Apoptosis signal-regulating kinase 1 (ASK1) is an evolutionarily conserved mitogen-activated protein 3-kinase that activates both Jnk and p38 mitogen-activated protein kinases. Here we used ASK1-deficient mice to show that ASK1 was selectively required for lipopolysaccharide-induced activation of p38 but not of Jnk or the transcription factor NF-kappaB. ASK1 was required for the induction of proinflammatory cytokines dependent on Toll-like receptor 4 (TLR4) but not TLR2 or other TLRs. Consistent with this, ASK1-deficient mice were resistant to lipopolysaccharide-induced septic shock. Lipopolysaccharide induced the production of intracellular reactive oxygen species, which was required for the formation of a complex of the adaptor molecule TRAF6 and ASK1 and subsequent activation of the ASK1-p38 pathway. Our data demonstrate that the reactive oxygen species-dependent TRAF6-ASK1-p38 axis is crucial for TLR4-mediated mammalian innate immunity.

Mutation in Cu/Zn-superoxide dismutase (SOD1) is a cause of familial amyotrophic lateral sclerosis (ALS).[Keywords: Amyotrophic lateral sclerosis; endoplasmic reticulum-associated degradation; endoplasmic reticulum stress; Derlin-1; ASK1] Supplemental material is available at http://www.genesdev.org.

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 H 2 O 2 . Furthermore, the H 2 O 2 -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.

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Apoptosis signal-regulating kinase 1 (ASK1) plays a pivotal role in oxidative stress-induced cell death. Reactive oxygen species disrupt the interaction of ASK1 with its cellular inhibitor thioredoxin and thereby activates ASK1. However, the precise mechanism by which ASK1 freed from thioredoxin undergoes oligomerization-dependent activation has not been fully elucidated. Here we show that endogenous ASK1 constitutively forms a high molecular mass complex including Trx (ϳ1,500 -2,000 kDa), which we designate ASK1 signalosome. Upon H 2 O 2 treatment, the ASK1 signalosome forms a higher molecular mass complex at least in part because of the recruitment of tumor necrosis factor receptor-associated factor 2 (TRAF2) and TRAF6. Consistent with our previous findings that TRAF2 and TRAF6 activate ASK1, H 2 O 2 -induced ASK1 activation and cell death were strongly reduced in the cells derived from Traf2؊/؊ and Traf6؊/؊ mice. A novel signaling complex including TRAF2, TRAF6, and ASK1 may thus be the key component in oxidative stress-induced cell death.Apoptosis signal-regulating kinase 1 (ASK1) 2 is a mitogen-activated protein (MAP) kinase kinase kinase (MAPKKK) family member that activates the JNK and p38 MAP kinase pathways and is activated by various stresses including oxidative stress, TNF␣, calcium overload, and endoplasmic reticulum stress (1-7). Recent analyses of ASK1-deficient mice have revealed that ASK1 is required for cell death induced by oxidative stress, TNF␣, and endoplasmic reticulum stress (4, 8).Among various stimuli tested, oxidative stress is one of the most potent activators of ASK1 (3). Thioredoxin (Trx), a reduction/oxidation (redox) regulatory protein, inhibits the kinase activity of ASK1 by its direct binding to the N-terminal noncatalytic region of ASK1 (6, 9). Reactive oxygen species (ROS) such as H 2 O 2 dissociate Trx from ASK1 and thereby activate ASK1 (6). Following the dissociation of Trx, autophosphorylation-dependent activation of ASK1 occurs most likely through mechanisms involving homo-oligomerization (10, 11); however, precise mechanisms of the oligomerization-dependent activation of ASK1 remained unknown. TNF receptor-associated factor 2 (TRAF2) has been shown to bind to and thereby activate ASK1 (5). TRAF2 is not only a pivotal intermediate in TNF␣-induced NF-B activation (12-16), but it also activates JNK in TNF␣ signaling through the association with MAPKKKs such as ASK1 and MEKK1 (5,17,18). A recent report has shown that AIP1/DAB2IP, a novel member of Ras-GAP family, regulates TRAF2-dependent activation of ASK1 in TNF␣-treated endothelial cells (19). In accordance with the findings that ROS function as second messengers in TNF␣ signaling (20, 21), TNF␣-induced association of ASK1 with TRAF2 and subsequent activation of ASK1 have been shown to depend largely on intracellular ROS production by TNF␣ (6,17).In addition to TRAF2, we have previously shown that among extensively characterized six members of the TRAF family (TRAF1-TRAF6) (22-24), TRAF5 and TRAF6 also interact with and ac...