In the budding yeast, mating pheromones trigger the arrest of the cell cycle and the induction of genes required for the mating response. Nutrient limitation changes the timing of cell division and induces genes required for the invasive growth pathway. These two distinct programs require the action of components of the same MAP kinase (MAPK) cascade. The MAPK pathway, like many other signal transduction pathways, is essential to various cell functions and its malfunction is associated with various diseases including cancers and several inflammatory conditions. Increasing evidence has implicated regulators of the cell cycle as either protooncogenes or as tumor suppressor genes. One of the general aims of this proposal is to help understand the mechanisms by which different signals may activate the same MAPK cascade, yet lead to entirely different cellular consequences. Yeast has illuminated numerous cellular processes common with mammalian ones and the study of MAPK signaling in yeast has often established the basis for understanding the homologous mechanisms in animal cells. In this respect, yeast may serve as a model system for the general process in mammalian cells, with the advantages of having its entire genome sequence available and in being much simpler and easier to manipulate, both genetically and biochemically. The two novel transcription regulators, Dig1 and Dig2 down regulate Ste 12, the downstream transcriptional transactivator of the MAPK pathway. It has also been suggested that Dig1 and Dig2 may also associate with cyclin- dependent cell cycle protein kinases, and thereby play a direct role in cell cycle control. The research proposed, aims to elucidate how Dig1 and Dig2 impose their negative effects on Ste-12 dependent gene expression. This will be addressed by performing biochemical assays, as well as by genetical means to map the sequences for Dig1 and Dig2 interaction with Ste 12. The effects of pheromone stimulation and nutrient limitation on Dig1 and Dig2 function and where it is mediated through MAPK-dependent phosphorylation will be assessed by in vitro phosphorylation and immunoprecipitation studies. Experiments will be also performed in order to determine whether Dig1 and Dig2 are phosphorylated by the cyclin- dependent kinases. Thus, the analysis of the role of Dig1 and Dig2 in the controlling of both signaling and cell cycle control in yeast may help to elucidate the kinds of molecular events that permit a single MAPK cascade to evoke different developmental outcomes.
