASK1 is required for the stability of ASK2. ASK2 in a heteromeric complex with a kinase-negative mutant of ASK1 (ASK1-KN) effectively activated MAP2K and was more competent to respond to oxidative stress than ASK2 alone. Knockdown of ASK2 revealed that ASK2 was required for oxidative stress-induced JNK activation. These results suggest that ASK2 forms a functional MAP3K complex with ASK1, in which ASK1 supports the stability and the active configuration of ASK2. Moreover, ASK2 was found to activate ASK1 by direct phosphorylation, suggesting that ASK1 and ASK2 in a heteromeric complex facilitate their activities to each other by distinct mechanisms. Such a formation of functional heteromeric complex between different MAP3Ks may be advantageous for cells to cope with a wide variety of stimuli by fine regulation of cellular responses.     

Cellular iron depletion stimulates the JNK and p38 MAPK signaling transduction pathways, dissociation of ASK1-thioredoxin, and activation of ASK1

The role of signaling pathways in the regulation of cellular iron metabolism is becoming increasingly recognized. Iron chelation is used for the treatment of iron overload but also as a potential strategy for cancer therapy, because iron depletion results in cell cycle arrest and apoptosis. This study examined potential signaling pathways affected by iron depletion induced by desferrioxamine (DFO) or di-2-pyridylketone-4,4-dimethyl-3-thiosemicarbazone (Dp44mT). Both chelators affected multiple molecules in the mitogen-activated protein kinase (MAPK) pathway, including a number of dual specificity phosphatases that directly de-phosphorylate MAPKs. Examination of the phosphorylation of major MAPKs revealed that DFO and Dp44mT markedly increased phosphorylation of stress-activated protein kinases, JNK and p38, without significantly affecting the extracellular signal-regulated kinase (ERK). Redox-inactive DFO-iron complexes did not affect phosphorylation of JNK or p38, whereas the redox-active Dp44mT-iron complex significantly increased the phosphorylation of these kinases similarly to Dp44mT alone. Iron or N-acetylcysteine supplementation reversed Dp44mT-induced up-regulation of phospho-JNK, but only iron was able to reverse the effect of DFO on JNK. Both iron chelators significantly reduced ASK1-thioredoxin complex formation, resulting in the increased phosphorylation of ASK1, which activates the JNK and p38 pathways. Thus, dissociation of ASK1 could serve as an important signal for the phosphorylation of JNK and p38 activation observed after iron chelation. Phosphorylation of JNK and p38 likely play an important role in mediating the cell cycle arrest and apoptosis induced by iron depletion.     

Type 1 insulin-like growth factor receptor (IGF-IR) signaling inhibits apoptosis signal-regulating kinase 1 (ASK1)

The type 1 insulin-like growth factor receptor (IGF-IR) is a receptor-tyrosine kinase that plays a critical role in signaling cell survival and proliferation. IGF-IR binding to its ligand, insulin-like growth factor (IGF-I) activates phosphoinositide 3-kinase (PI3K), promotes cell proliferation by activating the mitogen-activated protein kinase (MAPK) cascade, and blocks apoptosis by inducing the phosphorylation and inhibition of proapoptotic proteins such as BAD. Apoptosis signal-regulating kinase 1 (ASK1) is a MAP kinase kinase kinase (MAPKKK) that is required for c-Jun N-terminal kinase (JNK) and p38 activation in response to Fas and tumor necrosis factor (TNF) receptor stimulation, and for oxidative stress- and TNFalpha-induced apoptosis. The results presented here indicate that ASK1 forms a complex with the IGF-IR and becomes phosphorylated on tyrosine residue(s) in a manner dependent on IGF-IR activity. IGF-IR signaling inhibited ASK1 irrespective of TNFalpha-induced ASK1 activation and resulted in decreased ASK1-dependent JNK1 stimulation. Signaling through IGF-IR rescued cells from ASK1-induced apoptotic cell death in a manner independent of PI3K activity. These results indicate that IGF-IR signaling suppresses the ASK-1-mediated stimulation of JNK/p38 and the induction of programmed cell death. The simultaneous activation of MAP kinases and the inhibition of the stress-activated arm of the cascade by IGF-IR may constitute a potent proliferative signaling system and is possibly a mechanism by which IGF-I can stimulate growth and inhibit cell death in a wide variety of cell types and biological settings.     

Apoptosis signal-regulating kinase (ASK)-1 mediates apoptosis through activation of JNK1 following engagement of membrane immunoglobulin

Engagement of membrane immunoglobulin (mIg) on WEHI-231 mouse B lymphoma cells results in growth arrest at the G1 phase of the cell cycle, followed by a reduction of mitochondrial membrane potential (ΔΨm) and apoptosis. WEHI-231 cells resemble immature B cells in terms of the cell surface phenotype and sensitivity to mIg engagement. However, the molecular mechanisms underlying mIg-induced loss of ΔΨm and apoptosis have not yet been established. In this study, we show that apoptosis signal-regulating kinase 1 (ASK1)-c-Jun N-terminal kinase 1 (JNK1) signaling pathway participates in mIg-induced apoptosis through the generation of reactive oxygen species (ROS). Stimulation of WEHI-231 cells with anti-IgM induces phosphorylation and subsequent activation of ASK1, leading to JNK activation. Anti-IgM stimulation immediately (5 min) induces hydrogen peroxide (H₂O₂) production with a substantial increase during later time points (36-48 h), accompanied by loss of ΔΨm and an increase in cells with sub-G1 DNA content. The anti-IgM-induced late-phase H₂O₂ production, loss of ΔΨm, and increase in the sub-G1 fraction were all reduced substantially in WEHI-231 cells overexpressing a dominant-negative form of ASK1, compared with control vector alone, but enhanced substantially in cells overexpressing a constitutively active form of ASK1. These mIg-mediated events were also partially abrogated by ROS scavenger N-acetyl-l-cysteine (NAC). Taken together, these results suggest that mIg engagement induces H₂O₂ production leading to activation of ASK1-JNK1 pathway, creating a feedback amplification loop of ROS-ASK/JNK that leads to loss of ΔΨm and finally apoptosis.