Optogenetic tools for the dissection of oncogenic signaling mediated by kinases

用于解析激酶介导的致癌信号的光遗传学工具

基本信息

批准号：
9891973
负责人：
ANDREI V KARGINOV
金额：
$ 18.29万
依托单位：
UNIVERSITY OF ILLINOIS AT CHICAGO
依托单位国家：
美国
项目类别：
财政年份：
2018
资助国家：
美国
起止时间：
2018-04-01 至 2022-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9891973
关键词：
6-Phosphofructo-2-kinase Allosteric Regulation Biological Models Biological Process Cancer Biology Catalytic Domain Cell Compartmentation Cell Cycle Cell physiology Cells Closure by clamp Cyclic AMP-Dependent Protein Kinases Development Dissection Engineering Ensure Enzymes Event Glycolysis Guidelines Homodimerization Human Individual Light Light Cell Lighting Location Maintenance Malignant Neoplasms Masks Mediating Metabolic Methods Molecular Conformation Oncogenic Organ Pathologic Pathway interactions Pharmacology Phosphorylation Phosphotransferases Photoreceptors Physiological Physiological Processes Play Positioning Attribute Process Property Protein Engineering Protein Kinase Proteins Reagent Regulation Role Scientist Signal Pathway Signal Transduction Signaling Protein Site Specificity Structure System Testing Time Up-Regulation Warburg Effect base cancer cell cell motility cell transformation driving force fructose-6-phosphate genetic manipulation glucose metabolism inhibitor/antagonist novel novel strategies novel therapeutics optogenetics protein metabolite spatiotemporal src-Family Kinases tool tumor tumorigenesis

项目摘要

Aberrant signaling by protein kinases is one of the driving forces of tumorigenesis. Transition from physiological to oncogenic processes is often triggered by changes in temporal and spatial regulation of kinases. Dissection of these events is limited by the capabilities of current tools. It remains difficult to manipulate activity of a specific kinase with precise timing and localization in living cells. To overcome current limitations we propose to develop a novel broadly applicable optogenetic tool that will allow us to regulate kinase activity in living cells using light. To control kinase activity in time and space we will engineer a novel light-sensitive allosteric switch based on fungal photoreceptor Vivid that changes conformation upon illumination with blue light. Insertion of the engineered switch at a specific site within the catalytic domain of a kinase will allow us to achieve light-mediated regulation of the activity. This will enable tightly controlled, reversible and localized regulation of a specific kinase in living cells. To demonstrate broad applicability of this tool we will use it for regulation of oncogenic protein kinases Src, Abl and PKA. To further expand application of this strategy we propose to develop light regulated PFKFB3, a structurally different kinase that phosphorylates fructose 6-phosphate to promote glucose metabolism in cancer cells. The reagents used in this method will be genetically encoded enabling ready application in many systems. Using light-mediated regulation of tyrosine kinase Src we will determine its novel role in regulation of signaling pathways that stimulate glucose metabolism during oncogenic transformation. We will employ light-controlled PFKFB3 to identify its role in localized regulation of glycolysis in different subcellular compartments of the cell and its effect on oncogenic morphodynamic changes and cell cycle.

蛋白激酶的异常信号传导是肿瘤发生的驱动力之一。从生理学到致癌过程通常是由时间和空间调节的变化引起的激酶。这些事件的解剖受到当前工具的功能的限制。仍然很难操纵特定激酶的活性具有精确的时机和活细胞中的定位。克服电流我们建议开发一种新型的广泛适用的光遗传学工具，这将使我们能够调节活细胞中的激酶活性使用光。为了控制时间和空间中的激酶活性基于真菌感光体生动的光敏变构开关，它改变了构象用蓝光照明。插入工程开关在特定位置的催化域内激酶将使我们能够实现活性的光介导的调节。这将使受到严格控制，活细胞中特定激酶的可逆和局部调节。证明这一点的广泛适用性工具我们将使用它来调节致癌蛋白激酶SRC，ABL和PKA。进一步扩展申请在这种策略中，我们建议开发光调节的PFKFB3，这是一种结构上不同的激酶磷酸化6-磷酸果糖以促进癌细胞中的葡萄糖代谢。其中使用的试剂方法将是基因编码的，在许多系统中启用了现成的应用。使用光介导的调节酪氨酸激酶SRC的调节我们将确定其在调节信号通路中的新作用在致癌过程中刺激葡萄糖代谢。我们将使用光控制的PFKFB3到确定其在细胞不同亚细胞室中糖酵解局部调节中的作用及其作用在致癌形态动力学变化和细胞周期上。