Functional Characterization Of Ras2 Protein In Saccharomyces Cerevisiae

RAS gene family is highly conserved from yeast to mammals and has attracted great attention due to the important role of constitutively activated Ras in the development of human cancers. In the yeast Saccharomyces cerevisiae, Ras proteins are essential for cAMP signalling. Because they are highly homologous to their conterparts in higher eukaryotes, yeast Ras proteins have been studied intensively. This work focuses mainly on elucidating the role of yeast Ras2 protein in the feedback inhibition of the cAMP pathway by PKA. We also examined the effect of PKA activity on the localization of Ras2 and the maintenance of plasmid in yeast cells.Our results indicated that yeast cells carrying plasmids that harbours the dominant RAS2Val19 allele lost their plasmids quickly upon heat shock. The high PKA activity caused by this dominant Ras2 allele account for this phenomena, probably due to increased membrane permiability in such cells upon heat shock so that plasmids are excluded from cells. Alternatively, combination of heat shock and high PKA activity may render cells an ability to aquire specific enzymatic activity that causes degradation of the plasmids.A serine to alaline substitution at position of 214 in the yeast Ras2 protein, which is the PKA phosphorylation site, resulted in enhanced sensitivity to heat shock, reduced levels of storage carbon sources and higher levels of both basal cAMP and glucosed-induced cAMP signal compaired to wild type cells. Ras2Ala214 had a higher GTP-binding capability than wild type Ras2, and the amount of Cdc25 Co-Pulldowned with Ras2-GTP from cells expressing Ras2Ala214 was also higher than cells expressing wild type Ras2. The intracellular level of Ras2-GTP correlated very well with PKA activity in a reverse manner .Our result indicated that phosphorylation of Ras2 by PKA consitutes a feedback mechanism by which PKA down-regulate cAMP signalling. While phosphorylation of Ras2 did not cause an observable effect on its localization, changes in PKA activity resulted in plasmamembrane- cytoplasm redistribution of the protein. These results indicated that phosphorylation of Ras2 by PKA decreases the affinity of Ras2 for GTP and, therefore, reduces its ability to activate adenylate cyclase and, as a consequence, exserts feedback inhibition on the Ras-cAMP pathway. In addition, we also demonstrated that Cdc25 binds preferrentially to Ras2-GTP, the active form of the protein.Wild type cells growing on glycerol had a higher level of Ras2-GTP compaired to cells growing on glucose and the same was also true for Cdc25 Co-Pulldowned with Ras2-GTP. Cells carrying a null allele of SCH9 exibited a reduced level of Ras2-GTP when growing on glycerol, indicating that, in contrast to its role in cells growing on glucose, Sch9 inhibits PKA activity in glycerol growing cells.Ras1 and Ras2 have different effect on the Ras-cAMP pathway. Ras2 apparently plays a more prominent role in activating adenylyl cyclase than its conterpart dose. By exchanging their promoters, we found that the difference between these two proteins in terms of their ability to activate adenylyl cyclase was due mainly to the difference in their promotor strenth.