OPTICAL METHODS FOR MONITORING PROTEIN PHOSPHORYLATION

监测蛋白质磷酸化的光学方法

• 批准号: 7557306
• 负责人: KURT THORN
• 金额: $ 16.72万
• 依托单位: HARVARD UNIVERSITY
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7557306
• 关键词: biological signal transduction; bioluminescence; biotechnology; eukaryote; fluorescence; fluorescence microscopy; fluorescence resonance energy transfer; fungal proteins; image processing; method development; mitogen activated protein kinase; optics; pheromone; phosphopeptides; phosphorylation; polymerase chain reaction; protein binding; protein protein interaction; proteomics; time resolved data; yeasts

Protein phosphorylation is a key event in almost all signaling pathways, and mediates much of the information transfer and processing that occur in these pathways. Misregulation of phosphorylation has been implicated in a number of diseases, particularly in cancer. However, existing tools for measuring protein phosphorylation in vivo are unable to monitor phosphorylation of many proteins simultaneously and in real time. We aim to build a system to measure the phosphorylation of hundreds of proteins, in vivo, in real time. To do so, we are building a system that can detect phosphorylation of a YFP tagged protein by fluorescence resonance energy transfer to a CFP-tagged phosphopeptide binding domain. We will use this system to study phosphorylation events in the yeast mating pathway, a prototype eukaryotic signaling pathway. Real-time measurements of phosphorylation in this system will enable us to monitor the quantitative response of this pathway and its activation of downstream effectors.

蛋白质磷酸化是几乎所有信号传导途径中的关键事件，并介导这些途径中发生的大部分信息传递和处理。磷酸化的失调与许多疾病有关，特别是癌症。然而，现有的体内蛋白质磷酸化测量工具无法同时实时监测许多蛋白质的磷酸化。我们的目标是建立一个系统来实时测量体内数百种蛋白质的磷酸化。为此，我们正在构建一个系统，该系统可以通过荧光共振能量转移到 CFP 标记的磷酸肽结合域来检测 YFP 标记蛋白的磷酸化。我们将使用这个系统来研究 酵母交配途径中的磷酸化事件，这是一种原型真核信号传导途径。该系统中磷酸化的实时测量将使我们能够监测该途径的定量响应及其下游效应器的激活。