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

Abstract

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...

Human glutathione S-transferase P1-1 interacts with TRAF2 and regulates TRAF2–ASK1 signals

“…It has been well documented that TNF-a-induced TRAF2-ASK1 complex is required for ASK1 activation Liu et al, 2000;Noguchi et al, 2005). We and another group also found that GSTP1-1 inhibited the enzymatic activity of ASK1 in vivo (Yin et al, 2001) but not in vitro (Cho et al, 2001).…”

“…Previous studies suggested that TNF-atriggered TRAF2-ASK1 interaction was dependent on ROS. Some ROS scavengers, such as NAC, inhibited the disassociation of ASK1-Trx and thus suppressed TNF-a-elicited cell apoptosis Liu et al, 2000;Noguchi et al, 2005). However, our study demonstrated that the interaction of GSTP1-1 and TRAF2 was not influenced by NAC or BSO in resting or TNF-a-treated HeLa cells, and suggested that the mechanism of TNF-a-induced disassociation of TRAF2-GSTP1-1 and ASK1-Trx may be different.…”

“…Two TRAF family proteins, TRAF2 and TRAF6, have been demonstrated to bind to and thereby activate ASK1. Gene-knockout experiments showed that TRAF2 was essential for TNF-a-stimulated ASK1-JNK activation (Lee et al, 1997;Nishitoh et al, 1998;Noguchi et al, 2005), and TRAF6-ASK1 axis is selectively required for lipopolysaccharide (LPS)-induced activation of p38 . To explore the possibility that TRAF2 or TRAF6 was related with the inhibitory effect of GSTP1-1 on TNF-a triggered ASK1-JNK signaling cascade, endogenous TRAF2 and TRAF6 in HeLa cells were immunoprecipitated with respective antibodies.…”

“…As TRAF2-ASK1 axis is essential for mediating TNF-a and oxidative stresses stimulated apoptotic cell death (Tobiume et al, 2001;Noguchi et al, 2005), we also determined the effect of GSTP1-1 on TNF-ainduced HeLa cell apoptosis by microscopy ( Figure 7c) and flow cytometry using propidium iodide (PI) staining (Figure 7d). Exposure of GSTP1-1-knockdown HeLa cells to TNF-a plus CHX markedly enhanced apoptotic cell death.…”

Signaling pathways from membrane lipid rafts to JNK1 activation in reactive nitrogen species-induced non-apoptotic cell death

“…13 Role of ASK1 has also been well established for oxidative stressinduced JNK activation. 14 On the other hand, there are conflicting reports on whether RNS activate or inhibit ASK1. 7,15 Here we explored the involvement of ASK1 in RNS-induced JNK activation and cell death.…”