The aim of this proposal is to understand mechanisms that provide the specificity to converging MAP kinase pathways. The pheromone response pathway in Saccharomyces cerevisiae is responsible for transmitting extracellular mating signals into the nucleus during haploid conjugation. It includes pheromone receptors, a hetero-trimeric G protein, a highly conserved PAK/MEKK/MEK/MAPK kinase cascade (Ste20p, Ste11p, Ste7p, and Fus3p/Kss1p), and a transcription factor, Ste12p. Elements of the pathway, including Ste20p, Ste11p, Ste7p, and Ste12p, are also required for diploid filamentous growth (development of chains of elongated cells upon nitrogen starvation) and haploid invasive growth. Thus, one kinase cascade is used by different developmental pathways in haploids and diploids and the outputs of the pathways involve the same transcription factor, Ste12p. The proposed research uses molecular and genetic approaches to identify Ste12p regulators and targets, and to understand Ste 12p regulation in filamentous growth in comparison to its regulation in haploid mating. The first specific aim is to identify and characterize Ste12p regulated genes in filamentous diploid cells. Genes transcriptionally regulated by Ste12p during filamentous growth in diploids will be cloned with a yeast lacZ fusion library. Their expression pattern, protein localization and mutant phenotypes will be analyzed. Promoters of some of the cloned genes will be mapped to define the upstream activation sequences responsible for Ste 12p induced transcription.
The second aim i s to understand the mechanism of Ste12p regulation in filamentous growth and identify its regulators. We propose to study Ste12p phosphorylation under filamentation conditions. Genetic screens are designed to identify a Ste12p kinase in diploids because the MAP kinases Fus3p/Kss1 of the pheromone response pathway are not required for filamentous growth. We propose additional regulators functioning together with Stel2p provide the diploid specific regulation. Both genetic and biochemical experiments will be utilized to identify these regulators. Our research on Ste12p regulation by the conserved MAP kinase pathway addresses an interesting question concerning the regulation of specificity among these pathways. This study will enhance our understanding of the complicated regulation by MAP kinase pathways in mammalian cells. Investigation of dimorphic regulation in genetically tractable S. cerevisiae will also further our knowledge of fungal dimorphism, which is important in the pathogenesis of Candida albicans, a common fungal pathogen of humans.
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