Structural basis of autoregulatory scaffolding by apoptosis signal-regulating kinase 1

Abstract

Apoptosis signal-regulating kinases (ASK1-3) are apical kinases of the p38 and JNK MAP kinase pathways. They are activated by diverse stress stimuli, including reactive oxygen species, cytokines, and osmotic stress; however, a molecular understanding of how ASK proteins are controlled remains obscure. Here, we report a biochemical analysis of the ASK1 kinase domain in conjunction with its N-terminal thioredoxin-binding domain, along with a central regulatory region that links the two. We show that in solution the central regulatory region mediates a compact arrangement of the kinase and thioredoxin-binding domains and the central regulatory region actively primes MKK6, a key ASK1 substrate, for phosphorylation. The crystal structure of the central regulatory region reveals an unusually compact tetratricopeptide repeat (TPR) region capped by a cryptic pleckstrin homology domain. Biochemical assays show that both a conserved surface on the pleckstrin homology domain and an intact TPR region are required for ASK1 activity. We propose a model in which the central regulatory region promotes ASK1 activity via its pleckstrin homology domain but also facilitates ASK1 autoinhibition by bringing the thioredoxin-binding and kinase domains into close proximity. Such an architecture provides a mechanism for control of ASK-type kinases by diverse activators and inhibitors and demonstrates an unexpected level of autoregulatory scaffolding in mammalian stress-activated MAP kinase signaling.itogen-activated protein (MAP) kinase cascades transmit signals from membrane-associated receptors to intracellular targets to effect changes in cellular behavior. They form a hierarchical system in which activated upstream kinases (MAP3Ks) phosphorylate intermediate MAP kinase kinases (MAP2Ks), which in turn phosphorylate terminal MAP kinases, primarily ERK, p38, and JNK and their isoforms (1). Extensive studies have focused on the activation of RAS-RAF-MEK upstream in the ERK pathway and provided fertile ground for the discovery of new therapeutics (2). In contrast to the ERK pathway, which primarily promotes cellular proliferation, JNK and p38 phosphorylate a range of substrates to promote inflammation and cell death (1, 3). In addition, cross-regulation among the p38, JNK, and ERK pathways is important for the efficacy of various cancer therapies that are in use or in development (4, 5). Molecular details on the more diverse upstream regulation of the p38 and JNK pathways are currently less clear, however.Apoptosis signal-regulating kinases (ASK1-3) are MAP3Ks that trigger cellular responses to redox stress and inflammatory cytokines (6, 7) and play vital roles in innate immunity and viral infection (8-11). When activated, ASK1-3 activate JNK and p38 via phosphorylation of MAP2Ks (MKK3/4/6/7) (12). The key initiator role of ASK1-3 in this pathway means that either too much or too little ASK activity can have pathological effects. For instance, inhibiting ASK1 is beneficial against gastric cancer (13,14), but inactivating mutations in ASK1...

GPCR kinase 2–interacting protein‐1 protects against ischemia‐reperfusion injury of the spinal cord by modulating ASK1/JNK/p38 signaling

“…In fact, crosstalk between GIT1 and ASK1 signaling has not yet been reported. As the major apical kinase of the p38 and JNK signaling pathway (13,57), ASK1 plays key roles in the regulation of neuronal death by I/R injury (14)(15)(16)(17). Either excess or inadequate activation of ASK1 can disrupt homeostasis and lead to pathologic consequences, which indicates that keeping normal ASK1 activity is crucial.…”

“…ASK1 is an important apical kinase of the JNK and p38 signaling pathways (13,57) and plays key roles in the activation of JNK/p38 and the regulation of apoptosis in vivo and in vitro (6,7,46,58). Two of the three GIT1 shRNAs that target the human GIT1 sequence and that are carried by lentiviral vectors efficiently inhibited the expression of endogenous GIT1 in HEK293T cells (Supplemental Fig.…”

“…Apoptosis signal-regulating kinase-1 (ASK1), an MAPK kinase kinase (MKK), plays essential roles in stressinduced apoptosis (6,7). ASK1 is activated in response to a variety of stress-related stimuli, including proinflammatory factors, reactive oxygen species, endoplasmic reticulum stress, and calcium influx, through distinct mechanisms, and it activates MKK4 and MKK3, which, in turn, activate JNK and p38 (6,(8)(9)(10)(11)(12)(13). Previous studies have demonstrated that the ASK1/JNK/p38 pathway that is activated by stress-related stimuli was involved in neuronal apoptosis after SCI induced by I/R injury (14)(15)(16)(17).…”

Mechanism of preferential complex formation by Apoptosis Signal-regulating Kinases

“…With growing knowledge of the domain structures of ASK kinases the obvious challenge is understanding how oligomerisation by the SAM at the C-terminus integrates with the raft of other interactions through their N-termini. Previously, we reported the crystal structure of the central regulatory region of ASK1-found N-terminal to the kinase domain-which links the N-terminal thioredoxin-binding domain to the kinase domain (18). A pleckstrin-homology domain within this novel fold appears to promote substrate MAP2K phosphorylation, which could occur on an intra-or inter-molecular basis.…”

“…It is not clear if regulation of substrate recruitment and priming, through a domain just Nterminal to the kinase (18), occur in an intra-or inter-molecular manner. Likewise it is not known if dimers reported for the isolated kinase domain of ASK1 impact kinase function in the context of full-length protein (16).…”