Disulfide Bond-mediated Multimerization of Ask1 and Its Reduction by Thioredoxin-1 Regulate H₂O₂-induced c-Jun NH₂-terminal Kinase Activation and Apoptosis

Abstract

Apoptosis signal-regulated kinase-1 (Ask1) lies upstream of a major redox-sensitive pathway leading to the activation of Jun NH 2 -terminal kinase (JNK) and the induction of apoptosis. We found that cell exposure to H 2 O 2 caused the rapid oxidation of Ask1, leading to its multimerization through the formation of interchain disulfide bonds. Oxidized Ask1 was fully reduced within minutes after induction by H 2 O 2 . During this reduction, the thiol-disulfide oxidoreductase thioredoxin-1 (Trx1) became covalently associated with Ask1. Overexpression of Trx1 accelerated the reduction of Ask1, and a redox-inactive mutant of Trx1 (C35S) remained trapped with Ask1, blocking its reduction. Preventing the oxidation of Ask1 by either overexpressing Trx1 or using an Ask1 mutant in which the sensitive cysteines were mutated (Ask1-⌬Cys) impaired the activation of JNK and the induction of apoptosis while having little effect on Ask1 activation. These results indicate that Ask1 oxidation is required at a step subsequent to activation for signaling downstream of Ask1 after H 2 O 2 treatment. INTRODUCTIONH 2 O 2 is emerging as an important intracellular signaling molecule affecting a variety of cellular responses. In mammals, H 2 O 2 concentration transiently increases in response to different membrane-receptor agonists such as peptide growth factors, hormones, cytokines, and neurotransmitters. This increase of H 2 O 2 concentration is important for downstream signaling from the corresponding membrane receptors (Deyulia et al., 2005;Rhee et al., 2005;Stone and Yang, 2006). Some of these H 2 O 2 regulatory effects are mediated by protein-thiol oxidation, as shown for the oxidative inhibition of protein tyrosine phosphatase upon receptor-tyrosine kinases engagement. H 2 O 2 can oxidize cysteine residues to the sulfenic (protein-SOH), sulfinic (protein-SO 2 H), and sulfonic acid (protein-SO 3 H) forms, also leading to intra-or intermolecular disulfide bonds (protein-SS-protein) and to protein glutathionylation (protein-SSG). Similarly to protein posttranslational modification by serine, threonine, and tyrosine phosphorylation, oxidation of cysteine residues can trigger changes in protein conformation and activity (Filomeni et al., 2005). In bacteria, OxyR is an archetypical H 2 O 2 sensor at the top of a redox-sensitive pathway, being activated by H 2 O 2 through formation of an intramolecular disulfide bond and controlling the transcription of antioxidant defense genes (Storz and Tartaglia, 1992;Zheng et al., 1998). In yeast, H 2 O 2 sensing involves the thiol-based peroxidase Orp1/Gpx3, which, upon oxidation by H 2 O 2 , mediates the formation of an intramolecular disulfide bond in the Yap1 transcription factor, causing its activation (Lee et al., 1999;Delaunay et al., 2002).The mitogen-activated protein kinase kinase kinase apoptosis signal-regulated kinase-1 (Ask1) and its downstream stress-activated kinases p38 and Jun NH 2 -terminal kinase (JNK) constitute an important mammalian signaling pathway that can promote cell surviv...

A universal entropy-driven mechanism for thioredoxin–target recognition

“…Previously, it has been suggested that the interaction of Trx with g5p and ASK1 target proteins is critically dependent on the redox state of Trx, despite being independent of Trx oxido-reductase function (9,20). However, later studies have proposed alternative models possibly involving the oxidoreductase function of Trx in regulation of these proteins (36,37). In light of these studies and others, the absolute dependence of these interactions on the Trx redox-state may need to be reevaluated using a direct solution-based measurement of noncovalent interaction between Trx and these target proteins.…”

Oxidizable Residues Mediating Protein Stability and Cytoprotective Interaction of DJ-1 with Apoptosis Signal-regulating Kinase 1

“…Formation of DJ-1-ASK1-mixed Disulfides-The dependence of DJ-1 binding to ASK1 on the central redox residue prompted us to consider mixed disulfide formation between ASK1 and DJ-1, because it was recently shown for thioredoxin 1 (Trx1) (35). HEK293E cells were transiently co-transfected with ASK1/HA and Myc/DJ-1 variants as above, but in this experiment ASK1 complexes immunoprecipitated with anti-HA were resolved by non-reducing SDS-PAGE.…”

ASK1 and Its Role in Neurodegenerative Diseases

“…Studies conducted by Nadeau et al, revealed an alternative mechanism for the function of Trx1 in the regulation of ASK1 in response to H 2 O 2 . These studies demonstrated that H 2 O 2 induces ASK1 oxidation leading to the formation of interchain disulfide bond-linked ASK1 multimers [25,26]. These authors demonstrated that changing all oxidation sensitive cysteine residues responsible for disulfide bond-linked multimers, prevented H 2 O 2 -induced ASK1 proapototic activity.…”

“…Recently, redox sensitive molecules such as the Parkinson's associated protein DJ-1 (a.k.a PARK7) and Peroxiredoxin-2 have also been shown to attenuate ASK1 activity in response to toxic stress in dopaminergic neurons [27][28][29]. Interestingly, mutation analyses demonstrated, that the ER stress-inducing agent thapsigargin, while inducing ASK1 activation (as determined by Thr838 phosphorylation) was observed to be independent of Cys250 [25]. In attempting to address the role of Cys250 and the role of Trx1 in regulating Fig.…”

“…Beside ROS-induced ASK1 activation, Aβ, a toxic cleavage product of APP, was also demonstrated to activate ASK1 and subsequently JNK [68]. Whereas primary neuronal cultures derived from E14.5 ASK +/+ mice demonstrated an 80% reduction in cell viability after exposure to Aβ [25][26][27][28][29][30][31][32][33][34][35] , the survival in ASK -/-derived neurons treated with Aβ [25][26][27][28][29][30][31][32][33][34][35] was significantly elevated (only 30% reduction in viability). Furthermore, postmortem analysis of AD patient brains compared with age-matched controls revealed strong expression of the downstream ASK1 substrate MKK6 [69].…”