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Novel FRET-Based Src Biosensor Reveals Mechanisms of Src Activation and Its Dynamics in Focal Adhesions
L. Koudelková, AC. Pataki, O. Tolde, V. Pavlik, M. Nobis, J. Gemperle, K. Anderson, J. Brábek, D. Rosel,
Language English Country United States
Document type Journal Article, Research Support, Non-U.S. Gov't
- MeSH
- Biosensing Techniques methods MeSH
- Focal Adhesions metabolism MeSH
- Fluorescence Recovery After Photobleaching MeSH
- HEK293 Cells MeSH
- Humans MeSH
- Mutagenesis MeSH
- Fluorescence Resonance Energy Transfer MeSH
- src-Family Kinases antagonists & inhibitors genetics metabolism MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Src kinase plays an important role in a multitude of fundamental cellular processes and is often found deregulated in tumors. Active Src adopts an open conformation, whereas inactive Src is characterized by a very compact structure stabilized by inhibitory intramolecular interactions. Taking advantage of this spatial regulation, we constructed a fluorescence resonance energy transfer (FRET)-based Src biosensor and analyzed conformational changes of Src following Src activation and the spatiotemporal dynamics of Src activity in cells. We found that activatory mutations either in regulatory or kinase domains induce opening of the Src structure. Surprisingly, we discovered that Src inhibitors differ in their effect on the Src structure, some counterintuitively inducing an open conformation. Finally, we analyzed the dynamics of Src activity in focal adhesions by FRET imaging and found that Src is rapidly activated during focal adhesion assembly, and its activity remains steady and high throughout the life cycle of focal adhesion and decreases during focal adhesion disassembly.
Cancer Research UK Beatson Institute Glasgow G611BD UK
Charles University Faculty of Science BIOCEV Department of Cell Biology Vestec 252 50 Czech Republic
References provided by Crossref.org
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