Optogenetic tools for the dissection of oncogenic signaling mediated by kinases

蛋白激酶的异常信号传导是肿瘤发生的驱动力之一。过渡 从生理到致癌过程的变化通常是由肿瘤的时间和空间调节的变化触发的。 激酶。对这些事件的剖析受到当前工具的能力的限制。目前仍难以 在活细胞中以精确的时间和定位操纵特定激酶的活性。克服当前 限制，我们建议开发一种新的广泛适用的光遗传学工具，使我们能够调节 活细胞中的激酶活性。为了在时间和空间上控制激酶活性，我们将设计一种新的 基于真菌感光体Vivid的光敏变构开关， 用蓝光照明。将工程化的开关插入到多核苷酸的催化结构域内的特定位点， 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， 活细胞中特定激酶的可逆和局部调节。为了证明这一点的广泛适用性， 我们将用它来调节致癌蛋白激酶Src、Abl和PKA。为了进一步扩大应用 我们建议开发光调节的PFKFB 3，一种结构不同的激酶， 磷酸化果糖6-磷酸以促进癌细胞中的葡萄糖代谢。本实验中使用的试剂 该方法将被遗传编码，使得能够在许多系统中准备好应用。利用光介导的 调节酪氨酸激酶Src，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PFKFB 3， 确定其在细胞不同亚细胞区室糖酵解的局部调节中的作用及其影响 致癌形态动力学变化和细胞周期。

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.