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Encoding optical control in LCK kinase to quantitatively investigate its activity in live cells
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Liaunardy-Jopeace, Ardiyanto, Murton, Ben L, Mahesh, Mohan, Chin, Jason W and James, John R. (2017) Encoding optical control in LCK kinase to quantitatively investigate its activity in live cells. Nature Structural & Molecular Biology, 24 (12). pp. 1155-1163. doi:10.1038/nsmb.3492
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Official URL: http://dx.doi.org/10.1038/nsmb.3492
Abstract
LCK is a tyrosine kinase that is essential for initiating T-cell antigen receptor (TCR) signaling. A complete understanding of LCK function is constrained by a paucity of methods to quantitatively study its function within live cells. To address this limitation, we generated LCK*, in which a key active-site lysine is replaced by a photocaged equivalent, using genetic code expansion. This strategy enabled fine temporal and spatial control over kinase activity, thus allowing us to quantify phosphorylation kinetics in situ using biochemical and imaging approaches. We find that autophosphorylation of the LCK active-site loop is indispensable for its catalytic activity and that LCK can stimulate its own activation by adopting a more open conformation, which can be modulated by point mutations. We then show that CD4 and CD8, T-cell coreceptors, can enhance LCK activity, thereby helping to explain their effect in physiological TCR signaling. Our approach also provides general insights into SRC-family kinase dynamics.
| Item Type: | Journal Article | ||||||||
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| Divisions: | Faculty of Medicine > Warwick Medical School > Biomedical Sciences Faculty of Medicine > Warwick Medical School |
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| Journal or Publication Title: | Nature Structural & Molecular Biology | ||||||||
| Publisher: | Nature Publishing Group | ||||||||
| ISSN: | 1545-9993 | ||||||||
| Official Date: | December 2017 | ||||||||
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| Volume: | 24 | ||||||||
| Number: | 12 | ||||||||
| Page Range: | pp. 1155-1163 | ||||||||
| DOI: | 10.1038/nsmb.3492 | ||||||||
| Status: | Peer Reviewed | ||||||||
| Publication Status: | Published | ||||||||
| Access rights to Published version: | Restricted or Subscription Access | ||||||||
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