Version 1
: Received: 6 March 2024 / Approved: 11 March 2024 / Online: 11 March 2024 (13:29:50 CET)
How to cite:
Ouyang, M.; Xing, Y.; Zhang, S.; Li, L.; Pan, Y.; Deng, L. Development of FRET Biosensor to Characterize CSK Subcellular Regulations. Preprints2024, 2024030636. https://doi.org/10.20944/preprints202403.0636.v1
Ouyang, M.; Xing, Y.; Zhang, S.; Li, L.; Pan, Y.; Deng, L. Development of FRET Biosensor to Characterize CSK Subcellular Regulations. Preprints 2024, 2024030636. https://doi.org/10.20944/preprints202403.0636.v1
Ouyang, M.; Xing, Y.; Zhang, S.; Li, L.; Pan, Y.; Deng, L. Development of FRET Biosensor to Characterize CSK Subcellular Regulations. Preprints2024, 2024030636. https://doi.org/10.20944/preprints202403.0636.v1
APA Style
Ouyang, M., Xing, Y., Zhang, S., Li, L., Pan, Y., & Deng, L. (2024). Development of FRET Biosensor to Characterize CSK Subcellular Regulations. Preprints. https://doi.org/10.20944/preprints202403.0636.v1
Chicago/Turabian Style
Ouyang, M., Yan Pan and Linhong Deng. 2024 "Development of FRET Biosensor to Characterize CSK Subcellular Regulations" Preprints. https://doi.org/10.20944/preprints202403.0636.v1
Abstract
C-terminal Src kinase (CSK) is the major inhibitory kinase for Src family kinases (SFKs) through phosphorylation of their C-tail tyrosine site, and regulates various cellular activities in association with SFKs functions. As a cytoplasmic protein, CSK need be recruited to the plasma membrane to regulate SFKs activities. The regulatory mechanism for 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 CSK activity in live cells. The biosensor with optimized substrate peptide confirmed crucial Arg107 site in CSK SH2 domain, and displayed sensitivity and specificity to CSK activity while little responses to co-transfected Src and Fyn. FRET measurements showed CSK having relatively mild level of kinase activity in comparison to Src and Fyn ones 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 showed higher FRET level at non-lipid ones. Co-transfected receptor-type protein tyrosine phosphatase alpha (PTPα) had inhibitory effect on the CSK FRET response. The biosensor didn’t detect obvious changes of 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 while more present at non-lipid ones.
Keywords
C-terminal Src kinase (CSK), fluorescence resonance energy transfer (FRET), Src family kinase, PTPalpha, submembrane kinase activity
Subject
Biology and Life Sciences, Biochemistry and Molecular Biology
Copyright:
This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
