Chemical Rescue of Protein Kinases for Cell Signaling Applications

摘要 这是一个新的F32应用程序，用于开发和应用化学救援技术来进行功能分析 蛋白激酶。在健康和疾病中，蛋白激酶实际上调节细胞生理的方方面面 通过底物的磷酸化，通常是在Tyr和Ser/Thr残基上。尽管付出了巨大的努力 最“被充分研究”的蛋白激酶仍然没有完全被理解。蛋白激酶的研究方法 细胞环境中的信令是迫切需要的，但不幸的是，激活特定信号的方法很少 活细胞中的蛋白水解酶。可用于此目的的技术需要广泛的工程设计，这 引发了人们对自然监管机制保护的担忧，并可能限制了它们的广泛应用 申请。在此之前，我们的实验室开发了一种化学抢救方法，作为一种快速激活蛋白酪氨酸激酶的方法 在活细胞中。在催化部位产生一个单点突变体，使该激酶具有催化作用 不活跃的不扰乱其结构或规则的。然后加入一个小分子，以补充这一点 突变，并以可逆的方式迅速将该激酶重新启动。这项技术在概念上很简单 并已被证明在剖析酪氨酸蛋白激酶CSK、Src和Abl的细胞信号中是有用的。然而， 由于在哺乳动物细胞中产生点突变的历史困难，化学救援还没有 得到了广泛的应用。此外，还没有开发出针对丝氨酸/苏氨酸激酶的化学救援技术， 它们约占人类亲属组的80%。这项提议试图克服这两个问题。 限制。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 将使用化学拯救来确定两个神秘的Tyr激酶的底物和细胞信号通路。在AIM 1、ALK，一种在癌症中常见突变但生理功能尚不清楚的受体Tyr激酶将被 分析其野生型和致癌形式，以确定其细胞靶标。在AIM 2中，非受体酪氨酸ZAP70 T细胞激活所必需的、但底物很少的激酶将被研究。为了这两个目标， 我们将使用基于无偏向质谱学的磷酸蛋白质组学方法来鉴定底物蛋白质 并激活了信号通路。在目标3中，我们将为Ser/Thr的化学救援开发概念验证 使用模型蛋白Akt1的激活酶。总的来说，这项工作将为研究激酶信号转导打开新的大门 并加深了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 关于PI的化学生物学背景，并有望极大地拓宽PI在酶学方面的技能， 质谱学和细胞生物学。预计此处概述的培训计划将为 在独立的学术研究事业中取得成功。

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.