Single-molecule enzymology of protein kinases

项目摘要/摘要 蛋白激酶是普遍存在的信号酶，几乎调控细胞行为的方方面面。激活剂 在无数的人类疾病中，包括癌症、代谢性疾病和 神经和发育障碍。激酶受上游信号的调节，而上游信号调节的是激酶 通过一系列结合配偶体和翻译后修饰实现的酶活性。虽然在 体外重组可用于将结合配对和修饰与它们对激酶酶的影响联系起来 活性，体外方法是耗时的，应用于多个伙伴/修饰具有挑战性 同时，不适用于活体环境。这一限制使该领域无法 了解激酶如何整合上游信号以建立适当的活性水平。 拟议研究的长期目标是通过以下方式了解信号整合的生化基础 活体中的蛋白激酶。为了能够朝着这一目标取得进展，需要新的实验工具，因为 现有工具不能解析激酶分子的分子状态(即，它的补体 修饰和结合伙伴)，并同时测量其活性。这个项目的重点是 建立蛋白激酶活性的单分子酶分析方法。通过研究单分子， 申请者将克服总体平均的限制，否则将排除直接联系 由于蛋白质的异质性，激酶的修饰和结合伙伴对活性的变化 体内的复合体。单分子方法也将能够直接应用于细胞蛋白激酶， 消除了体外重建的需要。 这项工作的中心目标是获得新的技术知识，以便能够应用 单分子分析中基于荧光的激酶活性报告。申请者将探索三个 分离单个激酶分子的不同方法，测量它们的活性并同时 确定存在哪些结合伙伴和翻译后修饰(目标1)。方法是 然后将在细胞来源的激酶分子上进行测试，以确定是否应用 在体内进行单分子激酶检测是可行的(目标2)。本申请中提出的工作意义重大 因为它将建立一种新的方法来测量蛋白激酶活性的可行性， 有可能最终产生对细胞信号转导的基本见解。建议的工作是 申请人认为是创新的，因为它代表了一种全新的研究范式 通过蛋白激酶进行信号传递。通过建立有可能回答基本问题的新工具 关于细胞蛋白激酶的信号整合，这项工作将有助于基础研究的进展 生物医学研究。

Project Summary / Abstract Protein kinases are ubiquitous signaling enzymes that regulate nearly every aspect of cell behavior. Kinase activity is disrupted or misregulated in countless human diseases including cancer, metabolic diseases, and neurological and developmental disorders. Kinases are regulated by upstream signals, which tune kinase enzymatic activity by means of an array of binding partners and post-translational modifications. Although in vitro reconstitution can be used to link binding partners and modifications to their effects on kinase enzymatic activity, an in vitro approach is time consuming, challenging to apply to multiple partners/modifications simultaneously, and not applicable to in vivo settings. This limitation has prevented the field from understanding how kinases integrate upstream signals to establish an appropriate level of activity. The long-term goal of the proposed research is to understand the biochemical basis of signal integration by protein kinases in vivo. To enable progress towards this goal, new experimental tools are needed because existing tools are unable to resolve the molecular state of a kinase molecule (i.e., its complement of modifications and binding partners) and simultaneously measure its activity. This project focuses on developing a single-molecule enzymatic assay for protein kinase activity. By studying single molecules, the applicants will overcome the limitations of ensemble averaging, which would otherwise preclude directly linking kinase modifications and binding partners to changes in activity because of the heterogenous nature of protein complexes in vivo. A single-molecule approach will also enable direct application to cellular protein kinases, eliminating the need for in vitro reconstitution. The central objective of this work is to gain new technical knowledge that will enable application of fluorescence-based kinase activity reporters in single molecule assays. The applicants will explore three different approaches for isolating single kinase molecules, measuring their activity and simultaneously determining which binding partners and post-translational modifications are present (Aim 1). Approaches that appear promising will then be tested on cell-derived kinase molecules to determine whether application of single-molecule kinase assays in vivo is feasible (Aim 2). The work proposed in this application is significant because it will establish the feasibility of a novel approach to measuring protein kinase activity, with the potential to ultimately yield fundamental insights into cellular signal transduction. The proposed work is innovative, in the applicant’s opinion, because it represents a fundamentally new paradigm for studying signaling by protein kinases. By establishing new tools with the potential to answer fundamental questions about signal integration by cellular protein kinases, this work will contribute to the advancement of basic biomedical research.