Mutations in ras genes are involved in the pathogenesis of several human tumors, but relatively little is understood about the biochemical function or regulation of ras gene products in normal or tumor cells. In order to gain more insight into the control of the activity of Ras proteins, we propose to continue our search for novel regulators of Ras protein activity in the model eukaryotic cell, Saccharomyces cerevisiae. We will place special emphasis on the identification and characterization of novel positive regulators of Ras activity, by use of a genetic screen that has already identified a new gene required for Ras activation. Novel positive regulators of Ras, as well as the negative regulator we previously identified called Rpil, will be studied in depth by a combination of molecular genetic and biochemical approaches. We will utilize specific biochemical assays of Ras protein activation involving recombinant Cac25 and recombinant Ira2 proteins to explore the possible function of these novel regulators in light of what is already known about proteins that are involved in Ras regulation. Finally, we propose to study the requirement for specific regulators of Ras activity (e.g., Cdc25, Ira1, Ira2, and Rpil) in the transduction of specific nutrient signals to Ras proteins. Our long term goal is to gain a complete molecular picture of the gene products involved in the control of Ras activity and to determine their biochemical mode of action.
