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.     

Regulation of apoptosis signal-regulating kinase 1 by protein phosphatase 2Cepsilon

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 TNFalpha (tumour necrosis factor alpha). 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 PP2Cepsilon (protein phosphatase 2Cepsilon), a member of PP2C family, in the regulation of ASK1 signalling. Following expression in HEK-293 cells (human embryonic kidney cells), wild-type PP2Cepsilon inhibited ASK1-induced activation of an AP-1 (activator protein 1) reporter gene. Conversely, a dominant-negative PP2Cepsilon mutant enhanced AP-1 activity. Exogenous PP2Cepsilon associated with exogenous ASK1 in HEK-293 cells under non-stressed conditions, inactivating ASK1 by decreasing Thr845 phosphorylation. The association of endogenous PP2Cepsilon and ASK1 was also observed in mouse brain extracts. PP2Cepsilon directly dephosphorylated ASK1 at Thr845 in vitro. In contrast with PP5, PP2Cepsilon transiently dissociated from ASK1 within cells upon H2O2 treatment. These results suggest that PP2Cepsilon maintains ASK1 in an inactive state by dephosphorylation in quiescent cells, supporting the possibility that PP2Cepsilon and PP5 play different roles in H2O2-induced regulation of ASK1 activity.     

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.     

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.     

H(2)O(2) signals 5-HT-induced ERK MAP kinase activation and mitogenesis of smooth muscle cells

Our previous studies have shown that 5-hydroxytryptamine (5-HT) induces cellular hyperplasia/hypertrophy through protein tyrosine phosphorylation, rapid formation of superoxide (O(2)(-)), and extracellular signal-regulated kinase (ERK)1/ERK2 mitogen-activated protein (MAP) kinase activation. Intracellularly released O(2)(-) is rapidly dismuted by superoxide dismutase (SOD) to H(2)O(2), another possible cellular growth mediator. In the present study, we assessed whether H(2)O(2) participates in 5-HT-induced mitogenic signaling. Inactivation of cellular Cu/Zn SOD by copper-chelating agents inhibited 5-HT-induced DNA synthesis of bovine pulmonary artery smooth muscle cells (BPASMCs). Infection of BPASMCs with an adenovirus containing catalase inhibited both ERK1/ERK2 MAP kinase activation and DNA synthesis induced by 5-HT. Although we could not find evidence of p38 MAP kinase activation by 5-HT, SB-203580 and SB-202190, reported inhibitors of p38 MAP kinase, inhibited the 5-HT-induced growth of BPASMCs. However, these inhibitors also inhibited 5-HT-induced O(2)(-) release. Thus quenching of O(2)(-) may be their mechanism for inhibition of cellular growth unrelated to p38 MAP kinase inhibition. These data indicate that generation of O(2)(-) in BPASMCs in response to 5-HT is followed by an increase in intracellular H(2)O(2) that mediates 5-HT-induced mitogenesis through activation of ERK1/ERK2 but not of p38 MAP kinase.     

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.     

Identification of Op18/stathmin as a potential target of ASK1-p38 MAP kinase cascade

Apoptosis signal-regulating kinase 1 (ASK1) is a mitogen-activated protein (MAP) kinase kinase kinase that activates the JNK and p38 MAP kinase cascades and has a broad range of biological activities including cell differentiation and stress-induced apoptosis. However, effector molecules of ASK1-MAP kinase cascades that exert such activities have not been fully identified. Here we have identified oncoprotein 18 (Op18)/stathmin as a potential target of the ASK1-p38 cascade. By two-dimensional electrophoresis, phosphorylation of Op18/stathmin was found to be increased upon the expression of constitutively active ASK1 (ASK1DeltaN) in PC12 cells. The ASK1-dependent increase in the phosphorylation of Op18/stathmin was attenuated by the treatment with SB203580, suggesting that p38alpha and/or p38beta contribute to the phosphorylation of Op18/stathmin. Consistently, we found that all four isoforms of p38 directly phosphorylated Op18/stathmin primarily at serine 25 in vitro. Taken together with the quantitative RT-PCR data indicating that p38alpha was the dominantly expressed isoform in PC12 cells, ASK1-induced phosphorylation of Op18/stathmin appears to be mediated mainly through p38alpha in these cells. Given that the microtubule-destabilizing activity of Op18/stathmin is regulated by its phosphorylation, the ASK1-p38 cascade may regulate microtubule dynamics through Op18/stathmin.     

Involvement of ASK1 in Ca2+-induced p38 MAP kinase activation

The mammalian mitogen-activated protein (MAP) kinase kinase kinase apoptosis signal-regulating kinase 1 (ASK1) is a pivotal component in cytokine- and stress-induced apoptosis. It also regulates cell differentiation and survival through p38 MAP kinase activation. Here we show that Ca²⁺ signalling regulates the ASK1–p38 MAP kinase cascade. Ca²⁺ influx evoked by membrane depolarization in primary neurons and synaptosomes induced activation of p38, which was impaired in those derived from ASK1-deficient mice. Ca²⁺/calmodulin-dependent protein kinase type II (CaMKII) activated ASK1 by phosphorylation. Moreover, p38 activation induced by the expression of constitutively active CaMKII required endogenous ASK1. Thus, ASK1 is a critical intermediate of Ca²⁺ signalling between CaMKII and p38 MAP kinase.     

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.     

Inhibition of mammalian target of rapamycin activates apoptosis signal-regulating kinase 1 signaling by suppressing protein phosphatase 5 activity

Under serum-free conditions, rapamycin, an inhibitor of mammalian target of rapamycin (mTOR), induces a cellular stress response characterized by rapid and sustained activation of the apoptosis signal-regulating kinase 1 (ASK1) signaling pathway and selective apoptosis of cells lacking functional p53. Here we have investigated how mTOR regulates ASK1 signaling using p53-mutant rhabdomyosarcoma cells. In Rh30 cells, ASK1 was found to physically interact with protein phosphatase 5 (PP5), previously identified as a negative regulator of ASK1. Rapamycin did not affect either protein level of PP5 or association of PP5 with ASK1. Instead, rapamycin caused rapid dissociation of the PP2A-B" regulatory subunit (PR72) from the PP5-ASK1 complex, which was associated with reduced phosphatase activity of PP5. This effect was dependent on expression of eukaryotic initiation factor 4E-binding protein 1 (4E-BP1). Down-regulation of PP5 activity by rapamycin coordinately activated ASK1, leading to elevated phosphorylation of c-Jun. Amino acid deprivation, which like rapamycin inhibits mTOR signaling, also inhibited PP5 activity, caused rapid dissociation of PR72, and activated ASK1 signaling. Overexpression of PP5, but not the PP2A catalytic subunit, blocked rapamycin-induced phosphorylation of c-Jun, and protected cells from rapamycin-induced apoptosis. The results suggest that PP5 is downstream of mTOR, and positively regulated by the mTOR pathway. The findings suggest that in the absence of serum factors, mTOR signaling suppresses apoptosis through positive regulation of PP5 activity and suppression of cellular stress.     

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.     

S-nitrosylation of thioredoxin mediates activation of apoptosis signal-regulating kinase 1

Apoptosis signal-regulating kinase 1 (ASK1) was recently discovered as a typical member of the mitogen-activated protein (MAP) kinase kinase kinase family, which induces apoptosis by activation of c-Jun-N-terminal kinase/p38 MAP kinase pathways. In normal cells ASK1 is directly inhibited by thioredoxin (Trx), a 12-kDa protein ubiquitously expressed in all living cells, which has a variety of biological functions related to cell proliferation and apoptosis. Here we found that purified Trx is sensitive to S-nitrosylation. Stimulation of HEK-293 cells with S-nitrosoglutathione (GSNO) for 2, 4, 8, and 16h also caused Trx S-nitrosylation, which showed straight correlation with ASK1 activation based on Western blot detection of the enzyme, immunoprecipitation assay, and measurement of its catalytic activity. These results suggest that S-nitrosylation of Trx induces ASK1 activation. Treatment of cells with N-acetyl-cysteine for 2h after 8h of pretreatment with GSNO caused an increase in glutathione and nullified ASK1 activation.     

Heat shock protein hsp72 is a negative regulator of apoptosis signal-regulating kinase 1

Heat shock protein 72 (Hsp72) is thought to protect cells against cellular stress. The protective role of Hsp72 was investigated by determining the effect of this protein on the stress-activated protein kinase signaling pathways. Prior exposure of NIH 3T3 cells to mild heat shock (43 degrees C for 20 min) resulted in inhibition of H(2)O(2)-induced activation of apoptosis signal-regulating kinase 1 (ASK1). Overexpression of Hsp72 also inhibited H(2)O(2)-induced activation of ASK1 as well as that of downstream kinases in the p38 mitogen-activated protein kinase (MAPK) signaling cascade. Recombinant Hsp72 bound directly to ASK1 and inhibited ASK1 activity in vitro. Furthermore, coimmunoprecipitation analysis revealed a physical interaction between endogenous Hsp72 and ASK1 in NIH 3T3 cells exposed to mild heat shock. Hsp72 blocked both the homo-oligomerization of ASK1 and ASK1-dependent apoptosis. Hsp72 antisense oligonucleotides prevented the inhibitory effects of mild heat shock on H(2)O(2)-induced ASK1 activation and apoptosis. These observations suggest that Hsp72 functions as an endogenous inhibitor of ASK1.     

Activation of apoptosis signal-regulating kinase 1 by reactive oxygen species through dephosphorylation at serine 967 and 14-3-3 dissociation

Oxidative stress has been indicated in a variety of pathological processes such as atherosclerosis, diabetes, and neurodegenerative diseases. Understanding how intracellular signaling pathways respond to oxidative insults such as hydrogen peroxide (H(2)O(2)) would have significant therapeutic implications. Recent genetic studies have placed apoptosis signal-regulating kinase 1 (ASK1) in a pivotal position in transmitting H(2)O(2)-initiated signals. How ASK1 is activated by H(2)O(2), though, remains a subject of intense investigation. Here we report a mechanism by which H(2)O(2) induces ASK1 activation through dynamic control of its phosphorylation at serine 967. We found that treatment of COS7 cells with H(2)O(2) triggers dephosphorylation of Ser-967 through an okadaic acid-sensitive phosphatase, resulting in dissociation of the ASK1.14-3-3 complex with concomitant increase of ASK1 catalytic activity and ASK1-mediated activation of JNK and p38 pathways.     

Execution of macrophage apoptosis by Mycobacterium avium through apoptosis signal-regulating kinase 1/p38 mitogen-activated protein kinase signaling and caspase 8 activation

Macrophage apoptosis is an important component of the innate immune defense machinery (against pathogenic mycobacteria) responsible for limiting bacillary viability. However, little is known about the mechanism of how apoptosis is executed in mycobacteria-infected macrophages. Apoptosis signal-regulating kinase 1 (ASK1) was activated in Mycobacterium avium-treated macrophages and in turn activated p38 mitogen-activated protein (MAP) kinase. M. avium-induced macrophage cell death could be blocked in cells transfected with a catalytically inactive mutant of ASK1 or with dominant negative p38 MAP kinase arguing in favor of a central role of ASK1/p38 MAP kinase signaling in apoptosis of macrophages challenged with M. avium. ASK1/p38 MAP kinase signaling was linked to the activation of caspase 8. At the same time, M. avium triggered caspase 8 activation, and cell death occurred in a Fas-associated death domain (FADD)-dependent manner. The death signal induced upon caspase 8 activation linked to mitochondrial death signaling through the formation of truncated Bid (t-Bid), its translocation to the mitochondria and release of cytochrome c. Caspase 8 inhibitor (z-IETD-FMK) could block the release of cytochrome c as well as the activation of caspases 9 and 3. The final steps of apoptosis probably involved caspases 9 and 3, since inhibitors of both caspases could block cell death. Of foremost interest in the present study was the finding that ASK1/p38 signaling was essential for caspase 8 activation linked to M. avium-induced death signaling. This work provides the first elucidation of a signaling pathway in which ASK1 plays a central role in innate immunity.     

PP1/PP2A phosphatases inhibitors okadaic acid and calyculin A block ERK5 activation by growth factors and oxidative stress

Okadaic acid is an inhibitor of the protein Ser/Thr phosphatases PP1 and PP2A, which blocks the activation of extracellular signal-regulated protein kinase 5 (ERK5), a member of the MAP kinase family activated by growth factors and several types of stressors. The blocking of ERK5 activation by okadaic acid was observed in HeLa cells exposed to epidermal growth factor and H(2)O(2) as well as in PC12 cells stimulated by nerve growth factor and H(2)O(2). Calyculin A, another PP1 and PP2A inhibitor, behaved similarly although these compounds are not structurally related. This suggests that either PP1 or PP2A or both are necessary for ERK5 activation. Protein kinase C (PKC) acts as a negative regulator of the ERK5 activation pathway, however our data suggest that the effects of PKC and the phosphatase are unrelated.     

Mammalian thioredoxin is a direct inhibitor of apoptosis signal-regulating kinase (ASK) 1.

Apoptosis signal-regulating kinase (ASK) 1 was recently identified as a mitogen-activated protein (MAP) kinase kinase kinase which activates the c-Jun N-terminal kinase (JNK) and p38 MAP kinase pathways and is required for tumor necrosis factor (TNF)-alpha-induced apoptosis; however, the mechanism regulating ASK1 activity is unknown. Through genetic screening for ASK1-binding proteins, thioredoxin (Trx), a reduction/oxidation (redox)-regulatory protein thought to have anti-apoptotic effects, was identified as an interacting partner of ASK1. Trx associated with the N-terminal portion of ASK1 in vitro and in vivo. Expression of Trx inhibited ASK1 kinase activity and the subsequent ASK1-dependent apoptosis. Treatment of cells with N-acetyl-L-cysteine also inhibited serum withdrawal-, TNF-alpha- and hydrogen peroxide-induced activation of ASK1 as well as apoptosis. The interaction between Trx and ASK1 was found to be highly dependent on the redox status of Trx. Moreover, inhibition of Trx resulted in activation of endogenous ASK1 activity, suggesting that Trx is a physiological inhibitor of ASK1. The evidence that Trx is a negative regulator of ASK1 suggests possible mechanisms for redox regulation of the apoptosis signal transduction pathway as well as the effects of antioxidants against cytokine- and stress-induced apoptosis.     

Regulation of glucose transport and glycogen synthesis in L6 muscle cells during oxidative stress. Evidence for cross-talk between the insulin and SAPK2/p38 mitogen-activated protein kinase signaling pathways

We have investigated the cellular mechanisms that participate in reducing insulin sensitivity in response to increased oxidant stress in skeletal muscle. Measurement of glucose transport and glycogen synthesis in L6 myotubes showed that insulin stimulated both processes, by 2- and 5-fold, respectively. Acute (30 min) exposure of muscle cells to hydrogen peroxide (H(2)O(2)) blocked the hormonal activation of both these processes. Immunoblot analyses of cell lysates prepared after an acute oxidant challenge using phospho-specific antibodies against c-Jun N-terminal kinase (JNK), p38, protein kinase B (PKB), and p42 and p44 mitogen-activated protein (MAP) kinases established that H(2)O(2) induced a dose-dependent activation of all five protein kinases. In vitro kinase analyses revealed that 1 mM H(2)O(2) stimulated the activity of JNK by approximately 8-fold, MAPKAP-K2 (the downstream target of p38 MAP kinase) by approximately 12-fold and that of PKB by up to 34-fold. PKB activation was associated with a concomitant inactivation of glycogen synthase kinase-3. Stimulation of the p38 pathway, but not that of JNK, was blocked by SB 202190 or SB203580, while that of p42/p44 MAP kinases and PKB was inhibited by PD 98059 and wortmannin respectively. However, of the kinases assayed, only p38 MAP kinase was activated at H(2)O(2) concentrations (50 microM) that caused an inhibition of insulin-stimulated glucose transport and glycogen synthesis. Strikingly, inhibiting the activation of p38 MAP kinase using either SB 202190 or SB 203580 prevented the loss in insulin-stimulated glucose transport, but not that of glycogen synthesis, by oxidative stress. Our data indicate that activation of the p38 MAP kinase pathway plays a central role in the oxidant-induced inhibition of insulin-regulated glucose transport, and unveils an important biochemical link between the classical stress-activated and insulin signaling pathways in skeletal muscle.     

Activation of apoptosis signal-regulating kinase 1 by the stress-induced activating phosphorylation of pre-formed oligomer

Apoptosis signal-regulating kinase 1 (ASK1) is a MAPKKK family member which activates c-Jun N-terminal kinase (JNK) and p38. In non-stressed cells, ASK1 exists as an inactive complex with the reduced form of thioredoxin. Oxidative stress such as hydrogen peroxide (H2O2) disrupts the ASK1-thioredoxin complex by oxidization of thioredoxin and thereby activates ASK1. The precise mechanism by which ASK1 is activated after its release from thioredoxin is unknown. Here we show that phosphorylation of Thr845 at the activation loop is essential for ASK1 to be activated by H2O2. ASK1 appears to form a silent homo-oligomer through its C-terminal coiled-coil region in non-stressed cells. Following H2O2 treatment, pre-existing ASK1 oligomer undergoes conformational change and creates a new interface within an oligomer, which ultimately leads to trans-autophosphorylation of Thr845. Thus, direct interaction via the coiled-coil region is required for self-scaffolding but not sufficient for activation of ASK1. Importantly, Thr845 of ASK1 can also be trans-phosphorylated by an unidentified Thr845 kinase in response to H2O2 treatment. We propose that this potential Thr845 kinase may be an ignition kinase that triggers Thr845 phosphorylation in oligomerized and activation-competent forms of ASK1.