Ras signaling network and regulation in S. cerevisiae

Ras proteins mediate cellular growth and differentiation in response to external cues. Mammalian H-ras protein and its homologs in eukaryotes are central signaling hubs in coordinating cellular responses to changes in the environment, such as nutrient availability and a variety of stresses. This dissertation explores the topology of the Ras signaling network in yeast as well as its regulation.; Ras stimulates filamentous growth upon nutrient starvation in yeast. Previous work has suggested that filamentation occurs in part through Ras activation of a MAP kinase (MAPK) cascade, even though Ras promotes cell cycle progression solely through activation of protein kinase A (PKA). The first half of this dissertation, presented in Chapter 2, probes the nature of the Ras-MAPK relationship by studying the filamentation phenotypes of strains with Ras-unresponsive PKA (tpk2-w). We found that Ras stimulation of filamentation depended on its ability to activate PKA. Ras-induced transcriptional changes were completely eliminated in a tpk2-w strain but were fully intact in strains lacking the Fus3 and Kss1 MAP kinases. Consistent with this result, screening for mutations of RAS2G19V that would separate the filamentous growth stimulation activity from the PKA-dependent cell cycle function yielded no mutations that ablated filamentous growth without also eliminating cell cycle progression activity. From these studies, we propose that Ras stimulates filamentous growth in yeast solely through activation of PKA.; The second half of this dissertation, presented in Chapter 3, investigates a possible feedback control, analogous to that observed in mammalian cells, on the Ras-PKA signaling pathway, indicated by the transient increase of cAMP levels upon Ras activation. We observed that Ras2G19V protein levels were lower than Ras2 and investigated the role of PKA in the decrease. We showed that the decrease in Ras protein levels was dependent on Ras activation and PKA activity. We also showed that mutating the phosphorylation sites of Ras2WT and Ras2G19V restored protein levels and a further increase in levels was seen in a tpk2-w strain. We presented preliminary evidence suggesting that Ras may be degraded in the yeast vacuole. The proposed future experiments aim to show conclusively that Ras is under PKA feedback control.