Role of Ca2+/Calcineurin Signaling in S.Cerevisiae

描述（由申请人提供）：在哺乳动物中，钙调神经磷酸酶（Ca2 +/钙调蛋白依赖性蛋白磷酸酶）调节免疫细胞活性，促进心脏和血管发育，介导心肌对应激的反应，并调节大脑的学习和记忆。钙调磷酸酶抑制剂FK 506和环孢菌素A在临床上用作免疫抑制剂，并且在动物模型中减少心脏肥大。作为Ca2 +/钙调蛋白依赖性信号传导的关键效应子，钙调神经磷酸酶功能的详细分析具有影响人类健康和发育的许多方面的潜力。in s.在酿酒酵母中，钙调神经磷酸酶促进环境胁迫期间的存活和对细胞壁损伤的响应。钙调神经磷酸酶的主要作用是去磷酸化和激活Crz1p转录因子，其机制类似于钙调神经磷酸酶对哺乳动物转录因子NFAT的调节。在环境胁迫期间促进酵母存活的另外的钙调神经磷酸酶介导的事件不太好表征，并且是本申请的焦点。本研究旨在全面了解钙调神经磷酸酶依赖性信号通路的功能和组成。我们使用S来解决这些问题。酿酒酵母，因为这个简单的真核生物提供了许多实验优势，但努力建立适用于所有细胞中钙调神经磷酸酶依赖性信号传导的一般原则。在此之前，我们利用了钙调磷酸酶信号传导的一个特殊特征，即钙调磷酸酶需要通过与去磷酸化残基不同的对接位点直接与其底物相互作用，以确定钙调磷酸酶介导的信号传导途径的几个新组分，包括3种新底物：Slm1p，Slm2p和Hph1p。在这里，我们建议进一步表征钙调神经磷酸酶与其底物相互作用的机制，这是进化保守的。我们还将应用遗传学，基因组学和蛋白质组学的方法来鉴定钙调磷酸酶的其他底物和调节剂。具体来说，我们将1）表征底物中的钙调神经磷酸酶对接位点，并检查钙调神经磷酸酶-底物亲和力对信号传导的影响。2)检查钙调磷酸酶中特定点突变对底物相互作用的影响。3)确定钙调神经磷酸酶相互作用蛋白在钙调神经磷酸酶信号通路中的作用。4)使用新的蛋白质组学和基因组学筛选方法鉴定其他钙调磷酸酶底物。

DESCRIPTION (provided by applicant): In mammals, calcineurin, the Ca2+/calmodulin dependent protein phosphatase, regulates immune cell activity, promotes heart and blood vessel development, mediates cardiac muscle response to stress, and modulates learning and memory in the brain. Calcineurin inhibitors, FK506 and cyclosporin A, are used clinically as immunosupressants, and, in animal models, reduce cardiac hypertrophy. As a key effector of Ca2+/calmodulin dependent signaling, detailed analysis of calcineurin function has the potential to impact many aspects of human health and development. In S. cerevisiae, calcineurin promotes survival during environmental stress, and in response to cell wall damage. A major role of calcineurin is to dephosphorylate and activate the Crz1p transcription factor, using mechanism analogous to calcineurin regulation of the mammalian transcription factor, NFAT. Additional calcineurin-mediated events that promote yeast survival during environmental stress are less well characterized, and are the focus of this application. This research aims to identify comprehensively the functions and components of calcineurin-dependent signaling pathways. We address these questions using S. cerevisiae, because of many experimental advantages offered by this simple eukaryotic organism, but strive to establish general principles that apply to calcineurin-dependent signaling in all cells. Previously, we exploited a particular feature of calcineurin signaling, i.e. the requirement for calcineurin to interact directly with its substrates via a docking site that is distinct from residues that are dephosphorylated, to identify several new components of calcineurin-mediated signaling pathways including 3 novel substrates: Slm1p, Slm2p and Hph1p. Here we propose to characterize further the mechanism by which calcineurin interacts with its substrates, which is evolutionary conserved. We will also apply genetic, genomic, and proteomic approaches to the identification of additional substrates and regulators of calcineurin. Specifically we will 1) Characterize calcineurin-docking sites in substrates and examine the impact of calcineurin-substrate affinity on signaling. 2) Examine the effect of specific point mutations in calcineurin on substrate interaction. 3) Identify the role of calcineurin interacting proteins in calcineurin signaling pathways. 4) Identify additional calcineurin substrates using novel proteomic and genomic screening methods.