IDENTIFICATION OF PROTEIN KINASE SUBSTRATES

蛋白激酶底物的鉴定

• 批准号: 8363733
• 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/8363733
• 关键词: Address; Biochemical; Cell Communication; Cell Cycle; Cell physiology; Cells; Cellular Morphology; Chemicals; Engineering; Event; Exhibits; Funding; Genetic; Grant; Individual; Investigation; Mass Spectrum Analysis; Methods; National Center for Research Resources; PINK1 gene; Pathway interactions; Phosphorylation; Phosphotransferases; Play; Principal Investigator; Protein Kinase; Radiolabeled; Reaction; Regulation; Research; Research Infrastructure; Resources; Role; SRC gene; Source; Stress; Substrate Specificity; United States National Institutes of Health; cell motility; cost; in vivo; non-oncogenic; radiotracer; response

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. Kinase phosphorylation with a chemical tag to determine direct in vivo substrates. Eukaryotic protein kinases play a central role in controlling many cellular functions including cell-cell communication, cell-cycle entry/exit, cell morphology and motility, response to UV and O2 stress and many, many other functions. Often multiple kinases are involved in regulation of individual response pathways, making the assessment of the specific role of each kinase very difficult. This is due mainly to the fact that kinases exhibit overlapping substrate specificities which precludes the unambiguous assignment of the direct phosphorylation reaction catalyzed by each kinase in a given pathway. Recently, a new chemical method has been developed for directly tracing kinase substrates using an engineered kinase which accepts an unnatural phosphodonor with a [g-32P) radiolabel. This chemical approach to tracing pathways has been validated by its use in identification of the direct substrates of c-Src which has been under investigation for over 30 years using numerous genetic and biochemical methods. The same substrate tagging method can also be applied to the deconvolution of normal (non-oncogenic) kinase pathways in cells. The specific questions to be addressed are: 1) What substrates are phosphorylated by PINK1, GSK3beta, KSR1, RAF? 2) What are the functions of these phosphorylation events in their respective pathways?

该子项目是利用资源的众多研究子项目之一 由 NIH/NCRR 资助的中心拨款提供。子项目的主要支持 并且子项目的主要研究者可能是由其他来源提供的， 包括其他 NIH 来源。 子项目可能列出的总成本 代表子项目使用的中心基础设施的估计数量， NCRR 赠款不直接向子项目或子项目工作人员提供资金。 用化学标签进行激酶磷酸化以确定体内直接底物。真核蛋白激酶在控制许多细胞功能中发挥着核心作用，包括细胞间通讯、细胞周期进入/退出、细胞形态和运动、对紫外线和氧气应激的反应以及许多其他功能。通常多种激酶参与个体反应途径的调节，使得评估每种激酶的具体作用非常困难。这主要是由于激酶表现出重叠的底物特异性，这妨碍了给定途径中每种激酶催化的直接磷酸化反应的明确分配。最近，开发了一种新的化学方法，用于使用工程激酶直接追踪激酶底物，该激酶接受带有 [g-32P) 放射性标记的非天然磷酸供体。这种追踪途径的化学方法已通过其在 c-Src 直接底物鉴定中的应用得到验证，30 多年来，人们一直在使用多种遗传和生化方法对 c-Src 直接底物进行研究。相同的底物标记方法也可应用于细胞中正常（非致癌）激酶途径的解卷积。 需要解决的具体问题是： 1) PINK1、GSK3beta、KSR1、RAF 磷酸化哪些底物？ 2）这些磷酸化事件在各自途径中的功能是什么？