Protein Phosphatase 5-Recruiting Chimeras for Accelerating Apoptosis-Signal-Regulated Kinase 1 Dephosphorylation with Antiproliferative Activity.

A normal phosphorylation state is essential for the function of proteins. Biased regulation frequently results in morbidity, especially for the hyperphosphorylation of oncoproteins. The hyperphosphorylation of ASK1 at Thr838 leads to a persistently high activity state, which accelerates the course of gastric cancer. Under normal conditions, PP5 specifically dephosphorylates p-ASK1T838 in cells, thereby weakening ASK1 to a low-basal activity state. However, in tumor types, PP5 shows low activity with a self-inhibition mechanism, making p-ASK1T838 remain at a high level. Thus, we aim to design phosphatase recruitment chimeras (PHORCs) through a proximity-mediated effect for specifically accelerating the dephosphorylation of p-ASK1T838. Herein, we describe DDO3711 as the first PP5-recruiting PHORC, which is formed by connecting a small molecular ASK1 inhibitor to a PP5 activator through a chemical linker, to effectively decrease the level of p-ASK1T838 in vitro and in vivo. DDO3711 shows preferable antiproliferative activity (IC50 = 0.5 µM) against MKN45 cells through a direct binding and proximity-mediated mechanism, while the ASK1 inhibitor and the PP5 activator, used alone or in combination, exhibit no effect on MKN45 cells. Using DDO3711, PHORCs are identified as effective tools to accelerate the dephosphorylation of POIs and provide important evidence to achieve precise phosphorylation regulation, which will promote confidence in the further regulation of abnormally phosphorylated oncoproteins.

Redox-Inactive Peptide Disrupting Trx1-Ask1 Interaction for Selective Activation of Stress Signaling.

Thioredoxin 1 (Trx1) and glutaredoxin 1 (Grx1) are two ubiquitous redox enzymes that are central for redox homeostasis but also are implicated in many other processes, including stress sensing, inflammation, and apoptosis. In addition to their enzymatic redox activity, a growing body of evidence shows that Trx1 and Grx1 play regulatory roles via protein-protein interactions with specific proteins, including Ask1. The currently available inhibitors of Trx1 and Grx1 are thiol-reactive electrophiles or disulfides that may suffer from low selectivity because of their thiol reactivity. In this report, we used a phage peptide library to identify a 7-mer peptide, 2GTP1, that binds to both Trx1 and Grx1. We further showed that a cell-permeable derivative of 2GTP1, TAT-2GTP1, disrupts the Trx1-Ask1 interaction, which induces Ask1 phosphorylation with subsequent activation of JNK, stabilization of p53, and reduced viability of cancer cells. Notably, as opposed to a disulfide-derived Trx1 inhibitor (PX-12), TAT-2GTP1 was selective for activating the Ask1 pathway without affecting other stress signaling pathways, such as endoplasmic reticulum stress and AMPK activation. Overall, 2GTP1 will serve as a useful probe for investigating protein interactions of Trx1.

ASK1 regulates the survival of neuroblastoma cells by interacting with TLX and stabilizing HIF-1α.

Elevated expression of TLX (also called as NR2E1) in neuroblastoma (NB) correlates with unfavorable prognosis, and TLX is required for self-renewal of NB cells. Knockdown of TLX has been shown to reduce the NB sphere-forming ability. ASK1 (MAP3K5) and TLX expression are both enhanced in SP (side population) NB and patient-derived primary NB sphere cell lines, but the majority of non-SP NB lines express lower ASK1 expression. We found that ASK1 phosphorylated and stabilized TLX, which led induction of HIF-1α, and its downstream VEGF-A in an Akt dependent manner. In depleting ASK1 upon hypoxia, TLX decreased and the apoptosis ratio of NB cells was enhanced, while low-ASK1-expressing NB cell lines were refractory in TUNEL assay by using flow cytometry. Interestingly, primary NB spheres cell lines express only high levels of active pASK1Thr-838 but the established cell lines expressed inhibitory pASK1Ser-966, and both could be targeted by ASK1 depletion. We report a novel pro-survival role of ASK1 in the tumorigenic NB cell populations, which may be applied as a therapeutic target, inducing apoptosis specifically in cancer stem cells.

Cysteine residues mediate high-affinity binding of thioredoxin to ASK1.

Apoptosis signal-regulating kinase 1 (ASK1, MAP3K5) activates p38 mitogen-activated protein kinase and the c-Jun N-terminal kinase in response to proinflammatory and stress signals. In nonstress conditions, ASK1 is inhibited by association with thioredoxin (TRX) which binds to the TRX-binding domain (ASK1-TBD) at the N terminus of ASK1. TRX dissociates in response to oxidative stress allowing the ASK1 activation. However, the molecular basis for the ASK1:TRX1 complex dissociation is still not fully understood. Here, the role of cysteine residues on the interaction between TRX1 and ASK1-TBD in both reducing and oxidizing conditions was investigated. We show that from the two catalytic cysteines of TRX1 the residue C32 is responsible for the high-affinity binding of TRX1 to ASK1-TBD in reducing conditions. The disulfide bond formation between C32 and C35 within the active site of TRX1 is the main factor responsible for the TRX1 dissociation upon its oxidation as the formation of the second disulfide bond between noncatalytic cysteines C62 and C69 did not have any additional effect. ASK1-TBD contains seven conserved cysteine residues which differ in solvent accessibility with the residue C250 being the only cysteine which is both solvent exposed and essential for TRX1 binding in reducing conditions. Furthermore, our data show that the catalytic site of TRX1 interacts with ASK1-TBD region containing cysteine C200 and that the oxidative stress induces intramolecular disulfide bond formation within ASK1-TBD and affects its structure in regions directly involved and/or important for TRX1 binding.

Structural Insight into the 14-3-3 Protein-dependent Inhibition of Protein Kinase ASK1 (Apoptosis Signal-regulating kinase 1).

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 14-3-3ζ protein. Small angle x-ray scattering (SAXS) measurements and chemical cross-linking revealed that the pASK1-CD·14-3-3ζ 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 14-3-3ζ 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.

Identification of apoptosis signal-regulating kinase 1 (ASK1) inhibitors among the derivatives of benzothiazol-2-yl-3-hydroxy-5-phenyl-1,5-dihydro-pyrrol-2-one.

Apoptosis signal-regulating kinase 1 (ASK1) plays important roles in the pathogenesis of type 1 and type 2 diabetes, autoimmune disorders, cancer and neurodegenerative diseases suggesting that small compounds inhibiting ASK1 could be used for the treatment of these pathologies. We have identified novel chemical class of ASK1 inhibitors, namely benzothiazol-2-yl-3-hydroxy-5-phenyl-1,5-dihydro-pyrrol-2-one, using molecular modeling techniques. It was found that the most active compound 1-(6-fluoro-benzothiazol-2-yl)-3-hydroxy-5-[3-(3-methyl-butoxy)-phenyl]-4-(2-methyl-2,3-dihydro-benzofuran-5-carbonyl)-1,5-dihydro-pyrrol-2-one (BPyO-34) inhibits ASK1 with IC50 of 0.52µM in vitro in kinase assay. The structure-activity relationships of 34 derivatives of benzothiazol-2-yl-3-hydroxy-5-phenyl-1,5-dihydro-pyrrol-2-one have been studied and binding mode of this chemical class has been proposed.

Cigarette smoke extract-induced BEAS-2B cell apoptosis and anti-oxidative Nrf-2 up-regulation are mediated by ROS-stimulated p38 activation.

Cigarette smoke contains reactive oxygen (ROS) that can cause oxidative stress. It increases the number of apoptotic and necrotic lung cells and further induces the development of chronic airway disease. In this study, we investigated the effects of cigarette smoke extract (CSE) on apoptosis in human bronchial epithelial cells (BEAS-2B). CSE exposure induced ROS generation and p38 mitogen-activated protein kinase (MAPK) activation that are associated with the activation of apoptosis-regulating signal kinase 1 (ASK-1). N-acetylcysteine (a general antioxidant) attenuated the CSE-induced ASK-1 and p38 MAPK activation and cell apoptosis, suggesting a triggering role of ROS in ASK-1/p38 MAPK activation during apoptotic progression. In contrast, the inhibition and knockdown of p38 attenuated the expression of anti-oxidant master NF-E2-related factor 2 (Nrf-2) and CSE-induced apoptosis, suggesting that p38 MAPK modulates Nrf-2 expression and presumably prevents cell apoptosis. Taken together, the data presented in this manuscript demonstrate that the ROS-dependent ASK-1/p38 signaling cascade regulates CSE-induced BEAS-2B cell apoptosis. In addition, anti-oxidative Nrf-2 is also up-regulated by the ROS/p38 signaling cascade in this progression.

Kinase crystal identification and ATP-competitive inhibitor screening using the fluorescent ligand SKF86002.

The small kinase inhibitor SKF86002 lacks intrinsic fluorescence but becomes fluorescent upon binding to the ATP-binding sites of p38 mitogen-activated protein kinase (p38α). It was found that co-crystals of this compound with various kinases were distinguishable by their strong fluorescence. The co-crystals of SKF86002 with p38α, Pim1, ASK1, HCK and AMPK were fluorescent. Addition of SKF86002, which binds to the ATP site, to the co-crystallization solution of HCK promoted protein stability and thus facilitated the production of crystals that otherwise would not grow in the apo form. It was further demonstrated that the fluorescence of SKF86002 co-crystals can be applied to screen for candidate kinase inhibitors. When a compound binds competitively to the ATP-binding site of a kinase crystallized with SKF86002, it displaces the fluorescent SKF86002 and the crystal loses its fluorescence. Lower fluorescent signals were reported after soaking SKF86002-Pim1 and SKF86002-HCK co-crystals with the inhibitors quercetin, a quinazoline derivative and A-419259. Determination of the SKF86002-Pim1 and SKF86002-HCK co-crystal structures confirmed that SKF86002 interacts with the ATP-binding sites of Pim1 and HCK. The structures of Pim1-SKF86002 crystals soaked with the inhibitors quercetin and a quinazoline derivative and of HCK-SKF86002 crystals soaked with A-419259 were determined. These structures were virtually identical to the deposited crystal structures of the same complexes. A KINOMEscan assay revealed that SKF86002 binds a wide variety of kinases. Thus, for a broad range of kinases, SKF86002 is useful as a crystal marker, a crystal stabilizer and a marker to identify ligand co-crystals for structural analysis.

Crystal structures of ASK1-inhibtor complexes provide a platform for structure-based drug design.

ASK1, a member of the MAPK Kinase Kinase family of proteins has been shown to play a key role in cancer, neurodegeneration and cardiovascular diseases and is emerging as a possible drug target. Here we describe a 'replacement-soaking' method that has enabled the high-throughput X-ray structure determination of ASK1/ligand complexes. Comparison of the X-ray structures of five ASK1/ligand complexes from 3 different chemotypes illustrates that the ASK1 ATP binding site is able to accommodate a range of chemical diversity and different binding modes. The replacement-soaking system is also able to tolerate some protein flexibility. This crystal system provides a robust platform for ASK1/ligand structure determination and future structure based drug design.

GSK-3ß-induced ASK1 stabilization is crucial in LPS-induced endotoxin shock.

Glycogen synthase kinase-3ß (GSK-3ß), a multifunctional kinase, is a regulator of lipopolysaccharide (LPS)-mediated septic shock. Apoptosis signal-regulating kinase 1 (ASK1) is also required for LPS-induced activation of p38, which is a crucial determinant for the production of pro-inflammatory cytokines via Toll-like receptor 4 (TLR4) in endotoxemia. Here, we show that attenuation of endotoxemia induced by GSK-3 inhibition is caused by the ASK1 reduction-mediated inhibition of p38, a representative downstream kinase of ASK1. LPS-stimulated activation of p38 was blocked by the reduction of ASK1 via the knockdown of GSK-3ß. In addition, compared with L929 control cells, ASK1 protein was reduced in L929 cells stably expressing Wnt-3a and in which ß-catenin was active, due to the inhibition of GSK-3ß activity. GSK-3ß inhibition-mediated ASK1 reduction was also confirmed by reduced ASK1 in GSK-3ß-deficient mouse embryo fibroblasts (MEFs) and MCF7 GSK-3ß siRNA cells. Furthermore, ASK1 protein stability was also attenuated in MCF7 GSK-3ß siRNA cells compared with GFP control cells. Consistent with stability data, a much stronger ubiquitination of ASK1 was observed in cells in which GSK-3ß was knocked down. These findings suggest that GSK-3ß crosstalks with p38 kinase via the regulation of ASK1 protein stability in endotoxemia.

Heat shock protein 70 together with its co-chaperone CHIP inhibits TNF-alpha induced apoptosis by promoting proteasomal degradation of apoptosis signal-regulating kinase1.

Inducible heat shock protein70 (HSP70) is one of the most important HSPs for maintenance of cell integrity during normal cellular growth as well as pathophysiological conditions. Apoptosis signal-regulating kinase (ASK) 1, a mammalian MAPKKK, activates the JNK and p38 pathways. Here we report a novel function of HSP70 in regulating TNF-alpha-induced cell apoptosis. Our study demonstrated that HSP70 physically interacted with ASK1 and promoted the ubiquitin-dependent proteasomal degradation of ASK1. CHIP (carboxyl terminus of the HSC70-interacting protein) which acted as a co-chaperone of HSP70 cooperated with HSP70 in regulating ASK1. We also found that TNF-alpha stimulated HSP70/CHIP/ASK1 association and through cooperating with CHIP, HSP70 inhibits TNF-alpha-induced cell apoptosis both in over-expression and RNAi conditions. Structural analysis indicated that C-terminal domain of HSP70 was necessary for ASK1 degradation, and N- terminal domain of ASK1 was essential for its binding to HSP70. All these findings indicated that HSP70 and CHIP association is important for HSP70 in interacting with ASK1. Through forming the complex of HSP70/CHIP/ASK1, HSP70 promotes ASK1 proteasomal degradation and prevents TNF-alpha-induced cell apoptosis.

Reciprocal negative regulation of PDK1 and ASK1 signaling by direct interaction and phosphorylation.

Cell survival and death-inducing signals are tightly associated with each other, and the decision as to whether a cell survives or dies is determined by controlling the relationship between these signals. However, the mechanism underlying the reciprocal regulation of such signals remains unclear. In this study, we reveal a functional association between PDK1 (3-phosphoinositide-dependent protein kinase 1), a critical mediator of cell survival, and ASK1 (apoptosis signal-regulating kinase 1), an apoptotic stress-activated MAPKKK. The physical association between PDK1 and ASK1 is mediated through the pleckstrin homology domain of PDK1 and the C-terminal regulatory domain of ASK1 and is decreased by ASK1-activating stimuli, such as H(2)O(2), tumor necrosis factor alpha, thapsigargin, and ionomycin, as well as insulin, a PDK1 stimulator. Wild-type PDK1, but not kinase-dead PDK1, negatively regulates ASK1 activity by phosphorylating Ser(967), a binding site for 14-3-3 protein, on ASK1. PDK1 functionally suppresses ASK1-mediated AP-1 transactivation and H(2)O(2)-mediated apoptosis in a kinase-dependent manner. On the other hand, ASK1 has been shown to inhibit PDK1 functions, including PDK1-mediated regulation of apoptosis and cell growth, by phosphorylating PDK1 at Ser(394) and Ser(398), indicating that these putative phosphorylation sites are involved in the negative regulation of PDK1 activity. These results provide evidence that PDK1 and ASK1 directly interact and phosphorylate each other and act as negative regulators of their respective kinases in resting cells.

Serine-threonine kinase receptor-associated protein inhibits apoptosis signal-regulating kinase 1 function through direct interaction.

Serine-threonine kinase receptor-associated protein (STRAP) interacts with transforming growth factor beta (TGF-beta) receptors and inhibits TGF-beta signaling. Here, we identify STRAP as an interacting partner of ASK1 (apoptosis signal-regulating kinase 1). The association between ASK1 and STRAP is mediated through the C-terminal domain of ASK1 and the fourth and sixth WD40 repeats of STRAP. Using cysteine-to-serine amino acid substitution mutants of ASK1 (C1005S, C1351S, C1360S, and C1351S/C1360S) and STRAP (C152S, C270S, and C152S/C270S), we demonstrated that Cys(1351) and Cys(1360) of ASK1 and Cys(152) and Cys(270) of STRAP are required for ASK1-STRAP binding. ASK1 phosphorylated STRAP at Thr(175) and Ser(179), suggesting a potential role for STRAP phosphorylation in ASK1 activity regulation. Expression of wild-type STRAP, but not STRAP mutants (C152S/C270S and T175A/S179A), inhibited ASK1-mediated signaling to both JNK and p38 kinases by stabilizing complex formation between ASK1 and its negative regulators, thioredoxin and 14-3-3, or decreasing complex formation between ASK1 and its substrate MKK3. In addition, STRAP suppressed H(2)O(2)-mediated apoptosis in a dose-dependent manner by inhibiting ASK1 activity through direct interaction. These results suggest that STRAP can act as a negative regulator of ASK1.

A 39 kDa fragment of endogenous ASK1 suggests specific cleavage not degradation by the proteasome.

Transfected human apoptosis signal-regulating kinase 1 (ASK1) produces a 150 kDa protein. However, we have detected endogenous ASK1 predominantly as 39 and 50 kDa C-terminal and 75 and 110 kDa N-terminal fragments in a panel of nontransfected cancer cell lines and HUVEC endothelial cells. This suggests that in nonapoptotic cells, endogenous ASK1 protein is normally cleaved at a number of specific sites, some of which are in the kinase domain. Transfected ASK1 protein is known to be degraded by the proteasome. In contrast, the cleavage of endogenous ASK1 is independent of the proteasome as treatment with the proteasome inhibitor, lactacystin did not inhibit cleavage. Cisplatin treatment decreased the amount of 39 kDa C-terminal ASK1 fragments in mutant p53 cell lines suggesting a decrease in cleavage associated with apoptosis. Transfected ASK1 may, therefore, not accurately reflect the role of endogenous ASK1.

Thioredoxin and TRAF family proteins regulate reactive oxygen species-dependent activation of ASK1 through reciprocal modulation of the N-terminal homophilic interaction of ASK1.

Apoptosis signal-regulating kinase 1 (ASK1), a member of the mitogen-activated protein kinase kinase kinase family, plays pivotal roles in reactive oxygen species (ROS)-induced cellular responses. In resting cells, endogenous ASK1 constitutively forms a homo-oligomerized but still inactive high-molecular-mass complex including thioredoxin (Trx), which we designated the ASK1 signalosome. Upon ROS stimulation, the ASK1 signalosome unbinds from Trx and forms a fully activated higher-molecular-mass complex, in part by recruitment of tumor necrosis factor receptor-associated factor 2 (TRAF2) and TRAF6. However, the precise mechanisms by which Trx inhibits and TRAF2 and TRAF6 activate ASK1 have not been elucidated fully. Here we demonstrate that the N-terminal homophilic interaction of ASK1 through the N-terminal coiled-coil domain is required for ROS-dependent activation of ASK1. Trx inhibited this interaction of ASK1, which was, however, enhanced by expression of TRAF2 or TRAF6 or by treatment of cells with H2O2. Furthermore, the H2O2-induced interaction was reduced by double knockdown of TRAF2 and TRAF6. These findings demonstrate that Trx, TRAF2, and TRAF6 regulate ASK1 activity by modulating N-terminal homophilic interaction of ASK1.

Disulfide Bond-mediated multimerization of Ask1 and its reduction by thioredoxin-1 regulate H(2)O(2)-induced c-Jun NH(2)-terminal kinase activation and apoptosis.

Apoptosis signal-regulated kinase-1 (Ask1) lies upstream of a major redox-sensitive pathway leading to the activation of Jun NH(2)-terminal kinase (JNK) and the induction of apoptosis. We found that cell exposure to H(2)O(2) caused the rapid oxidation of Ask1, leading to its multimerization through the formation of interchain disulfide bonds. Oxidized Ask1 was fully reduced within minutes after induction by H(2)O(2). During this reduction, the thiol-disulfide oxidoreductase thioredoxin-1 (Trx1) became covalently associated with Ask1. Overexpression of Trx1 accelerated the reduction of Ask1, and a redox-inactive mutant of Trx1 (C35S) remained trapped with Ask1, blocking its reduction. Preventing the oxidation of Ask1 by either overexpressing Trx1 or using an Ask1 mutant in which the sensitive cysteines were mutated (Ask1-DeltaCys) impaired the activation of JNK and the induction of apoptosis while having little effect on Ask1 activation. These results indicate that Ask1 oxidation is required at a step subsequent to activation for signaling downstream of Ask1 after H(2)O(2) treatment.

Role of thioredoxin in cell growth through interactions with signaling molecules.

The thioredoxin system helps maintain a reducing environment in cells, but thioredoxin functions as more than simply an antioxidant. Thioredoxin functions depend on the protein's redox state, as determined by two conserved cysteines. Key biologic activities of thioredoxin include antioxidant, growth control, and antiapoptotic properties, resulting from interaction with target molecules including transcription factors. Mechanisms by which thioredoxin regulates cell growth include binding to signaling molecules such as apoptosis signal-regulating kinase-1 (ASK-1) and thioredoxin-interacting protein (Txnip). The molecular interplay between thioredoxin, ASK-1, and Txnip potentially influences cell growth and survival in diverse human diseases such as cancer, diabetes, and heart disease. In this review, we focus on the structure of thioredoxin and its functional regulation of cell growth through the interactions with signaling molecules.

Direct interaction and reciprocal regulation between ASK1 and calcineurin-NFAT control cardiomyocyte death and growth.

The calcium-calmodulin-activated protein phosphatase calcineurin functions as a key mediator of diverse biologic processes, including differentiation, apoptosis, growth, and adaptive responses, in part through dephosphorylation and activation of nuclear factor of activated T-cell (NFAT) transcription factors. Apoptosis signal-regulating kinase 1 (ASK1) is an upstream component of the mitogen-activated protein kinases that serves as a pivotal regulator of cytokine-, oxidative-, and stress-induced cell death. Here, we performed a yeast two-hybrid screen with calcineurin B as bait, which identified ASK1 as a direct physical interacting partner. The C-terminal 218 amino acids of ASK1 were sufficient to mediate interaction with calcineurin B in yeast, as well as in mammalian cell lysates. Importantly, endogenous calcium binding B subunit (CnB) protein interacted with endogenous ASK1 protein in cardiomyocytes at baseline, suggesting that the interaction observed in yeast was of potential biologic relevance. Indeed, calcineurin directly dephosphorylated ASK1 at serine 967 using purified proteins or mammalian cell lysates. Dephosphorylation of ASK1 serine 967 by calcineurin promoted its disassociation from 14-3-3 proteins, resulting in ASK1 activation. Calcineurin and ASK1 cooperatively enhanced cardiomyocyte apoptosis, while expression of a dominant negative ASK1 blocked calcineurin-induced apoptosis. Mouse embryonic fibroblasts deficient in ask1 were also partially resistant to calcineurin- or ionomycin-induced apoptosis. Finally, ASK1 negatively regulated calcineurin-NFAT signaling indirectly through c-Jun NH2-terminal kinase (JNK)- and p38-mediated phosphorylation of NFAT, which blocked calcineurin- and agonist-dependent hypertrophic growth of cardiomyocytes. Thus, ASK1 and calcineurin-NFAT constitute a feedback regulatory circuit in which calcineurin positively regulates ASK1 through direct dephosphorylation, while ASK1 negatively regulates calcineurin-NFAT signaling through p38- and JNK-mediated NFAT phosphorylation.

Functional analyses of mouse ASK, an activation subunit for Cdc7 kinase, using conditional ASK knockout ES cells.

ASK (activator of S phase kinase) is an activation subunit for mammalian Cdc7 kinase. We have generated mutant ES cell lines in which ASK can be conditionally inactivated. Upon loss of the ASK genes, the mutant ES cells rapidly cease cell growth. In keeping with its expected roles in activation of the essential S phase kinase, DNA synthesis is arrested and significant cell death is eventually induced in ASK-deficient cells, demonstrating essential roles of ASK for viability of ES cells. Using these mutant cells, we have set up a system where ASK molecules can be functionally dissected. In keeping with previous results from yeasts, conserved motif-M and motif-C were shown to be essential for in vivo functions of ASK, whereas a long C-terminal tail, found only in ASK-related molecules in higher eukaryotes, is not required. Unexpectedly, the motif-N, related to the BRCT motif and dispensable for viability in yeasts, is essential for the viability of ES cells. Further characterization reveals that motif-N is required for the maximum phosphorylation of MCM in cells, whereas the autophosphorylation activity of Cdc7 is not significantly affected by its loss. These results may suggest that motif-N of ASK may facilitate recruitment of substrates for Cdc7 kinase.

Thioredoxin-2 inhibits mitochondria-located ASK1-mediated apoptosis in a JNK-independent manner.

Apoptosis signal-regulating kinase 1 (ASK1) mediates cytokines and oxidative stress (ROS)-induced apoptosis in a mitochondria-dependent pathway. However, the underlying mechanism has not been defined. In this study, we show that ASK1 is localized in both cytoplasm and mitochondria of endothelial cells (ECs) where it binds to cytosolic (Trx1) and mitochondrial thioredoxin (Trx2), respectively. Cys-250 and Cys-30 in the N-terminal domain of ASK1 are critical for binding of Trx1 and Trx2, respectively. Mutation of ASK1 at C250 enhanced ASK1-induced JNK activation and apoptosis, whereas mutation of ASK1 at C30 specifically increased ASK1-induced apoptosis without effects on JNK activation. We further show that a JNK-specific inhibitor SP600125 completely blocks TNF induced JNK activation, Bid cleavage, and Bax mitochondrial translocation, but only partially inhibits cytochrome c release and EC death, suggesting that TNF induces both JNK-dependent and JNK-independent apoptotic pathways in EC. Mitochondria-specific expression of a constitutively active ASK1 strongly induces EC apoptosis without JNK activation, Bid cleavage, and Bax mitochondrial translocation. These data suggest that mitochondrial ASK1 mediates a JNK-independent apoptotic pathway induced by TNF. To determine the role of Trx2 in regulation of mitochondrial ASK1 activity, we show that overexpression of Trx2 inhibits ASK1-induced apoptosis without effects on ASK1-induced JNK activation. Moreover, specific knockdown of Trx2 in EC increases TNF/ASK1-induced cytochrome c release and cell death without increase in JNK activation, Bid cleavage, and Bax translocation. Our data suggest that ASK1 in cytoplasm and mitochondria mediate distinct apoptotic pathways induced by TNF, and Trx1 and Trx2 cooperatively inhibit ASK1 activities.