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Regulation of Ras and Rho Gtpases by Post-Translational Modification: Cysteine Oxidation and Ubiquitination

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REGULATION OF RAS AND RHO GTPASES BY POST-TRANSLATIONAL MODIFICATION: CYSTEINE OXIDATION AND UBIQUITINATION Guy Aaron Hobbs A dissertation submitted to the faculty of the University of North Carolina at Chapel Hill in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Biochemistry and Biophysics. Chapel Hill 2013 Approved by: Sharon Campbell Henrik Dohlman Brian Kuhlman Keith Burridge Andrew Lee © 2013 Guy Aaron Hobbs ALL RIGHTS RESERVED ii ABSTRACT Guy Aaron Hobbs: Regulation of Ras and Rho GTPases by Post-Translational Modification: Cysteine Oxidation and Ubiquitination (Under the direction of Sharon L. Campbell) Ras superfamily GTPases cycle between active GTP-bound and inactive GDP-bound forms to regulate a multitude of cellular processes, including cell growth, differentiation, and apoptosis. The activation state of Ras superfamily GTPases is regulated by protein modulatory agents that accelerate the slow intrinsic rates of GDP dissociation and GTP hydrolysis. In addition, evidence for the redox regulation of Ras superfamily GTPases is growing, and current data supports regulation by the generation of a thiyl radical. Oxidation and reduction events are critical to physiological and pathological processes and are highly regulated. In Ras GTPases, the redox-sensitive cysteine is in the NKCD motif, which is a motif that is critical for nucleotide binding. However, we show that oxidation that occurs absent of the generation of a thiyl radical does not result in Ras activation. In the Rho subfamily of GTPases, there is increasing in vitro and cell-based data supporting the oxidant-mediated regulation of Rho GTPases that contain a redox-sensitive cysteine at the end of the conserved phosphoryl-binding loop motif (GXXXXG[S/T]C). While this motif is distinct from Ras, our data suggest that Rho GTPases are regulated by free radical-generation as well as oxidation of the thiol. Specifically for RhoA and other Rho GTPases that have two cysteine residues within the phosphoryl-binding loop motif (GXXCXG[S/T]C), oxidation can result in internal disulfide bond formation that can inactivate these GTPases. In addition, we characterized relevant cellular oxidation states of RhoA and show that oxidation can result in the activation and deactivation of RhoA. iii Thus, from data presented herein, it is evident that Ras and Rho family GTPases are uniquely regulated by free radical and non-radical oxidants, and the modulation of activity of the GTPase due to oxidation is dependent on the position(s) of the redox-sensitive thiol. While Ras GTPases are only activated by free radicals because the redox-sensitive thiol does not make interactions with the bound nucleotide, in Rho GTPases, the redox-sensitive thiol makes direct interactions with the bound nucleotide, which results in activation upon modification of the redox-sensitive thiol in RhoA and related GTPases. iv ACKNOWLEDGEMENTS Foremost, I would like to acknowledge the individuals that have encouraged me always question the world around me. I would specifically like to acknowledge Dr. Gloria Lee for giving me a chance to work in her lab as an undergraduate. At the bench, Dr. Kiran Bhaskar and Dr. Daniel Ferraro were my first mentors in research while an undergraduate student at the University of Iowa. In addition, I would like to acknowledge Dr. Ramaswamy Subramanian and Dr. Bryce Plapp for their guidance and mentorship. I do not think I would be in the place I am today without the care and patience of these individuals. I would also like to acknowledge my mentor, Dr. Sharon Campbell, for always being there to listen to my wild ideas and provide feedback on new data. Her willingness to discuss anything will be greatly missed. While others got me excited to perform research, Sharon taught me how to think like a scientist. To specifically acknowledge every person that helped along the way would result in an acknowledgements section as long as the dissertation itself. Thus, I say thank you to current and previous lab members, classmates, and friends for asking questions, challenging my thoughts, and providing support when needed. Minh Huynh, who was an undergraduate student under my direction, served with me for two years and deserves a special acknowledgement. He ran every project during the last two years of my thesis by preparing proteins, buffers, and reagents, often with minimal direction, which allowed me the time to perform the most critical experiments as well as analyze and present the data. Without his help, the amount that I was able to accomplish would be significantly less. Lastly, I want to thank my parents and family for encouraging and fostering a desire to read, learn, and take my education seriously. Their unwavering support and v encouragement to achieve good grades, instill a solid work ethic, and do what I loved has led me to be who I am today. There are no words to describe the level of gratitude I have for my parents for teaching me right from wrong as well as the importance of never quitting and doing your best. I also wish the thank Heather. Although she joined me in my last two years of my journey at UNC, they were the most productive two years as well as the most exciting years. She taught me what school could never teach a person, led me out of the lab on some amazing adventures, and let me back into the lab to complete my work. While she has captured my heart, I will never be able to thank her enough for her help and support through some of the most demanding years in graduate school. Lastly, I must thank Josh Gough for providing me with one of my favorite sayings, “It sucks to suck.” It truly does suck to suck, and this saying has repeatedly served as motivation and as words of encouragement. Thank you. vi TABLE OF CONTENTS LIST OF FIGURES............................................................................................................... .... x LIST OF TABLES ...................................................................................................................xii LIST OF ABBREVIATIONS AND SYMBOLS ..................................................................... xiii Chapter 1: Introduction and Background ............................................................................... 1 Introduction ................................................................................................................. 1 Generation of ROS and RNS in cells and antioxidant defense ................................. 4 Reactive oxygen and nitrogen species as second messengers .................................. 9 Free radicals modify and activate Ras through a reactive cysteine in the NKCD motif ............................................................................................. 11 Redox regulation of Rho GTPases by 1e- and 2e- mechanisms through a redox-active cysteine in the phosphoryl-binding loop ................................................................................................................ 14 Chapter 2: Glutathiolated Ras: Characterization and Implications for Ras Activation ................................................................................................................... 17 Introduction ............................................................................................................... 17 Materials and Methods ............................................................................................ 20 Results ...................................................................................................................... 24 Discussion ................................................................................................................. 32 Conclusions ............................................................................................................... 37 Chapter 3: Characterization and Nitrosation of Oncogenic K-RasG12C ............................... 40 Introduction .............................................................................................................. 40 Methods .................................................................................................................... 42 vii Results ...................................................................................................................... 44 Discussion ................................................................................................................. 52 Chapter 4: Oxidation of RhoA at Cys20 regulates nucleotide binding ................................ 56 Introduction .............................................................................................................. 56 Methods .................................................................................................................... 60 Results ...................................................................................................................... 64 Discussion ................................................................................................................. 82 Chapter 5: Site-specific monoubiquitination activates Ras by impeding GTPase-activating protein function ......................................................................... 88 Introduction .............................................................................................................. 88 Commentary ............................................................................................................. 88 Chapter 6: Biophysical
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