YEAST PP2B PHOSPHATASES--ROLE IN PHEROMONE ADAPTATION

描述：钙调神经磷酸酶(或PP2B)，一种保守的蛋白质磷酸酶，是 在钙离子和钙调蛋白的激活下，通过 细胞内钙离子依赖性磷酸化状态的变化 蛋白质。钙调神经磷酸酶是哺乳动物T细胞的关键调节因子， 这种磷酸酶的抑制剂(FK506，环孢素A)很有效 免疫抑制剂。我们的目标是了解 酵母菌中的钙依赖信号转导，特别强调 钙调神经磷酸酶。我们对酵母菌的研究应该能让我们深入了解 钙依赖信号转导的机制和功能，尤其是 钙调神经磷酸酶介导的信号，在所有真核细胞中。 交配反应提供了一个机会来研究 酵母中钙离子依赖的信号转导机制。单倍体酵母中信息素的添加 细胞产生细胞内钙离子升高，钙调神经磷酸酶激活和 钙调素刺激的激酶(CaM)需要通过这种钙信号来实现 为了生存。钙调神经磷酸酶的一个基本功能是激活 Crz1p转录因子，尽管其关键靶点是 钙调神经磷酸酶/CRZ1依赖的转录调控尚未得到 已确认身份。钙调神经磷酸酶还具有额外的、尚未定性的 信息素处理细胞的基本功能。钙调素蛋白激酶在细胞周期调控中的作用 信息素的反应还不清楚。拟议中的实验将 钙调神经磷酸酶和钙调素蛋白在暴露于细胞中的作用 信息素。我们将：1)描述信息素诱导的机制 钙调神经磷酸酶和Crz1p介导的转录。Crz1p的模式 钙调神经磷酸酶的调节将被阐明，并将有更多的基因产品 参与钙调神经磷酸酶/CRZ1反应通路的将是 已确认身份。2)确定修改以下要求的基因产品 信息素孵育过程中钙离子依赖的信号转导。组件 钙调神经磷酸酶和钙调素依赖的反应都将被研究。3) 确定信息素诱导的钙调神经磷酸酶依赖转录的靶点 调节并确定这些基因对细胞活力的贡献。 微阵列技术将被用于检测所有6000个酵母基因 钙调神经磷酸酶依赖的基因表达变化。4)确定底物 通过鉴定与钙调神经磷酸酶物理相互作用的基因产物 钙调神经磷酸酶在体内和通过在体外测试特定的候选蛋白 钙调神经磷酸酶依赖的去磷酸化。

DESCRIPTION: 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.