INVESTIGATING THE ROLE OF POST-TRANSLATIONAL MODIFICATIONS IN THE CORE RAS GTPASE DOMAIN

Abstract

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, 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.Doctor of Philosoph