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

Area of Science:

• Immunology
• Molecular Biology
• Cell Signaling

Background:

• T-cell antigen receptor (TCR) signaling is crucial for adaptive immunity.
• LCK (lymphocyte-specific protein tyrosine kinase) is a key enzyme initiating TCR signaling.
• Current methods limit quantitative analysis of LCK function in live cells.

Purpose of the Study:

• To develop a novel method for precise temporal and spatial control of LCK activity in live cells.
• To quantitatively investigate LCK's role in TCR signaling initiation and regulation.
• To elucidate the mechanisms of LCK activation and its interaction with coreceptors.

Main Methods:

• Genetic code expansion was used to create a photocaged LCK variant (LCK*).
• LCK* allowed controlled activation of kinase activity using light.
• In situ biochemical and imaging techniques were employed to quantify phosphorylation kinetics.

Main Results:

• Autophosphorylation of the LCK active-site loop is essential for its catalytic function.
• LCK can enhance its own activation by adopting a more open conformation.
• T-cell coreceptors CD4 and CD8 were shown to potentiate LCK activity.

Conclusions:

• The developed photocaged LCK method provides unprecedented control over kinase activity for quantitative studies.
• LCK autophosphorylation and conformational changes are critical for its activation.
• CD4 and CD8 coreceptors play a significant role in modulating LCK activity during TCR signaling.
• This work offers generalizable insights into SRC-family kinase regulation and dynamics.