Orthogonally Gated Kinases and Phosphatases

正交门控激酶和磷酸酶

• 批准号: 9115644
• 负责人: INDRANEEL GHOSH
• 金额: $ 30.17万
• 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/9115644
• 关键词: Abscisic Acid; Address; Area; Biology; Catalytic Domain; Cell Adhesion; Cell Death; Cell division; Cells; Cellular biology; Chemicals; Chin; Collaborations; Communities; Complement; Complex; Control Groups; Cues; Development; Dimerization; Enzymes; Event; Family; Genetic; Hand; Health; Human; In Vitro; Individual; Kinetics; Knowledge; Length; Letters; Life; Ligands; Light; Location; Malignant Neoplasms; Methods; Modeling; Modification; Molecular; Other Genetics; Pathway interactions; Permeability; 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; Work; base; cell motility; chemical genetics; design; genetic approach; gibberellic acid; human disease; inorganic phosphate; insight; interest; knock-down; light gated; migration; mutation screening; novel therapeutics; optogenetics; research study; 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（由应用程序提供）：众多蛋白质的临时和位置特异性活动由无数的翻译后修饰控制。在这些独家化学修饰，磷酸化和去磷酸化中，分别由蛋白激酶和磷酸酶催化，调节了从细胞分裂到细胞死亡的细胞事件多样性。串联中的> 500个人类蛋白激酶和147种蛋白磷酸酶对各种细胞内环境线索均反应，并且据估计，至少有三分之一的蛋白质组能够被磷酸化。毫不奇怪，磷酸化的放松管制导致包括癌症在内的各种人类疾病。尽管磷酸化驱动信号传导的重要性，但解密特定激酶或磷酸酶的作用仍然受到巨大挑战。当前，几乎没有唯一选择性的小分子用于药物扰动的siRNA和其他遗传敲低方法，这些方法提供了有关特定酶功能的洞察力，无法提供临时控制和机械细节，并由补偿性细胞机制掩盖。已经开发了一些优雅而强大的方法，以提供对单个激酶的临时关闭或转启动控制，以研究细胞生物学。但是，没有任何方法可以控制多个用户定义的激酶和磷酸酶，这些激酶和磷酸酶通常在细胞信号传导途径中实现。我们假设可以通过开发一种方法来解决这种知识差距，该方法可以通过一种允许对分裂激酶（AIM 1）以及分裂磷酸酶（AIM 2）进行正交的小分子控制（AIM 2）。提出的三个目标旨在提供具有可互换零件（AIM 3）的经过验证的方法，以研究信号传导。我们建议证明一般性，量化动力学和底物特异性，并为研究特定激酶（AIM 1）和磷酸酶（AIM 2）（AIM 2）的临时方面提供验证方法。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，这些动态具有基本的兴趣，也是与合作者一起了解癌症中的迁移和转移途径的核心。