Weisheng Zhang, Nakayama Jun, Inomata Yukino, Higashiyama Shigeki, Hiratsuka Toru
Department of Molecular Oncology, Graduate School of Medicine, Osaka University.
Department of Oncogenesis and Growth Regulation, Research Center, Osaka International Cancer Institute.
Cell Struct Funct. 2025 Feb 7;50(1):15-24. doi: 10.1247/csf.24070. Epub 2024 Dec 18.
Extracellular signal-regulated kinase (ERK) regulates multiple cellular functions through distinct activation patterns. Genetically encoded fluorescent probes are instrumental in dissecting the ERK activity dynamics in living cells. Here we modified a previously reported Förster resonance energy transfer (FRET) probe for ERK, EKAREN5 by replacing its mTurquoise2 and YPet sequences with mTurquoise-GL and a synonymous codon variant of YPet, respectively. The modified biosensor, EKAREN5-gl, showed an increased sensitivity to EGF-induced ERK activation responding to a very low dose (20 pg/ml) of EGF stimulation. We quantitatively characterized two FRET-based ERK probes, EKAREN5 and EKAREN5-gl, and a subcellular kinase translocation-based probe, ERK-KTR. We found the three biosensors differently respond to EGF stimulations with different intensity, duration, and latency. Furthermore, we investigated how the minimal EGF-induced ERK activation affects the downstream transcription in HeLa cells by comprehensive transcriptional analysis. We found the minimal ERK activation leads to a distinct transcriptional pattern from those induced by higher ERK activations. Our study highlights the significance of sensitive fluorescent probes to understand cellular signal dynamics and the role of minimal ERK activation in regulating transcription.Key words: fluorescent probe, ERK, FRET, KTR.
细胞外信号调节激酶（ERK）通过不同的激活模式调节多种细胞功能。基因编码的荧光探针有助于剖析活细胞中的ERK活性动态。在此，我们对先前报道的用于ERK的荧光共振能量转移（FRET）探针EKAREN5进行了修饰，分别用mTurquoise-GL和YPet的同义密码子变体替换了其mTurquoise2和YPet序列。修饰后的生物传感器EKAREN5-gl对表皮生长因子（EGF）诱导的ERK激活表现出更高的敏感性，能响应极低剂量（20 pg/ml）的EGF刺激。我们对两种基于FRET的ERK探针EKAREN5和EKAREN5-gl以及一种基于亚细胞激酶易位的探针ERK-KTR进行了定量表征。我们发现这三种生物传感器对EGF刺激的反应在强度、持续时间和延迟方面各不相同。此外，我们通过全面的转录分析研究了最小的EGF诱导的ERK激活如何影响HeLa细胞中的下游转录。我们发现最小的ERK激活导致了与较高ERK激活诱导的转录模式不同的独特转录模式。我们的研究强调了灵敏的荧光探针对理解细胞信号动态的重要性以及最小的ERK激活在调节转录中的作用。关键词：荧光探针；ERK；FRET；KTR。
