IDENTIFICATION OF PROTEIN KINASE SUBSTRATES

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。次级项目的主要支助 子项目的主要研究者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。 激酶磷酸化与化学标签，以确定直接在体内底物。真核蛋白激酶在控制许多细胞功能中起核心作用，包括细胞-细胞通信、细胞周期进入/退出、细胞形态和运动性、对UV和O2应激的响应以及许多许多其他功能。通常，多种激酶参与调节个体反应途径，使得评估每种激酶的特定作用非常困难。这主要是由于激酶表现出重叠的底物特异性，这排除了在给定途径中由每个激酶催化的直接磷酸化反应的明确分配。最近，已经开发了一种新的化学方法，用于使用接受具有[g-32 P]放射性标记的非天然磷酸供体的工程化激酶直接追踪激酶底物。这种追踪途径的化学方法已经通过其在鉴定c-Src的直接底物中的使用而得到验证，c-Src的直接底物已经使用许多遗传和生物化学方法进行了30多年的研究。相同的底物标记方法也可以应用于细胞中正常（非致癌）激酶途径的去卷积。 需要解决的具体问题是： 1)PINK 1、GSK 3 β、KSR 1、RAF磷酸化的底物是什么？ 2)这些磷酸化事件在各自的通路中有什么功能？

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?