Kinetic Resolution of Ras and Arf Signaling Activation by GEFs on Lipid Membranes and in Live Cells

Supported Lipid Bilayers (SLBs) were used in conjunction with live cell measurements to better understand the activation of the small GTPases Ras and Arf by their respective GEFs SOS and ARNO. As membranes are crucial for proper activity of these proteins, membrane-mimic assays were developed to quantitatively measure kinetic activation and diffusion.Ras is a common oncogene that causes over 30% of all human cancers. SOS is one of Ras’s activators that is known to play a role in the determination of cell fate. The kinetics of SOS activation of Ras was explored in four of the following chapters. Firstly, a nanofabricated platform that incorporated liposome reaction chambers was developed to assess the kinetic activity of single SOS molecules with single nucleotide turnover resolution. Secondly, the measurement of SOS motility and localization in live cells contributed to an understanding of SOS regulation through a stable membrane-associated active state, resulting in SOS endocytosis. Thirdly, a small molecule inhibitor was shown to impact SOS binding to Ras in vitro and in vivo. Fourthly, the impact of oncogenic Ras mutations on SOS binding and catalytic rate was measured. These results provide insight in the tight regulation of SOS-mediated Ras activation, and also suggest future directions for rational drug design targeting oncogenic Ras.Arf is a small GTPase in the Ras family that regulates membrane traffic and morphology. In the final chapter, a SLB-based assay for GEF-mediated Arf recruitment studies was developed and diffusion rates for both Arf-GTP and Arf-GDP were measured, demonstrating a well-defined, transient membrane interaction for Arf in the GDP state. Additionally, autoinhibition of the GEF ARNO was confirmed. This yields new insight into the mechanism of Arf signaling initiation. Overall, it is demonstrated that in vitro membrane mimics, in combination with live cell measurements, provide a powerful tool to gain a deeper understanding about the activity and regulation of GTPases