Proteomics approaches for illuminating the functions of the dark kinases Nek6, Nek7 & Nek9

摘要 有丝分裂是一个动态的和严格调节的细胞周期阶段，跨越染色体凝聚，纺锤体， 形成，姐妹染色单体分离和分离，胞质分裂;细胞未能成功地 导航这些不同的生物功能是许多人类疾病的基础，包括出生缺陷和癌症。 这些功能的协调是由一系列复杂的蛋白激酶信号传导完成的，包括 未充分研究的NIMA相关激酶Nek 6、Nek 7和Nek 9。尽管基因缺失研究表明 这些Nek激酶在细胞分裂和胞质分裂中，它们发挥作用的生化机制仍然存在， 悬而未决因此，新的研究揭示了Nek 6，Nek 7和Nek 9的调节和下游效应子， 功能有可能提高我们对细胞分裂生物学的基本理解， 人类疾病治疗干预的新切入点。 在这里，我们建议开发和部署靶向蛋白质降解与质谱联用- 基于蛋白质组学，发现由Nek 6、Nek 7和Nek 9调控的新底物和信号网络 激酶活性。使用基于CRISPR/Cas9的靶向策略的组合， 和基因工程，我们将在这些激酶的内源基因座上敲入一个短的降解决定子标签， 不同的基因和组织来源。然后，我们将采用定量蛋白质组学方法来鉴定蛋白质 在各自Nek激酶快速耗尽后表现出不同占用的磷酸化位点。我们 预计这些实验将揭示新的机制，定义细胞分裂，并将另外 作为一个路线图，照亮其余的未充分研究的激酶组。

ABSTRACT Mitosis is a dynamic and tightly regulated cell cycle phase that spans chromosome condensation, spindle formation, sister chromatid separation and segregation, and cytokinesis; the failure of cells to successfully navigate these diverse biological functions underlies many human diseases including birth defects and cancer. The orchestration of these functions is accomplished by a complex array of protein kinase signaling, including the understudied NIMA-related kinases Nek6, Nek7 and Nek9. Although gene depletion studies have implicated these Nek kinases in cell division and cytokinesis, the biochemical mechanisms by which they function remain unresolved. Thus, new research that uncovers the regulation and downstream effectors of Nek6, Nek7 and Nek9 functions has the potential to both improve our basic understanding of cell division biology as well as identify new entry points for therapeutic intervention in human disease. Here, we propose to develop and deploy targeted protein degradation coupled with mass spectrometry- based proteomics to discover new substrates and signaling networks that are regulated by Nek6, Nek7 and Nek9 kinase activities. Using a combination of CRISPR/Cas9-based targeting strategies, homology-directed repair and genetic engineering, we will knock-in a short degron tag at endogenous loci for these kinases in cell lines of diverse genetic and tissue origin. We will then employ quantitative proteomics methods to identify protein phosphorylation loci that exhibit differential occupancy upon rapid depletion of the respective Nek kinase. We expect these experiments will shed new light on the mechanisms that define cell division and will additionally serve as a roadmap for illuminating the rest of the understudied kinome.