ASK3, a novel member of the apoptosis signal-regulating kinase family, is essential for stress-induced cell death in HeLa cells (original) (raw)
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Journal of Biological Chemistry, 2016
Apoptosis signal-regulating kinase 1 (ASK1, also known as MAP3K5), a member of the mitogen-activated protein kinase kinase kinase (MAP3K) family, regulates diverse physiological processes. The activity of ASK1 is triggered by various stress stimuli and is involved in the pathogenesis of cancer, neurodegeneration, inflammation, and diabetes. ASK1 forms a high molecular mass complex whose activity is, under non-stress conditions, suppressed through interaction with thioredoxin and the scaffolding protein 14-3-3. The 14-3-3 protein binds to the phosphorylated Ser-966 motif downstream of the ASK1 kinase domain. The role of 14-3-3 in the inhibition of ASK1 has yet to be elucidated. In this study we performed structural analysis of the complex between the ASK1 kinase domain phosphorylated at Ser-966 (pASK1-CD) and the 14-3-3 protein. Small angle x-ray scattering (SAXS) measurements and chemical cross-linking revealed that the pASK1-CD⅐14-3-3 complex is dynamic and conformationally heterogeneous. In addition, structural analysis coupled with the results of phosphorus NMR and time-resolved tryptophan fluorescence measurements suggest that 14-3-3 interacts with the kinase domain of ASK1 in close proximity to its active site, thus indicating this interaction might block its accessibility and/or affect its conformation. Apoptosis signal-regulating kinase 1 (ASK1, 3 also known as MAP3K5), a member of the mitogen-activated protein kinase kinase kinase (MAP3K) family, regulates diverse physiological * This work was supported by the Czech Science Foundation (Project 14-10061S) and the Czech Academy of Sciences (Research Project RVO: 67985823 of the Institute of Physiology). The authors declare that they have no conflicts of interest with the contents of this article. □ S This article contains supplemental Figs. S1-S3 and Tables S1 and S2.
ASK-1 (apoptosis signal-regulating kinase 1) is a direct E2F target gene
Biochemical Journal, 2006
In the present study, we show that E2Fs (E2 promoter-binding factors) regulate the expression of ASK-1 (apoptosis signal-regulating kinase 1), which encodes a mitogen-activated protein kinase kinase kinase, also known as MAP3K5. Its mRNA expression is cell-cycle-regulated in human T98G cells released from serum starvation. Moreover, overexpression and RNA interference experiments support the requirement of endogenous E2F/DP (E2F dimerization partner) activity for ASK-1 expression. Characterization of the human ASK-1 promoter demonstrates that the −95/+11 region is critical for E2F-mediated up-regulation. Chromatin immunoprecipitation assays show that E2F1–E2F4 are bound in vivo to the ASK-1 promoter in cycling cells, probably through a non-consensus E2F-binding site located 12 bp upstream of the transcription start site. Mutation of this site completely abolishes the ASK-1 promoter response to E2Fs as well as the E2F1 binding in electrophoretic mobility-shift experiments. Our result...
Regulation of apoptosis signal-regulating kinase 1 by protein phosphatase 2Cϵ
Biochemical Journal, 2007
ASK1 (apoptosis signal-regulating kinase 1), a MKKK (mitogen-activated protein kinase kinase kinase), is activated in response to cytotoxic stresses, such as H2O2 and TNFα (tumour necrosis factor α). ASK1 induction initiates a signalling cascade leading to apoptosis. After exposure of cells to H2O2, ASK1 is transiently activated by autophosphorylation at Thr845. The protein then associates with PP5 (protein serine/threonine phosphatase 5), which inactivates ASK1 by dephosphorylation of Thr845. Although this feedback regulation mechanism has been elucidated, it remains unclear how ASK1 is maintained in the dephosphorylated state under non-stressed conditions. In the present study, we have examined the possible role of PP2Cϵ (protein phosphatase 2Cϵ), a member of PP2C family, in the regulation of ASK1 signalling. Following expression in HEK-293 cells (human embryonic kidney cells), wild-type PP2Cϵ inhibited ASK1-induced activation of an AP-1 (activator protein 1) reporter gene. Conver...
Molecular and Cellular Biology, 2013
Cellular processes are tightly controlled through well-coordinated signaling networks that respond to conflicting cues, such as reactive oxygen species (ROS), endoplasmic reticulum (ER) stress signals, and survival factors to ensure proper cell function. We report here a direct interaction between inhibitor of B kinase (IKK) and apoptosis signal-regulating kinase 1 (ASK1), unveiling a critical node at the junction of survival, inflammation, and stress signaling networks. IKK can be activated by growth factor stimulation or tumor necrosis factor alpha engagement. IKK forms a complex with and phosphorylates ASK1 at a sensor site, Ser967, leading to the recruitment of 14-3-3, counteracts stress signal-triggered ASK1 activation, and suppresses ASK1-mediated functions. An inhibitory role of IKK in JNK signaling has been previously reported to depend on NF-B-mediated gene expression. Our data suggest that IKK has a dual role: a transcription-dependent and a transcription-independent action in controlling the ASK1-JNK axis, coupling IKK to ROS and ER stress response. Direct phosphorylation of ASK1 by IKK also defines a novel IKK phosphorylation motif. Because of the intimate involvement of ASK1 in diverse diseases, the IKK/ASK1 interface offers a promising target for therapeutic development.
Akt phosphorylates and negatively regulates apoptosis signal-regulating kinase 1
Science …, 2001
The Akt family of serine/threonine-directed kinases promotes cellular survival in part by phosphorylating and inhibiting death-inducing proteins. Here we describe a novel functional interaction between Akt and apoptosis signal-regulating kinase 1 (ASK1), a mitogen-activated protein kinase kinase kinase. Akt decreased ASK1 kinase activity stimulated by both oxidative stress and overexpression in 293 cells by phosphorylating a consensus Akt site at serine 83 of ASK1. Activation of the phosphoinositide 3-kinase (PI3-K)/Akt pathway also inhibited the serum deprivation-induced activity of endogenous ASK1 in L929 cells. An association between Akt and ASK1 was detected in cells by coimmunoprecipitation. Phosphorylation by Akt inhibited ASK1-mediated c-Jun N-terminal kinase and activating transcription factor 2 activities in intact cells. Finally, activation of the PI3-K/Akt pathway reduced apoptosis induced by ASK1 in a manner dependent on phosphorylation of serine 83 of ASK1. These results provide the first direct link between Akt and the family of stress-activated kinases.
Regulation of apoptosis signal-regulating kinase 1 by protein phosphatase 2C?
Biochem J, 2007
regulating kinase 1), a MKKK (mitogenactivated protein kinase kinase kinase), is activated in response to cytotoxic stresses, such as H 2 O 2 and TNFα (tumour necrosis factor α). ASK1 induction initiates a signalling cascade leading to apoptosis. After exposure of cells to H 2 O 2 , ASK1 is transiently activated by autophosphorylation at Thr 845. The protein then associates with PP5 (protein serine/threonine phosphatase 5), which inactivates ASK1 by dephosphorylation of Thr 845. Although this feedback regulation mechanism has been elucidated, it remains unclear how ASK1 is maintained in the dephosphorylated state under non-stressed conditions. In the present study, we have examined the possible role of PP2Cε (protein phosphatase 2Cε), a member of PP2C family, in the regulation of ASK1 signalling. Following expression in HEK-293 cells (human embryonic kidney cells), wild-type PP2Cε inhibited ASK1-induced activation of an AP-1 (activator protein 1) reporter gene. Conversely, a dominant-negative PP2Cε mutant enhanced AP-1 activity. Exogenous PP2Cε associated with exogenous ASK1 in HEK-293 cells under non-stressed conditions, inactivating ASK1 by decreasing Thr 845 phosphorylation. The association of endogenous PP2Cε and ASK1 was also observed in mouse brain extracts. PP2Cε directly dephosphorylated ASK1 at Thr 845 in vitro. In contrast with PP5, PP2Cε transiently dissociated from ASK1 within cells upon H 2 O 2 treatment. These results suggest that PP2Cε maintains ASK1 in an inactive state by dephosphorylation in quiescent cells, supporting the possibility that PP2Cε and PP5 play different roles in H 2 O 2-induced regulation of ASK1 activity. Key words: apoptosis, apoptosis signal-regulating kinase 1 (ASK1), hydrogen peroxide (H 2 O 2), mitogen-activated protein kinase (MAPK), protein phosphatase 2C (PP2C), protein serine/ threonine phosphatase 5 (PP5), stress-activated protein kinase (SAPK).
Journal of Biological Chemistry, 1998
Many growth factors and G protein-coupled receptors activate mitogen-activated protein (MAP) kinase pathways. The MAP kinase pathways are involved in the regulation of the ubiquitous process of apoptosis or programmed cell death. Two related MAP kinase kinase kinases, apoptosis-signal regulating kinase 1 (ASK1) and MAP kinase kinase kinase 1 (MEKK1), stimulate c-Jun kinase (JNK) activity and induce apoptosis. Transient transfection of dominant negative and constitutively active components of the JNK pathway in COS-7 cells showed that two G protein subunits, G␣12 and G␣13, stimulated the JNK pathway in a ASK1-and MEKK1-dependent manner. Moreover, the mutationally activated G␣12 and G␣13 stimulated the kinase activity of ASK1. Both G␣12 and G␣13 employ small GTPases, Cdc42 and Rac1, to transduce signal to MEKK1 and, subsequently, to JNK. However, activation of JNK by Cdc42 and Rac1 did not require ASK1. Additionally, ASK1 and MEKK1 are involved in the apoptosis induced by G␣12 and G␣13. We conclude that G␣12 and G␣13 can induce apoptosis using two separate MAP kinase pathways; one is initiated by ASK1, and the other is initiated by MEKK1. Furthermore, Bcl-2 can block apoptosis induced by G␣12 and G␣13. This death-sparing function was associated with increased Bcl-2 phosphorylation, suggesting that phosphorylation of Bcl-2 may be a critical mechanism protecting cells from G␣12-and G␣13-induced apoptosis.
Role of protein kinase activity in apoptosis
Experientia, 1996
The transmission of signals from the plasma membrane to the nucleus involves a number of different pathways all of which have in common protein modification. The modification is primarily in the form of phosphorylation which leads to the activation of a series of protein kinases. It is now evident that these pathways are common to stimuli that lead to mitogenic and apoptotic responses. Even the same stimuli under different physiological conditions can cause either cell proliferation or apoptosis. Activation of specific protein kinases can in some circumstances protect against cell death, while in others it protects the cell against apoptosis. Some of the pathways involved lead to activation of transcription factors and the subsequent induction of genes involved in the process of cell death or proliferation. In other cases, such as for the tumour suppressor gene product p53, activation may be initiated both at the level of gene expression or through pre-existing proteins. Yet in others, while the initial steps in the pathway are ill-defined, it is clear that downstream activation of a series of cysteine proteases is instrumental in pushing the cell towards apoptosis. In this report we review the involvement of protein kinases at several different levels in the control of cell behaviour.