Phosphoregulation of the Kinesin Motor Domain: Structure, Dynamics and Function

说明(申请人提供)：动蛋白是一种机械力化学酶，它利用三磷酸腺苷的水解作用沿微管晶格定向运输货物或调节微管的组装/拆卸。激动素的功能和在细胞内的定位是通过特定残基上的激酶介导的磷酸化机制受到严格调控的。虽然对肌动蛋白的磷调节已经研究了几十年，但分析几乎完全集中在保守的催化核心(运动区)之外的磷酸化位点上。然而，最近的研究表明，Kinesin的活性可以通过其运动域中高度保守的残基的磷酸化来调节。这一建议调查了中心假设，即运动蛋白运动域中几个高度保守的残基的磷酸化调节其与微管的结构、动态和功能相互作用。我们的目标是：1)确定运动蛋白运动域中与生理相关的新的磷酸化位点；2)确定由这些位点的磷酸化引起的结构变化；以及3)确定这些变化如何转化为运动蛋白催化活性和细胞功能的变化。将实现以下两个具体目标。目的1：验证一种假说，即整个超家族的运动蛋白运动域的调节是通过少数全局保守的磷酸化位点来实现的。这些位点的磷酸化迅速和可逆地改变了酶催化核心的关键结构特征。将要测试的结构/功能关系包括微管蛋白结合和ATP水解，这在所有发动机中都是常见的。目的2：验证一种假说，即运动蛋白家族特有的运动域的调控是通过少数家族保守的磷酸化位点来实现的。这些位点的磷酸化通过特定的结构修饰来调节家族特有的功能。为了实现这些目标，将寻求多学科的方法，包括电子顺磁共振(EPR)、单分子荧光光谱、低温电子显微镜、生物分子模拟、多维活细胞成像以及体外和体内功能分析。利用这些技术的协同整合，将从分子到细胞水平全面研究Kinesin运动区磷酸化的作用。相关性：本方案中要研究的动蛋白在细胞分裂、发育和功能中发挥关键作用，所有这些都受到磷酸化的调节。对这些过程的失控与许多人类疾病有关。这个项目与公共卫生相关，因为它将弥合我们对动蛋白功能的基本认识差距，并提供一个结构框架来指导针对这些疾病的新型治疗药物的开发。

DESCRIPTION (provided by applicant): Kinesins are mechanochemical enzymes which utilize ATP hydrolysis to transport cargos directionally along the microtubule lattice or to regulate microtubule assembly/disassembly. Kinesin function and localization within the cell are tightly regulated via a mechanism of kinase-mediated phosphorylation at specific residues. While the phosphoregulation of kinesins has been studied for decades, analyses have focused almost entirely on phosphorylation sites outside of the conserved catalytic core (motor domain). However, it has recently been shown that the activity of kinesins can be regulated through the phosphorylation of highly conserved residues within their motor domains. This proposal investigates the central hypothesis that phosphorylation of several highly conserved residues within the kinesin motor domain regulates its structural, dynamic and functional interactions with microtubules. Our objectives are to 1) identify new physiologically relevant phosphorylation sites on the kinesin motor domain; 2) identify structural changes induced by phosphorylation at these sites; and 3) determine how these changes are translated into alterations of kinesin catalytic activity and cellular function. The following two Specific Aims will be pursued. Aim 1: Test the hypothesis that regulation of the motor domains of kinesins across the superfamily is mediated through a small number of globally conserved phosphorylation sites. Phosphorylation of these sites rapidly and reversibly alters key structural features of the enzymes' catalytic core. Structure/function relationships that will be tested include tubulin binding and ATP hydrolysis, which are common to all motors. Aim 2: Test the hypothesis that regulation of the motor domains specific to each kinesin family is mediated through a small number of family-conserved phosphorylation sites. Phosphorylation of these sites regulates family-specific functions via specific structural modifications. A multidisciplinary approach to achieve these aims will be pursued, including Electron Paramagnetic Resonance (EPR), single molecule fluorescence spectroscopies, Cryo electron microscopy, biomolecular simulations, multidimensional live cell imaging, and in vitro and in vivo functional analysis. Using a synergistic integration of these techniques, the role of kinesin motor domain phosphorylation will be comprehensively investigated from the molecular to the cellular level. Relevance: The kinesins to be studied in this proposal perform key roles in cell division, development and function, all of which are subject to regulation by phosphorylation. Miss-regulation of these processes has been linked to numerous human diseases. This project is relevant to public health because it will bridge a fundamental gap in our knowledge of kinesin functionality and provide a structural framework to guide the development of novel therapeutic agents targeting these diseases.