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In vitro membrane reconstitution of the T-cell receptor proximal signaling network

Nature Structural & Molecular Biology volume 21, pages 133–142 (2014)Cite this article

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Abstract

T-cell receptor (TCR) phosphorylation is controlled by a complex network that includes Lck, a Src family kinase (SFK), the tyrosine phosphatase CD45 and the Lck-inhibitory kinase Csk. How these competing phosphorylation and dephosphorylation reactions are modulated to produce T-cell triggering is not fully understood. Here we reconstituted this signaling network using purified enzymes on liposomes, recapitulating the membrane environment in which they normally interact. We demonstrate that Lck's enzymatic activity can be regulated over an ~10-fold range by controlling its phosphorylation state. By varying kinase and phosphatase concentrations, we constructed phase diagrams that reveal ultrasensitivity in the transition from the quiescent to the phosphorylated state and demonstrate that co-clustering TCR and Lck or detaching Csk from the membrane can trigger TCR phosphorylation. Our results provide insight into the mechanism of TCR signaling as well as other signaling pathways involving SFKs.

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Figure 1: Lck-catalyzed phosphorylation of CD3ζ on liposomes.
Figure 2: Lck undergoes autophosphorylation on Y394 and Y505.
Figure 3: Enzyme kinetics analyses of Lck catalyzed phosphorylation of CD3ζ.
Figure 4: CD45 substrate specificity in the presence or absence of tSH2 of ZAP-70.
Figure 5: Phase behavior of the membrane reconstituted Lck-CD45-CD3ζ network.
Figure 6: Csk modulates the phosphorylation of Lck regulatory tyrosines and decreases CD3ζ phosphorylation.
Figure 7: Protein clustering influences the phase behavior of the TCR proximal signaling network.
Figure 8: Model for Lck regulation via tyrosine phosphorylations.

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Acknowledgements

We thank A. Weiss (University of California), J. Kuriyan (University of California), Y. Kaizuka (National Institute for Materials Science, Japan), I.A. Yudushkin (University of Vienna), J.R. James (University of Cambridge) and members of R.D.V.'s laboratory for comments and discussions. We acknowledge A. Chien and C. Adams of the Vincent Coates Foundation Mass Spectrometry Laboratory (Stanford University) for LC-MS analyses. R.D.V. is supported as an investigator of the Howard Hughes Medical Institute. E.H. is supported as a fellow of the Leukemia and Lymphoma Society.

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  1. The Howard Hughes Medical Institute, University of California, San Francisco, San Francisco, California, USA

    Enfu Hui & Ronald D Vale

  2. Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, California, USA

    Enfu Hui & Ronald D Vale

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E.H. and R.D.V. designed the study. E.H. collected the data and conducted the analyses. E.H. and R.D.V. wrote the manuscript.

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Correspondence to Ronald D Vale.

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Integrated supplementary information

Supplementary Figure 1 Characterization of His10-SNAP505–liposomes interaction and the effect of PS on Lck phosphorylation of CD3ζ.

(a, b) FRET based kinetic analysis of the interaction between His10-SNAP505 and Ni2+-NTA-containing liposomes. Purified His10-SNAP was fluorescently-labeled with SNAP-cell 505 (designated as His10-SNAP505), which served as a FRET donor for membrane-conjugated rhodamine. Panel a, shown in red is the representative time course of fluorescence (excitation: 504 nm; emission: 540 nm) change of 0.25 μM His10-SNAP505 upon mixing with 1.1 nM rhodamine-bearing Ni-NTA liposomes, as monitored by a plate reader. Single exponential fitting using Graphpad Prism 5.0 yielded an observed rate constant (kobs) of 0.08 s-1. The black trace corresponds to the fluorescence of His10-SNAP505 upon mixing buffer. Panel b, kobs measured in a plotted as a function of liposome concentration. Assuming pseudo first order kinetics, the elementary rate constants of the binding reaction was determined by linear regression, as previously described1. The on-rate (kon) and off-rate (koff) were 1.4 × 106 M-1s-1 and 0.0009 s-1, respectively, from which the dissociation constant (Kd) was computed (0.6 nM). Error bars represent s.e.m. from triplicate measurements. (c) Inclusion of PS in the membranes moderately accelerated the kinetics of Lck catalyzed phosphorylation of CD3ζ. The time course of CD3ζ phosphorylation was followed by FRET essentially as described in Fig. 1a,b. His10-Lck (~280 μm-2) and His10-CD3ζ (~1300 μm-2) were attached to liposomes that contained 10% DGS-NTA-Ni, 0.3% Rhod-PE and indicated molar fractions of PS (at the cost of PC). Shown are representative traces of SNAP505-tSH2 fluorescence before and after the addition of ATP, under the three indicated PS content. The inset shows the first 1 min linear phase of the fluorescence changes, allowing the estimation of initial rates of phosphorylation under different conditions. Increasing PS content from 0% to 10% accelerated CD3ζ phosphorylation by 1.8-fold. No further rate enhancement was observed when PS content increased from 10% to 20%. A similar result was obtained in an independent experiment.

Supplementary Figure 2 Characterization of Lck autophosphorylation and dephosphorylation reactions.

(a-c) Time course of autophosphorylation of liposome-bound Lck at different ATP concentrations. Panel a, immunoblots and a quantification plot showing the kinetics of Y394 phosphorylation at indicated ATP concentrations. Panel b, immunoblots and a quantification plot showing the kinetics of Y505 phosphorylation at indicated ATP concentrations. Panel c, immunoblots and a quantification plot showing the kinetics of Y505 phosphorylation at indicated ATP concentrations. Unphosphorylated Lck (WT) or Lck (Y394F) was pre-bound to liposomes at ~500 μm-2 density, and experiments were conducted at RT essentially as described in Online Methods. (d) An immunoblot showing the time-dependent dephosphorylation of Lck Y505 by the cytosolic portion of CD45. Freshly purified His10-Lck (10 μM) was incubated with 0.5 μM GST-CD45 on ice for indicated length of time, and then subjected to SDS-PAGE and WB using an mAb against pY505-Lck. (e,f) Immunoblots and quantification plots showing the time-dependent changes in phosphorylation of Lck. 5.04 μM His10-Lck (WT) was incubated with indicated concentrations of ATP in solution on ice. Aliquots of reactions were terminated with SDS sample buffer and indicated time points and analyzed for phosphorylation on both Y394 (panel e) and Y505 (panel f) using phosphospecifc antibodies (Online Methods).

Supplementary Figure 3 Quantification of Lck phosphorylation on both regulatory tyrosines.

(a-d) Representative fragment ion spectra for peptides containing phosphorylated Y394, unphosphorylated Y394, phosphorylated Y505, and unphosphorylated Y505. LC-MS was carried out as described in Online Methods. (e) Table summarizing relevant mass spectrometry parameters, which was obtained by using a 3 ppm peptide threshold and >2 Sequest Xcorr score. % occupancy was calculated by dividing the total ion current (TIC) for the phosphorylated peptide by the sum of TIC for both the phosphorylated and unphosphorylated cognate peptide. This TIC based quantification method has been shown to have greater dynamic range than both spectral counting and isotope labeling methods2,3.

Supplementary Figure 4 Enzyme kinetics for Lck on membranes using CD3ζ as a substrate.

(a) Representative traces for fluorescence changes of SNAP505-tSH2 at different CD3ζ concentrations. Phosphorylation assays were done essentially as described in Fig. 1a,b, using SNAP505-tSH2 (1.2 μM) as the FRET reporter for ITAM phosphorylation. (b) Coomassie-stained SDS-PAGE gels showing the SNAP505-tSH2 and CD3ζ in the total (liposome-bound + solution) and solution phase, upon the completion of FRET measurements (t = 100 min). Following FRET measurements, all samples were subjected to liposome sedimentation (278,000g, 20 min), and the supernatant fraction was collected. Equal fractions of the total and supernatant samples were subjected to SDS-PAGE. SNAP505-tSH2 was depleted from the supernatant as CD3ζ concentration increased. This experiment also suggests that 100% binding of SNAP505-tSH2 to pCD3ζ corresponds to ~28% fluorescence quenching. (c) The molar phosphorylation signal plotted as a function of time. The fluorescence signal in a was converted to molar phosphorylation signal, assuming each SNAP505-tSH2 binding reports phosphorylation of a single ITAM (hence, phospohrylation of a pair of tyrosine residues). (d) The initial rate (v0) of phosphorylation plotted as a function of CD3ζ concentration. v0 was calculated as the slope of the first 10% increase of the phosphorylation signal for each condition. Note: Lck density (~2.5 μm-2) is two orders of magnitude lower than in Fig. 2a, so that the effect of trans autophosphorylation on v0 is minimal.

Supplementary Figure 5 Further analyses of the phase diagrams of the membrane-reconstituted Lck-CD45-CD3ζ network.

(a-d) Phase diagrams of the membrane-reconstituted Lck-CD45-CD3ζ network measured at different time points. Liposome reconstitution of Lck (WT) with CD45 and CD3ζ and FRET assays were performed as described in Fig. 5a,b and the % donor quenching at 30 min, 60 min, 90 min and 120 min after ATP addition were used to construct heat maps. As in Fig. 5c, the black dashed lines indicate conditions with equal molar ratio of Lck and CD45. The red dashed boxes highlight the regime with physiological densities of Lck and CD45. Similar phase diagrams were acquired in another experiment using independent materials. (e-g) Dose response plots for Lck (Y394F). FRET data as shown in Fig. 5b,c for Lck (Y394F) were plotted in the same manner as for Lck (WT) in Fig. 5d-f. (h-j) Dose response plots for Lck (Y505F). FRET data as shown in Fig. 5b,c for Lck (Y505F) were plotted in the same manner as for Lck (WT) in Fig. 5d-f. All data shown in this figure were fit with sigmoidal dose response curves (variable slopes) using Graphpad Prism 5.0 and the Hill-coefficients (nH) are indicated with s.e.m. in brackets. Error bars represent s.e.m. from three independent measurements.

Supplementary Figure 6 Csk affects tyrosine phosphorylation of Lck in solution.

(a) Left, immunoblots showing the time course of ATP-triggered phosphorylation of Y394 and Y505 of 86 nM Lck in solution, with or without 86 nM Csk. Experiments shown here were essentially the same as Fig. 6a except in the absence of liposomes. Right, immunoblots quantified, normalized and plotted against time in a logarithmic scale. pY505 WB signals were and normalized to the last data point (90 min) of the “m-Lck + m-Csk” condition as shown in Fig. 6a, and plotted against time in a logarithmic scale; pY394 WB signals were quantified and normalized to the last data point (90 min) of the “m-Lck” condition as shown in Fig. 6a. (b) Left, immunoblots showing the time course of ATP-triggered phosphorylation of Lck at 8.6 nM concentration, with or without 86 nM Csk. Experiments shown here were essentially the same as Fig. 6b except in the absence of liposomes. Right, quantification plots of immunoblots shown on the left. pY505 WB signals were normalized to the last data point (90 min) of the “m-Lck + m-Csk” condition in Fig. 6b; pY394 WB signals were normalized to the last data point (90 min) of the “m-Lck” condition in Fig. 6b. For each condition, the WB signal at time zero was arbitrary plotted as a data point at 0.1 min. The dashed lines indicate 50% phosphorylation. All experiments shown here were performed side-by-side with experiments shown in Fig. 6a,b. Original images of blots can be found in Supplementary Fig. 9.

Supplementary Figure 7 Csk directly phosphorylates CD3ζ but retards CD3ζ phosphorylation in the context of Lck.

(a) Left, a cartoon showing a liposome membrane reconstituted with Csk and CD3ζ (~500 mm-2 each). Phosphorylation of CD3ζ was monitored by FRET using SNAP505-tSH2 (omitted in the cartoon) as described in Fig. 1a. Right, the time course of SNAP505-tSH2 fluorescence, before and after the addition of ATP. (b) The cartoon on the left shows a liposome membrane reconstituted with equal densities of Csk, CD3ζ and Lck (WT, Y394F, or Y505F mutant). Phosphorylation of CD3ζ was again monitored by FRET. The red traces in the plots corresponds to the the time course of SNAP505-tSH2 fluorescence before and after the addition of ATP, when both Lck and Csk were membrane reconstituted with CD3ζ; the black traces corresponds to conditions in which Csk omitted; the grey traces corresponds to conditions in which Lck was omitted. (c) Kinetic traces of SNAP505-tSH2 fluorescence when experiments as shown in b were repeated at lower surface density (~50 μm-2) of Lck. Results shown in this figure were repeatable in an independent measurement.

Supplementary Figure 8 Treatment of Jurkat cells with a Lck-specific inhibitor decreased the phosphorylation of Y505.

(a) Immunoblots showing the effects of Lck specific inhibitor on the phosphorylation of Y505 and Y394 of Lck. Jurkat cells were treated with either 0.2 μM or 20 μM cell-permeable Lck specific inhibitor (IC50: 0.016 μM for Lck and 5.18 μM for Csk4) or equal volume of DMSO (indicated as “0 μM”) at 37 °C for 0.5 h. Cells were harvested, lysed and subjected to SDS-PAGE, as described in Online Methods. Phosphorylation of Y505 and Y394 were immunoblotted using mouse anti-pY505-Lck mAb (558552, BD Phosflow) and rabbit anti-pY416-Src polyclonal Ab (2101S, Cell signaling), respectively. Total Lck and GAPDH levels from the same samples were determined by WB using rabbit anti-Lck polyclonal Ab (2984S, Cell Signaling) and mouse anti-GAPDH mAb (MAB374, Millipore). (b) A bar graph showing the quantification of immunoblots shown in a. pY505 and pY394 WB signals were quantified, and normalized to the DMSO control condition. Error bars represent s,d. from triplicate determination. Statistical significance was evaluated by one-sided Student's t test, *** p < 0.001.

Supplementary Figure 9 Uncropped immunoblots with molecular weight markers.

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Hui, E., Vale, R. In vitro membrane reconstitution of the T-cell receptor proximal signaling network. Nat Struct Mol Biol 21, 133–142 (2014). https://doi.org/10.1038/nsmb.2762

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  • DOI: https://doi.org/10.1038/nsmb.2762

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