IDENTIFICATION OF PROTEIN KINASE SUBSTRATES

这个子项目是利用资源的许多研究子项目之一。 由NIH/NCRR资助的中心拨款提供。对子项目的主要支持 子项目的首席调查员可能是由其他来源提供的， 包括美国国立卫生研究院的其他来源。为子项目列出的总成本可能 表示该子项目使用的中心基础设施的估计数量， 不是由NCRR赠款提供给次级项目或次级项目工作人员的直接资金。 用一个化学标记来确定直接体内底物的激酶磷酸化。真核蛋白激酶在许多细胞功能中起着核心作用，包括细胞间的通讯、细胞周期的进出、细胞的形态和运动、对紫外线和氧气胁迫的反应以及许多其他功能。通常情况下，多个激酶参与个体反应通路的调节，这使得评估每个激酶的具体作用非常困难。这主要是因为激酶表现出重叠的底物特异性，这排除了在给定的途径中每个激酶催化的直接磷酸化反应的明确分配。最近，一种新的化学方法已经被开发出来，使用一种接受带有[g-32P]放射性标记的非天然磷酸盐的工程激酶来直接示踪激酶底物。这种追踪途径的化学方法已经被用于鉴定c-Src的直接底物而得到验证，c-Src的直接底物已经使用多种遗传和生化方法进行了30多年的研究。同样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包括： 1)哪些底物被PINK1、GSK3β、KSR1、RAF磷酸化？ 2)这些磷酸化事件在各自的途径中有什么作用？

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?