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.

凋亡信号调节激酶（ASK1、ASK2 和 ASK3）是 p38 和 c-Jun N 端激酶 (JNK) 丝裂原活化蛋白激酶 (MAPK) 通路的激活剂。ASK1-3 形成称为 ASK 信号小体的寡聚复合物，可启动信号级联反应以响应不同的压力刺激。在这里，我们证明了 ASK 蛋白的寡聚化是由以前未表征的无菌 alpha 基序 (SAM) 域驱动的，这些域位于每个 ASK 蛋白的羧基末端。ASK1-3 的 SAM 结构域表现出不同的行为，ASK1 的 SAM 结构域形成不稳定的寡聚体，ASK2 的 SAM 结构域仍然主要是单体，而 ASK3 的结构域即使在低浓度下也形成稳定的寡聚体。与其孤立的行为相反，ASK1 和 ASK2 SAM 结构域优先形成稳定的异源复合物。ASK3 SAM 结构域的晶体结构、小角 X 射线散射和诱变表明 ASK3 寡聚体和 ASK1-ASK2 复合物通过一个分子的中环和末端螺旋之间的相互作用形成离散的准螺旋环。另一个分子。优先 ASK1-ASK2 结合与质谱法一致，表明全长 ASK1 形成了包含大量 ASK2 的异源寡聚复合物。因此，在 4-羟基-2-壬烯醛 (HNE) 诱导的亲电应激背景下，破坏 SAM 结构域之间的关联会损害 ASK 活性。这些发现为 ASK 蛋白如何组装驱动炎症信号的病灶提供了结构模板，并强化了靶向 ASK 蛋白的策略应考虑多个 ASK 家族成员的协同作用的观念。