Inducible activation of small GTPases reveals direct effector recruitment and signalling dynamics

Abstract

RAS GTPases regulate cellular activity through the selective activation of effectors, yet identifying proteins directly recruited by small GTPases in cells remains challenging. Current approaches rely on extracellular stimulation or prolonged expression of constitutively active mutants, which trigger secondary signalling and negative feedback pathways. Most of the RAS superfamily also lack known upstream activators, limiting investigation of their biological functions. Here we develop SPaRTa (Sequestered Protein activation through RAS-TEV actuation), an inducible system in which activated GTPases are maintained in a sequestered state by tethered effector-binding domains that can be released by rapamycin-induced reconstitution of split-TEV protease. We first applied SPaRTa to KRAS, as despite being one of the most intensely studied proteins in biology fundamental questions regarding its effector engagement remain unresolved. KRAS became activated within minutes of proteolytic release and stimulated rapid MAPK activity. Direct visualization of effector recruitment revealed distinct responses: BRAF was rapidly recruited to the plasma membrane (PM), whereas AFDN and RASSF5 accumulated gradually. In contrast, PI3K and SHOC2 were not recruited despite robust KRAS activation, though EGF stimulation efficiently re-localized PI3K to the membrane. This suggests that activation of KRAS alone is insufficient to stimulate PI3K. Sequestration shapes signalling as both AFDN and RASSF5 are subcellularly partitioned in the nucleus, and only prolonged KRAS activation re-located these effectors to the PM. Inducible activation of a designed RHOG SPaRTa resulted in ELMO1 recruitment and robust lamellipodia formation. Our system thus provides a broadly applicable platform for defining direct GTPase-effector interactions and signalling dynamics in cells.