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Investigating the Role of Post-Translational Modifications in the Core Ras Gtpase Domain

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Investigating the Role of Post-Translational Modifications in the Core Ras Gtpase Domain INVESTIGATING THE ROLE OF POST-TRANSLATIONAL MODIFICATIONS IN THE CORE RAS GTPASE DOMAIN Samantha Kathleen Kistler A dissertation submitted to the faculty at the University of North Carolina at Chapel Hill in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Chemical Biology and Medicinal Chemistry in the Eshelman School of Pharmacy. Chapel Hill 2019 Approved by: Sharon Campbell Michael Jarstfer Stephen Frye David Williams Adrienne Cox Eric Brustad © 2019 Samantha Kathleen Kistler ALL RIGHTS RESERVED ii ABSTRACT Samantha Kathleen Kistler: Investigating the Role of Post-Translational Modifications in the Core Ras GTPase Domain (Under the direction of Sharon Campbell) Ras proteins are the most commonly mutated oncoproteins in cancer (~30%). Oncogenic, activating Ras mutations are known drivers of the deadliest human cancers, including lung, pancreatic and colorectal cancers. Ras proteins function as critical regulators of cellular growth by acting as molecular switches, cycling between active, GTP- and inactive, GDP-bound states. In their active form, Ras proteins signal through downstream pathways that regulate cellular growth, differentiation and apoptosis. Early attempts to target Ras proteins (farnesyltransferase inhibitors) were directed toward inhibiting key carboxyl (C)-terminal lipid post-translational modifications (PTMs), which are crucial for proper Ras localization and function at the cellular membrane. Despite their failure, FTIs represent the first direct targeting efforts of Ras proteins. Promising new classes of anti-cancer drugs directed at targeting the dysregulation of PTM status in cancers (kinase inhibitors, histone deacetylase inhibitors, HDACi and methyltransferase inhibitors) have demonstrated multiple clinical successes in recent years. PTMs have been demonstrated to alter protein stability and localization as well as protein-protein interactions in several non-histone cancer-related proteins. While PTMs have been extensively studied in the C-terminus of Ras proteins, their role remains poorly understood in the core Ras guanine nucleotide binding domain (GTPase domain). Monoubiquitylation and acetylation within the core Ras GTPase domain have been demonstrated to modulate Ras protein activity, iii signaling and tumorigenesis, suggesting that PTMs in this region are capable of regulating Ras behavior. Further, aberrant dysregulation in the balance of PTMs has been characterized in several cancer types, including the Ras-driven pancreatic cancer. It is therefore reasonable that Ras PTMs may present a novel avenue for therapeutic targeting in cancer. Despite more than three decades of research, Ras has remained an elusive target for cancer therapy. We have recently identified novel sites of PTMs in Ras proteins at highly conserved residues within the core GTPase domain. Herein, we present highly innovative and novel methods of generating both acetyl- and methyl-lysine in intact Ras proteins. With the combined use of biochemical, structural, cellular and computational data, we provide mechanistic insight into the regulation Ras proteins by PTMs and also provide rationale for novel therapeutic targeting approaches in Ras-driven cancers. iv TABLE OF CONTENTS LIST OF TABLES ....................................................................................................................... vii LIST OF FIGURES .................................................................................................................... viii LIST OF ABBREVIATIONS ....................................................................................................... xi Chapter 1 – Introduction to the Ras superfamily of GTPases and Ras proteins ............................ 1 Ras Proteins as GTPases ............................................................................................................ 1 Ras History and Signaling in Cancer ......................................................................................... 2 Ras Structure and Dynamics ...................................................................................................... 6 Ras and Post-Translational Modifications ............................................................................... 11 Strategies to Therapeutically Target Ras Proteins: A Broad Overview .................................. 16 Chapter 2. – HDACi treatment causes Ras acetylation, directing signaling through the MAPK pathway through a reordering of the Ras:Raf binding interface ............................... 22 Introduction .............................................................................................................................. 22 Results ...................................................................................................................................... 25 Discussion ................................................................................................................................ 54 Materials and Methods ............................................................................................................. 57 Chapter 3. The ‘Ras-opathy’ mutant KRas K5N potentiates protein activation through destabilization of the GDP-bound state .......................................................................... 73 Introduction .............................................................................................................................. 73 Results ...................................................................................................................................... 76 Discussion ................................................................................................................................ 95 Materials and Methods ............................................................................................................. 98 v Chapter 4. A KRas GTPase K104Q Mutant Retains Downstream Signaling by Offsetting Defects in Regulation ............................................................................................... 105 Introduction ............................................................................................................................ 105 Results .................................................................................................................................... 108 Discussion .............................................................................................................................. 123 Materials and Methods ........................................................................................................... 126 Chapter 5. A Tool for Site-Specific Methyl-Lysine Generation and Selective Enrichment in Intact Proteins..................................................................................................... 135 Introduction ............................................................................................................................ 135 Site-specific methyl-lysine analogue alkylation reaction ...................................................... 137 Methyl-binding domain enrichment of methylated Ras ........................................................ 138 Results .................................................................................................................................... 140 Discussion .............................................................................................................................. 143 Materials and Methods ........................................................................................................... 146 Chapter 6. Conclusions and Future Directions .......................................................................... 149 Final Conclusions .................................................................................................................. 154 REFERENCES .......................................................................................................................... 156 vi LIST OF TABLES Table 2.1. KRas WT and KRas K5AcK rates of nucleotide exchange and hydrolysis ..................................................................................................................................... 37 Table 2.2. Thermal melting temperature for KRas WT and KRas acetylated protein .......................................................................................................................................... 38 Table 2.3. Calculated binding affinities of KRas WT, KRas G12V and KRas G12V-K5AcK to BRaf RBD, CRaf RBD and PI3Kα RBD ......................................................... 43 Table 3.1. Melting temperature of KRas K5N and wild-type protein. ......................................... 79 Table 3.2. Nucleotide exchange properties of KRas K5N and KRas WT proteins. .................... 80 Table 3.3. Nucleotide association rates for KRas WT and KRas K5N ........................................ 81 Table 3.4. Calculated binding affinities of KRas WT and KRas K5N to BRaf and CRaf RBDs .................................................................................................................................. 84 Table 4.1. Biochemical Properties of KRas WT and K104 mutant proteins. ........................... 110 vii LIST OF FIGURES Figure 1.1. Ras Regulation and Effector Binding .......................................................................... 2 Figure 1.2 Ras Mutations in Cancer. ............................................................................................
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