CHEMICAL GENETIC IDENTIFICATION OF DIRECT KINASE SUBSTRATES

直接激酶底物的化学遗传学鉴定

• 批准号: 8363761
• 负责人: KEVAN M. SHOKAT
• 金额: --
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-06-01 至 2012-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8363761
• 关键词: Affinity; Alkylation; Antibodies; Chemicals; Engineering; Epitopes; Funding; Grant; Individual; Label; Mass Spectrum Analysis; Methods; National Center for Research Resources; Phosphorylation; Phosphotransferases; Post-Translational Protein Processing; Principal Investigator; Protein Kinase; Research; Research Infrastructure; Resources; Signal Transduction; Source; United States National Institutes of Health; chemical genetics; cost; hydroxyl group; immunoaffinity chromatography; interest; phosphorothioate

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Protein phosphorylation is a major mechanism of post-translational protein modification used to control cellular signalling. A challenge in phosphoproteomics is to identify the direct substrates of each protein kinase. We have developed a chemical strategy for delivery of a bio-orthogonal affinity tag to the substrates of an individual protein kinase. The kinase of interest is engineered to transfer a phosphorothioate moiety to phosphoacceptor hydroxyl groups on direct substrates. In a second non-enzymatic step, the introduced phosphorothioate is alkylated with p-nitrobenzylmesylate (PNBM). Antibodies directed against the modified phosphorothioate epitope recognize these labelled substrates, but not alkylation products of other cellular nucleophiles. Immunoaffinity chromatography allows the purification of these substrates, and mass spectrometry provides an attractive method for their rapid identification.

这个子项目是许多利用资源的研究子项目之一 由NIH/NCRR资助的中心拨款提供。子项目的主要支持 而子项目的主要调查员可能是由其他来源提供的， 包括其它NIH来源。 列出的子项目总成本可能 代表子项目使用的中心基础设施的估计数量， 而不是由NCRR赠款提供给子项目或子项目工作人员的直接资金。 蛋白质磷酸化是用于控制细胞信号传导的翻译后蛋白质修饰的主要机制。 磷酸化蛋白质组学的一个挑战是确定每个蛋白激酶的直接底物。 我们已经开发了一种化学策略，用于将生物正交亲和标签递送到单个蛋白激酶的底物。 所关注的激酶经工程改造以将硫代磷酸酯部分转移至直接底物上的磷酸受体羟基。 在第二非酶促步骤中，引入的硫代磷酸酯用甲磺酸对硝基苄酯（PNBM）烷基化。 针对修饰的硫代磷酸酯表位的抗体识别这些标记的底物，但不识别其他细胞亲核试剂的烷基化产物。 免疫亲和层析允许这些底物的纯化，质谱法提供了一种有吸引力的方法，用于其快速鉴定。