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.     

ASK1 and its role in cardiovascular and other disorders: available treatments and future prospects

ABSTRACT

Introduction: Apoptosis signal-regulating kinase 1 (ASK1), also known as MAP3K5, is a member of mitogen-activated protein kinase kinase kinase (MAP3K) family and is well reported as crucial in the regulation of the JNK and P38 pathways. ASK1 is activated in response to a diverse array of stresses such as endoplasmic reticulum stress, lipopolysaccharides, tumor necrosis factor alpha, and reactive oxygen species. The activation of ASK1 induces various stress responses.

Areas covered: Considering ASK1 as an important therapeutic drug target, here we have discussed the role of ASK1 in the progression of various diseases. We have also provided an overview of the available inhibitors for ASK1. The success of computational-based approaches toward ASK1 inhibitor design has also been discussed.

Expert opinion: A number of reports have outlined the prominent role of ASK1 in the pathogenesis of several diseases. The discovery of novel ASK1 inhibitors would have a wide range of applications in medical science. In-silico techniques have been successfully used in the design of some novel ASK1 inhibitors. The use of machine learning-based approaches in combination with structure-based virtual screening (SBVS) and ligand-based virtual screening (LBVS) will be helpful toward the development of potent ASK1 inhibitors.     

ASK1 Negatively Regulates the 26 S Proteasome

The 26 S proteasome, composed of the 20 S core and 19 S regulatory particle, plays a central role in ubiquitin-dependent proteolysis. Disruption of this process contributes to the pathogenesis of the various diseases; however, the mechanisms underlying the regulation of 26 S proteasome activity remain elusive. Here, cell culture experiments and in vitro assays demonstrated that apoptosis signal-regulating kinase 1 (ASK1), a member of the MAPK kinase kinase family, negatively regulated 26 S proteasome activity. Immunoprecipitation/Western blot analyses revealed that ASK1 did not interact with 20 S catalytic core but did interact with ATPases making up the 19 S particle, which is responsible for recognizing polyubiquitinated proteins, unfolding them, and translocating them into the 20 S catalytic core in an ATP-dependent process. Importantly, ASK1 phosphorylated Rpt5, an AAA ATPase of the 19 S proteasome, and inhibited its ATPase activity, an effect that may underlie the ability of ASK1 to inhibit 26 S proteasome activity. The current findings point to a novel role for ASK1 in the regulation of 26 S proteasome and offer new strategies for treating human diseases caused by proteasome malfunction.     

ASK3, a novel member of the apoptosis signal-regulating kinase family, is essential for stress-induced cell death in HeLa cells

Apoptosis signal-regulating kinase 1 (ASK1) and ASK2 are both members of mitogen-activated protein kinase kinase kinase (MAP3K) family that are implicated in apoptotic cell death, stress responses, and various diseases. We have determined that NT2RI3007443, TESTI4031745, SGK341, and human MAP3K15 are all transcribed from the same genomic locus, which we designate “ASK3 gene” based on sequence homology to ASK1 and ASK2. NT2RI3007443, TESTI4031745, and SGK341 displayed distinct expression profiles among human tissues. TESTI4031745 was expressed in relatively high levels. The expression of TESTI4031745 was increased in rectum tumor and Alzheimer’s disease hippocampus and decreased in kidney tumor and Alzheimer’s disease frontal lobe. NT2RI3007443 showed moderate levels of ubiquitous expression in normal adult tissues. They did not drastically change in diseases except for increase in cirrhosis liver. Expression of SGK341 was restricted. It was highly expressed in fetal brain, and moderately expressed in normal hippocampus, pancreas, spleen, lung, and kidney. Further, its expression was dramatically increased in hepatic cirrhosis and decreased in lung tumor. Target proteins encoded by NT2RI3007443 and TESTI4031745 were translated in cell-free protein synthesis system. They exhibited protein kinase activity indicated by ATP consumption and phosphorylation of Syntide 2 as a substrate. We demonstrated that knockdown of ASK3 protected HeLa cells against cytotoxicity induced by anti-Fas monoclonal antibody, TNF-alpha, or oxidative stress. These findings suggest that “ASK3 gene” is a novel member of apoptosis signal-regulating kinases and that it plays a pivotal role in the signal transduction pathway implicated in apoptotic cell death triggered by cellular stresses. It can be a putative therapeutic drug target for multiple human diseases.     

Redox control of cell fate by MAP kinase: physiological roles of ASK1-MAP kinase pathway in stress signaling

The intracellular redox state is a key determinant of cell fate, such as cell survival, proliferation, differentiation, and apoptosis. Redox imbalance is closely linked to a variety of human diseases, so that the intracellular redox condition should be tightly regulated. The redox state of the cell is a consequence of the precise balance between the levels of oxidizing and reducing equivalents, such as reactive oxygen species (ROS) and endogenous antioxidants. ROS are not only toxicants to the cell, but also second messengers in intracellular signal transduction, and control the action of several signaling pathways, including mitogen-activated protein (MAP) kinases. Apoptosis signal-regulating kinase 1 (ASK1) is a MAP kinase kinase kinase of the c-Jun N-terminal kinase (JNK) and p38 MAP kinase pathways, which is preferentially activated in response to various types of stress such as oxidative stress and plays pivotal roles in a wide variety of cellular responses. Recent studies have revealed that ASK1 is also required for innate immune response through ROS production. In this review, we focus on redox control of cell function by MAP kinase signaling, and provide the advanced mechanism of redox-regulated ASK1 activation and physiological roles of the ASK1-MAP kinase pathway in stress signaling.     

Deletion of ASK1 Protects against Hyperoxia-Induced Acute Lung Injury

Apoptosis signal-regulating kinase 1 (ASK1), a member of the MAPK kinase kinase kinase (MAP3K) family, is activated by various stimuli, which include oxidative stress, endoplasmic reticulum (ER) stress, calcium influx, DNA damage-inducing agents and receptor-mediated signaling through tumor necrosis factor receptor (TNFR). Inspiration of a high concentration of oxygen is a palliative therapy which counteracts hypoxemia caused by acute lung injury (ALI)-induced pulmonary edema. However, animal experiments so far have shown that hyperoxia itself could exacerbate ALI through reactive oxygen species (ROS). Our previous data indicates that ASK1 plays a pivotal role in hyperoxia-induced acute lung injury (HALI). However, it is unclear whether or not deletion of ASK1 in vivo protects against HALI. In this study, we investigated whether ASK1 deletion would lead to attenuation of HALI. Our results show that ASK1 deletion in vivo significantly suppresses hyperoxia-induced elevation of inflammatory cytokines (i.e. IL-1β and TNF-α), cell apoptosis in the lung, and recruitment of immune cells. In summary, the results from the study suggest that deletion of ASK1 in mice significantly inhibits hyperoxic lung injury.     

Biophysical and Structural Characterization of the Thioredoxin-binding Domain of Protein Kinase ASK1 and Its Interaction with Reduced Thioredoxin

Apoptosis signal-regulating kinase 1 (ASK1), a mitogen-activated protein kinase kinase kinase, plays a key role in the pathogenesis of multiple diseases. Its activity is regulated by thioredoxin (TRX1) but the precise mechanism of this regulation is unclear due to the lack of structural data. Here, we performed biophysical and structural characterization of the TRX1-binding domain of ASK1 (ASK1-TBD) and its complex with reduced TRX1. ASK1-TBD is a monomeric and rigid domain that forms a stable complex with reduced TRX1 with 1:1 molar stoichiometry. The binding interaction does not involve the formation of intermolecular disulfide bonds. Residues from the catalytic WCGPC motif of TRX1 are essential for complex stability with Trp³¹ being directly involved in the binding interaction as suggested by time-resolved fluorescence. Small-angle x-ray scattering data reveal a compact and slightly asymmetric shape of ASK1-TBD and suggest reduced TRX1 interacts with this domain through the large binding interface without inducing any dramatic conformational change.     

Apoptosis signal-regulating kinase 1 attenuates atrial natriuretic peptide secretion

Atrial natriuretic peptide (ANP) is an endogenous peptide hormone that is synthesized and secreted by the myocardium in health and disease. Although the bioactivity of this molecule has been studied extensively, cellular mechanisms governing its processing and secretion are not fully understood. Through a yeast two-hybrid screen of a cDNA library made from tissue of a failing human heart, we have discovered that the precursor of ANP, natriuretic peptide precursor (NPPA), physically interacts with the N-terminus of apoptosis signal-regulating kinase 1 (ASK1), a kinase believed to be involved in the pathogenesis of heart failure. We demonstrated that NPPA is a substrate of ASK1 in an in vitro kinase assay. Indirect immunofluorescence microscopy shows that, when expressed in Hela cells, ASK1 and NPPA exhibit distinct, but overlapping, staining patterns, suggesting partial colocalization in cells. Additionally, coexpressing wild-type ASK1 with NPPA in Hela cells led to reduced levels of NPPA in the culture medium, suggesting that ASK1 negatively impacts NPPA processing and/or secretion. This negative effect was less pronounced when a dominant-negative allele of ASK1 with deficient kinase activity was coexpressed with NPPA. Because both ASK1 and ANP are associated with pathologic cardiac hypertrophy, their interaction may have pathophysiological and therapeutic relevance.     

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.     

ASK Family Proteins in Stress Response and Disease

Cells are continuously exposed to reactive oxygen species (ROS) generated by aerobic metabolism. Excessively generated ROS causes severe dysfunctions to cells as oxidative stress. On the other hand, there is increasing evidence that ROS plays important roles as a signaling intermediate that induces a wide variety of cellular responses such as proliferation, differentiation, senescence, and apoptosis. To transmit physiological ROS-mediated signals and to adapt to oxidative stress, cells are equipped with various intracellular signal transduction systems, represented by mitogen-activated protein kinase (MAPK) cascades. Apoptosis signal-regulating kinase 1 (ASK1) is an upstream regulator of the stress-activated MAPK cascades and has been shown to play critical roles in ROS-mediated cellular responses. Here, we highlight the roles of members of the ASK family, which consists of ASK1 and newly characterized ASK2, in ROS signaling with their possible involvement in human diseases.     

Structural and functional characterization of the human protein kinase ASK1

Apoptosis signal-regulating kinase 1 (ASK1) plays an essential role in stress and immune response and has been linked to the development of several diseases. Here, we present the structure of the human ASK1 catalytic domain in complex with staurosporine. Analytical ultracentrifugation (AUC) and crystallographic analysis showed that ASK1 forms a tight dimer (K(d) approximately 0.2 microM) interacting in a head-to-tail fashion. We found that the ASK1 phosphorylation motifs differ from known ASK1 phosphorylation sites but correspond well to autophosphorylation sites identified by mass spectrometry. Reporter gene assays showed that all three identified in vitro autophosphorylation sites (Thr813, Thr838, Thr842) regulate ASK1 signaling, but site-directed mutants showed catalytic activities similar to wild-type ASK1, suggesting a regulatory mechanism independent of ASK1 kinase activity. The determined high-resolution structure of ASK1 and identified ATP mimetic inhibitors will provide a first starting point for the further development of selective inhibitors.     

Involvement of ASK1–p38 pathway in the pathogenesis of diabetes triggered by pancreatic ß cell exhaustion

Background

Diabetes mellitus is characterized by high blood glucose levels. Pancreatic ß cell death contributes to type 1 and type 2 diabetes. Akita mice, which harbor a human permanent neonatal diabetes-linked mutation (Cys96Tyr) in the insulin gene, are well established as an animal model of diabetes caused by pancreatic ß cell exhaustion. Mutant Insulin 2 protein (Ins2^C96Y) induces endoplasmic reticulum (ER) stress and pancreatic ß cell death in Akita mice, although the molecular mechanism of Ins^C96Y-induced cell death remains unclear.

Methods

We investigate the mechanisms of Ins2^C96Y-induced pancreatic ß cell death in vitro and in vivo, using p38 inhibitor (SB203580), MIN6 cell (pancreatic ß cell line), Akita mice and apoptosis signal-regulating kinase 1 (ASK1) knockout mice.

Results

The expression of Ins^C96Y activated the ASK1–p38 pathway. Deletion of ASK1 mitigated Ins^C96Y-induced pancreatic ß cell death and delayed the onset of diabetes in Akita mice. Moreover, p38 inhibitor suppressed Ins^C96Y-induced MIN6 cell death.

Conclusions

These findings suggest that ER stress-induced ASK1–p38 activation, which is triggered by the accumulation of Ins^C96Y, plays an important role in the pathogenesis of diabetes.

General significance

Pancreatic ß cell death caused by insulin overload appears to be involved in the pathogenesis of type 1 and type 2 diabetes. Inhibition of the ASK1–p38 pathway may be an effective therapy for various types of diabetes.     

Apoptosis signal-regulating kinase 1 (ASK1) is an intracellular inducer of keratinocyte differentiation

Cells differentiate in response to various extracellular stimuli. This cellular response requires intracellular signaling pathways. The mitogen-activated protein (MAP) kinase cascade is a core signal transduction pathway that determines the fate of many kinds of cell. MAP kinase kinase kinase activates MAP kinase kinase, which in turn activates MAP kinase. Apoptosis signal-regulating kinase (ASK1) was identified as a MAP kinase kinase kinase involved in the stress-induced apoptosis-signaling cascade that activates the SEK1-JNK and MKK3/MKK6-p38 MAP kinase cascades. Expression of the constitutively active form of ASK1 (ASK1-DeltaN) in keratinocytes induced significant morphological changes and differentiation markers, transglutaminase-1, loricrin, and involucrin. A transient increase in p21(Cip1/WAF1) reduced DNA synthesis, and cell cycle analysis verified the differentiation. p38 MAP kinase inhibitors, SB202190 and SB203580, abolished the induction of differentiation markers, transglutaminase-1, loricrin, and involucrin. In turn, the induction of differentiation with ceramide in keratinocytes caused an increase in ASK1 expression and activity. Furthermore, normal human skin expresses ASK1 protein in the upper epidermis, implicating ASK1 in in vivo keratinocyte differentiation. We propose that the ASK1-p38 MAP kinase cascade is a new intracellular regulator of keratinocyte differentiation.     

The Cell Cycle-Regulatory CDC25A Phosphatase Inhibits Apoptosis Signal-Regulating Kinase 1

CDC25A phosphatase promotes cell cycle progression by activating G(1) cyclin-dependent kinases and has been postulated to be an oncogene because of its ability to cooperate with RAS to transform rodent fibroblasts. In this study, we have identified apoptosis signal-regulating kinase 1 (ASK1) as a CDC25A-interacting protein by yeast two-hybrid screening. ASK1 activates the p38 mitogen-activated protein kinase (MAPK) and c-Jun NH(2)-terminal protein kinase-stress-activated protein kinase (JNK/SAPK) pathways upon various cellular stresses. Coimmunoprecipitation studies demonstrated that CDC25A physically associates with ASK1 in mammalian cells, and immunocytochemistry with confocal laser-scanning microscopy showed that these two proteins colocalize in the cytoplasm. The carboxyl terminus of CDC25A binds to a domain of ASK1 adjacent to its kinase domain and inhibits the kinase activity of ASK1, independent of and without effect on the phosphatase activity of CDC25A. This inhibitory action of CDC25A on ASK1 activity involves diminished homo-oligomerization of ASK1. Increased cellular expression of wild-type or phosphatase-inactive CDC25A from inducible transgenes suppresses oxidant-dependent activation of ASK1, p38, and JNK1 and reduces specific sensitivity to cell death triggered by oxidative stress, but not other apoptotic stimuli. Thus, increased expression of CDC25A, frequently observed in human cancers, could contribute to reduced cellular responsiveness to oxidative stress under mitogenic or oncogenic conditions, while it promotes cell cycle progression. These observations propose a mechanism of oncogenic transformation by the dual function of CDC25A on cell cycle progression and stress responses.     

Identification of low-molecular inhibitors of proteinase ASK1

Protein kinase ASK1 (Apoptosis signal-regulating kinase 1) plays a key role in cell differentiation, aging and apoptosis. High activity of the kinase is associated with several pathologies. The ASK1 inhibitors might be therapeutic for patients with neurodegenerative, cardiovascular diseases and fibrous histiocytoma. In this work the identification of ASK1 inhibitors was performed by the methods of computer modeling and biochemical testing in vitro. The virtual screening experiments were carried out targeting the ATP binding site of ASK1 by browsing the database which contained 164 840 compounds of diverse chemical classes. The best-scored 300 ligands have been taken for the kinase assay analysis. In vitro tests revealed that derivatives of 2-thioxo-thiazolidin-4-one exhibited inhibitory activity against ASK1. The most active compound was 5-bromo-3-(4-oxo-2-thioxo-thiazolidin-5-ylidene)-1,3-dihydro-indol-2-one (IC50 = 2 microM). Binding mode for inhibitors of this class with ASK1 ATP-binding site was proposed. Our results can be used for further optimization and developing more potent and selective inhibitors of ASK1.     

Identification of novel ASK1 inhibitors using virtual screening

Apoptosis signal-regulating kinase 1 (ASK1, also called MAP3K5) is a mitogen-activated protein kinase kinase kinase (MAP3K) that plays important roles in stress-induced cell death and inflammation, and is expected as a new therapeutic target for cancer, cardiovascular diseases, and neurodegenerative diseases. We identified novel ASK1 inhibitors by virtual screening from the public chemical library collected by Chemical Biology Research Initiative (CBRI) at the University of Tokyo.     

The thioredoxin system in retroviral infection and apoptosis

Human thioredoxin (TRX) was first identified in human T-cell leukemia virus type I (HTLV-I)-positive T-cell lines and is associated with the pathophysiology of retroviral infections. TRX is a vital component of the thiol-reducing system and regulates various cellular function (redox regulation). Members of the TRX system regulate apoptosis through a wide variety of mechanisms. A family of thioredoxin-dependent peroxidases (peroxiredoxins) protects against apoptosis by scavenging hydrogen peroxide. Thioredoxin 2 is a critical regulator of cytochrome c release and mitochondrial apoptosis; transmembrane thioredoxin-related molecule (TMX) has a protective role in endoplasmic reticulum (ER) stress-induced apoptosis. TRX interacts with apoptosis signal-regulating kinase 1 (ASK1) and is a sensor of oxidative stress. Thioredoxin binding protein-2/vitamin D(3) upregulated protein 1 is a growth suppressor and its expression is suppressed in HTLV-I-transformed cells. Studies of these molecules of the TRX system provide novel insights into the apoptosis associated with retroviral diseases.