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

阐明暗激酶 Nek6、Nek7 功能的蛋白质组学方法

• 批准号: 10216469
• 负责人: Scott A. Gerber
• 金额: $ 16.4万
• 依托单位: DARTMOUTH COLLEGE
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-05-01 至 2023-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10216469
• 关键词: Address; Aspergillus; Aspergillus nidulans; Auxins; Biochemical; Biological Assay; Biological Process; Biology; Breast; CRISPR/Cas technology; Cell Cycle; Cell Line; Cell division; Cells; Chemicals; Chromosome Condensation; Colon; Communities; Complex; Congenital Abnormality; Coupled; Cytokinesis; Data; Diabetes Mellitus; Disease; Emerging Technologies; Exhibits; Failure; Family; Fibroblasts; Funding Mechanisms; G2 Phase; Gene Deletion; Gene Expression; Genes; Genetic; Genetic Engineering; Goals; Hela Cells; Hour; Human; In Vitro; Industry; Knock-in; Knowledge; Light; Link; Liver; Lung; Malignant Neoplasms; Mass Spectrum Analysis; Methods; Microtubules; Mitosis; Mitotic; Molecular Target; Mutation; Nerve Degeneration; Ovarian; PLK1 gene; Pathway interactions; Pharmacotherapy; Phase; Phenotype; Phosphorylation; Phosphotransferases; Post-Translational Protein Processing; Prostate; Protein Array; Protein Kinase; Proteins; Proteomics; RNA Interference; Regulation; Research; Research Personnel; Rest; Role; Severities; Severity of illness; Signal Pathway; Signal Transduction; Sister Chromatid; Stomach; Technology; Therapeutic Intervention; Time; Tissues; analog; base; chemical genetics; ciliopathy; cilium biogenesis; experimental study; genetic approach; genome sequencing; human disease; improved; inhibitor/antagonist; interest; loss of function; mutant; online resource; overexpression; phosphoproteomics; premature; protein degradation; repaired; segregation; small molecule inhibitor; therapy resistant

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.

抽象的 有丝分裂是一个动态且受到严格调节的细胞周期阶段，跨越染色体凝结，主轴 形成，姐妹染色单体分离和隔离以及细胞因子；细胞无法成功 导航这些多样化的生物学功能是许多人类疾病（包括先天缺陷和癌症）的基础。 这些功能的编排是通过一系列复杂的蛋白激酶信号传导来完成的 研究了与NIMA相关的激酶NEK6，NEK7和NEK9。尽管基因耗竭研究已牵涉 这些NEK激酶在细胞分裂和细胞因子中，其功能的生化机制保持 未解决。因此，揭示了NEK6，NEK7和NEK9的调节和下游效应子的新研究 功能有可能提高我们对细胞分裂生物学的基本理解，并确定 治疗干预人类疾病的新入口点。 在这里，我们建议开发和部署靶向蛋白质降解以及质谱 - 基于NEK6，NEK7和NEK9调节的新底物和信号网络的基于蛋白质组学 激酶活动。结合基于CRISPR/CAS9的靶向策略，以同源为导向的维修 和基因工程，我们将在这些激酶的细胞系中敲入内源性基因座的短degron标签 多样化的遗传和组织起源。然后，我们将采用定量蛋白质组学方法来识别蛋白质 相应NEK激酶快速耗竭时，磷酸化基因座表现出差异。我们 期望这些实验将为定义细胞分裂的机制提供新的启示 用作照亮其余研究的Kinome的路线图。