Structure-based mechanism of preferential complex formation by apoptosis signal–regulating kinases

Abstract

Apoptosis signal–regulating kinases (ASK1, ASK2, and ASK3) are activators of the p38 and c-Jun N-terminal kinase (JNK) mitogen-activated protein kinase (MAPK) pathways. ASK1–3 form oligomeric complexes known as ASK signalosomes that initiate signaling cascades in response to diverse stress stimuli. Here, we demonstrated that oligomerization of ASK proteins is driven by previously uncharacterized sterile-alpha motif (SAM) domains that reside at the carboxy-terminus of each ASK protein. SAM domains from ASK1–3 exhibited distinct behaviors, with the SAM domain of ASK1 forming unstable oligomers, that of ASK2 remaining predominantly monomeric, and that of ASK3 forming a stable oligomer even at a low concentration. In contrast to their behavior in isolation, the ASK1 and ASK2 SAM domains preferentially formed a stable heterocomplex. The crystal structure of the ASK3 SAM domain, small-angle x-ray scattering, and mutagenesis suggested that ASK3 oligomers and ASK1-ASK2 complexes formed discrete, quasi-helical rings through interactions between the mid-loop of one molecule and the end helix of another molecule. Preferential ASK1-ASK2 binding was consistent with mass spectrometry showing that full-length ASK1 formed hetero-oligomeric complexes incorporating large amounts of ASK2. Accordingly, disrupting the association between SAM domains impaired ASK activity in the context of electrophilic stress induced by 4-hydroxy-2-nonenal (HNE). These findings provide a structural template for how ASK proteins assemble foci that drive inflammatory signaling and reinforce the notion that strategies to target ASK proteins should consider the concerted actions of multiple ASK family members.

Cells recognize osmotic stress through liquid-liquid phase separation lubricated with poly(ADP-ribose)

“…S2A): two intrinsically disordered regions (IDRs; Fig. S2B), one coiled-coil domain (CC), one sterile alpha motif domain (SAM) ( 25 ) and one low complexity region (LCR). Between ASK3 CT mutants with these deletions, CTΔCC, CTΔSAM and CTΔLCR exhibited reduced condensation ability compared with the original CT (Fig.…”

β-adrenergic receptor signaling evokes the PKA-ASK axis in mature brown adipocytes

“…First, we coexpressed the KD of ASK1 with a PKA catalytic subunit, but the KD failed to be activated, whereas full-length ASK1 was activated, suggesting that the N-terminus and/or C-terminus are required for activation (Fig 2D). Next, we focused on the C-terminal region, which harbors a predicted C-terminal coiled-coil (CCC) domain [34] and a sterile-alpha motif (SAM) domain [45], which is essential for high-order oligomerization of ASK1. Interestingly, exogenously expressed PKA did not activate both ⊿C and ⊿CCC of ASK1 (Fig 2E), which indicates that the C-terminal regulatory region, specifically a domain for homo-oligomerization, is a prerequisite for PKA-dependent ASK1 activation.…”

“…In the previous analysis, we discovered that ASK1 forms a high molecular mass signaling complex (>MDa, compared to ~150 kDa for the monomer) [53], and subsequent studies have shed light on a wide variety of components of this complex [28,45], including ASK2, a member of the ASK family. ASK2 tightly forms a heteromeric complex with ASK1 and supports ASK1 kinase activity [25].…”

“…Ser 993 is retained in the ⊿CCC of ASK1, but the ⊿CCC loses its ability to be activated by PKA (Fig 2E), which suggests that both phospho-ASK1 Ser 993 and CCC-mediated homo-oligomerization are required for its activation; however, the mutual interaction of these two phenomena is still unknown. A recent study suggested that the SAM domain is located on the C-terminal side of the CCC and is critical for oligomerization [45]; hence, the effects of S993A and the ⊿CCC on the SAM domain structure should also be considered. Notably, we could not find similar sequences around mouse ASK1 Thr 99 or Ser 993 in mouse ASK2, suggesting that PKA differentially regulates ASK2.…”

β-adrenergic receptor signaling evokes the PKA-ASK axis in mature brown adipocytes

“…The apoptosis signal-regulating kinase (ASK) family, which consists of ASK1, ASK2, and ASK3, is a member of MAP3K and shares similar amino acid sequences throughout the kinase domain but functions differently in many cases [ 22 – 24 ]. Additionally, ASKs can form heteromeric complexes and are mutually interactive in some cases [ 25 – 28 ]. Although the key role of ASK1 is cell death regulation [ 29 – 33 ], we previously reported that ASK1 also functions as a differentiation regulator through gene induction in immature brown adipocytes and contributes to functional maturation [ 9 ].…”