Trans-kingdom coupling of redox signaling to environmental cell stress responses through multiphase partitioning.

Abstract

Reactive Oxygen Species (ROS) signaling is a conserved biological process with parallel functions in all evolutionary branches of life. Here, we identify Receptor for Activated C Kinase 1 (RACK1) as a conserved redox-regulated hub that integrates ROS signals to coordinate cellular stress responses. Using cysteine reactivity profiling in intestinal epithelial cells, we demonstrate that RACK1 undergoes NOX1-dependent oxidation at multiple residues, with C286 serving as a key regulatory site. Functional studies reveal that RACK1 negatively regulates NF{kappa}B signaling through redox-dependent interactions with upstream signaling complexes. Upon stress stimulation, RACK1 dynamically redistributes into membrane-less condensates that act as redox-privileged microenvironments enriched for hydrogen peroxide. We find that oxidized RACK1 condensates are conserved through evolution with analogous stress response behavior in bacteria and yeast indicating a ubiquitous and ancient stress sensor-effector system. Functionally, redox-dependent RACK1 activity links environmental stress to translational control, with oxidation promoting inhibition of protein synthesis. Furthermore, RACK1 mediates responses to diverse pathogen-associated stimuli, including viral and bacterial infection, highlighting its role in epithelial innate immune responses. Collectively, these findings establish RACK1 as a cellular node for redox signaling, operating within condensate-based microdomains to spatially encode oxidative signals and regulate environmental stress pathways in cells. Significance StatementThis study defines a mechanism by which cells achieve specificity in redox signaling through compartmentalization within condensates. By identifying RACK1 as a redox sensor-effector, we reveal an ancient and broadly conserved system linking environmental stress to innate immune signaling and translational control. The discovery of redox-privileged condensates provides a conceptual framework for understanding how transient ROS signals are stabilized and interpreted in epithelial biology, with implications for inflammatory diseases, host-pathogen interactions, and fundamental cellular stress responses.