Role of Ca2+/Calcineurin Signaling in S.Cerevisiae

描述(申请人提供)：在哺乳动物中，钙调神经磷酸酶是一种钙/钙调素依赖的蛋白磷酸酶，调节免疫细胞活动，促进心脏和血管发育，调节心肌对应激的反应，并调节大脑中的学习和记忆。钙调神经磷酸酶抑制剂FK506和环孢素A在临床上被用作免疫抑制剂，在动物模型中，它们可以减少心肌肥厚。钙调神经磷酸酶作为钙/钙调素依赖性信号转导的关键效应器，对其功能的详细分析有可能影响人类健康和发育的多个方面。在酿酒酵母中，钙调神经磷酸酶可促进在环境胁迫和对细胞壁损伤的反应中存活。钙调神经磷酸酶的主要作用是使Crz1p转录因子去磷酸化并激活，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其他钙调神经磷酸酶介导的事件还没有得到很好的描述，这是本应用的重点。本研究旨在全面鉴定钙调神经磷酸酶依赖的信号通路的功能和组成。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神经磷酸酶依赖的信号的一般原理。以前，我们利用钙调神经磷酸酶信号的一个特殊功能，即要求钙调神经磷酸酶通过不同于去磷酸化残基的对接位置直接与底物相互作用，鉴定了钙调神经磷酸酶介导的信号通路的几个新成分，包括3个新的底物：Slm1p、Slm2p和Hph1p。在这里，我们建议进一步描述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、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。具体地说，我们将1)表征底物中钙调神经磷酸酶的对接位置，并研究钙调神经磷酸酶与底物的亲和力对信号的影响。2)研究钙调神经磷酸酶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)使用新的蛋白质组和基因组筛选方法鉴定额外的钙调神经磷酸酶底物。

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.