Phosphorylation-dependent scaffolding role of JSAP1/JIP3 in the ASK1-JNK signaling pathway. A new mode of regulation of the MAP kinase cascade

JSAP1 (also termed JIP3) is a scaffold protein that interacts with specific components of the JNK signaling pathway. Apoptosis signal-regulating kinase (ASK) 1 is a MAP kinase kinase kinase that activates the JNK and p38 mitogen-activated protein (MAP) kinase cascades in response to environmental stresses such as reactive oxygen species. Here we show that JSAP1 bound ASK1 and enhanced ASK1- and H(2)O(2)-induced JNK activity. ASK1 phosphorylated JSAP1 in vitro and in vivo, and the phosphorylation facilitated interactions of JSAP1 with SEK1/MKK4, MKK7 and JNK3. Furthermore, ASK1-dependent phosphorylation was required for JSAP1 to recruit and thereby activate JNK in response to H(2)O(2). We thus conclude that JSAP1 functions not only as a simple scaffold, but it dynamically participates in signal transduction by forming a phosphorylation-dependent signaling complex in the ASK1-JNK signaling module.     

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.     

Ursolic acid induces ER stress response to activate ASK1–JNK signaling and induce apoptosis in human bladder cancer T24 cells

Here we studied the cellular mechanisms of ursolic acid's anti-bladder cancer ability by focusing on endoplasmic reticulum stress (ER stress) signaling. We show that ursolic acid induces a significant ER stress response in cultured human bladder cancer T24 cells. ER stress inhibitor salubrinal, or PERK silencing, diminishes ursolic acid-induced anti-T24 cell effects. Salubrinal inhibits ursolic acid-induced CHOP expression, Bim ER accumulation and caspase-3 activation in T24 cells. Ursolic acid induces IRE1–TRAF2–ASK1 signaling complex formation to activate pro-apoptotic ASK1–JNK signaling. We suggest that ER stress contributes to ursolic acid's effects against bladder cancer cells.     

Role of Bim in diallyl trisulfide-induced cytotoxicity in human cancer cells

The aim of this study was to investigate the effect of garlic constituent diallyl trisulfide (DATS) on the cell‐death signaling pathway in a human breast cell line (MDA‐MB‐231). We observed that DATS (10–100 µM) treatment resulted in dose‐ and time‐dependent cytotoxicity. Treatment of MDA‐MB‐231 cells with a cytotoxicity inducing concentration of DATS (50–80 µM) resulted in an increase in the intracellular level of reactive oxygen species (ROS). Data from assay with MitoSOX^TM Red reagent suggest that mitochondria are the main source of ROS generation during DATS treatment. DATS‐induced oxidative stress was detected through glutaredoxin (GRX), a redox‐sensing molecule, and subsequently GRX was dissociated from apoptosis signal‐regulating kinase 1 (ASK1). Dissociation of GRX from ASK1 resulted in the activation of ASK1. ASK1 activated a downstream signal transduction JNK (c‐Jun N‐terminal kinase)‐Bim pathway. SP600125, a JNK inhibitor, inhibited DATS‐induced Bim phosphorylation and protected cells from DATS‐induced cytotoxicity. Our results indicate that the cytotoxicity caused by DATS is mediated by the generation of ROS and subsequent activation of the ASK1‐JNK‐Bim signal transduction pathway in human breast carcinoma MDA‐MB‐231 cells. J. Cell. Biochem. 112: 118–127, 2011. © 2010 Wiley‐Liss, Inc.     

Murine protein serine/threonine kinase 38 activates apoptosis signal-regulating kinase 1 via Thr 838 phosphorylation

Murine protein serine/threonine kinase 38 (MPK38) is a member of the AMP-activated protein kinase-related serine/threonine kinase family that plays an important role in various cellular processes, including cell cycle, signaling pathways, and self-renewal of stem cells. Here we demonstrate a functional association between MPK38 and apoptosis signal-regulating kinase 1 (ASK1). The physical association between MPK38 and ASK1 was mediated through their carboxyl-terminal regulatory domains and was increased by H(2)O(2) or tumor necrosis factor alpha treatment. The use of kinase-dead MPK38 and ASK1 mutants revealed that MPK38-ASK1 complex formation was dependent on the activities of both kinases. Ectopic expression of wild-type MPK38, but not kinase-dead MPK38, stimulated ASK1 activity by Thr(838) phosphorylation and enhanced ASK1-mediated signaling to both JNK and p38 kinases. However, the phosphorylation of MKK6 and p38 by MPK38 was not detectable. In addition, MPK38-mediated ASK1 activation was induced through the increased interaction between ASK1 and its substrate MKK3. MPK38 also stimulated H(2)O(2)-mediated apoptosis by enhancing the ASK1 activity through Thr(838) phosphorylation. These results suggest that MPK38 physically interacts with ASK1 in vivo and acts as a positive upstream regulator of ASK1.     

MPTP-induced reactive oxygen species promote cell death through a gradual activation of caspase-3 without expression of GRP78/Bip as a preventive measure against ER stress in PC12 cells

Glucose-regulated protein 78 (GRP78)/Immunoglobulin binding protein (Bip) is a chaperone which functions to protect cells from endoplasmic reticulum (ER) stress. GRP78/Bip is expressed following ER stress induced by thapsigargin, tunicamycin or chemical factors. However, the mechanism of progression of ER stress against stress factors is still obscure. We examined whether reactive oxygen species (ROS) were involved in GRP78/Bip expression and caspase-3 activity was induced in PC12 cells using 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) to produce ROS. We report that PC12 cells lost viability in the presence of MPTP for 24 hours as a partial effect of ROS. We also show that N-acetyl-L-cysteine diminished the MPTP-induced apoptosis with expunction of ROS. Furthermore, we observed that GRP78/Bip was not up-regulated and the caspase-3 activity was increased in the presence of MPTP. These results suggest that insubstantial ROS do not contribute to the ER stress-mediated cell death while caspase-3 is involved in ROS-promoted cell death in MPTP-treated cells.     

PUMA overexpression dissociates thioredoxin from ASK1 to activate the JNK/BCL-2/BCL-XL pathway augmenting apoptosis in ovarian cancer

ASK1-JNK signaling promotes mitochondrial dysfunction-mediated apoptosis, but the bridge between JNK and apoptosis is not fully understood. PUMA induces apoptosis through BAX/BAK. Our previous study suggests a therapeutic potential of PUMA for ovarian cancer. However, whether and how PUMA activates ASK1 remains unclear. Here, we found for the first time that PUMA activated ASK1 by dissociating thioredoxin (TRX) from ASK1, however, it neither interacted with ASK1 nor TRX. Furthermore, PUMA overexpression caused ROS release from mitochondrial. H₂O₂ significantly impaired the interaction of ASK1 with TRX, whereas ROS scavenger NAC effectively abrogated the H₂O₂ effect, partly rescued PUMA-interfered interaction of ASK1 with TRX, and also abolished ASK1 phosphorylation. Interestingly, PUMA could not impair the association of ASK1 with TRX-C32S or TRX-C35S, two TRX mutants which are no longer oxidized in response to ROS. We further showed that PUMA activated ASK1-JNK axis to phosphorylate BCL-2 and BCL-XL, further augmenting apoptosis of ovarian cancer cells. In vivo, PUMA adenovirus combined with paclitaxel significantly inhibited intrinsically cisplatin-resistant ovarian cancer growth, and caused phosphorylation of BCL-2 and BCL-XL. Our results from human ovarian cancer TMA chips also revealed a positive correlation between PUMA expression and the phosphorylation of BCL-2 and BCL-XL. More importantly, all patients had no distal metastasis, implying a possibly clinical significance. Collectively, our results reveal a new pro-apoptotic signal amplification mechanism for PUMA by which PUMA overexpression first induces ROS-mediated dissociation of TRX from ASK1, and then causes JNK activation-triggering BCL-2/BCL-XL phosphorylation, ultimately augmenting apoptosis in ovarian cancer.     

Arrestin-3 Binds c-Jun N-terminal Kinase 1 (JNK1) and JNK2 and Facilitates the Activation of These Ubiquitous JNK Isoforms in Cells via Scaffolding

Non-visual arrestins scaffold mitogen-activated protein kinase (MAPK) cascades. The c-Jun N-terminal kinases (JNKs) are members of MAPK family. Arrestin-3 has been shown to enhance the activation of JNK3, which is expressed mainly in neurons, heart, and testes, in contrast to ubiquitous JNK1 and JNK2. Although all JNKs are activated by MKK4 and MKK7, both of which bind arrestin-3, the ability of arrestin-3 to facilitate the activation of JNK1 and JNK2 has never been reported. Using purified proteins we found that arrestin-3 directly binds JNK1α1 and JNK2α2, interacting with the latter comparably to JNK3α2. Phosphorylation of purified JNK1α1 and JNK2α2 by MKK4 or MKK7 is increased by arrestin-3. Endogenous arrestin-3 interacted with endogenous JNK1/2 in different cell types. Arrestin-3 also enhanced phosphorylation of endogenous JNK1/2 in intact cells upon expression of upstream kinases ASK1, MKK4, or MKK7. We observed a biphasic effect of arrestin-3 concentrations on phosphorylation of JNK1α1 and JNK2α2 both in vitro and in vivo. Thus, arrestin-3 acts as a scaffold, facilitating JNK1α1 and JNK2α2 phosphorylation by MKK4 and MKK7 via bringing JNKs and their activators together. The data suggest that arrestin-3 modulates the activity of ubiquitous JNK1 and JNK2 in non-neuronal cells, impacting the signaling pathway that regulates their proliferation and survival.     

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.     

The cadmium-induced death of mesangial cells results in nephrotoxicity

This study summarizes our most recent findings on the mechanisms underlying the cadmium-induced death of mesangial cells, which leads to nephrotoxicity. Multiple pathways participate in cadmium-induced nephrotoxicity. In the ROS-GSK-3β autophagy pathway, cadmium induces ROS most likely from the mitochondria, and the ROS consequently activate GSK-3β leading to autophagic cell death. In the calcium-ERK autophagy and apoptosis pathway, cadmium stimulates calcium release from the endoplasmic reticulum, which activates ERK leading to predominantly autophagic cell death and a minor level of apoptotic cell death. In the calcium-mitochondria-caspase apoptosis pathway, cadmium-induced elevation of calcium depolarizes the mitochondrial membrane potential and then activates caspase signaling leading to apoptosis. A proposed model for cadmium-induced autophagy and apoptosis leading to nephrotoxicity is summarized in Figure 1.     

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.     

Inhibition of endoplasmic reticulum stress counteracts neuronal cell death and protein aggregation caused by N-terminal mutant huntingtin proteins

Accumulation of abnormal proteins occurs in many neurodegenerative diseases including Huntington's disease (HD). However, the precise role of protein aggregation in neuronal cell death remains unclear. We show here that the expression of N-terminal huntingtin proteins with expanded polyglutamine (polyQ) repeats causes cell death in neuronal PC6.3 cell that involves endoplasmic reticulum (ER) stress. These mutant huntingtin fragment proteins elevated Bip, an ER chaperone, and increased Chop and the phosphorylation of c-Jun-N-terminal kinase (JNK) that are involved in cell death regulation. Caspase-12, residing in the ER, was cleaved in mutant huntingtin expressing cells, as was caspase-3 mediating cell death. In contrast, cytochrome-c or apoptosis inducing factor (AIF) was not released from mitochondria after the expression of these proteins. Treatment with salubrinal that inhibits ER stress counteracted cell death and reduced protein aggregations in the PC6.3 cells caused by the mutant huntingtin fragment proteins. Salubrinal upregulated Bip, reduced cleavage of caspase-12 and increased the phosphorylation of eukaryotic translation initiation factor-2 subunit-alpha (eIF2alpha) that are neuroprotective. These results show that N-terminal mutant huntingtin proteins activate cellular pathways linked to ER stress, and that inhibition of ER stress by salubrinal increases cell survival. The data suggests that compounds targeting ER stress may be considered in designing novel approaches for treatment of HD and possibly other polyQ diseases.     

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.     

NR4A1 counteracts JNK activation incurred by ER stress or ROS in pancreatic β‐cells for protection

Sustained hyperglycaemia and hyperlipidaemia incur endoplasmic reticulum stress (ER stress) and reactive oxygen species (ROS) overproduction in pancreatic β‐cells. ER stress or ROS causes c‐Jun N‐terminal kinase (JNK) activation, and the activated JNK triggers apoptosis in different cells. Nuclear receptor subfamily 4 group A member 1 (NR4A1) is an inducible multi‐stress response factor. The aim of this study was to explore the role of NR4A1 in counteracting JNK activation induced by ER stress or ROS and the related mechanism. qPCR, Western blotting, dual‐luciferase reporter and ChIP assays were applied to detect gene expression or regulation by NR4A1. Immunofluorescence was used to detect a specific protein expression in β‐cells. Our data showed that NR4A1 reduced the phosphorylated JNK (p‐JNK) in MIN6 cells encountering ER stress or ROS and reduced MKK4 protein in a proteasome‐dependent manner. We found that NR4A1 increased the expression of cbl‐b (an E3 ligase); knocking down cbl‐b expression increased MKK4 and p‐JNK levels under ER stress or ROS conditions. We elucidated that NR4A1 enhanced the transactivation of cbl‐b promoter by physical association. We further confirmed that cbl‐b expression in β‐cells was reduced in NR4A1‐knockout mice compared with WT mice. NR4A1 down‐regulates JNK activation by ER stress or ROS in β‐cells via enhancing cbl‐b expression.     

The beta1 integrin activates JNK independent of CagA, and JNK activation is required for Helicobacter pylori CagA+-induced motility of gastric cancer cells

The Helicobacter pylori CagA protein is translocated into gastric epithelial cells through a type IV secretion system (TFSS), and published studies suggest CagA is critical for H. pylori-associated carcinogenesis. CagA is thought to be necessary and sufficient to induce the motogenic response observed in response to CagA+ strains, as CagA interacts with proteins involved in adhesion and motility. We report that H. pylori strain 60190 stimulated AGS cell motility through a CagA- and TFSS-dependent mechanism, because strains 60190DeltacagA or 60190DeltacagE (TFSS-defective) did not increase motility. The JNK pathway is critical for H. pylori-dependent cell motility, as inhibition using SP600125 (JNK1/2/3 inhibitor) or a JNK2/3-specific inhibitor blocked motility. JNK mediates H. pylori-induced cell motility by activating paxillin, because JNK inhibition blocked paxillinTyr-118 phosphorylation, and paxillin expression knockdown completely abrogated bacteria-induced motility. Furthermore, JNK and paxillinTyr-118 were activated by 60190DeltacagA but not 60190DeltacagE, demonstrating CagA-independent signaling critical for cell motility. A beta1 integrin-blocking antibody significantly inhibited JNK and paxillinTyr-118 phosphorylation and cell scattering, demonstrating that CagA-independent signaling required for cell motility occurs through beta1. The requirement of both Src and focal adhesion kinase for signaling and motility further suggests the importance of integrin signaling in H. pylori-induced cell motility. Finally, we show that JNK activation occurs independent of known upstream kinases and signaling molecules, including Nod1, Cdc42, Rac1, MKK4, and MKK7, which demonstrates novel signaling leading to JNK activation. We report for the first time that H. pylori mediates CagA-independent signaling that promotes cell motility through the beta1 integrin pathway.