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Role of Ca2+/Calcineurin Signaling in S.Cerevisiae

Ca2/钙调磷酸酶信号在酿酒酵母中的作用

基本信息

批准号：
7930987
负责人：
Martha S. Cyert
金额：
$ 11.31万
依托单位：
STANFORD UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-09-30 至 2010-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7930987
关键词：
Address Affinity Alleles Animal Model Applied Genetics Biochemical Biological Assay Blood Vessels Brain Calcineurin Calcineurin inhibitor Calmodulin Catalytic Domain Cell Wall Cell physiology Cell surface Cells Cyclosporine Development Docking Event FK506 Family Funding Gene Expression Regulation Genetic Genomics Growth Health Heart Heart Hypertrophy Human Hybrids Immune Integral Membrane Protein Learning Mammals Mass Spectrum Analysis Mediating Membrane Memory Methods Mutation Myocardium Organism Point Mutation Property Protein Microchips Protein phosphatase Proteins Proteomics Regulation Research Research Proposals Role Saccharomyces cerevisiae Screening procedure Signal Pathway Signal Transduction Site Stress Substrate Interaction Testing Ubiquitin Variant Yeasts dosage in vivo mutant novel protein complex research study response scaffold success transcription factor

项目摘要

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.

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，介导心肌对压力的反应，并调节大脑的学习和记忆。钙调神经磷酸酶抑制剂 FK506 和环孢菌素 A 在临床上用作免疫抑制剂，并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器，对钙调神经磷酸酶功能的详细分析有可能影响人类健康和发育的许多方面。在酿酒酵母中，钙调神经磷酸酶可促进环境应激期间的存活以及对细胞壁损伤的反应。钙调磷酸酶的主要作用是利用类似于哺乳动物转录因子 NFAT 的钙调磷酸酶调节机制来去磷酸化并激活 Crz1p 转录因子。在环境应激期间促进酵母存活的其他钙调神经磷酸酶介导的事件尚未得到很好的表征，并且是本应用的重点。本研究旨在全面鉴定钙依赖磷酸酶依赖性信号通路的功能和成分。我们使用酿酒酵母来解决这些问题，因为这种简单的真核生物提供了许多实验优势，但我们努力建立适用于所有细胞中钙调磷酸酶依赖性信号传导的一般原则。此前，我们利用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通路的几个新组件，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，该机制是进化保守的。我们还将应用遗传、基因组和蛋白质组学方法来鉴定钙调神经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) 确定钙调磷酸酶相互作用蛋白在钙调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筛选方法鉴定其他钙调神经磷酸酶底物。