Biophysical Characterization of Map Kinase Activation and Regulation

The mitogen-activated protein kinase signaling pathways play a central role in the majority of cellular behaviors. The RAF/MKK/ERK pathway in particular is responsible for cellular proliferation and differentiation, and mutations that divorce downstream signaling from upstream regulation not only underlie the majority of melanoma cases, but play a significant role in a wide variety of other cancers as well. However, many questions about ERK still remain open in the field, including the differences between its two isoforms in humans, the precise nature of the activated state and the enzyme’s potential for autoactivation, and the effects of newly-developed inhibitors on ERK conformation and non-catalytic signaling. Hydrogen exchange mass spectrometry (HXMS) provides a unique approach to studying protein kinases in vitro through isotopic labeling and proteolytic digestion to provide peptide-level insight into solution-state protein dynamics that underlie protein-solvent interactions. This work applies HXMS to the study of the mitogen-activated protein kinase ERK2 in several contexts. First, HXMS is used to compare the binding effects of multiple different ligands on both the active and inactive forms of ERK2, and strong differential exchange behaviors throughout the enzyme are observed. The patterns generated by comparing multiple ligands allow mapping of regions of similarity within ERK2, suggesting the existence of allosteric networks within the kinase. These patterns can also be used to categorize both existing and novel inhibitors of ERK2 by their dynamic conformational effects, a unique approach that complements structural and affinity-based characterization. The ligands studied demonstrate an ability to coordinate long-range responses throughout ERK2 from a common binding site, and correlative analysis identifies a potential ligand structural motif that facilitates specific allosteric modulation. Further, this analysis shows that two ligands with similar affinities and binding modes produce strikingly different exchange behaviors in substrate binding regions of ERK2, correlating with activation-state differences observed by NMR. Second, the effects of an auto-activating mutation in ERK2 are investigated, comparing mutant and wild-type forms of both active and inactive enzyme, in apo and AMPPNP-bound states. In agreement with crystallographic data, HX data show the conformational dynamics of ERK2 are minimally affected by the autoactivating mutation. Autophosphorylation kinetic studies reveal substantial and differential effects of the mutation in ERK1 vs. ERK2 isoforms. Third, the two ERK isoforms -1 and -2 are mutated to introduce the alternate isoform’s residue at selected positions around the binding site, in order to determine whether these highly similar homologues have distinct internal allosteric regulation that can be modulated by sidechain identity. ERK1 and ERK2 show a complicated dependence on sequence identity with respect to auto- and substrate-phosphorylation, including significant effects on kinase activity by a highly conservative Leu-Ile mutation. Overall, this work investigates the role of sequence identity and ligand binding on conformational dynamics and autophosphorylation activity to provide insight into the activation and regulation of the critical signaling enzyme ERK2