Role of Ca2+/Calcineurin Signaling in S. Cerevisiae

说明(申请人提供)：钙调神经磷酸酶(CN)是一种高度保守的丝氨酸/苏氨酸蛋白磷酸酶，受钙离子和钙调蛋白调节，从而在体内介导钙依赖的磷酸化变化。在人类中，CN调节免疫细胞功能，促进肌肉、血管和胰腺组织的发育，并调节大脑中的学习和记忆。CN抑制剂FK506和环孢菌素A是临床上广泛使用的免疫抑制剂，CN依赖信号的干扰与唐氏综合征、心脏病、精神分裂症、癌症和糖尿病等许多病理生理疾病有关。CN底物的鉴定是理解和调节这一广泛的生理活动的关键。这项建议试图确定酵母中CN的功能和底物。先前的研究表明，CN促进了酵母细胞在环境胁迫下的存活，并确定了这一反应所需的几种底物。然而，还指出了CN的其他功能，这些功能的底物尚未确定。现已提出了酵母中CN底物的全球鉴定方法。这种简单的真核生物提供了许多实验优势；这些研究将为所有细胞相关的CN依赖信号提供洞察力，并将为在其他生物中全面鉴定磷酸酶底物铺平道路。拟议工作的具体目标是：1)使用两种互补的蛋白质组学方法全面鉴定CN底物。首先，被CN去磷酸化的蛋白质将通过一种使用磷酸化蛋白质微阵列的新的体外实验来鉴定。其次，富含在CN缺陷细胞提取液中的磷酸肽将通过无标记的定量质谱学进行鉴定。对所产生的底物集合的功能表征将扩大我们对CN调节活动的理解。对这些底物的生化和生物信息学分析将进一步确定与CN底物识别有关的序列。2)通过确定在体内改变其活性和与底物相互作用的突变来确定CN的关键区域。这些突变体将为研究许多生物体的CN功能和底物特异性提供新的工具。3)确定CN相互作用蛋白在CN信号通路中的作用。CN底物的特性是确定其生理功能的关键。将研究CN底物SLM1和SLM2在热应激期间依赖TOR激活Ypk1/2激酶中的作用，并研究CN与调节进入细胞周期的RNA结合蛋白Whi3的相互作用。初步观察表明，CN和Whi3在酵母应激颗粒中的作用，它是对葡萄糖饥饿的反应，含有与启动因子复杂的非翻译mRNAs。

DESCRIPTION (provided by applicant): Calcineurin (CN) is a highly conserved ser/thr protein phosphatase that is regulated by Ca2+ and calmodulin, and thus mediates Ca2+-dependent changes in phosphorylation in vivo. In humans, CN regulates immune cell function, promotes development of muscle, vascular and pancreatic tissues, and modulates learning and memory in the brain. CN inhibitors, FK506 and cyclosporin A, are in wide clinical use as immunosupressants, and perturbation of CN-dependent signaling is associated with many pathophyisological conditions including Down's syndrome, heart disease, schizophrenia, cancer, and diabetes. Identification of CN substrates is key to understanding and modulating this wide range of physiological activities. This proposal seeks to identify the functions and substrates of CN in yeast. Previous studies demonstrated that CN promotes yeast cell survival during environmental stress, and identified several substrates required for this response. However, additional functions of CN, for which substrates have not yet been identified, are also indicated. Now approaches for global identification of CN substrates in yeast are proposed. This simple eukaryote offers many experimental advantages; these studies will provide insights into CN-dependent signaling that are relevant to all cells and will pave the way for global identification of phosphatase substrates in other organisms. The specific aims of the proposed work are to: 1) Identify CN substrates comprehensively, using two complementary, proteomic approaches. First, proteins dephosphorylated by CN will be identified with a novel in vitro assay that uses phosphorylated protein microarrays. Second, phosphopeptides that are enriched in extracts of CN-deficient cells will be identified by label-free, quantitative mass spectrometry. Functional characterization of the resulting collection of substrates will expand our understanding of CN regulatory activities. Biochemical and bioinformatic analyses of these substrates will further define sequences involved in CN substrate recognition. 2) Identify critical regions of CN by identifying mutations that alter its activity and interactions with substrates in vivo. These mutants will provide new tools for investigating CN function and substrate specificity in many organisms. 3) Identify the role of CN-interacting proteins in CN signaling pathways. Characterization of CN substrates is essential for defining its physiological functions. The role of CN substrates, Slm1 and Slm2, in TOR-dependent activation of Ypk1/2 kinases during heat stress, will be investigated, and the interaction of CN with Whi3, an RNA binding protein that regulates entry into the cell cycle will be examined. Preliminary observations suggest roles for CN and Whi3 in yeast stress granules, which form in response to glucose starvation and contain non-translating mRNAs complexed with initiation factors.