Genetically-encoded biosensors based on Förster/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.

中文翻译：

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基于KISS技术识别的共有底物序列的岩石FRET生物传感器。

基于Förster/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以及组织内信号分子的时空调控的多功能工具。开发用于蛋白激酶的FRET生物传感器的最艰巨任务可能是确定要在FRET生物传感器中使用的激酶特异性底物肽。为解决此问题，我们利用了激酶相互作用底物筛选（KISS）技术，该技术推导了目标蛋白激酶的共有底物序列。在这里，我们显示了由KISS鉴定的ROCK的共有底物序列产生了ROCK的FRET生物传感器，名为Eevee-ROCK，具有高灵敏度和特异性。通过用针对ROCK的抑制剂或siRNA处理HeLa细胞，我们显示Eevee-ROCK的基础FRET信号的很大一部分来自ROCK1和ROCK2的活动。Eevee-ROCK容易检测到表皮生长因子，溶血磷脂酸和血清对ROCK的活化作用。时延成像稳定表达Eevee-ROCK的细胞三天后，发现胞质分裂完成后ROCK活性增加，并伴有细胞扩散。Eevee-ROCK还显示出凋亡过程中ROCK活性的逐渐增加。因此，由KISS技术预测的底物序列开发的Eevee-ROCK将为更好地了解ROCK在生理环境中的功能铺平道路。溶血磷脂酸和血清。时延成像稳定表达Eevee-ROCK的细胞三天后，发现胞质分裂完成后ROCK活性增加，并伴有细胞扩散。Eevee-ROCK还显示出凋亡过程中ROCK活性的逐渐增加。因此，由KISS技术预测的底物序列开发的Eevee-ROCK将为更好地了解ROCK在生理环境中的功能铺平道路。溶血磷脂酸和血清。时延成像稳定表达Eevee-ROCK的细胞三天后，发现胞质分裂完成后ROCK活性增加，并伴有细胞扩散。Eevee-ROCK还显示出凋亡过程中ROCK活性的逐渐增加。因此，由KISS技术预测的底物序列开发的Eevee-ROCK将为更好地了解ROCK在生理环境中的功能铺平道路。