Institute of Biomedical Engineering and Health Sciences, School of Medical and Health Engineering, Changzhou University, Changzhou 213164, China.
School of Pharmacy, Changzhou University, Changzhou 213164, China.
Biosensors (Basel). 2024 Apr 20;14(4):206. doi: 10.3390/bios14040206.
C-terminal Src kinase (CSK) is the major inhibitory kinase for Src family kinases (SFKs) through the phosphorylation of their C-tail tyrosine sites, and it regulates various types of cellular activity in association with SFK function. As a cytoplasmic protein, CSK needs be recruited to the plasma membrane to regulate SFKs' activity. The regulatory mechanism behind CSK activity and its subcellular localization remains largely unclear. In this work, we developed a genetically encoded biosensor based on fluorescence resonance energy transfer (FRET) to visualize the CSK activity in live cells. The biosensor, with an optimized substrate peptide, confirmed the crucial Arg site in the CSK SH2 domain and displayed sensitivity and specificity to CSK activity, while showing minor responses to co-transfected Src and Fyn. FRET measurements showed that CSK had a relatively mild level of kinase activity in comparison to Src and Fyn in rat airway smooth muscle cells. The biosensor tagged with different submembrane-targeting signals detected CSK activity at both non-lipid raft and lipid raft microregions, while it showed a higher FRET level at non-lipid ones. Co-transfected receptor-type protein tyrosine phosphatase alpha (PTPα) had an inhibitory effect on the CSK FRET response. The biosensor did not detect obvious changes in CSK activity between metastatic cancer cells and normal ones. In conclusion, a novel FRET biosensor was generated to monitor CSK activity and demonstrated CSK activity existing in both non-lipid and lipid raft membrane microregions, being more present at non-lipid ones.
C 端Src 激酶（CSK）通过磷酸化 Src 家族激酶（SFKs）的 C 端酪氨酸残基来抑制 SFKs 的活性，它与 SFK 功能相关，调节各种类型的细胞活动。作为细胞质蛋白，CSK 需要被募集到质膜来调节 SFKs 的活性。CSK 活性及其亚细胞定位的调节机制在很大程度上仍不清楚。在这项工作中，我们开发了一种基于荧光共振能量转移（FRET）的遗传编码生物传感器，用于可视化活细胞中的 CSK 活性。该生物传感器使用优化的底物肽，证实了 CSK SH2 结构域中的关键 Arg 位点，并对 CSK 活性具有敏感性和特异性，而对共转染的Src 和 Fyn 的反应较小。FRET 测量显示，与 Src 和 Fyn 相比，CSK 在大鼠气道平滑肌细胞中的激酶活性相对较弱。带有不同亚细胞膜靶向信号的生物传感器在非脂筏和脂筏微区都检测到了 CSK 活性，而在非脂筏区的 FRET 水平更高。共转染的受体型蛋白酪氨酸磷酸酶α（PTPα）对 CSK 的 FRET 反应有抑制作用。生物传感器未检测到转移性癌细胞和正常细胞之间 CSK 活性的明显变化。总之，生成了一种新的 FRET 生物传感器来监测 CSK 活性，并证明 CSK 活性存在于非脂筏和脂筏膜微区，在非脂筏区更为明显。
