Paul Joseph W, Muratcioğlu Serena, Kuriyan John
Department of Molecular and Cell Biology, University of California, Berkeley, CA, 94720 USA.
California Institute for Quantitative Bioscience (QB3), University of California, Berkeley, CA, 94720 USA.
bioRxiv. 2023 Dec 8:2023.12.07.570636. doi: 10.1101/2023.12.07.570636.
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 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 monitor the fluorescence of kinases fused to a fluorescent protein relative to that of a co-expressed reference fluorescent protein. 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 (SH2) and Src-homology 3 (SH3) 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.
致癌突变可使信号蛋白不稳定，导致活性增加或不受调控。因此，人们对绘制突变与蛋白质稳定性之间的关系非常感兴趣，以便更好地理解致癌突变的后果，并可能为新疗法的开发提供信息。在这里，我们开发了一种工具来研究活的哺乳动物细胞中蛋白激酶的稳定性以及热休克蛋白90（HSP90）伴侣系统对这些激酶稳定性的影响。我们监测与荧光蛋白融合的激酶相对于共表达的参考荧光蛋白的荧光。我们使用这个工具来研究Src和Raf家族激酶对HSP90系统的依赖性。我们证明这种传感器报告了这些激酶中致癌突变诱导的不稳定。我们还表明，Src家族激酶自身抑制所需的Src同源2（SH2）和Src同源3（SH3）结构域可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。
