A computationally engineered RAS rheostat reveals RAS/ERK signaling dynamics

Synthetic protein switches controlled with user-defined inputs are powerful tools for studying and controlling dynamic cellular processes. To date, these approaches have relied primarily on intermolecular regulation. Here, we report a computationally-guided framework for engineering intramolecular regulation of protein function. We utilize this framework to develop Chemically Inducible Activator of RAS (CIAR), a single-component RAS rheostat that directly activates endogenous RAS in response to a small molecule. Using CIAR, we show that direct RAS activation elicits markedly different RAS/ERK signaling dynamics compared to growth factor stimulation, and that these dynamics differ between cell types. We also found that the clinically-approved RAF inhibitor vemurafenib potently primes cells to respond to direct wild-type RAS activation. These results demonstrate the utility of CIAR for quantitatively interrogating RAS signaling. Finally, we demonstrate the general utility of our approach to design intramolecularly-regulated protein tools by applying this methodology to the Rho Family GEFs.