FINDING NEK2 KINASE AUTO AND SUBSTRATE PHOSPHORYLATION SITES IN HUMAN CELLS

寻找人类细胞中的 NEK2 激酶自动和底物磷酸化位点

• 批准号: 8363809
• 负责人: Jack Taunton
• 金额: --
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-06-01 至 2012-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8363809
• 关键词: Cells; Enzymes; Epitopes; Event; Funding; Grant; Human; Link; Mass Spectrum Analysis; Mediating; Metabolic Pathway; Methods; National Center for Research Resources; Phosphorylation; Phosphorylation Site; Phosphotransferases; Principal Investigator; Protein Kinase; Proteins; Research; Research Infrastructure; Resistance; Resources; Sampling; Signal Transduction; Source; United States National Institutes of Health; autophosphorylation-dependent multifunctional protein kinase; cell behavior; cost; inhibitor/antagonist; interest; mutant; small molecule

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Protein kinases are a diverse set of regulatory enzymes responsible for signaling in a majority of metabolic pathways and cell behaviors. Kinases control signaling events within the cell through phosphorylation of substrate proteins. Identification of a given kinases' substrate proteins and their specific phosphorylation sites is essential to understanding kinases mediated signaling events. However, there is no general or trivial method for such identifications. We are investigating a method of elucidating kinase phosphorylation sites using small molecule inhibitors of a specific kinase, Nek2, as well as an inhibitor resistant mutant of Nek2 kinase. The method involves over-expressing the kinase of interest (Nek2) in human cells with an epitope tag that can be used to purify Nek2 and associated proteins from these cells. Mass Spec analysis can then be used to identify phosphorylation sites on these purified proteins. By making a quantitative comparison between samples treated with out Nek2 inhibitor and samples expressing the inhibitor resistant Nek2 mutant we can determine phosphorylation sites specifically linked to Nek2 kinase activity.

该子项目是利用资源的众多研究子项目之一 由 NIH/NCRR 资助的中心拨款提供。子项目的主要支持 并且子项目的主要研究者可能是由其他来源提供的， 包括其他 NIH 来源。 子项目可能列出的总成本 代表子项目使用的中心基础设施的估计数量， NCRR 赠款不直接向子项目或子项目工作人员提供资金。 蛋白激酶是一组多样化的调节酶，负责大多数代谢途径和细胞行为中的信号传导。 激酶通过底物蛋白的磷酸化控制细胞内的信号传导事件。 识别给定激酶的底物蛋白及其特定磷酸化位点对于了解激酶介导的信号转导事件至关重要。 然而，没有通用或简单的方法来进行此类识别。 我们正在研究一种使用特定激酶 Nek2 的小分子抑制剂以及 Nek2 激酶抑制剂抗性突变体来阐明激酶磷酸化位点的方法。 该方法涉及在人类细胞中过度表达目标激酶 (Nek2)，并带有表位标签，可用于从这些细胞中纯化 Nek2 和相关蛋白。 然后可以使用质谱分析来识别这些纯化蛋白质上的磷酸化位点。 通过对未经 Nek2 抑制剂处理的样品和表达抑制剂抗性 Nek2 突变体的样品进行定量比较，我们可以确定与 Nek2 激酶活性特异性相关的磷酸化位点。