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

摘要 蛋白激酶介导的磷酸化广泛调节正常和疾病中的细胞反应 各州。细胞信号网络中的激酶扮演着微处理器的角色，将不同的刺激耦合到 不同的信号输出。它们细胞功能的多功能性和特异性源于 几个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，而不涉及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，它可以监测和/或控制这些蛋白质 相互作用，以提供对任何一种激酶的细胞功能的详细的机械理解。此外, 激酶工具箱克服了识别小分子/治疗药物的现有技术的局限性 它们区分了紧密相关的激酶。我们已经开发并测试了Focus的激活剂工具箱 黏附激酶(FAK)和蛋白激酶C(PKC)。我们建议实行三个相辅相成和并行的原则 目标，以实现这一新技术的变革潜力，同时分配风险。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亚型在空间和时间上的调节 心肌肥厚和糖尿病视网膜病变的细胞模型。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激酶超家族的新的概念性见解 (60名成员)。我们的第二个目标是利用这些见解来理解 5种密切相关的AGC激酶(PKA、Akt/PKB、PKC、PDK1和S6K1)的调节。我们的第三个目标是 基于激酶工具箱，对三种新方法进行初步研究，以设计特定的异构体 AGC激酶的抑制剂。综上所述，拟议的研究是迈向我们长期- 设计和表征重要药物--AGC激酶的高特异性抑制剂的学期目标 糖尿病、心力衰竭和癌症等疾病状态的靶点。圆满完成概述 研究将改变我们对激酶的总体理解，同时为研究人员提供新的工具和 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.