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

LCK is a tyrosine kinase 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 photo-caged equivalent, using genetic code expansion. This enabled fine temporal and spatial control over kinase activity, allowing us to quantify phosphorylation kinetics in situ using biochemical and imaging approaches. We find that auto-phosphorylation 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, the T cell coreceptors, can enhance LCK activity, helping to explain their effect in physiological TCR signaling. Our approach also provides general insights into SRC-family kinase dynamics.