UC Berkeley

Ras GTPase activation is part of an important class of signaling reactions that take place on membrane surfaces in cells. This membrane-anchored protein functions as binary switch that relays information to many essential intracellular signaling pathways. Some of the most aggressive cancers are caused by mutations in Ras, making it a clinical target of paramount importance. Cells have evolved multiple layers of regulation that tightly control Ras signaling, including enzymes that control the activation state of Ras, membrane specificity, allosteric mechanisms, and two-dimensional protein phase transitions. At the center of this regulation is the guanine nucleotide exchange factor (GEF), Son of Sevenless (SOS). SOS is a key Ras activator that is autoinhibited in the cytosol and activates upon membrane recruitment in response extracellular stimuli. In addition to its GEF activity, SOS facilitates a phosphotyrosine-driven phase transition by crosslinking proteins at the membrane.

This dissertation aims to delve into the molecular processes and mechanisms behind the multilayered regulation of Ras activation by SOS. First, we extended an in vitro reconstitution platform of Ras on supported lipid bilayers (SLB) to include a real-time imaging readout of Ras activation. We engineered the Ras binding domain (RBD) of Raf1 kinase, which exhibits a natural selectivity for the active Ras-GTP state, to have rapid and fully reversible binding to Ras-GTP. This platform was then applied to the investigation of the allosteric activation of SOS and positive feedback. We found that allosteric Ras binding is a necessary, but not exclusive feature of a processive activation state observed in SOS. However, the nucleotide specificity in allosteric Ras binding promoted SOS processive activity, suggesting an alternate pathway to establish positive feedback in Ras activation. Finally, the Ras SLB platform was extended to include membrane inputs necessary to reconstitute the receptor-mediated recruitment of SOS and formation of two-dimensional protein assemblies. This work identified that the receptor-mediated membrane recruitment and release of autoinhibition introduces a kinetic threshold to the duration of SOS membrane dwells for effective signal propagation. The phosphotyrosine-driven phase transition modulates signaling by increasing the probability of SOS enzymes crossing this kinetic threshold. Collectively, these findings are suggestive of a kinetic proofreading mechanism in Ras signaling, where spurious signals from short dwells are filtered, and true signals from long dwells are propagated and amplified by the processive activity of SOS.