Role of Ca2+/Calcineurin Signaling in S. cerevisiae

描述(申请人提供)：钙/钙调蛋白依赖的蛋白磷酸酶，钙调神经磷酸酶，是钙依赖的信号转导的关键成分。在哺乳动物中，钙调神经磷酸酶调节免疫细胞功能，促进心脏和血管发育，允许心脏细胞对压力做出反应，并调节学习和记忆。在体外，钙调神经磷酸酶的催化活性只需要添加钙离子和钙调素。然而，在体内，钙调神经磷酸酶与其底物和调节蛋白的相互作用对其活性有深远的影响。在萌芽酵母中，钙调神经磷酸酶允许细胞适应几种环境压力，包括暴露于离子(Na+，Mn2+，OH-)，细胞壁破坏，以及与交配因子的长时间孵育。在这些条件下，钙调神经磷酸酶使Crz1p转录因子去磷酸化，并导致其从胞浆快速移位到细胞核，在那里它激活了基因表达。钙调神经磷酸酶依赖的反应在体内是如何调节的，它们如何与其他信号机制协调，以实现对特定环境条件的适当生理反应？为了解决这些问题，我们将研究钙调神经磷酸酶和/或Crz1功能的调节机制，研究其他信号通路与Crz1/Calcineurin的相互作用，并表征钙调神经磷酸酶和Crz1p在信号传递过程中与其他蛋白质的联系。具体地说，我们将1)识别使Crz1p磷酸化的蛋白激酶并确定这些蛋白激酶在Crz1p调控中的作用；2)识别和突变Crz1p磷酸化位点并确定它们在调节Crz1p定位和活性中的作用；3)研究酵母对氧化应激反应中的关键蛋白Snu7p影响儿茶酚/Crz1p功能以调节这些信号通路之间的串扰的机制；以及4)表征含钙调神经磷酸酶和Crz1p的蛋白质复合体，以确定特定的蛋白质-蛋白质相互作用在调节体内钙调神经磷酸酶和Crz1依赖的信号通路中的作用。本研究的主要目的是阐明钙/钙调神经磷酸酶依赖的信号转导的机制和功能。我们使用酿酒酵母来解决这些问题，因为这种微生物提供了许多实验优势。然而，我们相信，我们的发现将建立适用于所有细胞中钙调神经磷酸酶依赖信号的一般原则。

DESCRIPTION (provided by applicant): The Ca2+/calmodulin dependent protein phosphatase, calcineurin, is a critical component of Ca2+-dependent signal transduction. In mammals, calcineurin regulates immune cell function, promotes heart and blood vessel development, allows heart cells to respond to stress, and modulates learning and memory. In vitro, calcineurin catalytic activity requires only the addition of Ca2+ and calmodulin. However, in vivo the interaction of calcineurin with its substrates and with regulatory proteins has a profound impact on its activity. In budding yeast, calcineurin allows cells to adapt to several environmental stresses, including exposure to ions (Na+, Mn2+, OH-), cell wall damage, and prolonged incubation with mating factor. Under these conditions, calcineurin dephosphorylates the Crz1p transcription factor, and causes its rapid translocation from the cytosol to the nucleus where it activates gene expression. How are calcineurin-dependent responses modulated in vivo, and how are they coordinated with other signaling mechanisms to achieve the proper physiological response to a particular environmental condition? To address these questions, we will examine mechanisms that regulate calcineurin and/or Crz1 function, study the interactions of other signaling pathways with Crz1/calcineurin, and characterize the association of calcineurin and Crz1 p with other proteins during signaling. Specifically, we will 1) Identify protein kinases that phosphorylate Crz1 p and determine the role of these kinases in Crz1p regulation, 2) Identify and mutagenize Crz1p phosphorylation sites and determine their roles in regulating Crz1p localization and activity, 3) Examine the mechanism by which Skn7p, a key protein in the yeast response to oxidative stress, influences catcineurin/Crz1p function to mediate cross-talk between these signaling pathways, and 4) Characterize calcineurin- and Crz1 p-containing protein complexes to define the role of specific protein-protein interactions in modulating calcineurin and Crz1 p-dependent signaling in vivo. The broad goal of this research is to elucidate the mechanisms and functions of Ca2+/calcineurin dependent signal transduction. We address these questions using S. cerevisiae, because of the many experimental advantages offered by this organism. However, we believe our findings will establish general principles that will apply to calcineurin-dependent signaling in all cells.