FINDING NEK2 KINASE AUTO AND SUBSTRATE PHOSPHORYLATION SITES IN HUMAN CELLS

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 蛋白激酶是一组不同的调节酶，在大多数代谢途径和细胞行为中负责信号传递。激酶通过底物蛋白的磷酸化控制细胞内的信号事件。识别一种特定蛋白的底物蛋白及其特定的磷酸化位置对于理解蛋白激酶介导的信号事件是至关重要的。然而，没有通用的或微不足道的方法来确定这种身份。我们正在研究一种使用特定激酶的小分子抑制剂Nek2以及耐药突变的Nek2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的蛋白激酶(Nek2)，并使用表位标签从这些细胞中纯化Nek2及其相关蛋白。质谱仪分析然后可以用来鉴定这些纯化蛋白质上的磷酸化位点。通过对用Out Nek2抑制剂处理的样品和表达具有抑制剂抗性的Nek2突变体的样品进行定量比较，我们可以确定与Nek2激酶活性特异关联的磷酸化位点。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. 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.