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控的细胞周期阶段，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；细胞不能成功地 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。 这些功能的协调是通过一系列复杂的蛋白激酶信号来完成的，包括 未被充分研究的NIMA相关激酶Nek6、Nek7和Nek9。尽管基因枯竭研究表明 这些Nek激酶参与细胞分裂和胞质分裂，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Nek6、Nek7和Nek9的调控和下游效应 功能有可能提高我们对细胞分裂生物学的基本理解，以及识别 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定向蛋白质降解与质谱学相结合- 基于蛋白质组学发现受Nek6、Nek7和Nek9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、同源定向修复 和基因工程，我们将在这些细胞系的内源性基因座上敲入一个短的退化标签。 不同的遗传和组织来源。然后，我们将使用定量蛋白质组学方法来鉴定蛋白质 当各自的Nek激酶迅速耗尽时，表现出不同占有率的磷酸化基因座。我们 预计这些实验将为定义细胞分裂的机制提供新的线索，并将进一步 作为一个路线图，照亮其余未被研究的亲缘关系。

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.