ZAK activation at the collided ribosome

Ribosome collisions activate the ribotoxic stress response (RSR) mediated by the MAP3K ZAKα (ZAK), which in turn regulates downstream phosphorylation of the MAPKs JNK and p38 and cell fate consequences. Despite this critical role during cellular stress, a structural and mechanistic understanding of the ZAK-ribosome interaction and activation have remained elusive. Here, we combine biochemistry with cryo-electron microscopy (cryo-EM) to dissect ZAK interactions with ribosomes in various states both in vivo and in vitro. We identified regions of ZAK necessary for binding to ribosomes under normal and under stress-induced conditions. Moreover, we discovered how interactions between ZAK and the ribosomal protein RACK1 mediate ZAK activation by recognizing the collision-interface. Further, we provide insight into how this process can be negatively regulated to prevent constitutive ZAK activation. Characterizing novel variants as well as a known pathogenic variant of ZAK’s SAM domain supports a key role of this conserved domain in regulating kinase activity on and off the ribosome. Collectively, we show that ZAK directly binds at the collided ribosome interface giving molecular insight into kinase activity regulation at the ribosome. CLIP-seq of ZAK-KO HEK293T cells transfected with different ZAK constructs (Full-length (WT), Activation Loop mutant (ActLoopD), 1-649 truncation (RBRdel), overexpressed Activation Loop Mutant (OX-ActLoopD), or mock (ZAK-KO), treated with or without Anisomycin. Two replicates for each group.核糖体碰撞可激活由丝裂原活化蛋白激酶激酶激酶（MAP3K）ZAKα（ZAK）介导的核糖体毒性应激反应（ribotoxic stress response, RSR），该反应进而调控丝裂原活化蛋白激酶（MAPK）家族成员JNK与p38的下游磷酸化过程及细胞命运结局。尽管该反应在细胞应激过程中发挥关键调控作用，但学界对ZAK与核糖体的相互作用及激活机制的结构与分子解析仍未取得清晰进展。本研究将生物化学技术与冷冻电子显微镜（cryo-electron microscopy, cryo-EM）相结合，系统解析了ZAK在体内、体外多种状态下与核糖体的相互作用模式。我们鉴定出ZAK在正常生理及应激诱导条件下结合核糖体所必需的结构区域。此外，我们揭示了ZAK与核糖体蛋白RACK1之间的相互作用如何通过识别碰撞界面介导ZAK的激活过程。进一步，我们阐明了该过程如何被负向调控以避免ZAK的组成型激活。我们对ZAK的SAM结构域的新型变体及已知致病变体进行功能表征，证实了这一保守结构域在调控核糖体上及核糖体外的激酶活性中发挥核心作用。综上，本研究证明ZAK可直接结合至碰撞核糖体的界面，为核糖体处的激酶活性调控提供了分子层面的机制解析。我们对转染不同ZAK构建体（全长野生型（WT）、激活环突变体（ActLoopD）、1-649截短体（RBRdel）、过表达激活环突变体（OX-ActLoopD））或空载体（ZAK敲除（ZAK-KO））的ZAK敲除人类胚胎肾293T（HEK293T）细胞进行紫外交联免疫沉淀测序（cross-linking immunoprecipitation sequencing, CLIP-seq），各组细胞均施加或不施加茴香霉素（Anisomycin）处理，且每组设置两个生物学重复。