A Bio-orthogonal Approach for Identifying Direct Kinase Substrates and their phosphorylation sites

通过这一奖项，化学系的生命过程化学项目为普渡大学的Kavita Shah博士提供资金。这位研究人员正在开发一种高通量的化学工具，以识别和验证一类名为激酶的酶的直接靶标。激酶是与细胞中的蛋白质进行磷酸化反应的关键细胞调节因子。它们在支持每个组织独特生理学的生物学结果中起着重要作用。据估计，一个细胞中约有33,000种蛋白质被激酶磷酸化。目前，要识别518个人类激酶中的每一个都是一项艰巨的努力。该项目正在开发特殊的探针，具有极高的灵敏度，可以专门标记和分离激酶靶标。该方法具有广泛的适用性，可用于鉴定和验证细胞中任何所需的激酶的靶标。该项目允许研究生、本科生和博士后研究员获得高度跨学科的培训。培训的科学领域包括化学合成、蛋白质工程和酶分析。该项目纳入了一个由跨学科化学生物学课程组成的外展计划。本课程是为化学专业本科生和研究生开设的。它还包括普渡大学通过特定项目招收的未被充分代表的本科生的研究培训计划的一部分。化学生物学课程也被用作高中教师的专业发展机会。总体目标是增加对高中生、代表性不足的少数族裔和研究生的STEM教育和保留。这项提议的目标是开发一种高通量生物正交化学方法，能够在蛋白质组范围内快速鉴定低丰度的激酶底物及其磷酸化位点。“捕捉和释放”策略，与创新的化学遗传方法相结合，被用来特别地标记独特的磷酸标记，任何所需的感兴趣的激酶的底物。这种方法的精致敏感性来自于磷酸标签，它将用于选择性地从整个蛋白质组中分离底物，而不需要任何其他分离技术。这些底物使用CRISPR设计的细胞系和正交抑制剂在高通量筛选中得到验证。一些选定的底物被用作揭示这些激酶调节各种细胞过程的机制的线索，可能导致发现新的途径和网络。快速而全面地分析所需的激酶底物及其磷酸化位置对于剖析该激酶在激活不同的信号级联中的作用是至关重要的。

With this award, the Chemistry of Life Processes Program in the Chemistry Division is funding Dr. Kavita Shah from Purdue University. The investigator is developing a high-throughput chemical tool to identify and validate the direct targets of a class of enzymes called kinases. Kinases are key cell regulators that perform phosphorylation reactions with proteins in the cell. They have an important role in biological outcomes that support each tissue's unique physiology. It is estimated that ~33,000 proteins are phosphorylated by kinases in a cell. At present, it is a formidable endeavor to identify each of the 518 human kinases. This project is developing special probes, with exquisite sensitivity for specifically tagging and isolating kinase targets. This method is widely applicable and could be used to identify and validate the targets of any desired kinase in cells. This project allows graduate students, undergraduate students, and postdoctoral fellows to acquire highly interdisciplinary training. The scientific areas of training include chemical synthesis, protein engineering, and enzymatic assays. This project is integrated into an outreach program which consists of an interdisciplinary Chemical Biology course. This course is designed for Chemistry majors and graduate students. It also comprises part of a research training program for under-represented undergraduates recruited through specific programs at Purdue University. The Chemical Biology course is also used as a professional development opportunity for high school teachers. The overall goal is to increase STEM education and retention for high school students, under-represented minorities, and graduate students.The goal of this proposal is to develop a high-throughput bio-orthogonal chemical approach that enables rapid identification of low abundance kinase substrates and their phosphorylation sites on a proteome-wide scale. A "catch and release" strategy, in combination with an innovative chemical genetic approach, is used to specifically label with a unique phospho-tag, the substrates of any desired kinase of interest. The exquisite sensitivity of this approach stems from the phospho-tag, which will be used to selectively isolate substrates from the whole proteome, without the need for any other separation techniques. These substrates are validated in a high throughput screen using CRISPR-engineered cell lines and orthogonal inhibitors. A few chosen substrates are used as cues to uncover the mechanisms by which these kinases regulate various cellular processes, potentially leading to the discovery of novel pathways and networks. A rapid and comprehensive analysis of a desired kinase's substrates and their phosphorylation sites is crucial for dissecting the contribution of the kinase in activating distinct signaling cascades.