Encoding optical control in LCK kinase to quantitatively investigate its activity in live cells.

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.

SUBMITTER: Liaunardy-Jopeace A 

PROVIDER: S-EPMC5736103 | biostudies-literature | 2017 Dec

REPOSITORIES: biostudies-literature