Regulation of Calcineurin

描述（由申请人提供）：钙调神经磷酸酶是一种丝氨酸/苏氨酸磷酸酶， 主要调节几个信号转导级联，包括那些 参与免疫细胞活化、骨骼肌发育、记忆 形成和心肌肥大。这个项目的假设是 决定钙调磷酸酶底物的蛋白质间相互作用 特异性和在体外使钙调神经磷酸酶失活的氧化应激， 与Ca 2+和钙调蛋白结合以确保适当的钙调神经磷酸酶功能 在完整的细胞中。虽然钙调神经磷酸酶需要Ca 2+和钙调蛋白 活动，其他监管机制是必不可少的适当方向， 酶，并作为制动器的活动。这些相对重要性 机制是未知的，没有什么是已知的相互作用， 整合监管机制。该项目的目标是 确定钙调磷酸酶靶向蛋白如何指导酶，以及 程度氧化剂在完整细胞中起“关闭开关”的作用。为此 用于表达钙调神经磷酸酶蛋白的构建体 将制备钙调素指示蛋白（CN/CaM-Ind）。这些CN/CaM-Ind将 在具有稳定整合的NFAT报告基因的细胞中使用， 钙调磷酸酶活性，以确定细胞定位和钙调蛋白 在各种条件下与钙调磷酸酶结合。该项目的目标是：1） CN/CaM-Ind系统的制作和测试。2)确定CN-A的作用 酶活化、CN-A/CN-B相互作用和NFAT底物中的氨基末端 约束力3)为了确定钙调磷酸酶靶向是否是Ca 2+依赖性的， VP/IT钙调磷酸酶结合结构域指导钙调磷酸酶活性朝向特异性 无论是距离还是方向。4)为了确定是否钙调神经磷酸酶在 完整细胞被氧化剂可逆地抑制，如果靶向蛋白 结合或钙调蛋白依赖性活化增加对氧化剂的敏感性。 由于钙调神经磷酸酶在NFAT信号传导和靶向中起着关键作用， 是信号级联中钙调神经磷酸酶所必需的， 实验将使我们能够设计底物特异性钙调磷酸酶抑制剂， 这将是上级免疫抑制剂。此外，贡献 钙调神经磷酸酶活性的生理和病理反应， 氧化应激将被定义和干预策略，以尽量减少 会发现氧化剂对健康的不良影响。

DESCRIPTION (provided by applicant): Calcineurin is a Ser/Thr phosphatase and a primary regulator for several signal transduction cascades, including those involved in immune cell activation, skeletal muscle development, memory formation, and cardiac muscle hypertrophy. The hypothesis for this project is that protein-protein interactions, which determine calcineurin substrate specificity, and oxidative stress, which inactivates calcineurin in vitro, act in conjunction with Ca2+ and calmodulin to ensure proper calcineurin function in intact cells. Although Ca2+ and calmodulin are required for calcineurin activity, other regulatory mechanisms are essential for proper direction of the enzyme and to act as a brake on activity. The relative importance of these mechanisms is unknown and nothing is known about interactions among and integration of the regulatory mechanisms. The objective of this project is to determine how calcineurin targeting proteins direct the enzyme and to what extent oxidants function as an "off switch" in intact cells. To this end constructs for expression of calcineurin proteins as fusions with fluorescent calmodulin indicator proteins (CN/CaM-Ind) will be made. These CN/CaM-Ind will be used in cells with a stably integrated NFAT reporter, which measures calcineurin activity, to determine the cellular localization and calmodulin binding to calcineurin under various conditions. Aims of the project are: 1) To produce and test the CN/CaM-Ind system. 2) To determine the role of the CN-A amino terminus in enzyme activation, CN-A/CN-B interaction and NFAT substrate binding. 3) To determine if calcineurin targeting is Ca2+-dependent and if the VP/IT calcineurin binding domain directs calcineurin activity toward specific residues either by distance or direction. 4) To determine if calcineurin in intact cells is reversibly inhibited by oxidants and if targeting protein binding or calmodulin-dependent activation increase susceptibility to oxidants. Because calcineurin plays such a pivotal role in NFAT signaling and targeting is essential for calcineurin in that signaling cascade, the results of these experiments will enable us to design substrate specific calcineurin inhibitors which would be superior immunosuppressive agents. In addition, the contribution of calcineurin activity to the physiological and pathological responses to oxidative stress will be defined and interventive strategies to minimize the adverse health effects of oxidants will be found.