Spatio-temporal imaging of EGF-induced activation of protein kinase A by FRET in living cells

Jin Jun Wang; Xiao-Chuan Chen; Da Xing

doi:10.1117/12.572440

29 July 2004 Spatio-temporal imaging of EGF-induced activation of protein kinase A by FRET in living cells

Jin Jun Wang, Xiao-Chuan Chen, Da Xing

Proceedings Volume 5486, ALT'03 International Conference on Advanced Laser Technologies: Biomedical Optics; (2004) https://doi.org/10.1117/12.572440
Event: ALT'03 International Conference on Advanced Laser Technologies: Biomedical Optics, 2003, Silsoe, United Kingdom

Abstract

Intracellular molecular interaction is important for the study of cell physiology, yet current relevant methods require fixation or microinjection and lack temporal or spatial resolution. We introduced a new method -- fluorescence resonance energy transfer (FRET) to detect molecular interaction in living cells. On the basis of FRET principle, A-kinase activity reporter (AKAR) protein was designed to consist of the fusions of cyan fluorescent protein (CFP), a phosphoamino acid binding domain, a consensus substrate for protein kinase-A (PKA), and yellow fluorescent protein (YFP). In this study, the designed pAKAR plasmid was used to transfect a human lung cancer cell line (ASTC-a-1). When the AKAR-transfected cells were treated by forskolin (Fsk), we were able to observe the efficient transfer of energy from excited CFP to YFP within the AKAR molecule by fluorescence microcopy, whereas no FRET was detected in the transfected cells without the treatment of Fsk. When the cells were treated by Epidermal growth factor (EGF), the change of FRET was observed at different subcellular locations, reflecting PKA activation inside the cells upon EGF stimulation. The successful design of a fluorescence reporter of PKA activation and its application demonstrated the superiority of this technology in the research of intracellular protein-protein interaction.

Citation Download Citation

Jin Jun Wang, Xiao-Chuan Chen, and Da Xing "Spatio-temporal imaging of EGF-induced activation of protein kinase A by FRET in living cells", Proc. SPIE 5486, ALT'03 International Conference on Advanced Laser Technologies: Biomedical Optics, (29 July 2004); https://doi.org/10.1117/12.572440

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KEYWORDS

Fluorescence resonance energy transfer

Proteins

Fluorescent proteins

Lung cancer

Digital cameras

Luminescence

Radium

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