Calcineurin (or PP2B), a conserved protein phosphatase that is activated by Ca2+ and calmodulin, participates in signal transduction by effecting Ca2+-dependent changes in the phosphorylation state of cellular proteins. Calcineurin is a critical regulator of mammalian T-cells, and inhibitors of this phosphatase (FK506, cyclosporinA) are powerful immunosuppressants. Our goal is to understand the functions of Ca2+-dependent signaling in yeast, with particular emphasis on the role of calcineurin. Our studies of yeast should provide insight into the mechanisms and functions of Ca2+-dependent signaling, especially calcineurin-mediated signaling, in all eukaryotic cells. The mating response provides an opportunity to examine the role of Ca2+-dependent signaling in yeast. Addition of pheromone to haploid yeast cells produces a rise in cytosolic Ca2+, and activation of calcineurin and calmodulin-stimulated kinase (CaM kinase) by this Ca2+ signal is required for viability. One essential function of calcineurin is to activate the Crz1p transcription factor, although the critical targets of calcineurin/CRZ1-dependent transcriptional regulation have not yet been identified. Calcineurin also performs additional, as yet uncharacterized, essential functions in pheromone-treated cells. The role of CaM kinase in the pheromone response is not understood. The proposed experiments will examine the functions of calcineurin and CaM kinase in cells exposed to pheromone. We will: 1) Characterize the mechanism of pheromone-induced transcription mediated by calcineurin and Crz1p. The mode of Crz1p regulation by calcineurin will be elucidated and additional gene products that participate in the calcineurin/CRZ1 response pathway will be identified. 2) Identify gene products that modify the requirement for Ca2+-dependent signaling during incubation with pheromone. Components of both calcineurin and CaM kinase-dependent responses will be studied. 3) Identify targets of pheromone-induced calcineurin-dependent transcriptional regulation and determine the contribution of these genes to cell viability. Microarray technology will be employed to examine all 6000 yeast genes for calcineurin-dependent changes in gene expression. 4) Identify substrates of calcineurin by identifying gene products that physically interact with calcineurin in vivo and by testing specific candidate proteins in vitro for calcineurin-dependent dephosphorylation.
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