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UBC Theses and Dissertations

The Ras subfamily protein, RasC, is required for the aggregation of Dictyostelium discoideum Lim, Chinten James

Abstract

The Ras subfamily proteins are monomeric GTPases that function as molecular switches in cellular signal transduction. Dictyostelium discoideum possesses at least six ras subfamily genes, each with a unique expression profile during vegetative growth and multicellular development. Gene disruption analyses have identified important roles for Dictyostelium Ras proteins in the regulation of cellular processes including growth, motility, cytokinesis, karyokinesis, and cell type specific functions during multicellular development. Previously, disruption of genes that encode putative regulators and effectors of Ras protein function have indicated that a Ras signaling pathway is involved in the regulation of the cAMP relay and in the chemotactic response to cAMP during aggregation. This thesis describes the genetic and biochemical evidence that RasC is the Ras protein that had been previously implicated in the aggregation processes. Replacement of the genomic rasC allele with a disrupted copy completely abolished RasC protein expression and resulted in cells that were incapable of aggregation. Ectopic expression of RasC from the rasC promoter restored the development of rasC- cells, thus confirming that the aggregation deficient phenotype was a consequence of rasC disruption. Periodic application of exogenous cAMP, or chimeric development in mixes with wildtype cells, effectively circumvented the developmental defect of rasC- cells and resulted in normally proportioned terminal fruiting bodies which contained viable, mature spores. cAMP was not produced in rasC- cells stimulated in vivo with 2'-deoxy-cAMP, indicating that RasC is required for coupling cAMP receptor to adenylyl cyclase activation. The effects of GTPγS on reduced cAMP production, and high affinity cAMP receptor binding sites in lysates of rasC- cells relative to that in wildtype cells suggested that RasC modulates the dissociation of the subunits of the heterotrimeric G-protein complex. cAMP induced ERK2 phosphorylation was unaffected in rasC- cells, indicating RasC is not an upstream activator of the MAPK required for cAMP relay. cAMP stimulation of rasC- cells resulted in a dramatically reduced transient PKB phosphorylation and activation. However, cAMP induced translocation of PKB to the cell membrane was unaffected, suggesting that RasC is not necessary for PI3-kinase dependent generation of PH-domain binding sites, but is required for optimal PKB activation. Despite the reduced activation of PKB, rasC- cells that had been pulsed with cAMP exhibited robust chemotaxis, indicating that RasC is not required for chemotaxis. However, RasC is required for cellular motility, as indicated by the reduced random motility and folate chemotaxis of vegetative rasC- cells. RasC appears to act downstream of cAMP receptor stimulation and is required for two distinct effector pathways, the activation of PKB and the activation of adenylyl cyclase. The key role played by the RasC protein in aggregation appears to be in the sensing of and response to chemotactic signals.

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