Design, Synthesis, and Application of a Real-Time MAPK Activity Sensor

描述(由申请人提供)：本提案详细介绍了选择性实时丝裂原激活蛋白激酶(MAPK)活性探针的设计、合成和应用。磷酸化在几乎所有细胞过程的激活和失活中起着核心作用。特别是，MAPK信号级联通过激活转录因子来响应细胞外刺激，从而改变细胞的可观察表型。最近的工作表明，使用螯合增强的荧光可以产生基于合成肽的磷酸化传感器，当磷酸化时，其荧光增加2-10倍。这些探针的设计是模块化的，由一个可互换的激酶识别基序、一个磷酸化位点和一个非天然氨基酸组成。这些探针的模块化性质允许它们延伸到任何激酶，只要使用了适当的识别元件。我们的具体目标如下：1)设计、合成和评价一种选择性实时MAPK活性传感器；2)将该传感器送入活细胞。因此，这项提议将提供一套完整的模块化合成的激酶活性探针，用于活细胞的实时活性实验。公共卫生相关性丝裂原活化蛋白激酶(MAPK)途径可以刺激从细胞分裂到细胞死亡的一系列不同的细胞反应；因此，即使该途径调控的细微变化也可能导致获得癌症等疾病状态。MAPK是这一途径的末端调节蛋白，因此引起了科学界和医学界的极大兴趣。开发一种选择性的实时MAPK活性探针将有助于评估MAPK活性对外界因素的反应时间和强度。

DESCRIPTION (provided by applicant): This proposal details the design, synthesis and application of a selective real-time Mitogen-Activated Protein Kinase (MAPK) activity probe. Phosphorylation plays a central role in the activation and deactivation of virtually all cellular processes. In particular, the MAPK signaling cascade produces a change in the observable phenotype of a cell by activating transcription factors in response to an extracellular stimulus. Recent work has shown that the use of CHelation-Enhanced Fluorescence can produce synthetic peptide-based phosphorylation sensors which undergo a 2- to 10-fold increase in fluorescence upon phosphorylation. These probes are modular in design and consist of an interchangeable kinase recognition motif, a phosphorylation site, and an unnatural amino acid. The modular nature of these probes allows for their extension to any kinase provided an appropriate recognition element is employed. Our specific aims are as follows: 1) Design, synthesis, and evaluation of a selective real-time MAPK activity sensor and 2) Delivery of the sensor into living cells. Thus this proposal will provide a fully modular synthetic set of kinase activity probes for use in real-time activity experiments in living cells. PUBLIC HEALTH RELEVANCE The Mitogen-Activated Protein Kinase (MAPK) pathway can stimulate a diverse set of cellular responses ranging from cell division to cell death; therefore even subtle changes in the regulation of this pathway can result in the acquisition of a disease state such as cancer. MAPKs are the terminal regulatory proteins in this pathway and as such are of great interest to the scientific and medical communities. The development of a selective real-time MAPK activity probe would aid in the evaluation of the duration and intensity of MAPK activity in response to external factors.