Thioredoxin and TRAF Family Proteins Regulate Reactive Oxygen Species-Dependent Activation of ASK1 through Reciprocal Modulation of the N-Terminal Homophilic Interaction of ASK1

Abstract

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.

CIB1 functions as a Ca 2+ -sensitive modulator of stress-induced signaling by targeting ASK1

“…Activation of ASK1 in cellular response to various stimuli including ROS requires ASK1 homo-oligomerization, recruitment of TRAF2 or TRAF6 to ASK1, and the autophosphorylation of a threonine residue in ASK1 (Thr 838 or Thr 845 of human or mouse ASK1, respectively) (19)(20)(21)(22)(23). These events may be potentially susceptible to modulation by ASK1-interacting proteins, among which thioredoxin was the first identified ASK1-inhibitory protein (4,7).…”

“…Activation of ASK1 requires ASK1 homo-oligomerization, recruitment of TRAF family proteins to ASK1, and the subsequent autophosphorylation of a threonine residue in the kinase domain of ASK1 (Thr 838 in the human ASK1) (19)(20)(21)(22)(23). We therefore examined a possible effect of CIB1 on those processes required for ASK1 activation.…”

“…Interestingly, CIB1 directly bound to an ASK1 deletion mutant comprising amino acids 378 to 648 (Fig. 1 A), which is the same region of ASK1 that binds TRAF2 (23). Indeed, CIB1 inhibited the binding between TRAF2 and ASK1 (1-936) in vitro, whereas myelin basic protein did not (Fig.…”

ASK1 Negatively Regulates the 26 S Proteasome

“…of an inhibitory N-terminal domain, an internal kinase domain, and a C-terminal regulatory domain (5,21,22). To determine which domain within ASK1 is crucial for 26 S proteasome inhibition, we expressed Ub G76V GFP along with full-length ASK1 (amino acids 1-1375), ASK1…”

“…Immunoblot analyses of the eluted fractions with anti-20 S core, anti-19 S Rpt2, and anti-HA antibodies revealed that the components of the 20 S and 19 S proteasome co-eluted over the same range of high molecular weight fractions (fraction numbers [13][14][15][16][17][18][19][20][21][22][23][24][25], whereas ASK1 was present in the lower molecular weight fraction (fraction number [43][44][45][46][47][48][49][50][51][52][53][54][55] (Fig. 5F).…”