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）：驱动蛋白是机械化学酶，其利用ATP水解来沿着微管晶格定向运输货物或调节微管组装/分解。细胞内的驱动蛋白功能和定位通过激酶介导的特定残基磷酸化机制受到严格调节。虽然驱动蛋白的磷酸调节已经研究了几十年，但分析几乎完全集中在保守催化核心（运动结构域）之外的磷酸化位点。然而，最近的研究表明，驱动蛋白的活性可以通过其运动域内高度保守的残基的磷酸化来调节。该提案研究了一个中心假设，即驱动蛋白运动域内几个高度保守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) 确定驱动蛋白运动域上新的生理相关磷酸化位点； 2) 鉴定这些位点磷酸化引起的结构变化； 3) 确定这些变化如何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：检验整个超家族驱动蛋白运动域的调节是通过少数全局保守的磷酸化位点介导的假设。这些位点的磷酸化快速且可逆地改变酶催化核心的关键结构特征。将测试的结构/功能关系包括微管蛋白结合和 ATP 水解，这对所有电机来说都是常见的。目标 2：检验以下假设：每个驱动蛋白家族特有的运动结构域的调节是通过少数家族保守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调节家族特异性功能。将采取多学科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驱动蛋白运动结构域磷酸化的作用。相关性：本提案中要研究的驱动蛋白在细胞分裂、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。这些过程的失调与许多人类疾病有关。该项目与公共卫生相关，因为它将弥补我们对驱动蛋白功能知识的根本差距，并提供一个结构框架来指导针对这些疾病的新型治疗剂的开发。 公共健康相关性：该项目的目标是从分子到细胞水平了解运动域内驱动蛋白的磷酸调节机制。这项研究与 NIH 的使命相关，它将弥合我们在驱动蛋白功能知识方面的根本差距，并提供一个结构框架来指导针对多种人类疾病的新型治疗药物的开发。