Probing the Efficiency of Activated MEK Phosphotransfer Using ERK Variants

Abstract

The Mitogen-Activated Protein Kinase (MAPK) pathway is a signal transduction pathway that is essential in regulating cellular processes involved in growth and development. Proteins in this pathway are conserved across all species, and mutations in these proteins have been linked to abnormal cell growth in cancer and other developmental disorders [5]. Components of the MAPK pathway are thus frequent targets of inhibitors used to treat these diseases. However, it is not well-understood how specific reactions associated with the MAPK pathway are affected by pathological mutations.

This thesis uses two proteins in the MAPK pathway (MEK1 and ERK2) as a model to investigate the biochemical effects of specific mutations on the reactions that are associated with MEK phosphotransfer. Since MEK acts as both a kinase that successfully phosphorylates ERK and an ATPase that nonproductively hydrolyzes ATP, measuring ADP production as a function of protein substrate concentration gives insight to catalytic efficiency in the MEK-ERK reaction. There are several conclusions that can be drawn from this work: First, because there is ATPase activity in MEK without a protein substrate, it is likely that ATPase activity also occurs in reactions containing both MEK and ERK. The rate of this ATPase activity is observed in the context of mutations made to ERK that affect its phosphorylation capabilities (dual-, mono-, or binding defective phosphorylation). Differences in ADP production, and consequently the kinetics of kinase and ATPase reactions with MEK and ERK, can be explained by conformational and functional changes caused by the mutations. Furthermore, results suggest that a constitutively active mutation made to MEK may speed up the second step of phosphorylation, relative to the first step. These findings are represented in a mathematical model describing the relevant reactions. By exploring the impacts of MEK and ERK mutations on specific reactions in vitro, this thesis broadens our understanding of the biochemical interactions in the MAPK pathway that exhibit promising potential for developing targeted MEK inhibitors.