DThe kinase toolbox: Mapping the spatial and temporal regulation of cell signaling

摘要 激酶介导的蛋白质磷酸化广泛调节正常和疾病中的细胞反应 states.细胞信号网络中的激酶起着“微处理器”的作用， 不同的信号输出。它们细胞功能的多样性和特异性来自于 几种分子内和分子间的蛋白质相互作用。然而，目前探测激酶的方法 将它们视为简单的“开-关”开关，而不解决它们在细胞中复杂的空间和时间调节。 我们已经开发了一种技术，称为激酶工具箱，它监测和/或控制这些蛋白质 本发明的目的在于提供对任何激酶的细胞功能的详细机制理解。此外，本发明还提供了一种方法， 激酶工具箱克服了现有技术的局限性， 区分密切相关的激酶。我们已经开发并测试了激酶工具箱， 粘附激酶（FAK）和蛋白激酶C（PKC）。我们建议， 目标，以实现这一新技术的变革潜力，同时分散风险。我们的第一个目标 是使用PKC工具箱绘制两种密切相关的PKC亚型的时空调控， 心脏肥大和糖尿病视网膜病变的细胞模型。除了概念验证外，PKC 工具箱已经为我们提供了广泛应用于AGC激酶超家族的新概念见解 (60成员）。我们的第二个目标是利用这些见解来理解 调节五种密切相关的AGC激酶（PKA、Akt/PKB、PKC、PDK 1和S6 K1）。我们的第三个目标是 基于激酶工具箱，对三种新方法进行试点研究，以设计亚型特异性 AGC激酶抑制剂。总的来说，拟议的研究是迈向我们长期目标的重要的第一步- 设计和表征AGC激酶高特异性抑制剂的长期目标是重要的药物 糖尿病、心力衰竭和癌症等疾病状态中的靶点。成功完成概述的 研究将改变我们对激酶的理解，同时为研究人员提供新的工具， 研究它们细胞功能的路线图。

Abstract Kinase mediated phosphorylation of proteins broadly regulates cellular responses in normal and disease states. Kinases in cellular signaling networks play the role of `micro-processors' that couple different stimuli to distinct signaling outputs. The versatility and specificity of their cellular function arise from the coordination of several intra-molecular and inter-molecular protein interactions. However, current approaches to probe kinases treat them as simple `on-off' switches and do not address their complex spatial and temporal regulation in cells. We have developed a technology, termed the kinase toolbox, which monitors and/or controls these protein interactions to provide a detailed mechanistic understanding of the cellular function of any kinase. In addition, the kinase toolbox overcomes the limitations of existing techniques to identify small molecules/therapeutics that differentiate between closely related kinases. We have developed and tested kinase toolboxes for focal adhesion kinase (FAK) and protein kinase C (PKC). We propose to pursue three complementary and parallel goals in order to realize the transformative potential of this new technology, while distributing risk. Our first goal is to use the PKC toolbox to map the spatial and temporal regulation of two closely related PKC isoforms in cellular models of cardiac hypertrophy and diabetic retinopathy. In addition to proof-of-concept, the PKC toolbox has already provided us with new conceptual insights that broadly apply to the AGC kinase superfamily (60 members). Our second goal is to use these insights to understand the similarities and differences in the regulation of five closely related AGC kinases (PKA, Akt/PKB, PKC, PDK1 and S6K1). Our third goal is to conduct pilot studies of three new approaches, based on the kinase toolbox, to design isoform-specific inhibitors of AGC kinases. Taken together, the proposed research is an essential first step towards our long- term goal of designing and characterizing high-specificity inhibitors of AGC kinases, which are important drug targets in disease states such as diabetes, heart failure and cancer. Successful completion of the outlined studies will transform our understanding of kinases in general, while providing researchers with new tools and a roadmap to study their cellular function.