Explore the vast range of titles available.

The MAPK pathway is a crucial cell-signaling cascade that is composed of RAS, MEK, BRAF, and ERK, which serves to connect extracellular signals to intracellular responses. Over-activating mutations in the MAPK pathway can lead to uncontrolled cell growth ultimately resulting in various types of cancer. While this pathway has been heavily studied using a battery of techniques, herein we employ native mass spectrometry (MS) to characterize the MAPK pathway, including nucleotide, drug, and protein interactions. We utilize native MS to provide detailed insights into nucleotide and drug binding to BRAF complexes, such as modulation of nucleotide binding in the presence of MEK1. We then demonstrate that different CRAF segments vary in their complex formation with KRAS, with the addition of the cysteine rich domain (CRD) enhancing complex formation compared to Ras binding domain (RBD) alone. We report differences in KRAS GTPase activity in the presence of different RAF segments, with KRAS exhibiting significantly enhanced nucleotide turnover when bound to CRAF fragments. We use ERK2 as a downstream readout to monitor the MAPK phosphorylation cascade. This study demonstrates the utility of native MS to provide detailed characterization of individual MAPK pathway components and monitor the phosphorylation cascade in real time. Native mass spectrometry enables real time characterization of MAPK phosphorylation cascade and impact of oncogenic RAS mutants on kinetics.

Ras is regulated by a specific guanine nucleotide exchange factor Son of Sevenless (SOS), which facilitates the exchange of inactive, GDP-bound Ras with GTP. The catalytic activity of SOS is also allosterically modulated by an active Ras (Ras–GTP). However, it remains poorly understood how oncogenic Ras mutants interact with SOS and modulate its activity. Here, native ion mobility–mass spectrometry is employed to monitor the assembly of the catalytic domain of SOS (SOScat) with KRas and three cancer-associated mutants (G12C, G13D, and Q61H), leading to the discovery of different molecular assemblies and distinct conformers of SOScat engaging KRas. We also find KRasG13D exhibits high affinity for SOScat and is a potent allosteric modulator of its activity. A structure of the KRasG13D•SOScat complex was determined using cryogenic electron microscopy providing insight into the enhanced affinity of the mutant protein. In addition, we find that KRasG13D–GTP can allosterically increase the nucleotide exchange rate of KRas at the active site more than twofold compared to KRas–GTP. Furthermore, small-molecule Ras•SOS disruptors fail to dissociate KRasG13D•SOScat complexes, underscoring the need formore potent disruptors. Taken together, a better understanding of the interaction between oncogenic Ras mutants and SOS will provide avenues for improved therapeutic interventions.

Mutations in RAS are associated with many different cancers, and RAS has been a therapeutic target for more than three decades. Studying the biochemical properties of RAS mutants and most importantly the interaction with activator and downstream effectors lay a foundation to better understand how RAS functions in signaling pathway. In chapter 2, high-resolution native mass spectrometry (MS) was used to determine the kinetics and transition state thermodynamics of intrinsic hydrolysis for KRAS and oncogenic mutants. MS data reveal heterogeneity where both 2’-deoxy and 2’-hydroxy forms of GDP (Guanosine diphosphate) and GTP (Guanosine triphosphate) are bound to the recombinant enzyme. In addition, MS results show that the transition state thermodynamics for the intrinsic GTPase activity of KRAS is both enthalpically and entropically unfavorable. The oncogenic mutants, G12C, Q61H and G13D unexpectedly exhibit a higher 2’-deoxy GTP intrinsic hydrolysis rate compared to that for GTP.In chapter 3, the interaction of RAS mutants with a specific guanine nucleotide exchange factor, Son of Sevenless (SOS), is studied. Native ion mobility-mass spectrometry was employed to monitor the assembly of the catalytic domain of SOS (SOScat) with KRAS and three cancer-associated mutants (G12C, G13D, and Q61H), leading to the discovery of different molecular assemblies and distinct conformers of SOScat engaging KRAS. Also, KRASG13D exhibits high affinity for SOScat and is a potent allosteric modulator of its activity. A structure of the KRASG13D•SOScat complex was determined using cryogenic electron microscopy providing insight into the enhanced affinity of the mutant protein. In addition, we find that KRASG13D-GTP can allosterically increase the nucleotide exchange rate of KRAS at the active site more than two-fold compared to KRAS-GTP. Furthermore, small molecule RAS•SOS disruptors fail to dissociate KRASG13D•SOScat complexes underscoring the need for more potent disruptors.Lastly, chapter 4 is focused on the biochemical characterization of BRAF, a downstream effector of RAS. Our results indicate that BRAF populates different stoichiometries with MEK1 and 14-3-3 dimers. In the presence of ATP, BRAF undergoes dimerization suggesting that ATP promotes the activation of BRAF. In addition, copper stimulates the dimerization of BRAF in MEK1-independent manner.