Negative feedback regulation of ASK1 by protein phosphatase 5 (PP5) in response to oxidative stress.

Apoptosis signal-regulating kinase 1 (ASK1) is a MAP kinase kinase kinase (MAPKKK) that activates the JNK and p38 MAP kinase cascades and is activated in response to oxidative stress such as hydrogen peroxide (H(2)O(2)). A yeast two-hybrid screening identified a serine/threonine protein phosphatase 5 (PP5) as a binding partner of ASK1. PP5 directly dephosphorylated an essential phospho-threonine residue within the kinase domain of ASK1 and thereby inactivated ASK1 activity in vitro and in vivo. The interaction between PP5 and ASK1 was induced by H(2)O(2) treatment and was followed by the decrease in ASK1 activity. PP5 inhibited not only H(2)O(2)-induced sustained activation of ASK1 but also ASK1-dependent apoptosis. Thus, PP5 appears to act as a physiological inhibitor of ASK1-JNK/p38 pathways by negative feedback.     

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.     

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.     

Protein phosphatase with EF-hand domains 2 (PPEF2) is a potent negative regulator of apoptosis signal regulating kinase-1 (ASK1)

The function of protein phosphatases with EF-hand domains (PPEF) in mammals is not known. Large-scale expression profiling experiments suggest that PPEF expression may correlate with stress protective responses, cell survival, growth, proliferation, or neoplastic transformation. Apoptosis signal regulating kinase-1 (ASK1) is a MAP kinase kinase kinase implicated in cancer, cardiovascular and neurodegenerative diseases. ASK1 is activated by oxidative stress and induces pro-apoptotic or inflammatory signalling, largely via sustained activation of MAP kinases p38 and/or JNK. We identify human PPEF2 as a novel interacting partner and a negative regulator of ASK1. In COS-7 or HEK 293A cells treated with H₂O₂, expression of PPEF2 abrogated sustained activation of p38 and one of the JNK p46 isoforms, and prevented ASK1-dependent caspase-3 cleavage and activation. PPEF2 efficiently suppressed H₂O₂-induced activation of ASK1. Overexpessed as well as endogenous ASK1 co-immunoprecipitated with PPEF2. PPEF2 was considerably more potent both as a suppressor of ASK1 activation and as its interacting partner as compared to protein phosphatase 5 (PP5), a well-known negative regulator of ASK1. PPEF2 was found to form complexes with endogenous Hsp70 and to a lesser extent Hsp90, which are also known interacting partners of PP5. These data identify, for the first time, a possible downstream signalling partner of a mammalian PPEF phosphatase, and suggest that, despite structural divergence, PPEF and PP5 phosphatases may share common interacting partners and functions.     

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.     

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.     

Stress kinase MKK7: savior of cell cycle arrest and cellular senescence

The c-Jun N-terminal kinase (JNK/SAPK) signaling cascade controls a spectrum of cellular processes, including cell growth, differentiation, transformation, and apoptosis. We recently demonstrated that stress kinase MKK7, a direct activator of JNKs, couples stress signaling to G2/M cell cycle progression, CDC2 expression, and cellular senescence. We further explored other molecules involved in JNK pathway and found that both MKK4, another direct activator of JNK, and c-Jun, a direct substrate of JNK, have similar roles to MKK7. Here we discuss the importance of the MKK4/MKK7-JNK-c-Jun pathway linking stress and developmental signals to cell proliferation, cell cycle progression, cellular senescence, and apoptosis including recent unpublished data from our lab.     

Apoptosis signal-regulating kinase 1 (ASK1) induces neuronal differentiation and survival of PC12 cells

Apoptosis signal-regulating kinase 1 (ASK1) is a ubiquitously expressed mitogen-activated protein kinase kinase kinase that activates the c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase signaling cascades. We report here that expression of constitutively active ASK1 (ASK1DeltaN) induces neurite outgrowth in the rat pheochromocytoma cell line PC12. We found that p38 and to a lesser extent JNK, but not ERK, were activated by the expression of ASK1DeltaN in PC12 cells. ASK1DeltaN-induced neurite outgrowth was strongly inhibited by treatment with the p38 inhibitor SB203580 but not with the MEK inhibitors, suggesting that activation of p38, rather than of ERK, is required for the neurite-inducing activity of ASK1 in PC12 cells. We also observed that ASK1DeltaN induced expression of several neuron-specific proteins and phosphorylation of neurofilament proteins, confirming that PC12 cells differentiated into mature neuronal cells by ASK1. Moreover, ASK1DeltaN-expressing PC12 cells survived in serum-starved condition. ASK1 thus appears to mediate signals leading to both differentiation and survival of PC12 cells. Together with previous reports indicating that ASK1 functions as a pro-apoptotic signaling intermediate, these results suggest that ASK1 has a broad range of biological activities depending on cell types and/or cellular context.     

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.     

Oncogenic potential of a C.elegans cdc25 gene is demonstrated by a gain-of-function allele

In multicellular organisms, developmental programmes must integrate with central cell cycle regulation to co-ordinate developmental decisions with cell proliferation. Hyperplasia caused by deregulated proliferation without significant change to other aspects of developmental behaviour is a probable step towards full oncogenesis in many malignancies. CDC25 phosphatase promotes progression through the eukaryotic cell cycle by dephosphorylation of cyclin-dependent kinase and, in humans, different cdc25 family members have been implicated as potential oncogenes. Demonstrating the direct oncogenic potential of a cdc25 gene, we identify a gain-of-function mutant allele of the Caenorhabditis elegans gene cdc-25.1 that causes a deregulated proliferation of intestinal cells resulting in hyperplasia, while other aspects of intestinal cell function are retained. Using RNA-mediated interference, we demonstrate modulation of the oncogenic behaviour of this mutant, and show that a reduction of the wild-type cdc-25.1 activity can cause a failure of proliferation of intestinal and other cell types. That gain and loss of CDC-25.1 activity has opposite effects on cellular proliferation indicates its critical role in controlling C.elegans cell number.     

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.     

Tumor necrosis factor alpha-induced desumoylation and cytoplasmic translocation of homeodomain-interacting protein kinase 1 are critical for apoptosis signal-regulating kinase 1-JNK/p38 activation

The apoptosis signal-regulating kinase 1 (ASK1)-JNK/p38 signaling pathway is pivotal component in cell apoptosis and can be activated by a variety of death stimuli including tumor necrosis factor (TNF) alpha and oxidative stress (reactive oxygen species). However, the mechanism for ASK1 activation is not fully understood. We have recently identified ASK1-interacting protein (AIP1) as novel signal transducer in TNFalpha-induced ASK1 activation by facilitating dissociation of ASK1 from its inhibitor 14-3-3. In the present study, we employed yeast two-hybrid system using the N-terminal domain of AIP1 as bait and identified homeodomain-interacting protein kinase 1 (HIPK1) as an AIP1-associated protein. Interestingly, we showed that TNFalpha induced HIPK1 desumoylation concomitant with a translocation from nucleus to cytoplasm at 15 min followed by a return to nucleus by 60 min. The kinetics of HIPK1 translocation correlates with those of stress-induced ASK1-JNK/P38 activation. A specific JNK inhibitor blocked the reverse but not the initial translocation of HIPK1, suggesting that the initial translocation is an upstream event of ASK1-JNK/p38 signaling and JNK activation regulates the reverse translocation as a feedback mechanism. Consistently, expression of HIPK1 increased, whereas expression of a kinase-inactive form (HIPK1-D315N) or small interference RNA of HIPK1 decreased stress-induced ASK1-JNK/P38 activation without effects on IKK-NF-kappaB signaling. Moreover, a sumoylation-defective mutant of HIPK1 (KR5) localizes to the cytoplasm and is constitutively active in ASK1-JNK/P38 activation. Furthermore, HIPK1-KR5 induces dissociation of ASK1 from its inhibitors 14-3-3 and thioredoxin and synergizes with AIP1 to induce ASK1 activation. Our study suggests that TNFalpha-induced desumoylation and cytoplasmic translocation of HIPK1 are critical in TNFalpha-induced ASK1-JNK/p38 activation.     

A novel CDC25A/DYRK2 regulatory switch modulates cell cycle and survival

The cell division cycle 25A (CDC25A) phosphatase is a key regulator of cell cycle progression that acts on the phosphorylation status of Cyclin–Cyclin-dependent kinase complexes, with an emergent role in the DNA damage response and cell survival control. The regulation of CDC25A activity and its protein level is essential to control the cell cycle and maintain genomic integrity. Here we describe a novel ubiquitin/proteasome-mediated pathway negatively regulating CDC25A stability, dependent on its phosphorylation by the serine/threonine kinase DYRK2. DYRK2 phosphorylates CDC25A on at least 7 residues, resulting in its degradation independent of the known CDC25A E3 ubiquitin ligases. CDC25A in turn is able to control the phosphorylation of DYRK2 at several residues outside from its activation loop, thus affecting DYRK2 localization and activity. An inverse correlation between DYRK2 and CDC25A protein amounts was observed during cell cycle progression and in response to DNA damage, with CDC25A accumulation responding to the manipulation of DYRK2 levels or activity in either physiological scenario. Functional data show that the pro-survival activity of CDC25A and the pro-apoptotic activity of DYRK2 could be partly explained by the mutual regulation between both proteins. Moreover, DYRK2 modulation of CDC25A expression and/or activity contributes to the DYRK2 role in cell cycle regulation. Altogether, we provide evidence suggesting that DYRK2 and CDC25A mutually control their activity and stability by a feedback regulatory loop, with a relevant effect on the genotoxic stress pathway, apoptosis, and cell cycle regulation.Subject terms: Proteins, Cell biology, Proteomics     

C-terminus of heat shock protein 70-interacting protein facilitates degradation of apoptosis signal-regulating kinase 1 and inhibits apoptosis signal-regulating kinase 1-dependent apoptosis

Apoptosis signal-regulating kinase 1 (ASK1) is a mitogen-activated protein kinase kinase kinase (MAPKKK) that is regulated under conditions of cellular stress. ASK1 phosphorylates c-Jun N-terminal kinase (JNK) and elicits an apoptotic response. ASK1 activity is regulated at multiple levels, 1 of which is through inhibition by cytosolic chaperones of the heat shock protein (Hsp) 70 family. Among the proteins that determine Hsp70 function, CHIP (C-terminus of Hsp70-interacting protein) is a cochaperone and ubiquitin ligase that interacts with Hsp70 through an amino-terminal tetratricopeptide repeat (TPR) domain. Prominent among the cellular functions mediated by CHIP is protection against physiologic stress. Because ASK1 is known to contain a TPR-acceptor site, we examined the role of CHIP in regulating ASK1 function. CHIP interacted with ASK1 in a TPR-dependent fashion and induced ubiquitylation and proteasome-dependent degradation of ASK1. Targeting of ASK1 by CHIP inhibited JNK activation in response to oxidative challenge and reduced ASK1-dependent apoptosis, whereas short interfering RNA (siRNA)-dependent depletion of CHIP enhanced JNK activation. Consistent with its ability to reduce cytoplasmic ASK1 levels, CHIP triggered the translocation of ASK1 partner protein death-associated protein (Daxx) into the nucleus, where it is known to activate an antiapoptotic response. These results indicate that CHIP regulates ASK1 activity by inducing its ubiquitylation and degradation, which, together with its effects on Daxx localization, provides a mechanism for the antiapoptotic effects of CHIP observed in the face of cellular and physiologic stress.     

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.     

p38γ Mitogen-activated Protein Kinase Signals through Phosphorylating Its Phosphatase PTPH1 in Regulating Ras Protein Oncogenesis and Stress Response

Phosphatase plays a crucial role in determining cellular fate by inactivating its substrate kinase, but it is not known whether a kinase can vice versa phosphorylate its phosphatase to execute this function. Protein-tyrosine phosphatase H1 (PTPH1) is a specific phosphatase of p38γ mitogen-activated protein kinase (MAPK) through PDZ binding, and here, we show that p38γ is also a PTPH1 kinase through which it executes its oncogenic activity and regulates stress response. PTPH1 was identified as a substrate of p38γ by unbiased proteomic analysis, and its resultant phosphorylation at Ser-459 occurs in vitro and in vivo through their complex formation. Genetic and pharmacological analyses showed further that Ser-459 phosphorylation is directly regulated by Ras signaling and is important for Ras, p38γ, and PTPH1 oncogenic activity. Moreover, experiments with physiological stimuli revealed a novel stress pathway from p38γ to PTPH1/Ser-459 phosphorylation in regulating cell growth and cell death by a mechanism dependent on cellular environments but independent of canonical MAPK activities. These results thus reveal a new mechanism by which a MAPK regulates Ras oncogenesis and stress response through directly phosphorylating its phosphatase.