A biochemical system for rapid on/off switching of ras activity

Ras protein is widely considered a critical pro-mitogenic signalling molecule with an essential role in multiple stages of the cell cycle, probably through the induction of Cyclin-D (one of the key drivers of G1/S transition). Owing to the lack of experimental approaches for the acute inhibition of Ras, the precise role of Ras-dependent signalling during the cell cycle could not be proven to date. We developed a novel biochemical tool that enables direct, acute interference with Ras activity at will. The system consists of two protein partners: an anchor unit confined to the plasma membrane (PM) and an effector unit coupled to the catalytic domain of the Ras-GAP NF1. The latter is recruited to the PM upon addition of the small iDimerizer, rendering it close to PM-bound Ras. Biochemical analysis proved that EGF-dependent activation of Ras and its downstream effector Erk was fully abrogated by short-term (15 min) pre-treatment with the iDimerizer. By examining the activity of other small GTPases, largely unaffected in presence of the iDimeriser, we prove that the RasOFF system is accurate and selective in inhibiting Ras alone. Using this approach, we demonstrate a critical role for Ras in cell cycle entry till deep G1 of serum-stimulated quiescent cells and G1/S transition through the regulation of cyclin-D1. Further analysis in continuously proliferating cells indicated that the absence of Ras function in G2 and early G1 phase led to a decrease in the ensuing S phase entry accompanied by a reduction of cyclin-D1. Dissection of signalling downstream of Ras identified Erk and excluded PI3K/Akt and Ral as the mediator of its signals to the cell cycle machinery. In conclusion, we have charted the temporal signalling program of Ras during G1 phase employing a new methodology for the acute and specific inhibition of Ras that may have broad applicability in many areas of signal transduction and molecular oncology research.