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

Nature Structural & Molecular Biology volume 24, pages 11551163 (2017) | Download Citation

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.

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Acknowledgements

This work was supported by a Sir Henry Dale Fellowship jointly funded by the Wellcome Trust and the Royal Society (Grant Number: 099966/Z/12/Z to J.R.J.) and by the Medical Research Council, UK (MC_U105181009 and MC_UP_A024_1008 to J.W.C.). We would like to thank the engineering workshop at the MRC-LMB for manufacturing the illumination device used in this study.

Author information

Affiliations

  1. Molecular Immunity Unit, Department of Medicine, University of Cambridge, MRC-LMB, Cambridge, UK.

    • Ardiyanto Liaunardy-Jopeace
    • , Ben L Murton
    •  & John R James
  2. Medical Research Council Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge, UK.

    • Mohan Mahesh
    •  & Jason W Chin

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Contributions

A.L.-J., B.L.M. and J.R.J. designed and performed all of the experiments in the study. A.L.-J. and J.R.J. analyzed the data. M.M. synthesized the unnatural amino acid pc-Lys. J.W.C. provided scientific input and helped revise the manuscript, which was written by A.L.-J. and J.R.J. All authors contributed to the final manuscript. J.R.J. oversaw and supervised the research.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to John R James.

Integrated supplementary information

Supplementary information

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    Supplementary Text and Figures

    Supplementary Figures 1–5

  2. 2.

    Life Sciences Reporting Summary

  3. 3.

    Supplementary Data Set 1

    Uncropped grayscale images of Western blots shown in the main figures. The Western blots in the main figures are presented as merged images of the 700 nm channel (red) and 800 nm channel (green).

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Videos

  1. 1.

    Differential rate of ZAP70 membrane translocation after uncaging of LCK* mutants.

    A combined video showing ZAP70-mRuby2 translocation from cytosol to phosphorylated ITAMs at the plasma membrane of HEKTCRH cells by wildtype or mutant LCK* as indicated in the video. LCK* photo-uncaging by illumination for 2 s at 5 mW/cm2 (t = 0 s) is marked by the white box. Video is shown at 10× real-time (7.1 fps) and scale bar represents 5 μm. Video is related to Fig. 2e.

  2. 2.

    ZAP70 recruitment to a spatio-temporally defined sub-cellular region of a cell conjugate.

    Representative time lapse video of ZAP70-mRuby2 translocation from cytosol to phosphorylated ITAMs at a specific cell conjugate region between HEK-TCRH and Raji B cell expressing pMHC-BFP after uncaging of wildtype LCK*. A diffraction-limited spot in the upper conjugate shown was exposed to focused 405 nm laser pulses (10×100 μs) to uncage LCK* in that region only, as marked by the appearance of a white arrow in ZAP70 panel at -1 s. Colored labels denote protein representation in the overlay. Video is shown at 10× real-time (10 fps) and scale bar represents 5 μm. Video is related to Fig. 6.

  3. 3.

    ZAP70 recruitment to cell conjugate region after wildtype LCK* uncaging.

    Representative time-lapse video of ZAP70-mRuby2 translocation from cytosol to phosphorylated ITAMs at the cell conjugate region between HEK-TCRH and Raji B cell expressing pMHC-BFP after uncaging of wildtype LCK*. LCK* photo-uncaging by illumination for 2 s at 5 mW/cm2 (t = 0 s) is marked by the white box. Colored labels denote protein representation in the overlay. Video is shown at 10× real-time (7.1 fps) and scale bar represents 5 μm. Video is related to Fig. 7a.

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DOI

https://doi.org/10.1038/nsmb.3492

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