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

• 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
• 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

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.

摘要 这是一个新的F32应用开发和应用化学救援技术的功能分析， 蛋白激酶蛋白激酶几乎调节健康和疾病中细胞生理学的各个方面 通过其底物的磷酸化，通常在Tyr和Ser/Thr残基上。尽管付出了巨大的努力， 大多数“充分研究的”蛋白激酶仍然没有完全了解。研究蛋白激酶的方法 细胞环境中的信号传导是迫切需要的，但不幸的是，很少有方法存在激活特异性的， 活细胞中的激酶。可用于此目的的技术需要广泛的工程， 引起了人们对保护自然调节机制的担忧，并可能限制了它们的广泛传播。 应用程序.在此之前，我们的实验室开发了化学救援作为快速激活蛋白质Tyr激酶的方法 在活细胞中。在催化位点内产生单点突变体，这使得激酶催化 不活跃而不干扰其结构或调节。然后加入一种小分子， 突变并以可逆的方式迅速将激酶重新打开。这种技术在概念上很简单 并且已被证明可用于剖析蛋白质Tyr激酶Csk、Src和Tyr的细胞信号传导。然而， 由于在哺乳动物细胞中产生点突变体的历史困难， 被广泛应用。此外，尚未开发用于Ser/Thr激酶的化学拯救技术， 占人类激酶组的80%这项建议旨在克服这两个问题 局限性。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， 将使用化学拯救来识别两种神秘的Tyr激酶的底物和细胞信号通路。在Aim中 1，ALK，一种在癌症中通常突变但生理功能不清楚的受体Tyr激酶，将被用于治疗癌症。 分析其野生型和致癌形式以鉴定其细胞靶点。在Aim 2中，ZAP 70，一种非受体Tyr 将研究T细胞活化所必需的激酶，但已知底物很少。为了这两个目标， 我们将使用基于无偏质谱的磷酸蛋白质组学方法来鉴定底物蛋白质， 并激活信号通路。在目标3中，我们将开发Ser/Thr的化学拯救的概念验证 使用模型蛋白Akt 1的激酶。总的来说，这项工作将为研究激酶信号转导打开新的大门 并促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 对PI的化学生物学背景，并承诺大大拓宽PI的酶学技能， 质谱和细胞生物学。预计此处概述的培训计划将使PI定位于 在独立的学术研究生涯中取得成功。