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

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.

Figure 1. Lck catalyzed phosphorylation of CD3ζ on liposomes

(a) A cartoon depicting the kinetic, FRET-based assay for monitoring the phosphorylation of CD3ζ on liposomes. The energy donor, SNAP-cell-505, is labeled to SNAP-tag fusion tandem SH2 domain of ZAP70 (SNAP_505-tSH2). Liposomes harbor polyhistidine chelating lipid DGS-NTA-Ni, and fluorescently labeled lipid Rhod-PE, which serves as the energy acceptor. Phosphorylation of ITAMs recruits SNAP₅₀₅-tSH2 to the membrane, leading to FRET. (b) Time course of the fluorescence intensity of SNAP₅₀₅-tSH2 (0.34 μM, 2-fold of CD3ζ bulk concentration) upon incubation with liposome-bound His₁₀-Lck (~12 μm⁻²) and His₁₀-CD3ζ (~500 μm⁻²) and addition of 1 mM ATP. (c) Western-blot (WB) showing the time course of Lck-catalyzed CD3ζ phosphorylation, in the presence or absence of SNAP₅₀₅-tSH2. Experiment condition was identical to b except using immunoblotting rather than fluorescence to follow CD3ζ phosphorylation. Samples were derived from the same experiment and the blots were processed in parallel. Original images of blots are shown in Supplementary Fig. 9. (d) Comparison of the phosphorylation kinetics in solution (“—liposomes”, cyan trace) versus on liposomes with three indicated surface densities for Lck and CD3ζ. CD3ζ was labeled with tetramethylrhodamine (FRET donor) via an engineered cysteine (Online Methods), and Rhod-PE was omitted from the liposomes. Data shown were successfully repeated in another independent experiment.

Figure 2. Lck undergoes autophosphorylation on both Y394 and Y505

(a) Immunoblot analysis of Lck autophosphorylation on membranes versus in solution using WB. Left, kinetics of Lck phosphorylation at Y394 and Y505 upon ATP addition, with Lck either in solution (86 nM) or attached on liposomes (~500 Lck per μm², see Online Methods). Right, quantification of the immunoblots. Optical density (OD) for each band quantified, normalized to the last time point (180 min) under each condition, and plotted against time. (b) Time course of ATP-triggered phosphorylation of WT and kinase-dead Lck (K273R), when both proteins were attached to the same membrane. Left, cartoon showing the two proteins of interested reconstituted at the same density. The 13-kDa FKBP (inserted between His₁₀ and Lck) was introduced for electrophoretic separation of WT from kinase-dead Lck. Middle, immunoblots showing the kinetics of Y505 phosphorylation upon ATP addition and the quantification plot. Right, immunoblots showing the kinetics of Y394 phospohorylation upon ATP addition and the quantification plot. (c) Immunoblots for measuring the ATP K_M of Lck autophosphorylation at Y394 and Y505 (Lck density: 500 μm⁻²). The normalized WB signals at 5 min after ATP addition was plotted against ATP concentration, and fit using Michaelis-Menten model, yielding K_M values. For immunoblots shown in each panel, samples were derived from the same experiment and the blots were processed in parallel. Original images of blots are shown in Supplementary Fig. 9.

Figure 3. Enzyme kinetics analyses of Lck catalyzed phosphorylation of CD3ζ

(a) Initial rates (v₀) of CD3ζ phosphorylation (determined as shown in Supplementary Fig. 4) plotted against CD3ζ surface density for both unphosphorylated Lck (Apo) and the double tyrosine mutant (Y394F, Y505F) of Lck. (b) v₀-density plots for monophosphorylated Lck-pY394 (Y505F), Lck-pY505 (Y394F), and doubly phosphorylated Lck-pY394-pY505. Data in a and b were fit by the “allosteric sigmoidal enzyme kinetics” equation using Graphpad Prism 5.0; kinetic parameters were summarized in Table 1, error bars: s.e.m., n = 4. (c,d) An electrophoretic mobility assay for studying the kinetic mechanism of Lck-catalyzed multisite phosphorylation on CD3ζ, as described in Online Methods. Shown are Coomassie-stained SDS-PAGE gels for the time-dependent mobility decrease of CD3ζ, upon addition of 1 mM ATP, indicative of ITAM phosphorylation by Lck. Panel c and d correspond to two parallel experiments in which WT Lck and Lck (ΔSH2) were used, respectively. All samples were derived from the same experiment and the gels were processed in parallel.

Figure 4. CD45 substrate specificity in the presence or absence of tSH2 of ZAP70

(a) Immunoblots showing the time course of CD45 catalyzed dephosphorylation of prephosphorylated Lck (pLck) and prephosphorylated CD3ζ (pCD3ζ), in the presence of indicated concentrations of tSH2 of ZAP70, at RT (Online Methods) All samples were obtained from the same experiment and the blots were processed in parallel. Original images of blots can be found in Supplementary Fig. 9. (b, c) Quantification of the immunoblots shown in a. The OD of each band normalized to time zero of each condition, and plotted as a function of time. Shown in b is a plot comparing the dephosphorylation kinetics of three potential substrates of CD45, in the absence of tSH2. Shown in c is a plot comparing the dephosphorylation kinetics of pY-142 of CD3ζ, in the presence of different tSH2 concentrations.

Figure 5. Phase behavior of the membrane reconstituted Lck-CD45-CD3ζ network

(a) Cartoon depicting the liposome-reconstituted Lck-CD45-CD3ζ network. The dashed arrows indicate potential phosphorylation (“→”) and dephosphorylation (“—|”) reactions. (b) A representative set of time-dependent SNAP₅₀₅-tSH2 fluorescence changes at 84 combinations of Lck and CD45 densities, after ATP addition. (c) Heat maps of the Lck-CD45-CD3ζ network with the indicated form of Lck used. % fluorescence quenching (–Δ(%FI)) at 1 h after ATP addition plotted against both Lck and CD45 densities in MATLAB R2012b (Online Methods). Black dashed lines: equal Lck and CD45 densities. Red dashed boxes: physiological Lck and CD45 densities. (d) % fluorescence quenching for each well pooled, and plotted against the molar ratio of WT Lck over CD45. (e) % donor quenching plotted against WT Lck densities for two fixed CD45 densities. (f) % donor quenching plotted against CD45 density for two WT Lck densities. (g) Reactions as shown in b were performed with or without SNAP₅₀₅-tSH2 with 200 μm⁻² Lck and varying CD45. Phosphorylation of Y142-CD3ζ was measured by WB at 1 h, quantified, normalized and plotted against CD45 density. Black dashed line: best-fit of the FRET data in panel f (open circles). All data were normalized and fit with sigmoidal dose response curves (variable slopes) with the n_H values indicated (s.e.m. in brackets). Error bars in d-f: s.e.m, n = 3. All samples in g were derived from the same experiments. Original blots are shown in Supplementary Fig. 9.

Figure 6. Csk modulates the phosphorylation of Lck regulatory tyrosines and decreases CD3ζ phosphorylation

(a) Left, immunoblots showing the time course of ATP-triggered phosphorylation of Y394 and Y505 of liposome-bound Lck (~500 μm⁻²), with or without liposome-bound Csk (~500 μm⁻²). For experiment details, see Online Methods. Right, immunoblots quantified, normalized to the last data points (90 min) and plotted against time in a logarithmic scale. The starting signal (time zero) was arbitrary plotted as a data point at 0.1 min. (b) Left, immunoblots showing the time course of ATP-triggered phosphorylation of liposome-bound Lck (~50 μm⁻²), with or without liposome-bound Csk (~500 μm⁻²). Right, quantification plots of immunoblots shown on the left, as described in a. All samples in a,b were derived from the same experiments. Original images of blots are shown in Supplementary Fig. 9. (c) Left, a phase diagram for membrane-reconstituted Lck-CD45-CD3ζ network determined as described in Fig. 4b, except in the presence of Csk (~150 μm⁻²). Right, phase diagram in the absence of Csk (identical to Fig. 4c). Black dashed lines: equal levels of Lck and CD45; red dashed boxes: physiological densities of Lck and CD45. (d) The time course of SNAP₅₀₅-tSH2 (omitted in the cartoon) fluorescence changes upon sequential addition of ATP (1 mM) and His₁₀-TVMV (1 μM). Protein densities on membranes: ~1490 μm⁻² for CD3ζ, ~290 μm⁻² each for Lck, CD45 and Csk.

Figure 7. Protein clustering influences the phase behavior of the TCR proximal signaling network

(a) A cartoon depicting the experimental system for inducing co-clustering of Lck and CD3ζ on liposome membranes. (b) Phase diagrams of the membrane-reconstituted kinasephosphatase-CD3ζ network measured under either unclustered (– rapamycin) or Lck-CD3ζ co-clustered (+ rapamycin) conditions. For experiment details, see Online Methods. (c) Phase diagrams of the membrane-reconstituted kinase-phosphatase-CD3ζ network measured under either unclustered (– rapamycin) or CD3ζ clustered conditions (+ rapamycin). Red dashed boxes denote physiological densities of Lck and CD45.

Figur 8. Model for Lck regulation via tyrosine phosphorylations

A summary of the relative catalytic activity (logarithmic scale) of Lck for different tyrosine phosphorylation states (the activity of Apo, unphosphorylated Lck was set at “1”). The kinases and phosphatase that promote the reactions are shown. See the Discussion section for details of Lck regulation by phosphorylation.