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. 2020 Jan 14;7:938. doi: 10.3389/fchem.2019.00938

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Copyright © 2020 Thirukkumaran, Wang, Asouzu, Fron, Rocha, Hofkens, Lavis and Mizuno.

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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Dissociation of PKA subunits observed by BRET. (A) Interaction of NL-RS and CS-HT modeled with the crystal structures (PDB files: 2QBVS, 2F7E, 5IBO, 5Y2Y). The regulatory subunit is shown in purple, catalytic subunit in wheat, NanoLuc in cyan, and HaloTag in greenish-yellow. (B) Schematic explaining the decrease in BRET upon dissociation of PKA subunits. The tetrameric structure of tagged NL-RS and CS-HT under resting condition (left) is labeled with HaloTag ligands (middle) and stimulated with forskolin to dissociate the interaction of RS and CS (right). (C) Luminescence images of NIH3T3 cells co-expressing NL-RS and CS-HT. Images were acquired after loading respective HaloTag ligands (JF525 and Halo618). All images were acquired with identical camera settings. Scale bar = 10 μm. (D) Representative intensity traces of donor and acceptor signals and (E) Mean SNR ± SEM for cells labeled with either JF525 or Halo618. (F) Time traces of acceptor/donor emission ratios showing dissociation of PKA subunits NL-RS and CS-HT upon forskolin stimulation (indicated by black arrow). (G) Box plot showing ΔR_A/D values for cells labeled with respective HaloTag ligands. n = 8.