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

鉴定直接激酶底物及其磷酸化位点的生物正交方法

• 批准号: 1708823
• 负责人: Kavita Shah
• 金额: $ 50万
• 依托单位: Purdue University
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-08-01 至 2022-07-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1708823&HistoricalAwards=false
• 关键词: Bio; orthogonal; Approach; Identifying; Direct

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.

有了这个奖项，化学部门的生命过程化学项目资助了普渡大学的Kavita Shah博士。研究者正在开发一种高通量的化学工具来识别和验证一类被称为激酶的酶的直接靶标。激酶是细胞中与蛋白质发生磷酸化反应的关键细胞调节剂。它们在支持每个组织独特生理机能的生物学结果中起着重要作用。据估计，在一个细胞中约有33,000种蛋白质被激酶磷酸化。目前，鉴定518种人类激酶是一项艰巨的任务。该项目正在开发特殊探针，具有特异性标记和分离激酶靶点的灵敏度。该方法广泛适用，可用于识别和验证细胞中任何所需激酶的靶标。该项目允许研究生、本科生和博士后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。这个项目被整合到一个由跨学科化学生物学课程组成的外展计划中。本课程专为化学专业学生和研究生设计。它还包括普渡大学通过特定项目招募的代表性不足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专业发展的机会。总体目标是提高高中学生、少数族裔和研究生的STEM教育和保留率。本提案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激酶底物及其磷酸化位点。一种“捕获和释放”策略，结合一种创新的化学遗传方法，被用来特异性地标记一个独特的磷酸标签，任何感兴趣的激酶的底物。这种方法的灵敏度源于磷酸标签，它将被用来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。这些底物在高通量筛选中使用crispr工程细胞系和正交抑制剂进行验证。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。快速和全面分析所需激酶的底物及其磷酸化位点对于解剖激酶在激活不同信号级联反应中的作用至关重要。

项目成果

The significant others: Global search for direct kinase substrates using chemical approaches

其他重要的：使用化学方法全球搜索直接激酶底物

• DOI: 10.1002/iub.2023
• 发表时间: 2019
• 期刊: IUBMB Life
• 影响因子: 4.6
• 作者: Shah, Kavita;Kim, Hyunjin
• 通讯作者: Kim, Hyunjin

LIMK2-NKX3.1 Engagement Promotes Castration-Resistant Prostate Cancer.

• DOI: 10.3390/cancers13102324
• 发表时间: 2021-05-12
• 期刊: Cancers
• 影响因子: 5.2
• 作者: Sooreshjani MA;Nikhil K;Kamra M;Nguyen DN;Kumar D;Shah K
• 通讯作者: Shah K

Reciprocal deregulation of NKX3.1 and AURKA axis in castration-resistant prostate cancer and NEPC models.

• DOI: 10.1186/s12929-021-00765-z
• 发表时间: 2021-10-08
• 期刊: Journal of biomedical science
• 影响因子: 11
• 作者: Sooreshjani MA;Kamra M;Zoubeidi A;Shah K
• 通讯作者: Shah K

Aurora Kinase A-YBX1 Synergy Fuels Aggressive Oncogenic Phenotypes and Chemoresistance in Castration-Resistant Prostate Cancer

• DOI: 10.3390/cancers12030660
• 发表时间: 2020-03-01
• 期刊: CANCERS
• 影响因子: 5.2
• 作者: Nikhil, Kumar;Raza, Asif;Shah, Kavita
• 通讯作者: Shah, Kavita