A FRET Biosensor for ROCK Based on a Consensus Substrate Sequence Identified by KISS Technology
A FRET Biosensor for ROCK Based on a Consensus Substrate Sequence Identified by KISS Technology
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DOI：
10.1247/csf.16016
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发表时间：
2017-01-01
影响因子：
1.5
通讯作者：
Matsuda, Michiyuki
中科院分区：
文献类型：
--
作者：
Li, Chunjie;Imanishi, Ayako;Matsuda, Michiyuki
基于Frster/荧光共振能量转移（FRET）的基因编码生物传感器是研究细胞内和组织内信号分子时空调控的通用工具。也许最困难的任务，在发展的FRET生物传感器的蛋白激酶是确定的激酶特异性底物肽的FRET生物传感器中使用。为了解决这个问题，我们利用了激酶相互作用底物筛选（KISS）技术，该技术推导出感兴趣的蛋白激酶的共有底物序列。在这里，我们表明，由KISS确定的ROCK的共识底物序列产生了一个FRET生物传感器的ROCK，命名为Eevee-ROCK，具有高灵敏度和特异性。通过用ROCK抑制剂或siRNA处理HeLa细胞，我们发现Eevee-ROCK的基础FRET信号的相当大一部分来自ROCK 1和ROCK 2的活性。Eevee-ROCK很容易检测到表皮生长因子、溶血磷脂酸和血清对ROCK的激活。当对稳定表达Eevee-ROCK的细胞进行三天的延时成像时，发现ROCK活性在胞质分裂完成后增加，伴随着细胞的扩散。Eevee-ROCK还显示在细胞凋亡过程中ROCK活性逐渐增加。因此，从KISS技术预测的底物序列开发的Eevee-ROCK将为更好地理解ROCK在生理环境中的功能铺平道路。
Genetically-encoded biosensors based on Frster/fluorescence resonance energy transfer (FRET) are versatile tools for studying the spatio-temporal regulation of signaling molecules within not only the cells but also tissues. Perhaps the hardest task in the development of a FRET biosensor for protein kinases is to identify the kinase-specific substrate peptide to be used in the FRET biosensor. To solve this problem, we took advantage of kinase-interacting substrate screening (KISS) technology, which deduces a consensus substrate sequence for the protein kinase of interest. Here, we show that a consensus substrate sequence for ROCK identified by KISS yielded a FRET biosensor for ROCK, named Eevee-ROCK, with high sensitivity and specificity. By treating HeLa cells with inhibitors or siRNAs against ROCK, we show that a substantial part of the basal FRET signal of Eevee-ROCK was derived from the activities of ROCK1 and ROCK2. Eevee-ROCK readily detected ROCK activation by epidermal growth factor, lysophosphatidic acid, and serum. When cells stably-expressing Eevee-ROCK were time-lapse imaged for three days, ROCK activity was found to increase after the completion of cytokinesis, concomitant with the spreading of cells. Eevee-ROCK also revealed a gradual increase in ROCK activity during apoptosis. Thus, Eevee-ROCK, which was developed from a substrate sequence predicted by the KISS technology, will pave the way to a better understanding of the function of ROCK in a physiological context.