Orthogonally Gated Kinases and Phosphatases

正交门控激酶和磷酸酶

• 批准号: 9242030
• 负责人: INDRANEEL GHOSH
• 金额: $ 30.2万
• 依托单位: UNIVERSITY OF ARIZONA
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-08-01 至 2019-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9242030
• 关键词: Abscisic Acid; Address; Alpha Cell; Area; Biology; Catalytic Domain; Cell Adhesion; Cell Death; Cell division; Cells; Cellular biology; Chemicals; Chin; Collaborations; Communities; Complement; Complex; Control Groups; Cues; Development; Dimerization; Enzyme Inhibitor Drugs; Enzymes; Event; Family; Genetic; Hand; Heterodimerization; Human; In Vitro; Individual; Kinetics; Knowledge; Length; Letters; Ligands; Light; Location; Malignant Neoplasms; Methods; Modeling; Modification; Molecular; Other Genetics; Pathway interactions; Permeability; Pharmacology; Phosphoric Monoester Hydrolases; Phosphorylation; Phosphotransferases; Post-Translational Protein Processing; Protein Dephosphorylation; Protein Kinase; Protein Tyrosine Phosphatase; Protein phosphatase; Proteins; Proteome; Reporting; Research; Role; Scheme; Signal Pathway; Signal Transduction; Small Interfering RNA; Specificity; Substrate Specificity; System; Tertiary Protein Structure; Testing; Trimethoprim; Up-Regulation; Validation; Vitronectin; Work; base; cell motility; chemical genetics; design; dimer; experimental study; genetic approach; gibberellic acid; human disease; inorganic phosphate; insight; interest; knock-down; light gated; migration; mutation screening; novel therapeutics; optogenetics; portability; public health relevance; small molecule; small molecule inhibitor; temporal measurement; tool; tumor progression

 DESCRIPTION (provided by applicant): The temporal and location specific activity of numerous proteins is controlled by myriad posttranslational modifications. Of these exquisite chemical modifications, phosphorylation and dephosphorylation, catalyzed by protein kinases and phosphatases, respectively, regulate a diversity of cellular events from cell division to cell death. The >500 human protein kinases and 147 protein phosphatases in tandem respond to a variety of both intra- and extra-cellular environmental cues and it has been estimated that at least a third of the proteome is capable of being phosphorylated. Not surprisingly, the deregulation of phosphorylation leads to a variety of human diseases including cancer. Despite the importance of phosphorylation driven signaling, decrypting the role of a specific kinase or phosphatase remains enormously challenging. Currently there are almost no uniquely selective small molecules for pharmacological perturbation of native kinases or phosphatases. Prevailing siRNA and other genetic knockdown methods, which provide insight regarding the function of a specific enzyme, cannot afford temporal control and mechanistic details are obscured by compensatory cellular mechanisms. A few elegant and powerful methods have been developed to provide temporal turn-off or turn-on control over a single kinase to study cell biology. However, there are no methods that allow for controlling multiple user-defined kinases and phosphatases that are often implicated in a cell signaling pathway. We hypothesize that this knowledge gap can be addressed by developing an approach that allows for orthogonal small molecule control over split-kinases (Aim 1) as well as split-phosphatases (Aim 2), which we have recently designed. The three proposed aims are designed to provide validated methods with interchangeable parts (Aim 3) for studying signaling. We propose to demonstrate generality, quantify kinetics and substrate specificity, and provide validate methods for studying temporal aspects of specific kinases (Aim 1) and phosphatases (Aim 2) in live cells. These methods will perhaps be particularly relevant for studying the dynamics of cell motility and cell adhesion, which are of fundamental interest as well as central to understanding migration and metastatic pathways in cancer with collaborators.

 描述（由申请人提供）：许多蛋白质的时间和位置特异性活性受无数翻译后修饰控制。在这些精致的化学修饰中，分别由蛋白激酶和磷酸酶催化的磷酸化和去磷酸化调节从细胞分裂到细胞死亡的多种细胞事件。超过500种人类蛋白激酶和147种蛋白磷酸酶串联响应多种细胞内和细胞外环境线索，并且据估计至少三分之一的蛋白质组能够被磷酸化。毫不奇怪，磷酸化的失调导致包括癌症在内的各种人类疾病。尽管磷酸化驱动信号的重要性，解密特定激酶或磷酸酶的作用仍然具有巨大的挑战性。目前，几乎没有独特的选择性小分子用于天然激酶或磷酸酶的药理学扰动。流行的siRNA和其他基因敲除方法，提供了关于特定酶的功能的见解，不能提供时间控制和机制的细节是模糊的补偿性细胞机制。已经开发了一些优雅而强大的方法来提供对单个激酶的时间关闭或打开控制，以研究细胞生物学。然而，没有允许控制通常涉及细胞信号传导途径的多种用户定义的激酶和磷酸酶的方法。我们假设，这种知识差距可以通过开发一种方法来解决，该方法允许正交小分子控制分裂激酶（目标1）以及分裂磷酸酶（目标2），我们最近设计的。提出的三个目标的目的是提供有效的方法与可互换的部分（目标3）研究信令。我们建议证明的一般性，定量动力学和底物特异性，并提供验证方法，用于研究活细胞中的特定激酶（目的1）和磷酸酶（目的2）的时间方面。这些方法可能与研究细胞运动和细胞粘附的动力学特别相关，这是根本的兴趣，也是与合作者一起理解癌症迁移和转移途径的核心。