Recruitment of Tumor Necrosis Factor Receptor-associated Factor Family Proteins to Apoptosis Signal-regulating Kinase 1 Signalosome Is Essential for Oxidative Stress-induced Cell Death

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 (∼1,500-2,000 kDa), which we designate ASK1 signalosome. Upon H2O2 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, H2O2-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) 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 (∼1,500-2,000 kDa), which we designate ASK1 signalosome. Upon H2O2 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, H2O2-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) 2The abbreviations used are: ASK1, apoptosis signal-regulating kinase 1; ROS, reactive oxygen species; TNF, tumor necrosis factor; TRAF, TNF receptor-associated factor; MAP, mitogen-activated protein; MAPKKK, MAP kinase kinase kinase; JNK, c-Jun N-terminal kinase; Trx, thioredoxin; MEKK, MAP kinase/extracellular signal-regulated kinase kinase kinase; MEF, mouse embryonic fibroblast; HA, hemagglutinin; Chaps, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; HMM, high molecular mass; IKK, IκB kinase. 2The abbreviations used are: ASK1, apoptosis signal-regulating kinase 1; ROS, reactive oxygen species; TNF, tumor necrosis factor; TRAF, TNF receptor-associated factor; MAP, mitogen-activated protein; MAPKKK, MAP kinase kinase kinase; JNK, c-Jun N-terminal kinase; Trx, thioredoxin; MEKK, MAP kinase/extracellular signal-regulated kinase kinase kinase; MEF, mouse embryonic fibroblast; HA, hemagglutinin; Chaps, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; HMM, high molecular mass; IKK, IκB kinase. 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, TNFα, calcium overload, and endoplasmic reticulum stress (1Takeda K. 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Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). molecular mass of ASK1 is in we that a high molecular mass complex in with high activity by the second A of ASK1 also complex not that the kinase activity of ASK1 is not a for the complex the a complex but with activity is that a part of also complex with kinase activity that oligomerization-dependent of the complex is required for the kinase activity of ASK1. ROS a of the of endogenous ASK1 by of the from cells through a revealed that to (∼1,500-2,000 kDa), endogenous ASK1 a complex as by A of complex in of the cells we tested, and not that the complex is a of endogenous ASK1. the kinase activity of ASK1 is by its association with Trx (6Saitoh M. Nishitoh H. Fujii M. Takeda K. Tobiume K. Sawada Y. Kawabata M. Miyazono K. Ichijo H. EMBO J. 1998; 17: 2596-2606Crossref PubMed Scopus (2045) Google Scholar). Trx is a component of the to in were and to with Trx to be with ASK1 that endogenous ASK1 forms a complex Trx, which may function as a signalosome to activation of ASK1. cells were with the ASK1 signalosome in higher molecular mass second and to that of that a of ASK1 activated complex of the by the of cells with such as and not in the of the ASK1 complex with the activation of ASK1 and its JNK and p38 as by the of cell for the that recruitment of to the signaling complex of ASK1 may be in H2O2-induced activation of ASK1. the binding Trx and ASK1 ROS cells to the previously in cells (6Saitoh M. Nishitoh H. Fujii M. Takeda K. Tobiume K. Sawada Y. Kawabata M. Miyazono K. Ichijo H. 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In of it has been that TRAF2 is required for H2O2-induced JNK activation and cell death H.M. Lin Y. Choksi S. Tran J. T. L. Karin M. Zhang J. Liu Z.G. Mol. Cell. Biol. 2004; PubMed Scopus Google Scholar). TRAF2 is in H2O2-induced activation of ASK1. In with the of H2O2-induced activation of ASK1 ASK1 with TRAF2 in and ASK1 and thus activated strongly that the molecular for H2O2-induced complex of the ASK1 signalosome TRAF2 and TRAF6 but TRAF5 for of the the TRAF family not only TRAF2 but also TRAF5 and TRAF6 activate ASK1 (5Nishitoh H. Saitoh M. Mochida Y. Takeda K. Nakano H. Rothe M. Miyazono K. Ichijo H. Mol. Cell. 1998; 2: 389-395Abstract Full Text Full Text PDF PubMed Scopus (566) Google Scholar). our recent report that interaction of TRAF6 and ASK1 is for activation of the A. Saegusa K. T. C. Nishitoh H. S. S. Matsumoto K. Takeda K. Ichijo H. PubMed Scopus Google Scholar). thus the of TRAF2, and TRAF6 in ASK1 the of for ASK1 we for TRAF2 A. D. J. A. D. B. N. Rothe M. Goeddel D.V. 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TRAF6 for of interaction be induced by ASK1 with TRAF6 in and ASK1 with TRAF6 endogenous interaction of TRAF6 and ASK1 also in cells that TRAF2 and TRAF6 likely for the of H2O2-induced higher molecular mass complex of ASK1. with were with H2O2 and to the as cells in that H2O2 induced the of to the higher molecular mass also in of the with the revealed that H2O2-induced of ASK1 in the higher molecular mass complex of revealed that the complex of TRAF6 and ASK1 were in the higher molecular 1 and that higher molecular mass complex is the complex of ASK1 and that the recruitment of TRAF6 is at least in part for of higher molecular mass have not been in TRAF2 in the higher molecular mass because of the of TRAF2 the of endogenous TRAF2 with TRAF6 in not the of TRAF6 for of the higher molecular mass were not of the ASK1 signalosome in Traf6-/- that recruitment of TRAF6 is required for H2O2-induced higher molecular mass complex of the ASK1 signalosome. of and Traf6-/- to H2O2-induced Cell that ASK1 is required for H2O2-induced cell death K. 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