FINDING NEK2 KINASE AUTO AND SUBSTRATE PHOSPHORYLATION SITES IN HUMAN CELLS

这个子项目是许多利用资源的研究子项目之一 由NIH/NCRR资助的中心拨款提供。子项目的主要支持 而子项目的主要调查员可能是由其他来源提供的， 包括其它NIH来源。 列出的子项目总成本可能 代表子项目使用的中心基础设施的估计数量， 而不是由NCRR赠款提供给子项目或子项目工作人员的直接资金。 蛋白激酶是一组不同的调节酶，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来控制细胞内的信号事件。 识别给定激酶的底物蛋白及其特异性磷酸化位点对于理解激酶介导的信号传导事件是必不可少的。 然而，没有一般的或平凡的方法，这样的标识。 我们正在研究一种使用特定激酶Nek 2的小分子抑制剂以及Nek 2激酶的抑制剂抗性突变体来阐明激酶磷酸化位点的方法。 该方法涉及在具有表位标签的人细胞中过表达感兴趣的激酶（Nek 2），该表位标签可用于从这些细胞中纯化Nek 2和相关蛋白。 然后可以使用质谱分析来鉴定这些纯化蛋白质上的磷酸化位点。 通过在用Nek 2抑制剂处理的样品和表达抑制剂抗性Nek 2突变体的样品之间进行定量比较，我们可以确定与Nek 2激酶活性特异性相关的磷酸化位点。

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.