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

驱动蛋白运动域的磷酸调节：结构、动力学和功能

• 批准号: 8238617
• 负责人: GARY J. GERFEN
• 金额: $ 46.78万
• 依托单位: ALBERT EINSTEIN COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-05-01 至 2016-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8238617
• 关键词: ATP Hydrolysis; Binding; Biochemistry; Biological Assay; Catalytic Domain; Cell division; Cell physiology; Cells; Cellular biology; Cryoelectron Microscopy; Development; Disease; Electron Spin Resonance Spectroscopy; Enzymes; Family; Family member; Fluorescence Spectroscopy; Goals; In Vitro; Kinesin; Knowledge; Life; Link; Mediating; Microscopy; Microtubules; Mission; Modification; Molecular; Motor; Phosphorylation; Phosphorylation Site; Phosphotransferases; Principal Investigator; Process; Protein Array; Public Health; Regulation; Research; Role; Sampling; Serine; Site; Spin Labels; Structure; Structure-Activity Relationship; Techniques; Testing; Therapeutic Agents; Translating; Tubulin; United States National Institutes of Health; base; cell motility; cellular imaging; depolymerization; human disease; in vitro activity; in vivo; interdisciplinary approach; member; molecular domain; molecular dynamics; novel therapeutics; protein function; protein structure; simulation; single molecule; spatiotemporal

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. PUBLIC HEALTH RELEVANCE: The goal of this project is to understand the mechanism of phosphoregulation of kinesins within their motor domain from the molecular to the cellular level. This research is relevant to the mission of NIH in that 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 numerous human diseases.

描述（由申请人提供）：动力素是机械化学酶，利用ATP水解沿微管晶格方向转运肉类或调节微管组装/拆卸。细胞内的动力素功能和定位通过特定残基处的激酶介导的磷酸化机制严格调节。虽然已经研究了驱动蛋白的磷酸化数十年，但分析几乎完全集中在保守的催化核心（运动结构域）之外的磷酸化位点上。然而，最近已经显示，可以通过在其运动结构域内高度保守的残基来调节驱动蛋白的活性。该提案调查了一个中心假设，即在驱动蛋白运动结构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）在驱动蛋白运动域上确定新的生理相关磷酸化位点； 2）确定这些位点磷酸化引起的结构变化； 3）确定如何将这些变化转化为驱动蛋白催化活性和细胞功能的改变。将追求以下两个具体目标。目标1：检验以下假设：通过少数全球保守的磷酸化位点，介导了超家族的驱动蛋白的运动结构域。这些位点的磷酸化迅速，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/功能关系包括所有电动机共有的小管蛋白结合和ATP水解。 AIM 2：检验以下假设：每个驱动蛋白家族特有的运动域的调节是通过少数家庭保存的磷酸化位点介导的。这些位点的磷酸化通过特定的结构修饰来调节家庭特异性功能。将采用一种多学科的方法来实现这些目标，包括电子顺磁共振（EPR），单分子荧光光谱，冷冻电子显微镜，生物分子模拟，多维活细胞成像以及体外和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进行全面研究驱动蛋白运动结构域磷酸化的作用。相关性：该提案中要研究的动力素在细胞分裂，发育和功能中起关键作用，所有这些都受磷酸化的调节。对这些过程的失误与许多人类疾病有关。该项目与公共卫生有关，因为它将弥合我们对动力素功能知识的根本差距，并提供一个结构性框架，以指导针对这些疾病的新型治疗剂的发展。 公共卫生相关性：该项目的目的是了解从分子到细胞水平的运动结构域内驱动蛋白磷酸化的机制。这项研究与NIH的使命相关，这将弥合我们对动力素功能的知识的根本差距，并提供了一个结构性框架，以指导针对多种人类疾病的新型治疗剂的发展。