Mechanism of RACK1-dependent ZAKα activation at stalled and collided ribosomes

Despite a growing interest in the ribotoxic stress response (RSR), it remains unknown how the upstream p38- and JNK-activating MAP3 kinase ZAKα senses translational impairment. Combining AlphaFold3 prediction and RNA crosslinking and immunoprecipitation (CLIP), we uncover that ZAKα dynamically monitors the mRNA exit channel of elongating ribosomes. This is accomplished by ZAKα via direct interactions with the ribosomal proteins RACK1 and RPS27 as well as 18S rRNA helix-26. In this conformation, the RNA-binding S (sensing) and C-terminal domain of ZAKα span across the mRNA exit channel. Loss of ribosome processivity and mRNA stasis stabilizes the interaction allowing for kinase activation. Prolonged binding of ZAKα to stalled and collided ribosomes is associated with sequestration of the sterile alpha-motif (SAM) domain on RACK1, which allows for transient ZAKα dimerization, activation loop trans-autophosphorylation, and RSR activation. Our findings highlight how ZAKα senses both stalled and collided ribosomes in human cells through overlapping mechanisms.