Designed sensors reveal normal and oncogenic Ras signaling in endomembranes and condensates

Abstract

AbstractWhile the ability of Ras to dynamically shuttle around the cell is well characterized, the activity, mechanism of activation, and function of non-plasma membrane-localized Ras is less well understood due to lack of suitable tools. Here, we describe the use of the Latching Orthogonal Cage-Key pRotein (LOCKR) switch platform to generate first-in-class intracellular sensors of endogenous Ras activity (Ras-LOCKR-S) and signaling environment (Ras-LOCKR-PL). By targeting these tools to endomembranes and oncogenic condensates, we defined subcellular Ras activity and identified upstream Ras effectors (guanine exchange factors and SAM68) responsible for signaling in these locations. We also found that Major Vault Protein drives RasG12Cinhibitor resistance by enhancing wild type Ras-mediated signaling at the golgi and mutant Ras signaling at mitochondria. Together, these results highlight the importance of non-plasma membrane Ras signaling (endomembranes and condensates), and our new sensors should accelerate the discovery of new therapeutic targets.