Optogenetically controlled RAF to characterize BRAF and CRAF protein kinase inhibitors.
Optogenetically controlled RAF to characterize BRAF and CRAF protein kinase inhibitors.
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DOI：
10.1038/srep23713
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发表时间：
2016-03-30
影响因子：
4.6
通讯作者：
Radziwill G
中科院分区：
文献类型：
--
作者：
Chatelle CV;Hövermann D;Müller A;Wagner HJ;Weber W;Radziwill G
在这里，我们应用了optoRAF，这是一种光遗传学工具，用于光控聚类和激活RAF蛋白，模拟自然发生的ras介导的二聚化。这个多功能工具允许研究对BRAF和CRAF同二聚体以及异二聚体诱导的RAF信号的影响。Vemurafenib和dabrafenib是两种临床批准的BRAF抑制剂，可有效抑制致癌BRAF （V600E）的激酶活性。然而，在野生型表达BRAF的细胞中，BRAF抑制剂可以发挥野生型CRAF的矛盾激活。使用optoRAF， vemurafenib被鉴定为BRAF和CRAF同源和异源二聚体的矛盾激活剂。低剂量Dabrafenib增强光刺激CRAF活性，高剂量Dabrafenib抑制CRAF信号传导。此外，dabrafenib增加了CRAF蛋白的蛋白水平，但没有增加BRAF蛋白的蛋白水平。CRAF水平的增加与RAF信号的升高相关，以dabrafenib依赖的方式，独立于光激活。
Here, we applied optoRAF, an optogenetic tool for light-controlled clustering and activation of RAF proteins that mimics the natural occurring RAS-mediated dimerization. This versatile tool allows studying the effect on BRAF and CRAF homodimer- as well as heterodimer-induced RAF signaling. Vemurafenib and dabrafenib are two clinically approved inhibitors for BRAF that efficiently suppress the kinase activity of oncogenic BRAF (V600E). However in wild-type BRAF expressing cells, BRAF inhibitors can exert paradoxical activation of wild-type CRAF. Using optoRAF, vemurafenib was identified as paradoxical activator of BRAF and CRAF homo- and heterodimers. Dabrafenib enhanced activity of light-stimulated CRAF at low dose and inhibited CRAF signaling at high dose. Moreover, dabrafenib increased the protein level of CRAF proteins but not of BRAF proteins. Increased CRAF levels correlate with elevated RAF signaling in a dabrafenib-dependent manner, independent of light activation.