Abstract:
Oncogenic mutations can destabilize signaling proteins, resulting in increased or unregulated activity. Thus, there is considerable interest in mapping the relationship between mutations and the stability of signaling proteins, to better understand the consequences of oncogenic mutations and potentially inform the development of new therapeutics. Here, we develop a tool to study protein-kinase stability in live mammalian cells and the effects of the HSP90 chaperone system on the stability of these kinases. We determine the expression levels of protein kinases by monitoring the fluorescence of fluorescent proteins fused to those kinases, normalized to that of co-expressed reference fluorescent proteins. We used this tool to study the dependence of Src- and Raf-family kinases on the HSP90 system. We demonstrate that this sensor reports on destabilization induced by oncogenic mutations in these kinases. We also show that Src-homology 2 and Src-homology 3 domains, which are required for autoinhibition of Src-family kinases, stabilize these kinase domains in the cell. Our expression-calibrated sensor enables the facile characterization of the effects of mutations and small-molecule drugs on protein-kinase stability.
摘要:
致癌突变可以破坏信号蛋白的稳定性,导致活动增加或不受调节。因此,有相当大的兴趣在映射突变和信号蛋白的稳定性之间的关系,为了更好地了解致癌突变的后果,并有可能为新疗法的开发提供信息。这里,我们开发了一种工具来研究活哺乳动物细胞中蛋白激酶的稳定性以及HSP90伴侣系统对这些激酶稳定性的影响。我们通过监测与这些激酶融合的荧光蛋白的荧光来确定蛋白激酶的表达水平,归一化为共表达的参考荧光蛋白。我们使用此工具来研究Src和Raf家族激酶对HSP90系统的依赖性。我们证明该传感器报告了由这些激酶中的致癌突变诱导的去稳定。我们还显示了Src-同源性2和Src-同源性3结构域,是自身抑制Src家族激酶所必需的,稳定细胞中的这些激酶结构域。我们的表达校准传感器能够轻松表征突变和小分子药物对蛋白激酶稳定性的影响。