Defining human kinase-substrate networks and their dynamic regulation

项目摘要 蛋白质磷酸化是一种重要的翻译后修饰（PTM）， 流程.在人类细胞中，超过四分之三的蛋白质在一个或多个位点被磷酸化。 系统基因组测序、基因表达和RNAi研究表明， 在许多人类疾病中起作用，包括癌症、糖尿病和神经变性。但这种 这些方法不能揭示特定的信号通路和分子靶点。因此，存在未满足的需求， 对人类激酶-底物关系的系统研究。我们研究的长期目标是 在基础生物学和疾病中破译激酶信号。为了实现这一目标，我们开发并应用了 定量磷酸化蛋白质组学策略，以连接特定的激酶，其底物，包括波罗- 如激酶1（Plk 1）。Plk 1是Plk家族的创始成员，从酵母到人类都是保守的。 Plk 1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表达。Plk 1是真正的致癌基因，Plk 2和Plk 3 作为肿瘤抑制因子，保护细胞免受DNA损伤，并为其他G1和S期所需 过程，虽然这些功能的机制基本上是未知的。传统战略 选择性研究激酶功能，例如基因缺失、缺失或过表达改变激酶丰度 在几小时到几天的时间尺度上，这通常排除了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， 他们ATP类似物敏感已被实施，以克服一般缺乏选择性抑制剂， 时间控制问题。然而，这些突变通常降低激酶活性和稳定性，限制了激酶的功能。 普遍采用这种方法。因此，需要新的策略来连接激酶及其受体。 印刷受体.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品 蛋白质组学策略，使特定的激酶底物的鉴定。诱导性蛋白质降解是 这是一种直接操纵蛋白质丰度的新兴技术。我们假设 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 用于鉴定特异性激酶底物和阐明磷酸化信号传导的策略 密切相关的酶的网络。在这项建议中，我们提供了一个蓝图， 在激酶组水平上的激酶-底物关系。这是绘制细胞信号通路的关键， 识别因突变或药物治疗和耐药性破坏后的激酶途径重编程，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.