OPTICAL METHODS FOR MONITORING PROTEIN PHOSPHORYLATION

蛋白质磷酸化是几乎所有信号通路中的一个关键事件，并介导了这些通路中发生的许多信息传递和处理。磷酸化的错误调控与许多疾病有关，特别是在癌症中。然而，现有的测量体内蛋白质磷酸化的工具无法同时和实时地监测许多蛋白质的磷酸化。我们的目标是建立一个系统，实时测量体内数百种蛋白质的磷酸化。为此，我们正在建立一个系统，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标记的蛋白质的磷酸化。我们将利用这个系统来研究 酵母交配途径中的磷酸化事件，这是一个典型的真核信号传递途径。实时测量该系统中的磷酸化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.