Research ArticleImmunology

The tyrosine phosphatase SHP-1 promotes T cell adhesion by activating the adaptor protein CrkII in the immunological synapse

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Science Signaling  08 Aug 2017:
Vol. 10, Issue 491, eaal2880
DOI: 10.1126/scisignal.aal2880
  • Fig. 1 Activated Rap1 is recruited to the immunological synapse.

    (A) Jurkat cells were cotransfected with plasmids encoding GFP-RalGDS-RBD (the probe for activated Rap1) and Cherry-Rap1, cocultured with Raji cells (APCs) preloaded with SEE (1.5 μg/ml), and subjected to live imaging. Images are representative of at least 50 cells from each of three independent experiments. DIC, differential interference contrast; AU, arbitrary units. Line profile analysis is quantified as fluorescent intensity units over distance. (B) OT-II T cells were cotransfected with plasmids encoding GFP-RalGDS-RBD and Cherry-Rap1, cocultured with DCs pulsed with OVA peptide (100 μg/ml), and subjected to live imaging. Line profile analysis is quantified as fluorescent intensity units over distance. (C to E) Primary human T cells were transfected with plasmids encoding GFP-tagged Rap1, N-Ras, RalGDS-RBD, or Raf1-RBD (the probe for activated Ras), as indicated. The cells were then introduced onto SLBs containing anti-CD3 antibody (5 μg/ml) labeled with Alexa Fluor 568 (to label the TCR) and ICAM-1 (250 molecules/mm2) tagged with Alexa Fluor 405. Live cells were imaged by TIRF microscopy at the indicated times. (D and E) Quantification of the distribution of the GFP-tagged proteins within the indicated compartments of the immunological synapse at the 20-min time point. Data are means ± SEM of at least 50 cells from each of three experiments. **P < 0.01, ***P < 0.001, ****P < 0.0001 by unpaired t test. Scale bars, 10 μm. IRM, interference reflection microscopy.

  • Fig. 2 Analysis of the factors regulating Rap1 localization in the immunological synapse.

    (A) Primary human T cells expressing GFP-tagged Rap1 (wild type), GFP-Rap1CVVM (Rap1 with N-Ras tail), GFP-Rap1G12V (GTP-loaded Rap1), GFP-Rap1S17N (GDP-loaded Rap1), GFP-C3G, GFP-C3GY504D (phosphomimetic), or GFP-CalDAG-GEFIII were introduced onto SLBs containing anti-CD3 antibody (5 μg/ml) labeled with Alexa Fluor 568 and ICAM-1 (250 molecules/mm2) tagged with Alexa Fluor 405 and subjected to live imaging by TIRF microscopy at the indicated times. (B to D) Quantification of the distribution of the indicated GFP-tagged proteins within the different compartments of the immunological synapse at the 20-min time point. Data are means ± SEM of at least 50 cells from more than three experiments. **P < 0.01, ***P < 0.001, by two-way analysis of variance (ANOVA) and unpaired t tests. Scale bar, 10 μm.

  • Fig. 3 CrkII phosphorylation is required for its proper localization in the immunological synapse.

    (A) Domain structures of active (dephosphorylated) and inactive (phosphorylated) CrkII. (B) Primary human T cells were transfected with plasmids encoding GFP-tagged wild-type CrkII, phosphodeficient CrkII (Y221A), or phosphomimetic CrkII (Y221D). The cells were injected onto SLBs containing fluorescently labeled anti-CD3 antibody (5 μg/ml) and ICAM-1 (250 molecules/mm2) and subjected to live imaging at different time points. (C) Quantification of the relative distribution of the different versions of CrkII in the immunological synapse. (D) Primary human T cells expressing GFP-CrkII were plated on SLBs containing fluorescently labeled anti-CD3 antibody (5 μg/ml) and ICAM-1 (250 molecules/mm2), treated with 50 μM pervanadate, and subjected to live imaging. (E) Quantification of the relative distribution or CrkII before and after treatment with pervanadate within the different compartments of the immunological synapse. Data are means ± SEM of at least 25 cells from each of three experiments. *P < 0.05, by unpaired t tests. NS, not significant. Scale bar, 10 μm.

  • Fig. 4 CrkII dephosphorylation leads to T cell activation.

    (A) Jurkat cells were stimulated with anti-CD3 antibody (5 μg/ml) for 2 to 15 min, lysed, and then analyzed by Western blotting with antibodies against phosphorylated and total CrkII proteins. (B) Quantification of the ratio between phosphorylated CrkII and total CrkII after stimulation with anti-CD3 antibody (5 μg/ml). (C) Jurkat cells expressing the PICCHUx construct (YFP-CrkII-CFP-CAAX) were treated with anti-CD3 antibody (5 μg/ml) and fixed with 2% paraformaldehyde. FRET emission was then recorded (excitation at 433 nm; emission at 530 nm). (D) Quantification of FRET efficiency in the indicated cells. (E) Jurkat cells expressing GFP-CrkII (wild type), GFP-CrkIIY221A, or GFP-CrkIIY221D were stimulated with anti-CD3 antibody (5 μg/ml), and the amounts of activated Rap1 were measured by glutathione S-transferase (GST) pull-down assay. (F) Jurkat cells expressing GFP-CrkII (wild type), GFP-CrkIIY221A, or GFP-CrkIIY221D were stimulated with anti-CD3 antibody (5 μg/ml), and the percentages of cells that exhibited adhesion to ICAM-1–coated wells were measured. (G) Jurkat cells expressing GFP-CrkII, GFP-CrkIIY221A, or GFP-CrkIIY221D were stimulated with anti-CD3 antibody (1 μg/ml), plated overnight on wells coated with ICAM-1 (2 μg/ml), and then subjected to flow cytometry analysis of the relative amounts of cell surface CD69. MFI, mean fluorescence intensity. (H) Jurkat cells expressing GFP-CrkII, GFP-CrkIIY221A, or GFP-CrkIIY221D were stimulated with anti-CD3 antibody (1 μg/ml) and plated on wells coated with ICAM-1 (2 μg/ml). Forty-eight hours later, the amounts of IL-2 in the cell culture medium were measured. Data in all panels are means ± SEM of three experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, by unpaired t tests and two-way ANOVA. Scale bar, 10 μm.

  • Fig. 5 SHP-1 dephosphorylates CrkII downstream of TCR activation.

    (A) Jurkat cells expressing GFP-CrkII were pretreated with SHP-1/2 inhibitor (20 mM NSC-87877) or PP2A inhibitor (400 mM cantharidic acid) for 30 min, stimulated with anti-CD3 antibody (5 μg/ml) for 2 min, lysed, and subjected to Western blotting analysis of the extent of CrkII phosphorylation. The ratios of the relative amount of phosphorylated CrkII to that of total CrkII were determined. Data are means ± SEM of three experiments. (B) Jurkat cells expressing GFP or GFP-CrkIIY221A were pretreated with 20 nM NSC-87877 for 30 min before being stimulated with anti-CD3 antibody (1 μg/ml) and plated on ICAM-1–coated wells. The percentages of adherent cells after 15 min were measured. (C) Jurkat cells were infected with lentiviruses encoding scrambled shRNA or shRNAs targeting SHP-1 or SHP-2. The relative amounts of SHP-1 and SHP-2 were analyzed by Western blotting. (D) Jurkat cells expressing GFP-CrkII and the indicated shRNAs were treated with anti-CD3 antibody (5 μg/ml) for 5 min. Cell lysates were then subjected to immunoprecipitation (IP) with anti-GFP antibody and Western blotting (IB) analysis with anti–p-Tyr antibody. (E) Quantification of the indicated band intensities from three Western blotting experiments. (F and G) Jurkat cells expressing the indicated shRNAs (F) or primary human T cells treated with control, SHP-1–specific, or SHP-2–specific siRNAs (G) were treated with anti-CD3 antibody (5 μg/ml), and the fold change in the numbers of cells that adhered to ICAM-1–coated wells was measured. (H) SHP-1 was either knocked down with shRNA (SHP-1 KD) in Jurkat cells expressing GFP or rescued by overexpressing (OE) CrkIIY221A (GFP-CrkIIY221A), CrkIIY221D (GFP-CrkIIY221D), or an alternative SHP-1 construct (GFP-SHP-1). The cells were then treated with anti-CD3 antibody (5 μg/ml), and adhesion to ICAM-1–coated wells was measured. (I) Primary human T cells treated with the indicated siRNAs were stimulated with soluble anti-CD3 antibody (5 μg/ml) and assessed for motility on ICAM-1–coated plates. Between 20 and 50 cells were counted per field. (J) Jurkat cells expressing GFP-RalGDS-RBD and either shSHP-1 or shSHP-2 constructs were cocultured with Raji cells preloaded with SEE (1.5 μg/ml) and subjected to live imaging. (K) Primary human T cells expressing GFP-RalGDS-RBD and treated with the indicated siRNAs were cocultured with SEE-preloaded Raji cells and subjected to live imaging. The percentages of cells of the predominant phenotype are shown. (L) Primary human T cells were treated with the indicated siRNAs and cocultured with SEE-preloaded Raji cells. The percentages of cells that formed transient or stable conjugates were recorded. Data in all panels are means ± SEM of three or four independent experiments. *P < 0.05, **P < 0.01, ****P < 0.0001, by unpaired t tests. Scale bars, 10 μm.

  • Fig. 6 Model for the localization and function of CrkII within the immunological synapse.

    Stimulation of the TCR leads to the SHP-1–mediated dephosphorylation of CrkII, which then travels from the c-SMAC to the p-SMAC. Here, CrkII binds to the exchange factor C3G, which directly activates Rap1, leading to LFA-1 activation and enhanced T cell adhesion.

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/10/491/eaal2880/DC1

    Fig. S1. Conjugate formation between Jurkat cells and SEE-pulsed Raji cells.

    Fig. S2. The different compartments of the immunological synapse.

    Fig. S3. GFP-CAAX is not excluded from the c-SMAC.

    Fig. S4. The hypervariable tail of Rap1 replaced with the N-Ras tail.

    Fig. S5. Analysis of the amounts of GFP-Rap1G12V and GFP-Rap1S17N in Jurkat cells.

    Fig. S6. CrkII interacts with C3G.

    Fig. S7. Pervanadate results in the phosphorylation of CrkII at Tyr221.

    Fig. S8. Stimulation of the TCR leads to the recruitment of CrkII to the plasma membrane.

    Fig. S9. The PICCHUxY221D construct is folded before stimulation.

    Fig. S10. Analysis of the quantities of SHP-1 and SHP-2 in Jurkat cells.

    Fig. S11. CrkII interacts with CasL.

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