OPTICAL METHODS FOR MONITORING PROTEIN PHOSPHORYLATION

蛋白质磷酸化是几乎所有信号通路中的关键事件，并介导这些通路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，特别是癌症有关。然而，用于测量体内蛋白质磷酸化的现有工具不能同时且真实的监测许多蛋白质的磷酸化。我们的目标是建立一个系统来测量数百种蛋白质的磷酸化，在体内，在真实的时间。为此，我们正在建立一个系统，可以检测磷酸化的YFP标记的蛋白质的荧光共振能量转移到一个CFP标记的磷酸肽结合域。我们将用这个系统来研究 酵母交配途径（真核细胞信号传导途径的原型）中的磷酸化事件。在这个系统中的磷酸化的实时测量将使我们能够监测这一途径的定量反应及其下游效应物的激活。

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.