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

描述（由申请人提供）：激酶介导的蛋白质磷酸化广泛调节正常和疾病状态下的细胞反应。细胞信号网络中的激酶扮演“微处理器”的角色，将不同的刺激配对到不同的信号输出。其细胞功能的多功能性和特异性源于多种分子内和分子间蛋白质相互作用的协调。然而，目前探测激酶的方法将它们视为简单的“开-关”开关，并没有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/或控制这些蛋白质相互作用，以提供任何激酶的细胞功能的详细机制理解。此外，激酶工具箱克服了现有技术的局限性，可以识别小分子/治疗方法，区分密切相关的激酶。我们开发并测试了局灶黏附激酶（FAK）和蛋白激酶C （PKC）的激酶工具箱。我们建议追求三个互补和平行的目标，以实现这项新技术的变革潜力，同时分散风险。我们的第一个目标是使用PKC工具箱来绘制两个密切相关的PKC亚型在心脏肥大和糖尿病视网膜病变细胞模型中的时空调节。除了概念验证之外，PKC工具箱已经为我们提供了广泛适用于AGC激酶超家族（60个成员）的新概念见解。我们的第二个目标是利用这些见解来了解五种密切相关的AGC激酶（PKA, Akt/PKB， PKC， PDK1和S6K1）调控的异同。我们的第三个目标是开展基于激酶工具箱的三种新方法的试点研究，以设计AGC激酶的异构体特异性抑制剂。综上所述，拟议的研究是我们设计和表征AGC激酶高特异性抑制剂的长期目标的重要的第一步，AGC激酶抑制剂是糖尿病、心力衰竭和癌症等疾病状态的重要药物靶点。成功完成概述的研究将改变我们对激酶的一般理解，同时为研究人员提供研究其细胞功能的新工具和路线图。

DESCRIPTION (provided by applicant): 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 an 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.