Department of Chemistry, Graduate School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
Department of Molecular Neuroscience, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, 2630 Sugitani, Toyama 930-0194, Japan.
Photochem Photobiol Sci. 2019 Nov 1;18(11):2740-2747. doi: 10.1039/c9pp00249a. Epub 2019 Oct 1.
Cyclic adenosine monophosphate (cAMP) response element-binding protein (CREB) is associated with memory formation and controls cell survival and proliferation via regulation of downstream gene expression in tumorigenesis. As a transcription factor, CREB binds to cAMP response elements. Phosphorylation of CREB triggers transcriptional activation of CREB downstream genes following the interaction of the kinase-inducible domain (KID) of CREB with the KID interaction domain (KIX) of CREB-binding protein. Nevertheless, because of the lack of single-cell analytical techniques, little is known about spatiotemporal regulation of CREB phosphorylation. To analyze CREB activation in single living cells, we developed genetically encoded bioluminescent sensors using luciferase-fragment complementation: the sensors are designed based on KID-KIX interaction with a single-molecule format. The luminescence intensity of the sensor, designated as CREX (a sensor of CREB activation based on KID(CREB)-KIX interaction), increased by phosphorylation of CREB. Moreover, the luminescence intensity of CREX was sufficient to detect CREB activation in live-cell bioluminescence imaging for single-cell analysis because of the higher sensitivity. CREX sensor is expected to contribute to elucidation of the spatiotemporal regulation of CREB phosphorylation by applying single-cell analysis.
环磷酸腺苷反应元件结合蛋白（CREB）与记忆形成有关，并通过调节肿瘤发生过程中下游基因的表达来控制细胞存活和增殖。作为一种转录因子，CREB 与 cAMP 反应元件结合。磷酸化的 CREB 触发 CREB 下游基因的转录激活，随后 CREB 的激酶诱导结构域（KID）与 CREB 结合蛋白的 KID 相互作用结构域（KIX）相互作用。然而，由于缺乏单细胞分析技术，关于 CREB 磷酸化的时空调控知之甚少。为了在单个活细胞中分析 CREB 的激活，我们使用荧光素酶片段互补开发了遗传编码的生物发光传感器：该传感器是基于 KID-KIX 相互作用设计的，具有单分子格式。该传感器的发光强度，命名为 CREX（基于 KID（CREB）-KIX 相互作用的 CREB 激活传感器），通过 CREB 的磷酸化而增加。此外，由于更高的灵敏度，CREX 的发光强度足以在活细胞生物发光成像中单细胞分析中检测到 CREB 的激活。CREX 传感器有望通过单细胞分析为阐明 CREB 磷酸化的时空调控做出贡献。
