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. 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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Figure 1: Engineering of a photocaged LCK kinase using UAA incorporation.
Figure 2: Phosphorylation of LCK active-site loop is essential for its kinase activity.
Figure 3: Intramolecular SH3 interaction restrains LCK kinase activity.
Figure 4: Active LCK conformation increases substrate availability for autophosphorylation at Y394.
Figure 5: Differential phosphorylation kinetics between Y319 and Y493 of ZAP70.
Figure 6: Precise spatiotemporal control of LCK kinase activity in live cells.
Figure 7: T-cell coreceptors CD4 and CD8 directly enhance LCK activity.

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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.

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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.

Corresponding author

Correspondence to John R James.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Incorporation of photo-caged lysine (pc-Lys) at K273 of human LCK allows specific control of the enzyme activity.

(a) Modeling of the kinase domain of LCK with ATP (left) or pc-Lys at position 273 (right) in the native active site, based on a crystal structure of the kinase domain alone (1QPC). For the ATP-bound structure, the original AMP-PNP ligand has been modeled as ATP, with K273 also shown. For the pc-Lys model, the incorporated unnatural amino acid was built into the active site using PyMol. Rendering was performed in Chimera. (b) A representative western blot to show that the ZAP70 pY319 signal observed is specifically due to LCK* uncaging by UV illumination (350-400 nm). HEK-TCR cells were transfected with WT LCK* or kinase inactive LCK (K273R), ZAP70-mRuby2 and the components for pc-Lys incorporation, and illuminated with 350-400 nm or 500-550 nm light for 30 s as indicated in the left panel. Right panel shows the quantification of the western blot, data are normalized relative to final time point (15 min) of wildtype LCK* 350-400 nm and are presented as mean ± s.e.m. from independent experiments (n = 3). Uncropped blot images are shown in Supplementary Data Set 1.

Supplementary Figure 2 Expression of proteins in various cell lines.

(a) Histogram of flow cytometry data showing expression of cell surface TCR in cell lines indicated in the panel using a fluorescently conjugated anti-TCRβ antibody. (b) Histogram of flow cytometry data showing expression of endogenous CD45 phosphatase in HEK293T and Jurkat T cells using a fluorescently conjugated anti-CD45 antibody. (c) Normalized cytoplasmic densities of CSK, SHP-1 and SHP-2 expressed endogenously in cell lines indicated in the panels. Protein expression was detected by western blot using monoclonal antibodies specific to the proteins and data are normalized relative to the protein expression in CD4+ T cells. Quantification of the western blot is described in the Methods section. (d-f) Histograms of flow cytometry data showing cell surface expression of TCR (d), CD8 (e) and CD4 (f) in various HEK-derived cell lines using fluorescently conjugated antibodies against TCRβ, CD8β or CD4, respectively.

Supplementary Figure 3 Kinase activities of WT and mutant LCK* proteins on ZAP70 phosphorylation and recruitment to plasma membrane after uncaging.

(a) Plot of ZAP70 Y319 phosphorylation kinetics by wildtype LCK* from two identical sets of experiments with different numbers of time points collected, as indicated within the figure. (b) Plot of the kinetics of ZAP70 Y319 phosphorylation by wildtype or Y394F LCK* after uncaging, in the presence or absence of dominant negative version of CSK (K222R). Data in a and b are relative to final time point (15 min) of wildtype LCK* in each experiment and were fit using a 3-parameter logistic function, and are presented as mean ± s.e.m. from independent experiments (n = 4 in a [22 time points], n = 12 in a [6 time points] and n = 3 in b). (c,d) Quantification of the activities of wildtype and mutant LCK* kinases derived from the phosphorylation and recruitment kinetic curves. (c) Initial reaction rates (V0) of ZAP70 Y319 phosphorylation by LCK* mutants relative to WT LCK* after uncaging (related to Fig. 2b-d and 3a-d). (d) Time to achieve half-maximal recruitment of ZAP70 to plasma membrane by WT LCK* or indicated mutants after UV illumination at 5 mW/cm2 (related to Fig. 2f-h). For this measure of LCK* reaction rate, a lower value indicates more efficient kinase activity, which was not detectable (n.d.) for the Y394F mutant. Data are presented as mean ± s.e.m. from independent experiments (n = 4). (e) Identification of proline in the PxxP motif in the linker region of HCK (PDB ID: 1QCF) that binds intramolecularly to the SH3 domain. The amino acid numbering used here is based on human LCK sequence.

Supplementary Figure 4 Plot of the kinetics of ZAP70 Y319 phosphorylation by wildtype LCK*, FYN*, or SRC* after uncaging.

Data are relative to final time point (15 min) of LCK* and were fit using a 3-parameter logistic function. Data are presented as mean ± s.e.m. from independent experiments (n = 3).

Supplementary Figure 5 Time to achieve half-maximal recruitment of ZAP70 to phosphorylated ITAMs by wildtype LCK* in the presence of CD4 or CD8 co-receptors.

(a) Time to achieve half-maximal recruitment of ZAP70 to conjugate interface by WT LCK* in CD8+ HEK-TCRH and in HEK-TCRH cells after UV illumination at 5 mW/cm2 (related to Fig. 7b). For this measure of LCK* reaction rate, a lower value indicates more efficient kinase activity. (b,c) Time to achieve half-maximal recruitment of ZAP70 to plasma membrane by WT LCK* in the absence or presence of CD8 (b) or CD4 (c) in HEK-TCRH cells after UV illumination at 5 mW/cm2 for 2 s (related to Fig. 7e,f). (d,e) Time to achieve half-maximal recruitment of ZAP70 to plasma membrane by WT LCK* in CD8+ and CD86ExCD8Int+ (d), or CD4+ and CD86ExCD4Int+ (e) HEK‑TCRH cells relative to that in HEK‑TCRH cells after UV illumination at 5 mW/cm2 for 2 s (related to Fig. 7g,h). (f,g) Microscopy image quantification showing ZAP70 recruitment to plasma membrane after LCK* uncaging in the presence or absence of CD8 (f) or CD4 (g) in HEK-TCRH cells after UV illumination at 500 mW/cm2 for 2 s. Data are normalized to maximum asymptote values for each dataset. Lines show data smoothed used a moving-average filter, data points represent mean and filled areas represent s.e.m. from independent experiments (n = 3), where 4-8 cells were used in each independent experiment. (h,i) Time to achieve half-maximal recruitment of ZAP70 to plasma membrane by WT LCK* in the absence or presence of CD8 (h) or CD4 (i) in HEK-TCRH cells after UV illumination at 500 mW/cm2 for 2 s (related to Supplementary Fig. 5f,g). Data in a-e,h,i are presented as mean ± s.e.m. from independent experiments (n=4 in a-c; 3 in d,e,h,i).

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–5 (PDF 706 kb)

Life Sciences Reporting Summary (PDF 130 kb)

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). (PDF 852 kb)

Supplementary Data Set 2

Source data for all figures. (XLSX 378 kb)

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. (MP4 1391 kb)

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. (MP4 5125 kb)

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. (MP4 1871 kb)

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Liaunardy-Jopeace, A., Murton, B., Mahesh, M. et al. Encoding optical control in LCK kinase to quantitatively investigate its activity in live cells. Nat Struct Mol Biol 24, 1155–1163 (2017). https://doi.org/10.1038/nsmb.3492

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