A Comprehensive Characterization of ERK Mutations in the Drosophila Embryo

Abstract

Developmental disorders present themselves early in life and cause substantial limitations to human functionality. These diseases tend to have a genetic basis involving DNA mutations to genes integral to development, and many of the most potent mutations involve disruptions to cellular signal transduction paths [29]. Transduction occurs when a signal activates a receptor located in the plasma membrane of a cell. This in turn triggers a cascade of proteins that interact with each other in series. In this thesis we focus on a specific transduction network, the Ras/MAPK path, whose final kinase is translocated into the nucleus to activate the transcription of hundreds of developmental genes. The Ras network’s components interact with many substrates in parallel, so a single mutation can percolate to cause innumerable and varied effects. This makes decoding the causes of a genetic disease quite difficult. In this thesis, we mutate the terminal kinase in the Ras communication pathway, ERK. We introduce the mutant into a living system, the model organism Drosophila, to study the effects of such a perturbation. Through imaging phenotypic changes, quantifying active ERK levels, and measuring downstream substrate concentrations, we learn about the molecular interactions between ERK and its substrates. Our results suggest many impressive in vitro studies cannot be recreated in a living organism. Our work also provides new insights into the importance of phosphatases, which exert considerable control over ERK through negative regulation. Substrate binding events can be modeled using a simple ODE kinetics model, replicating results for our most important mutants. With the knowledge of Ras protein interactions, mechanisms, and biochemistry, we can learn how Ras interactions effect different tissues, and can provide help to others designing more effective tumor-suppression medication.