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.     

CaMKII activates ASK1 and NF-kappaB to induce cardiomyocyte hypertrophy

Ca2+/calmodulin-dependent protein kinase (CaMK) is an important downstream target of Ca2+ in the hypertrophic signaling pathways. We previously showed that the activation of apoptosis signal-regulating kinase 1 (ASK1) or NF-kappaB is sufficient for cardiomyocyte hypertrophy. Infection of isolated neonatal cardiomyocytes with an adenoviral vector expressing CaMKIIdelta3 (AdCaMKIIdelta3) induced the activation of ASK1, while KN93, an inhibitor of CaMKII, inhibited phenylephrine-induced ASK1 activation. Overexpression of CaMKIIdelta3 induced characteristic features of in vitro cardiomyocyte hypertrophy. Infection of cardiomyocytes with an adenoviral vector expressing a dominant negative mutant of ASK1 (AdASK(KM)) inhibited the CaMKIIdelta3-induced hypertrophic responses. Overexpression of CaMKIIdelta3 increased the kappaB-dependent promoter/luciferase activity and induced IkappaBalpha degradation. Coinfection with AdCaMKIIdelta3 and AdASK(KM), and pre-incubation with KN93 attenuated CaMKIIdelta3- and phenylephrine-induced NF-kappaB activation, respectively. Expression of a degradation resistant mutant of IkappaBalpha inhibited CaMKIIdelta3-induced hypertrophic responses. These results indicate that CaMKIIdelta3 induces cardiomyocyte hypertrophy mediated through ASK1-NF-kappaB signal transduction pathway.     

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.     

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 β (TGF-β) receptors and inhibits TGF-β 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 Cys¹³⁵¹ and Cys¹³⁶⁰ of ASK1 and Cys¹⁵² and Cys²⁷⁰ of STRAP are required for ASK1-STRAP binding. ASK1 phosphorylated STRAP at Thr¹⁷⁵ and Ser¹⁷⁹, 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 H₂O₂-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 non-covalent interaction between small ubiquitin-like modifier-1 and Zac1 regulates Zac1 cellular functions

Zac1, a zinc-finger protein that regulates apoptosis and cell cycle arrest 1, such as p53, can induce cell-cycle arrest and apoptosis. The transactivation and coactivation functions of Zac1 may occur at non-promyelocytic leukemia nuclear body (PML-NB) sites in the presence of other PML-NB components, including ubiquitin-conjugating 9 (Ubc9). It is unclear whether post-translational modification of Zac1 by the small ubiquitin-like modifier SUMO plays a role in the coactivation functions of Zac1 for the regulation of the p21 gene. Mutagenesis experiments revealed that the two SUMO-binding lysine residues of Zac1, K237 and K424, repress the transactivation activity of Zac1. Studies using a SUMO-1 C-terminal di-glycine motif mutant that is deficient in the ability to form covalent bonds with lysines, SUMO-1 (GA), and a dominant-negative Ubc9 construct (C93S) indicated that SUMO-1 might regulate Zac1 transactivation and coactivation via a non-covalent interaction. Unlike the wild-type Zac1, which induced apoptosis, the Zac1 (K237/424R) double mutant had the ability to induce autophagy. The functional role of p21 remains to be investigated. SUMO-1 selectively suppressed the induction of the p21 gene and protein by wild-type Zac1 but not by the Zac1 (K237/424R) double mutant. Moreover, wild-type Ubc9 but not Ubc9 (C93S) further potentiated the suppression of SUMO-1 in all Zac1-induced p21 promoter activities. Our data reveal that p21 may be an important factor for the prevention of Zac1-induced apoptosis without affecting autophagosome formation. This work indicates that Zac1 functions are regulated, at least in part, via non-covalent interactions with SUMO-1 for the induction of p21, which is important for the modulation of apoptosis.     

Critical role of ASK1 in the 6-hydroxydopamine-induced apoptosis in human neuroblastoma SH-SY5Y cells

6-hydroxydopamine (6-OHDA)-induced apoptosis in dopaminergic neuronal cells is a common cell model of Parkinson's disease (PD). The role of apoptosis signal-regulating kinase 1 (ASK1) in this model has not been well studied. We observed significant activation of ASK1, p38 and JNK, as well as apoptosis in human dopaminergic neuroblastoma SH-SY5Y cells exposed to 6-OHDA. Over-expressing kinase-dead mutant ASK1(K709M) or knock-down of endogenous ASK1 by its small interfering RNA (siRNA) greatly suppressed activation of these kinases and apoptosis in the cells. It was found that the activation of p38 and JNK was suppressed to almost the same extent as that of ASK1 in the ASK1-knock-down cells, suggesting that activated ASK1 is almost totally responsible for activation of p38/JNK. It was also observed that the 6-OHDA-induced cell apoptosis could be effectively prevented by over-expressing the dominant-negative mutant of p38 or p38 inhibitor SB203580, demonstrating that activation of p38/JNK signalling is required for initiating the programmed cell death. Furthermore, suppression of the 6-OHDA-generated reactive oxygen species (ROS) by pre-incubation of cells with N-acetyl-L-cysteine effectively inhibited the 6-OHDA-induced activation of ASK1, p38 and JNK, and protected the cells from apoptosis. This study clearly shows the route from ROS generation by 6-OHDA to initiation of p38/JNK signalling via activation of ASK1 in the studied PD model.     

Daxx deletion mutant (amino acids 501-625)-induced apoptosis occurs through the JNK/p38-Bax-dependent mitochondrial pathway

Death-associated protein (Daxx) deletion mutant (aa 501-625) has been known to be an inducer of apoptosis. In this study, we observed that the Bax-dependent mitochondrial death signaling pathway plays an important role in Daxx501-625-induced apoptosis. Daxx fragment-induced activation of caspase-9 and -3 was mediated through the apoptosis signal-regulating kinase 1 (ASK1)-MEK-c-Jun-N-terminal kinase (JNK)/p38-Bax pathway. By overexpressing JNK-binding domain (JBD) of JIP1, a JNK-inhibitory protein, and treatment with SB203580, a specific p38 inhibitor, DU-145 cells were made resistant to Daxx501-625-induced apoptosis. Capase-3 deficiency, Bax deficiency, or overexpression of a dominant-negative caspase-9 mutant prevented apoptosis, even though the Daxx501-625 fragment still activated the ASK1-MEK-MAPK pathway. Interestingly, Daxx501-625-induced Bcl-2 interacting domain (Bid) cleavage was suppressed in the dominant-negative caspase-9 mutant cells, whereas Bim was still phosphorylated in these cells. These results suggest that cleavage of Bid occurs downstream of caspase-9 activation. In contrast, phosphorylation of Bim is upstream of caspase-9 activation. Taken together, our results suggest that Daxx501-625-induced apoptosis is mediated through the ASK1-MEK-JNK/p38-Bim-Bax-dependent caspase pathway.     

Interaction of apoptosis signal-regulating kinase 1 with isoforms of 14-3-3 proteins

Apoptosis signal-regulating kinase 1 (ASK1) is a critical mediator of apoptotic signaling pathways initiated by a variety of death stimuli. Its activity is tightly controlled by various mechanisms such as covalent modification and protein-protein interaction. One of the proteins that control ASK1 function is 14-3-3zeta, a member of the 14-3-3 protein family. Here, we report that ASK1 is capable of binding to other isoforms of 14-3-3, suggesting that binding ASK1 is a general property of the 14-3-3 family. In support of this notion, mutational analysis revealed that the ASK1/14-3-3 interaction was mediated by the conserved amphipathic groove of 14-3-3 with some residue selectivity. Functionally, expression of various isoforms of 14-3-3 suppressed ASK1-induced apoptosis. To understand how 14-3-3 controls the ASK1 activity, we examined intracellular localization of ASK1 upon 14-3-3 co-expression. We found that 14-3-3 co-expression is correlated with the translocation of ASK1 from the cytoplasm to a perinuclear localization, likely the ER compartment. Consistent with this notion, ASK1(S967A), a 14-3-3 binding defective mutant of ASK, showed no change in intracellular distribution upon 14-3-3 co-expression. These data support a model that 14-3-3 proteins regulate the proapoptotic function of ASK1 in part by controlling its subcellular distribution.     

Inhibition of apoptosis signal-regulating kinase 1 by nitric oxide through a thiol redox mechanism

Nitric oxide is an endogenous thiol-reactive molecule that modulates the functions of many regulatory proteins by a thiol-redox mechanism. NO has now been shown to inhibit the activation of apoptosis signal-regulating kinase 1 (ASK1) in murine fibrosarcoma L929 cells through such a mechanism. Exposure of L929 cells to interferon-gamma resulted in the endogenous production of NO and in inhibition of the activation of ASK1 by hydrogen peroxide. The interferon-gamma-induced inhibition of ASK1 activity was blocked by N(G)-nitro-l-arginine, an inhibitor of NO synthase. Furthermore, the NO donor S-nitro-N-acetyl-dl-penicillamine (SNAP) inhibited ASK1 activity in vitro, and this inhibition was reversed by thiol-reducing agents such as dithiothreitol and beta-mercaptoethanol. SNAP did not inhibit the kinase activities of MKK3, MKK6, or p38 in vitro. The inhibition of ASK1 by interferon-gamma was not changed by 1H- (1,2,4)oxadiazolo[4,3-alpha]quinoxalin-1-one, an inhibitor of guanylyl cyclase nor was it mimicked by 8-bromo-cyclic GMP. Site-directed mutagenesis revealed that replacement of cysteine 869 of ASK1 by serine rendered this protein resistant to the inhibitory effects both of interferon-gamma in intact cells and of SNAP in vitro. Co-immunoprecipitation data showed that NO production inhibited a binding of ASK1, but not ASK1(C869S), to MKK3 or MKK6. Moreover, interferon-gamma induced the S-nitrosylation of endogenous ASK1 in L929 cells. Together, these results suggest that NO mediates the interferon-gamma-induced inhibition of ASK1 in L929 cells through a thiolredox mechanism.     

Regulation of apoptosis signal-regulating kinase 1 (ASK1) by polyamine levels via protein phosphatase 5

Recent evidence has implicated the protein phosphatase PP5 in a variety of signaling pathways. Whereas several proteins have been identified that interact with PP5 and regulate its activity, a possibility of its regulation by second messengers remains speculative. Activation of PP5 in vitro by polyunsaturated fatty acids (e.g. arachidonic acid) and fatty acyl-CoA esters (e.g. arachidonoyl-CoA) has been reported. We report here that PP5 is strongly inhibited by micromolar concentrations of a natural polyamine spermine. This inhibition was observed both in assays with a low molecular weight substrate p-nitrophenyl phosphate as well as phosphocasein and apoptosis signal-regulating kinase 1 (ASK1), thought to be a physiological substrate of PP5. Furthermore, a decrease in polyamine levels in COS-7 cells induced by alpha-difluoromethylornithine (DFMO), an inhibitor of ornithine decarboxylase, led to accelerated dephosphorylation of oxidative stress-activated ASK1. This effect was suppressed by okadaic acid and by siRNA-mediated PP5 depletion, indicating that the effect of polyamine levels on ASK1 dephosphorylation was mediated by PP5. In line with the decreased ASK1 activation, polyamine depletion in COS-7 cells abrogated oxidative stress-induced activation of caspase-3, which executes ASK1-induced apoptosis, as well as caspase-3 activation induced by ASK1 overexpression, but had no effect on basal caspase-3 activity. These results implicate polyamines, emerging intracellular signaling molecules, as potential physiological regulators of PP5. Our findings also suggest a novel mechanism of the anti-apoptotic action of a decrease in polyamine levels via de-inhibition of PP5 and accelerated dephosphorylation and deactivation 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.     

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.     

Apoptosis signal-regulating kinase 1 mediates denbinobin-induced apoptosis in human lung adenocarcinoma cells

In the present study, we explore the role of apoptosis signal-regulating kinase 1 (ASK1) in denbinobin-induced apoptosis in human lung adenocarcinoma (A549) cells. Denbinobin-induced cell apoptosis was attenuated by an ASK1 dominant-negative mutant (ASK1DN), two antioxidants (N-acetyl-L-cysteine (NAC) and glutathione (GSH)), a c-Jun N-terminal kinase (JNK) inhibitor (SP600125), and an activator protein-1 (AP-1) inhibitor (curcumin). Treatment of A549 cells with denbinobin caused increases in ASK1 activity and reactive oxygen species (ROS) production, and these effects were inhibited by NAC and GSH. Stimulation of A549 cells with denbinobin caused JNK activation; this effect was markedly inhibited by NAC, GSH, and ASK1DN. Denbinobin induced c-Jun phosphorylation, the formation of an AP-1-specific DNA-protein complex, and Bim expression. Bim knockdown using a bim short interfering RNA strategy also reduced denbinobin-induced A549 cell apoptosis. The denbinobin-mediated increases in c-Jun phosphorylation and Bim expression were inhibited by NAC, GSH, SP600125, ASK1DN, JNK1DN, and JNK2DN. These results suggest that denbinobin might activate ASK1 through ROS production to cause JNK/AP-1 activation, which in turn induces Bim expression, and ultimately results in A549 cell apoptosis.     

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.     

Sema4D/plexin-B1 activates GSK-3beta through R-Ras GAP activity, inducing growth cone collapse

Plexins are receptors for the axonal guidance molecules known as semaphorins, and the semaphorin 4D (Sema4D) receptor plexin-B1 induces repulsive responses by functioning as an R-Ras GTPase-activating protein (GAP). Here we characterized the downstream signalling of plexin-B1-mediated R-Ras GAP activity, inducing growth cone collapse. Sema4D suppressed R-Ras activity in hippocampal neurons, in parallel with dephosphorylation of Akt and activation of glycogen synthase kinase (GSK)-3beta. Ectopic expression of the constitutively active mutant of Akt or treatment with GSK-3 inhibitors suppressed the Sema4D-induced growth cone collapse. Constitutive activation of phosphatidylinositol-3-OH kinase (PI(3)K), an upstream kinase of Akt and GSK-3beta, also blocked the growth cone collapse. The R-Ras GAP activity was necessary for plexin-B1-induced dephosphorylation of Akt and activation of GSK-3beta and was also required for phosphorylation of a downstream kinase of GSK-3beta, collapsin response mediator protein-2. Plexin-A1 also induced dephosphorylation of Akt and GSK-3beta through its R-Ras GAP activity. We conclude that plexin-B1 inactivates PI(3)K and dephosphorylates Akt and GSK-3beta through R-Ras GAP activity, inducing growth cone collapse.