Defining human kinase-substrate networks and their dynamic regulation

项目概要 蛋白质磷酸化是一种重要的翻译后修饰 (PTM)，可控制大多数生物 流程。超过四分之三的蛋白质在人体细胞的一个或多个位点被磷酸化。 系统的基因组测序、基因表达和 RNAi 研究表明激酶的失调 在许多人类疾病中发挥作用，包括癌症、糖尿病和神经退行性疾病。然而，这样的 方法没有揭示特定的信号传导途径和分子靶点。因此，存在未满足的需求 对人类激酶-底物关系的系统询问。我们研究的长期目标是 破译基础生物学和疾病中的激酶信号传导。为了实现这一目标，我们开发并应用了 将特定激酶与其底物连接的定量磷酸化蛋白质组学策略，包括 Polo- 如激酶 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 DNA 损伤恢复和有丝分裂进入、有丝分裂进展和 胞质分裂，并且在癌症中经常过度表达。虽然 Plk1 是真正的癌基因，但 Plk2 和 Plk3 起作用 作为肿瘤抑制因子，保护细胞免受 DNA 损伤，并且是其他 G1 和 S 期所需的 尽管这些功能背后的机制很大程度上是未知的。传统策略 选择性研究激酶功能，例如基因缺失、耗尽或过度表达改变激酶丰度 在数小时至数天的时间范围内，这通常会妨碍直接激酶底物的分配。优雅 化学遗传学方法将突变引入保守的催化激酶结构域以呈现 它们对 ATP 类似物敏感，以克服普遍缺乏选择性抑制剂的问题， 时间控制问题。然而，这些突变通常会降低激酶活性和稳定性，从而限制了 普遍实施这一方法。因此，需要新的策略来连接激酶及其 基材。为了解决这一能力差距，我们建议建立一个通用的定量化学 蛋白质组学策略能够识别特定激酶底物。诱导蛋白降解是 一种直接操纵蛋白质丰度的新兴技术。我们假设组合 可诱导的、快速的蛋白质降解（< 10 分钟半衰期）和基于质谱的蛋白质组学是一种可行的方法 识别特定激酶底物和阐明磷酸化信号传导的策略 密切相关的酶网络。在本提案中，我们提供了全面研究的蓝图 激酶组水平上的激酶-底物关系。这对于绘制细胞信号通路至关重要， 识别因突变或药物治疗和耐药性而破坏的激酶途径重编程，以及 确定临床相关抑制剂的脱靶效应。超过一半的人类激酶组是非或 特征不足；这里概述的实验代表了填补这一知识空白的路线图。

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.