Dissecting the in vivo role of glycogen synthase kinase-3 beta (GSK3b) in the function of kinesin-1 using CRISPR/cas-1

尽管 25 年前就发现分子马达被磷酸化，但关于分子马达的基本问题 蛋白激酶或特定磷酸化位点的身份，以及它们如何发挥控制电机的作用 仍然没有得到答复。由于激酶级联显示出相当大的串扰并在细胞归巢中发挥多种作用 由于稳态，破译哪种激酶参与特定过程一直很困难。此外，还有一些 关于磷酸化抑制或刺激细胞内运输的程度、调节程度的争论 机制在物种之间是保守的，以及体外机制如何转化为体内系统。因此， 所缺乏的是一个有凝聚力的策略来成功地揭示磷酸化如何促进空间和 控制体内细胞内转运的时间调节机制，如果没有该机制，则无法靶向有效的治疗 对癌症或神经退行性疾病等疾病早期可能被破坏的途径进行治疗是不可能的。 长期目标是确定参与细胞内调节的细胞/分子机制。 体内运输。总体目标是开发一个体内平台来弄清楚特定激酶如何控制 通过识别所涉及的精确功能位点，并通过将调节步骤与 复杂的机制网络。中心假设是激酶糖原合酶激酶-3beta (GSK3b) 差异磷酸化驱动蛋白-1 上的特定位点以控制体内细胞内转运。的 拟议研究的基本原理是，一旦 GSK3b 参与驱动蛋白的体内机制 介导的运输是已知的，该领域将更接近确定控制的复杂机制 MT 轨道上大量细胞货物的运动性，用于将其运送到远端站点。在强有力的预先指导下 没有数据，这个假设将通过追求特定目标来检验；确定 GSK3b 调节驱动蛋白-1 体内细胞内运输过程中的功能。将追求两个目标；生成可遗传的 GSK3b 磷酸- 使用 CRISPR/Cas 系统（目标 1）对磷酸化缺陷/活性 KHC/KLC 蝇系进行分析（目标 1），并识别其中的 GSK3b 介导的磷酸化在细胞内控制驱动蛋白 1 功能的体内机制 运输（目标 2）。所使用的实验策略采用了已经经过验证的体内方法，结合 果蝇遗传学，与生化分析和生物物理范式相结合。该方法论是 申请人认为这是创新的，因为它通过对特定的分析进行分析而脱离了现状 体内驱动蛋白 1 亚基上的 GSK3b 磷酸化事件，这将有助于更好地了解 kinesin-1 功能的机制细节；看起来与现在的情况有很大不同 由体外研究可知。拟议的研究意义重大，因为它有望纵向推进 并在生理条件下，在整个有机体环境中改变目前已知的东西。的 获得的知识将极大地推动精确药理学/基因修饰剂的发展 对抗该途径的缺陷，这将有利于癌症和神经退行性疾病的治疗。

Despite the discovery that molecular motors are phosphorylated 25 years ago, fundamental questions on the identity of the protein kinase(s) or the particular phosphorylation sites, and how they function to control motors remain unanswered. Since kinase cascades display considerable crosstalk and play multiple roles in cell home- ostasis, deciphering which kinase is involved in a particular process has been difficult. Further, there is some debate as to the extent to which phosphorylation inhibits or stimulates intracellular transport, the extent regulatory mechanisms are conserved between species, and how in vitro mechanisms translate to in vivo systems. Thus, what is lacking is a cohesive strategy to successfully unravel how phosphorylation contributes to the spatial and temporal regulatory mechanisms that control intracellular transport in vivo, without which targeting effective treat- ments to a pathway that is likely disrupted early in disease such as cancer or neurodegeneration is unattainable. The long-term goal is to identify the cellular/molecular mechanisms involved in the regulation of intracellular transport in vivo. The overall objective is to develop an in vivo platform to tease out how a specific kinase controls motor function by identifying the precise functional sites involved, and by isolating the regulatory steps from a complex network of mechanisms. The central hypothesis is that the kinase glycogen synthase kinase-3beta (GSK3b) differentially phosphorylates particular sites on kinesin-1 to control intracellular transport in vivo. The rationale for the proposed research is that once the in vivo mechanisms of how GSK3b is involved in kinesin- mediated transport are known, the field will be a step closer to identifying the complex mechanisms that govern the motility of numerous cellular cargoes on MT tracks for their delivery to distal sites. Guided by strong prelimi- nary data, this hypothesis will be tested by pursuing the specific aim; identify that GSK3b-regulates kinesin-1 function during intracellular transport in vivo. Two objectives will be pursued; generate heritable GSK3b phos- phorylation defective/active KHC/KLC fly lines using the CRISPR/Cas system (Objective 1), and identify the in vivo mechanisms of how GSK3b-mediated phosphorylation controls kinesin-1 function during intracellular transport (Objective 2). The experimental strategy used employs an already proven in vivo approach, coupled with Drosophila genetics, integrated with biochemical analysis and biophysical paradigms. This methodology is innovative in the applicant’s opinion, because it departs from the status quo by enabling the analysis of particular GSK3b-phosphorylation events on kinesin-1 subunits in vivo, which will lead to a better understanding of the mechanistic details of how kinesin-1 functions; which appear to be considerably different from what is currently known from in vitro studies. The proposed research is significant, because it is expected to vertically advance and transform what is currently known, under physiological conditions, in a whole organism setting. The knowledge acquired will dramatically propel the development of precise pharmacological/genetic modifiers against defects in this pathway which will benefit the treatment of cancer and neurodegeneration.