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The identification of Dictyostelium phosphoproteins altered in response to the activation of RasG Secko, David Matthew


Dictyosteliiim RasG has been implicated in the regulation of a variety of cellular processes, including the initiation of development, cell movement, and cytokinesis, but the molecular components of the signaling pathways involved are largely unknown. This thesis describes the search for putative downstream targets of the RasG signaling pathway through the identification of proteins whose level of phosphorylation were changed in response to the induction of a gene encoding an activated form of RasG, RasG(G12T). Two expression systems were tested to increase the level of RasG(G12T) in the cell. One system used the rasG(G12T) gene fused to the ribonucleotide reductase (rnrB) promoter. Induction of RasG(G12T) expression from this promoter was accompanied by a decrease in discoidin mRNA levels, a proposed negatively regulated target of RasG. Regulation of Discoidin expression was also down regulated in rasG null cells under all experimental conditions, but the response to well established discoidin regulators still occurred in this strain. These results revealed a role for RasG in modulating discoidin gene expression. A tetracycline-regulated expression system was used to study the effect of increasing the level of RasG(G12T) on the phosphorylation state of Dictyostelium proteins. Over 70 phosphorylated proteins in vegetative cells were resolved by 2D immunoblot analysis and thirteen proteins, which reproducibly changed in response to RasG(G12T) expression, were recovered from 2D gels and identified by mass spectrometry. The proteins identified were: the signaling proteins RasGEF-R and protein kinase B (PKB), the adhesion protein DdCAD-1, the cytoskeletal protein actin, the mitochondrial division protein FtsZA, as well as several proteins involved in protein translation or metabolism. An additional set of experiments showed that phosphorlyation of the vacuolar H+-ATPase component VatA upon folate stimulation was modulated by RasG. Two of the proteins whose phosphorylation levels were affected by RasG(G12T), DdCAD-1 and PKB, were analyzed further. Cells expressing RasG(G12T) exhibited increased cohesion and this increase correlated with DdCAD-1 localization and dephosphorylation. Induction of RasG(G12T) was also found to up-regulate both the basal and folate-induced level of phosphorylation of PKB, and cells expressing RasG(G12T) contained slightly more membrane bound PKB. Together the presented work has identified several proteins whose phosphorylation state was affected by RasG(G12T) and additional regulatory roles for RasG.

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