The cAMP-dependent protein kinase inhibitor H-89 attenuates the bioluminescence signal produced by Renilla Luciferase.
The cAMP-dependent protein kinase inhibitor H-89 attenuates the bioluminescence signal produced by Renilla Luciferase.
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DOI：
10.1371/journal.pone.0005642
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发表时间：
2009-05-21
期刊：
影响因子：
3.7
通讯作者：
Zhang J
中科院分区：
文献类型：
--
作者：
Herbst KJ;Allen MD;Zhang J
对激酶（例如 cAMP 依赖性蛋白激酶 (PKA)）的调节和功能作用的研究越来越依赖于细胞测定。目前，有许多基于生物发光的检测，例如报告基因检测，可以研究 PKA 在细胞环境中的调节、活性和功能效应。此外，人们还在不断努力设计改进的生物传感器，使其能够检测细胞中实时 PKA 信号动态。这些基于细胞的测定通常用于通过使用 H-89（一种 PKA 可逆竞争性抑制剂）来测试 PKA 依赖性过程的参与情况。我们在此提供的数据表明，H-89 除了是一种竞争性 PKA 抑制剂之外，还能减弱细胞群和单个细胞中海肾荧光素酶 (RLuc) 变体产生的生物发光信号。使用 10 µM 荧光素酶底物和 10 µM H-89，我们观察到细胞中来自 RLuc 和 RLuc8（RLuc 的八点突变变体）的信号分别降低至仅暴露于媒介物的对照的 50% (±15%) 和 54% (±14%)。在体外，我们发现H-89降低了RLuc8生物发光信号，但不与腔肠素-h竞争RLuc8活性位点，并且也不影响萤火虫荧光素酶的活性。相比之下，另一种 PKA 竞争性抑制剂 KT5720 并不影响 RLuc8 的活性。 H-89 对 RLuc 信号不利影响的识别和表征将有助于对之前基于 RLuc 的检测生成的数据进行解卷积，以研究 PKA 信号传导的功能影响。此外，对于生物发光检测的当前应用和未来发展，KT5720被认为是更适合与基于RLuc的检测结合使用的PKA抑制剂。这些主要发现还提供了一个重要的教训，即在从数据中得出结论之前，充分考虑实验条件对基于细胞的测定读数的所有潜在影响。
Investigations into the regulation and functional roles of kinases such as cAMP-dependent protein kinase (PKA) increasingly rely on cellular assays. Currently, there are a number of bioluminescence-based assays, for example reporter gene assays, that allow the study of the regulation, activity, and functional effects of PKA in the cellular context. Additionally there are continuing efforts to engineer improved biosensors that are capable of detecting real-time PKA signaling dynamics in cells. These cell-based assays are often utilized to test the involvement of PKA-dependent processes by using H-89, a reversible competitive inhibitor of PKA. We present here data to show that H-89, in addition to being a competitive PKA inhibitor, attenuates the bioluminescence signal produced by Renilla luciferase (RLuc) variants in a population of cells and also in single cells. Using 10 µM of luciferase substrate and 10 µM H-89, we observed that the signal from RLuc and RLuc8, an eight-point mutation variant of RLuc, in cells was reduced to 50% (±15%) and 54% (±14%) of controls exposed to the vehicle alone, respectively. In vitro, we showed that H-89 decreased the RLuc8 bioluminescence signal but did not compete with coelenterazine-h for the RLuc8 active site, and also did not affect the activity of Firefly luciferase. By contrast, another competitive inhibitor of PKA, KT5720, did not affect the activity of RLuc8. The identification and characterization of the adverse effect of H-89 on RLuc signal will help deconvolute data previously generated from RLuc-based assays looking at the functional effects of PKA signaling. In addition, for the current application and future development of bioluminscence assays, KT5720 is identified as a more suitable PKA inhibitor to be used in conjunction with RLuc-based assays. These principal findings also provide an important lesson to fully consider all of the potential effects of experimental conditions on a cell-based assay readout before drawing conclusions from the data.
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