Tetracysteine exposure to track open and closed Src family kinase conformations in the cell  — ASN Events
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  2. Session Seven - Poster Session B includes Happy Hour & Trade Display
  3. Tetracysteine exposure to track open and closed Src family kinase conformations in the cell 

Tetracysteine exposure to track open and closed Src family kinase conformations in the cell  (#243)

Rebecca Wood 1 2 , Sevgi Irtegun 1 2 , Jörg Schweiggert 1 2 , Leigh A Johnston 3 , Terrence D Mulhern 1 2 , Danny M Hatters 1 2
  1. Department of Biochemistry and Molecular Biology, University of Melbourne, Parkville, VIC, Australia
  2. Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, Parkville, VIC, Australia
  3. Department of Electrical & Electronic Engineering, University of Melbourne, Parkville, VIC, Australia

Src family kinases are an important group of signalling regulators that are overactive in a wide variety of human cancers, making them important therapeutic targets. High-resolution crystal structures have provided a good understanding of the structure and regulation of this family, which adopts active “open” and inactive “closed” conformations. However, studying structural changes in the native environment remains challenging, and typically requires incorporation of large tags, such as GFP. To study Src family kinase conformation in live cells, we applied the genetically encoded small tag tetracysteine/FlAsH technology developed by Griffin et al. (Science (1998) 281, 269-272). By substituting just four residues with cysteines, we developed a binary biosensor for open and closed conformations of Src family kinases using differential biarsenical dye reactivity.  Using confocal microscopy or flow cytometry, the biosensor distinguished open and closed conformations of c-Src, Lck and Lyn.  Furthermore, the sensor detected shifts in conformational equilibrium, due to overexpression of the regulatory protein Csk or the inhibitor PP2. Thus, the biosensor is a non-invasive high-throughput platform for measuring c-Src conformation in individual cells.