Elucidating the Ras activation pathways in response to cyclic AMP in the aggregation of Dictyostelium discoideum

• Main Author: Kae, Helmut
• Language: English
• Published: University of British Columbia 2011
• Online Access: http://hdl.handle.net/2429/31357

Ras proteins are highly conserved molecular switches that regulate cellular responses to external stimuli. Dictyostelium discoideum contains an extensive family of Ras proteins that function in growth, cell type differentiation, and more. While there is much genetic evidence for the involvement of Ras proteins in these behaviours, little is known about the events that lead to their activation. The primary objective of this thesis project was to develop an efficient and rapid assay that allowed the measurement of activated Dictyostelium Ras proteins. Modification of an assay used successfully to measure activated Ras in mammalian cells demonstrated that the Ras Binding Domain (RBD) of the Schizosaccharomyces pombe Ras effector protein, Byr2, was able to bind to RasB, RasC, and RasG. Furthermore, the use of dominant negative Ras mutants and nucleotide loading of cellular lysates showed that Byr2 (RBD) bound preferentially to Ras-GTP (activated Ras) over Ras-GDP (quiescent Ras). The 'RBD binding assay' was applied to elucidate the Ras activation pathways stimulated by cAMP during Dictyostelium aggregation. Previous experiments have suggested that RasC is involved in cAMP stimulated signalling events during Dictyostelium aggregation, and this was confirmed using the RBD binding assay, as levels of RasC-GTP displayed a rapid and transient increase upon cAMP stimulation. Similarly, activated RasG also showed an increase in activation in response to cAMP. Through the use of various mutant strains, it was demonstrated that RasC and RasG activation is downstream of the cAMP receptor and its associated heterotrimeric G-protein. This thesis also provides evidence that the Dictyostelium Ras guanine nucleotide exchange factor (RasGEF) proteins can each act on specific Ras proteins. RasC activation was abolished in a gefA - mutant, while RasG activation was normal, indicating that RasGEFA activates RasC, but not RasG. Conversely, RasC activation was normal in gefM - and gefR - mutants, whereas RasG activation was reduced in each strain, indicating that RasGEFM and RasGEFR activate RasG. Analysis of PKB activation suggests that RasGEFM is necessary for the activation of RasG in the pathways leading to PKB activation. Observing the streaming behaviour of aggregating gefR - cells has suggested that RasGEFR may play a role in regulating chemotaxis. === Science, Faculty of === Microbiology and Immunology, Department of === Graduate