Chemical Rescue of Protein Kinases for Cell Signaling Applications

用于细胞信号传导应用的蛋白激酶的化学救援

基本信息

批准号：
10331818
负责人：
Brad Andrew Palanski
金额：
$ 6.67万
依托单位：
BRIGHAM AND WOMEN'S HOSPITAL
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-02-01 至 2022-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10331818
关键词：
Active Sites Address Adopted Area Back Biology Biomedical Research CRISPR/Cas technology Catalytic Domain Cell Line Cell model Cell physiology Cells Cellular biology Chemicals Complement Cyclic AMP-Dependent Protein Kinases Development Disease Engineering Enzymatic Biochemistry Enzymes Functional disorder Genes Goals Health Human Imidazole Impairment In Vitro Jurkat Cells Kinetics Laboratories Laboratory Chemicals Life Ligands Malignant Neoplasms Mammalian Cell Mass Spectrum Analysis Methods Modeling Modernization Mutate Mutation Neuroblastoma Oncogenic Pathway interactions Phenotype Phosphorylation Phosphotransferases Physiological Physiology Play Point Mutation Positioning Attribute Protein Analysis Protein Kinase Proteins Proteomics Publishing Receptor Protein-Tyrosine Kinases Regulation Research Role Signal Pathway Signal Transduction Signaling Protein Specificity Structure System T-Cell Activation T-Lymphocyte Techniques Technology Training Work ZAP-70 Gene anaplastic lymphoma kinase base career cellular targeting comparative experimental study extracellular human disease interest mutant phosphoproteomics preservation protein-tyrosine kinase c-src rapid technique rational design receptor skills small molecule small molecule libraries success virtual

项目摘要

ABSTRACT This is a new F32 application to develop and apply chemical rescue technology to the functional analysis of protein kinases. Protein kinases regulate virtually every aspect of cellular physiology in health and disease through phosphorylation of their substrates, typically on Tyr and Ser/Thr residues. Despite intense efforts even the most “well-studied” protein kinases remain incompletely understood. Methods to study protein kinase signaling in cellular contexts are urgently needed, but unfortunately, few methods exist for activating specific kinases in living cells. The techniques that are available for this purpose require extensive engineering, which raises concerns about preservation of natural regulatory mechanisms and has likely limited their widespread application. Previously, our lab developed chemical rescue as a method for rapidly activating protein Tyr kinases in living cells. A single point mutant is generated within the catalytic site, which renders the kinase catalytically inactive without disturbing its structure or regulation. A small molecule is then added which complements this mutation and rapidly switches the kinase back on in a reversible manner. This technique is conceptually simple and has been proven useful in dissecting cell signaling of the protein Tyr kinases Csk, Src, and Abl. However, due to the historical difficulty with generating point mutants in mammalian cells, chemical rescue has not yet been widely applied. Additionally, a chemical rescue technique has not been developed for Ser/Thr kinases, which make up approximately 80% of the human kinome. This proposal seeks to overcome both of these limitations. By using CRISPR/Cas9 gene editing to introduce the required point mutations in model cell lines, we will use chemical rescue to identify substrates and cell signaling pathways of two enigmatic Tyr kinases. In Aim 1, ALK, a receptor Tyr kinase commonly mutated in cancer but with unclear physiological function will be analyzed in its wild-type and oncogenic forms to identify its cellular targets. In Aim 2, ZAP70, a nonreceptor Tyr kinase essential for T-cell activation but with very few known substrates will be investigated. For these two aims, we will use an unbiased mass spectrometry based phosphoproteomic approach to identify substrate proteins and activated signaling pathways. In Aim 3, we will develop proof-of-concept for chemical rescue of Ser/Thr kinases using the model protein Akt1. Collectively, this work will open new doors to investigating kinase signaling and advance our understanding of three key enzymes in biomedical research. In addition, this proposal builds on the chemical biology background of the PI and promises to greatly broaden the PI's skills in enzymology, mass spectrometry, and cell biology. It is anticipated that the training plan outlined here will position the PI for success in an independent academic research career.

抽象的这是一个新的 F32 应用程序，旨在开发化学救援技术并将其应用于功能分析蛋白激酶几乎调节健康和疾病中细胞生理学的各个方面。尽管付出了巨大的努力，但它们的底物（通常是 Tyr 和 Ser/Thr 残基）被磷酸化。研究得最充分的蛋白激酶仍然不完全清楚研究蛋白激酶的方法。迫切需要细胞环境中的信号传导，但不幸的是，很少有方法可以激活特定的信号活细胞中的激酶可用于此目的的技术需要大量的工程设计。人们对自然调节机制的保护提出了担忧，并可能限制了其广泛应用此前，我们的实验室开发了化学救援作为快速激活蛋白酪氨酸激酶的方法。在活细胞中，催化位点内产生单点突变体，从而使激酶具有催化作用。然后添加一个小分子来补充它，使其失去活性。突变并以可逆的方式快速重新开启激酶，这种技术在概念上很简单。并已被证明可用于剖析蛋白质 Tyr 激酶 Csk、Src 和 Abl 的细胞信号传导。由于在哺乳动物细胞中产生点突变体的历史困难，化学救援尚未此外，尚未开发出针对 Ser/Thr 激酶的化学救援技术。约占人类激酶组的 80%。该提案旨在克服这两个问题。通过使用 CRISPR/Cas9 基因编辑在模型细胞系中引入所需的点突变，我们 In Aim 将使用化学救援来鉴定两种神秘 Tyr 激酶的底物和细胞信号通路。 1、ALK，一种在癌症中常见突变但生理功能尚不清楚的受体酪氨酸激酶在 Aim 2 中，对其野生型和致癌形式进行分析，以确定其细胞靶点 ZAP70（一种非受体 Tyr）。为了这两个目的，我们将研究 T 细胞激活所必需的激酶，但已知的底物很少。我们将使用基于磷酸化蛋白质组学方法的无偏质谱来鉴定底物蛋白在目标 3 中，我们将开发 Ser/Thr 化学救援的概念验证。总的来说，这项工作将为研究激酶信号传导打开新的大门。并增进我们对生物医学研究中三种关键酶的理解。此外，该提案还建立了。基于 PI 的化学生物学背景，并有望大大拓宽 PI 在酶学方面的技能，预计此处概述的培训计划将为 PI 定位。在独立学术研究生涯中取得成功。