Optogenetic tools for the dissection of oncogenic signaling mediated by kinases

蛋白激酶信号的异常是肿瘤发生的驱动力之一。从 生理致癌过程通常由时间和空间调节的变化触发 激活剂。对这些事件的剖析受到当前工具功能的限制。仍然很难做到 在活细胞中通过精确的时间和定位来操纵特定的激酶的活性。克服洋流 局限性我们建议开发一种新的广泛适用的光遗传工具，使我们能够 利用光来激活活细胞中的激酶活性。为了在时间和空间上控制激酶的活性，我们将设计一种新的 基于真菌感光体Vivid的构象变化的光敏变构开关 用蓝光照明。将工程设计的开关插入到A的催化区域内的特定位置 激酶将使我们能够实现光介导的活性调节。这将使严格控制、 在活细胞中对特定的激酶进行可逆的和局部的调节。为了展示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，Ab1和PKA。进一步扩大应用范围 在这一策略中，我们建议开发光调控的PFKFB3，一种结构不同的激酶，它 使6-磷酸果糖磷酸化以促进癌细胞中的葡萄糖代谢。本实验中使用的试剂 方法将被遗传编码，从而能够在许多系统中随时应用。使用光中介 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 在致癌转化过程中刺激葡萄糖代谢。我们将采用光控PFKFB3来 确定其在细胞不同亚细胞间糖酵解的局部调节中的作用及其作用 关于致癌形态动态变化和细胞周期的研究。

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.