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年前发现了分子马达是磷酸化的，但关于 蛋白激酶(S)或特定的磷酸化位点的特性，以及它们如何作用于控制马达 仍未得到答复。由于激活级联信号表现出相当大的串扰，并在细胞内扮演多种角色- 在静止期，破译哪一种激酶参与了特定的过程一直是困难的。此外，还有一些 关于磷酸化在多大程度上抑制或刺激细胞内运输的争论，调控的程度 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， 缺乏的是一种连贯的策略，以成功地解开磷酸化如何对空间和 控制体内细胞内运输的时间调控机制，如果没有这种机制，就可以有效地靶向治疗- 对于癌症或神经退行性变等疾病早期可能被破坏的途径，是无法实现的。 长期的目标是确定参与细胞内调节的细胞/分子机制。 体内转运。总体目标是开发一个体内平台，以梳理出特定的激酶是如何控制的 通过确定涉及的精确功能部位，并通过将调节步骤与 复杂的机械网络。中心假说是激酶-糖原合成酶-3β (GSK3b)通过差异磷酸化Kinesin-1上的特定位点来控制体内的细胞内转运。这个 这项拟议研究的基本原理是，一旦GSK3b参与运动蛋白的体内机制- 介导性转运是已知的，该领域将向确定支配 MT轨道上大量移动的蜂窝货物将它们运送到远端站点。在强劲的前期指导下- 几乎没有数据，这一假说将通过追求特定的目标来检验；确定GSK3b调节kinesin-1 在体内的细胞内转运过程中发挥作用。将实现两个目标：产生可遗传的GSK3b PHO- 使用CRISPR/CAS系统的磷酸化缺陷/活性KHC/KLC飞行系(目标1)，并鉴定In GSK3b介导的磷酸化调控细胞内激动素-1功能的体内机制 运输(目标2)。使用的实验策略采用了已经在体内得到验证的方法，再加上 与果蝇遗传学相结合，结合生化分析和生物物理范式。这种方法论是 申请人的观点具有创新性，因为它通过能够分析特定的 GSK3b-体内Kinesin-1亚基的磷酸化事件，这将有助于更好地理解 Kinesin-1如何发挥作用的机械细节；这似乎与目前的情况有很大不同 从体外研究得知。这项拟议的研究意义重大，因为预计它将垂直推进 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。这个 所获得的知识将极大地推动精确的药理/遗传修饰剂的发展 对抗这一通路的缺陷，这将有利于癌症和神经退行性变的治疗。

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.