Defining human kinase-substrate networks and their dynamic regulation

定义人类激酶底物网络及其动态调节

• 批准号: 9980956
• 负责人: Scott A. Gerber
• 金额: $ 33.32万
• 依托单位: DARTMOUTH COLLEGE
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-09-15 至 2022-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9980956
• 关键词: Address; Auxins; Binding; Biological Process; Biology; Cell Line; Cells; Centrioles; Chemicals; Cytokinesis; DNA Damage; Diabetes Mellitus; Disease; Emerging Technologies; Enzymes; Family; Family member; G1 Phase; Gene Deletion; Gene Expression; Goals; Half-Life; Hour; Human; In Vitro; Individual; Knowledge; Label; Malignant Neoplasms; Mass Spectrum Analysis; Methods; Mitosis; Mitotic; Molecular Target; Mutation; Nerve Degeneration; Oncogenes; PLK1 gene; Pathway interactions; Pharmacotherapy; Phosphorylation; Phosphorylation Site; Phosphotransferases; Polo-Box Domain; Post-Translational Protein Processing; Process; Proteins; Proteomics; RNA Interference; Recovery; Regulation; Research; Research Personnel; Signal Pathway; Signal Transduction; Site; Structure; Substrate Interaction; Testing; Time; Tumor Suppressor Proteins; Yeasts; analog; base; chemical genetics; clinical effect; clinically relevant; experimental study; genetic approach; genome sequencing; human disease; inhibitor/antagonist; interest; member; mutant; nanomolar; overexpression; phosphoproteomics; protein degradation; response; small molecule; small molecule inhibitor; therapy resistant

PROJECT SUMMARY Protein phosphorylation is an essential post-translational modification (PTM) that controls most biological processes. More than three-quarters of all proteins are phosphorylated at one or more sites in human cells. Systematic genome sequencing, gene expression and RNAi studies have implicated deregulation of kinase function in many human diseases, including cancer, diabetes, and neurodegeneration. However, such approaches do not reveal specific signaling pathways and molecular targets. Thus, there is an unmet need for the systematic interrogation of human kinase-substrate relationships. The long-term goal of our research is to decipher kinase signaling in basic biology and disease. To accomplish this, we have developed and applied quantitative phosphoproteomics strategies to connect specific kinases to their substrates, including for Polo- like kinase 1 (Plk1). Plk1 is the founding member of the Plk family and is conserved from yeast to humans. Plk1 is an essential regulator of recovery from DNA damage and mitotic entry, mitotic progression and cytokinesis, and is frequently overexpressed in cancer. While Plk1 is a bona fide oncogene, Plk2 and Plk3 act as tumor suppressors, protect cells against DNA damage, and are required for other G1 and S-phase processes, although the mechanisms that underlie these functions are largely unknown. Traditional strategies to selectively study kinase function such as gene deletion, depletion, or overexpression alter kinase abundance on a time scale of hours to days which often precludes assignment of direct kinase substrates. Elegant chemical genetics approaches that introduce mutations into the conserved catalytic kinase domain to render them ATP analog-sensitive have been implemented to overcome the general lack of selective inhibitors and the temporal control problem. However, these mutations often reduce kinase activity and stability, limiting the universal implementation of this approach. Thus, new strategies are needed for connecting kinases and their substrates. To address this gap in capability, we propose to establish a general quantitative chemical proteomics strategy to enable the identification of specific kinase substrates. Inducible protein degradation is an emerging technology for directly manipulating protein abundance. We hypothesize that the combination of inducible, rapid protein degradation (< 10 min half-life) and mass spectrometry based proteomics is a viable strategy for the identification of specific kinase substrates and elucidation of phosphorylation signaling networks of closely related enzymes. In this proposal, we provide a blueprint for comprehensive studies of kinase–substrate relationships on a kinome-wide level. This is pivotal for mapping cellular signaling pathways, identifying kinase pathway reprogramming upon disruption by mutations or drug treatment and resistance, and determining off-target effects of clinically relevant inhibitors. More than half of the human kinome is un- or under-characterized; experiments outlined here represent a roadmap for filling this gap in knowledge.

项目摘要 蛋白质磷酸化是一种基本的翻译后修饰（PTM），可控制大多数生物学 过程。所有蛋白质的四分之三以上的四分之三在人类细胞中的一个或多个部位被磷酸化。 系统的基因组测序，基因表达和RNAi研究已经实施了激酶的失调 在许多人类疾病中起作用，包括癌症，糖尿病和神经变性。但是，这样 方法没有揭示特定的信号通路和分子靶标。那是没有满足的需求 人类激酶 - 基底关系的系统审问。我们研究的长期目标是 基本生物学和疾病中的破译激酶信号传导。为此，我们已经开发和应用 定量的磷酸蛋白质组学策略，将特定激酶连接到其底物，包括polo- 像激酶1（PLK1）。 PLK1是PLK家族的创始成员，从酵母到人类都是保守的。 PLK1是从DNA损伤和有丝分裂进入，有丝分裂进展和 细胞因子，并且经常在癌症中过表达。虽然PLK1是真正的癌基因，PLK2和PLK3 ACT 作为肿瘤补充剂，保护细胞免受DNA损伤，是其他G1和S期所需的 尽管这些功能的基础的机制在很大程度上未知。传统策略 有选择地研究激酶功能，例如基因缺失，耗竭或过表达改变激酶抽象 在小时到几天的时间范围内，通常排除了直接激酶底物的分配。优雅的 化学遗传学方法将突变引入配置的催化激酶结构域以渲染 他们已经实施了ATP模拟敏感的，以克服总体缺乏选择性抑制剂和 临时控制问题。但是，这些突变通常会降低激酶活性和稳定性，从而限制 这种方法的普遍实施。这是需要新的策略来连接激酶及其 基材。为了解决能力的差距，我们建议建立一般的定量化学物质 蛋白质组学策略可以鉴定特定的激酶底物。诱导蛋白降解是 一种直接操纵蛋白质丰度的新兴技术。我们假设结合 可诱导的快速蛋白质降解（<10分钟的半衰期）和质谱基蛋白质组学是可行的 鉴定特定激酶底物和阐明磷酸化信号传导的策略 密切相关酶的网络。在此提案中，我们为全面研究提供了蓝图 激酶 - 基底关系在整个范围内。这对于映射细胞信号通路的关键是 在突变，药物治疗和耐药时识别激酶途径重编程，以及 确定临床相关抑制剂的脱靶作用。超过一半的人类组合是没有或 特征不足；此处概述的实验代表了填补知识差距的路线图。