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

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Kinetic Resolution of Ras and Arf Signaling Activation by Gefs on Lipid Membranes and in Live Cells Kinetic Resolution of Ras and Arf Signaling Activation by GEFs on Lipid Membranes and in Live Cells By Meredith Gahn Triplet A dissertation submitted in partial satisfaction of the requirements for the degree of Doctor of Philosophy in Chemistry in the Graduate Division of the University of California, Berkeley Committee in charge: Professor Jay T. Groves, Chair Professor Ke Xu Professor James H. Hurley Summer 2017 Kinetic Resolution of Ras and Arf Signaling Activation by GEFs on Lipid Membranes and in Live Cells By Meredith Gahn Triplet University of California, Berkeley Copyright 2017 Abstract Kinetic Resolution of Ras and Arf Signaling Activation by GEFs on Lipid Membranes and in Live Cells by Meredith Gahn Triplet Doctor of Philosophy in Chemistry University of California, Berkeley Professor Jay T. Groves, Chair 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. 1 TABLE OF CONTENTS CHAPTER 1: Introduction ...........................................................................................................1 Lipid membranes ..........................................................................................................................1 Protein interactions with membranes ........................................................................................1 Theoretical and experimental exploration of 2D diffusion .......................................................1 Lipid rafts: a theory reconsidered .............................................................................................3 Membrane-bound signaling proteins ............................................................................................3 The Ras superfamily .................................................................................................................3 Arf biology ................................................................................................................................4 Ras: a highly studied oncogene ................................................................................................5 Ras: a molecular switch in signal transduction .........................................................................6 Technical advances in membrane studies ....................................................................................7 SLBs .........................................................................................................................................7 Nanodiscs ..................................................................................................................................8 TIRF microscopy ......................................................................................................................9 Micro- and nano-fabrication .....................................................................................................9 Live cell imaging ....................................................................................................................10 Figures .......................................................................................................................................11 CHAPTER 2: Monitoring the waiting time sequence of single Ras GTPase activation events using liposome functionalized Zero-Mode Waveguides ...............................................17 Abstract .....................................................................................................................................18 Main Text ..................................................................................................................................18 Methods .....................................................................................................................................22 Fabrication and functionalization of ZMWs for liposome immobilization ............................22 Protein purification and labeling ............................................................................................23 Optical microscopy .................................................................................................................23 Liposome preparation .............................................................................................................23 Ras activation assay ................................................................................................................24 Fitting of single turnover traces ..............................................................................................24 Simulations of the excitation field inside a ZMW ..................................................................25 i Estimating the effective excitation flux impinging on fluorophores diffusing at the membrane of a ZMW immobilized liposome ...................................................................26 Figures .......................................................................................................................................28 CHAPTER 3: One-way membrane trafficking of SOS in receptor-triggered Ras activation ........................................................................................................................................................37 Abstract .....................................................................................................................................38 Introduction ..............................................................................................................................38 Results .......................................................................................................................................40 Supported-lipid-bilayer SOS-activation assay ........................................................................40 Allosteric activation of SOS via altered membrane recruitment ............................................40 Regulation of membrane binding by N-terminal domains .....................................................41 Multicomponent analysis of SOS-Ras-ERK signaling ...........................................................42 Timely signaling requires SOSCat-flanking domains ............................................................42 SOS autoinhibition prevents spontaneous activation .............................................................43 Positive regulation of SOS activity in stimulated cells ..........................................................44 Regulation of super-processive SOS by endocytosis .............................................................44 Discussion ..................................................................................................................................46 Methods .....................................................................................................................................48 Proteins and reagents ..............................................................................................................48 Protein labeling and benchmarking ........................................................................................48 Ras-decorated supported lipid bilayers for in vitro assays .....................................................48 Antibody-functionalized supported lipid bilayers for live cell imaging .................................49 Stopped flow supported lipid bilayer assay ............................................................................49 Maintenance and transfection of DT40 and Jurkat cell lines .................................................50 Live cell imaging ....................................................................................................................50
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