Feedback circuits monitor and adjust basal Lck-dependent events in T cell receptor signaling

Abstract

The Src family kinase Lck is crucial for the initiation of TCR signaling. The activity of Lck is tightly controlled to prevent erroneous immune activation, yet it enables rapid cellular responses over a range of sensitivities to antigens. Here, in experiments with an analog-sensitive variant of the tyrosine kinase Csk, we report that Lck in T cells is dynamically controlled by an equilibrium between Csk and the tyrosine phosphatase CD45. By rapidly inhibiting Csk, we showed that changes in this equilibrium were sufficient to activate canonical TCR signaling pathways independently of ligand binding to the TCR. The activated signaling pathways showed sustained and enhanced phosphorylation compared to that in TCR-stimulated cells, revealing a feedback circuit that was sensitive to the basal signaling machinery. We identified the inhibitory adaptor molecule Dok-1 (downstream of kinase 1) as a candidate that may respond to alterations in basal signaling activity. Our results also suggest a role for Csk in the termination or dampening of TCR signals.

Figure 1. Generation and characterization of the Csk^AS system

(A) Mutation of the conserved gatekeeper residue of Csk (T266G) permits access to larger analogs of PP1. The eleven amino-terminal residues of Lck were fused to Csk^AS to enforce its localization to lipid rafts in the plasma membrane. (B) Structure of 3-IB-PP1. (C) Jurkat T cells were transiently cotransfected with either cytoplasmic- or membrane-Csk^AS and a GFP cotransfection marker. Cells were serum-starved, then stimulated with anti-TCR for 5 min in the presence of DMSO or 3-IBPP1 at doses shown, and analyzed for phospho-ERK. Data show % of live, GFP⁻ untransfected cells or live GFP⁺ transfected cells that are phospho-ERK⁺. Data are representative of 3 independent experiments.

Figure 2. Effect of Csk^AS on Lck activity

(A) Jurkat T cells expressing empty vector, cytoplasmic- or membrane-Csk^AS were serum-starved, pretreated with DMSO or 3-IB-PP1 for 25 minutes, then lysates were prepared directly or following TCR stimulation. Data are representative of three independent experiments. (B) Vector or Csk^AS cells were serum-starved, then pretreated with DMSO or 3-IB-PP1 for 20 minutes. Cells were then stimulated with anti-TCR or vehicle for 2 min prior to fixation and staining for ζ-chain phosphorylation. Histograms show ζ-chain phosphorylation in live, transfected cells. (C) Bar graphs of (B), representing the mean fluorescence intensity (MFI) of phospho-ζ-chain in GFP⁺ transfected cells. (D) Control or membrane-Csk^AS cells were serum-starved and fixed directly or following anti-TCR stimulation or 3-IB-PP1 treatment. Data represent the MFI of phospho-ζ-chain in GFP⁺ transfected cells. Data are representative of at least three independent experiments.

Figure 3. Sustained signaling in presence of Csk^AS inhibition

Control or membrane-Csk^AS T cells were serum-starved, then lysed directly or following stimulation. Control cells were stimulated with anti-TCR (left) and membrane-Csk^AS cells were treated with 3-IB-PP1 (right) for the times indicated. (A) Total tyrosine phosphorylation, and (B) phosphorylation of Lck, ZAP-70, LAT and ERK were assessed by immunoblotting. Data are representative of at least three independent experiments.

Figure 4. Distal TCR signaling and cell activation induced by Csk^AS inhibition

(A) Calcium release is triggered by Csk^AS inhibition alone in T cells expressing cytoplasmic- (top) or membrane- Csk^AS (bottom). Jurkat T cells transiently cotransfected with Csk^AS and CD16 constructs. The ratios between Fluo-3 and FuraRed is shown for CD16⁻ untransfected cells or for CD16⁺ Csk^AS transfected cells in response to DMSO, 3-IB-PP1, anti-TCR and ionomycin. B) Jurkat T cells expressing membrane-Csk^AS have impaired ERK phosphorylation that is overcome by inhibition of Csk^AS alone. Control or Csk^AS cells were serum-starved, pretreated with DMSO or 3-IB-PP1 for 15 minutes. Cells were then harvested directly (unstim) or after 2 minutes of TCR stimulation. Plots show total live cells, with GFP⁻ untransfected cells in the bottom quadrants and GFP⁺ Csk^AS–transfected cells in the upper quadrants. (C) Upregulation of CD69 is impaired in TCR-stimulated membrane-Csk^AS cells, and is induced in response to Csk^AS inhibition alone. Transiently transfected Jurkat T cells were treated with either 3-IB-PP1 or DMSO, and were TCR stimulated for 18 hours prior to surface staining for CD69. Data are representative of two (A) or three (B, C) independent experiments.

Figure 5. Requirement of TCR signaling components for activation upon Csk^AS inhibition

(A) Untransfected or (B) membrane-Csk^AS transfected Jurkat signaling mutant cells were purified, serum-starved and stimulated with anti-TCR or 3-IB-PP1 for 10 minutes and lysed. Total tyrosine phosphorylation and phosphorylation of Lck, LAT and ERK were assessed by immunoblotting. (C, D) Membrane-Csk^AS cells were treated for 2 hours with either vehicle or MEK inhibitor U0126. Cells were serum-starved for 30 minutes in the presence of vehicle or U0126, and then stimulated with anti-TCR or 3-IB-PP1 for 5 minutes as noted. Lysates were blotted for total tyrosine phosphorylation, Csk, and phosphorylation of Lck and ERK. The Csk antibody recognizes both endogenous Csk (denoted **) and Csk^AS (denoted *, larger MW due to Myc tag, linker sequences and membrane targeting motif). Data are representative of at least 3 independent experiments.

Figure 6. Dok-1 is uniquely phosphorylated upon Csk^AS inhibition and interacts with Csk

(A) Jurkat T cells expressing vector, membrane-Csk^AS or SH2 domain R107K mutant (SH2^mut) membrane-Csk^AS were serum-starved, then lysed directly or following treatment with 3-IB-PP1 for the times indicated. Total tyrosine phosphorylation and levels of Csk and ERK were assessed by immunoblotting. (B) Myc immunoprecipiation of wild-type and SH2^mut membrane-Csk^AS demonstrates p65 interacts with Csk in an SH2-dependent manner. Control or membrane-Csk^AS cells were treated with 3-IB-PP1 or DMSO for 10 minutes. Lysates were immunoprecipitated for the times indicated with anti-Myc and blotted for pTyr and Csk. (C) Silver stain of Myc immunoprecipitates of control or membrane-Csk^AS cells. Dok-1 is denoted with arrow. (d) Dok-1 IP of control or membrane-Csk^AS cells. Cells were treated with 3-IB-PP1 or DMSO for 10 minutes. Lysates were immunoprecipitated with anti-Dok-1 and blotted for Dok-1, pTyr, and Csk. Note: WCL and post-IP lysates are probed with anti-Csk which recognizes both wild type (marked with **) and membrane-Csk^AS (marked with *), while Dok-1 IP was probed with anti-Myc, which recognizes only membrane-Csk^AS. Data are representative of at least two (C) or three (A, B) independent experiments.