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. 2007 Mar;27(5):1960-73.
doi: 10.1128/MCB.01983-06. Epub 2007 Jan 8.

PAG-associated FynT regulates calcium signaling and promotes anergy in T lymphocytes

Dominique Davidson  1 , Burkhart Schraven, André Veillette
Affiliations

PAG-associated FynT regulates calcium signaling and promotes anergy in T lymphocytes

Dominique Davidson et al. Mol Cell Biol. 2007 Mar.

Abstract

Phosphoprotein associated with glycolipid-enriched membranes (PAG), also named Csk-binding protein (Cbp), is a transmembrane adaptor associated with lipid rafts. It is phosphorylated on multiple tyrosines located in the cytoplasmic domain. One tyrosine, tyrosine 314 (Y314) in the mouse, interacts with Csk, a protein tyrosine kinase that negatively regulates Src kinases. This interaction enables PAG to inhibit T-cell antigen receptor (TCR)-mediated T-cell activation. PAG also associates with the Src-related kinase FynT. Genetic studies indicated that FynT was required for PAG tyrosine phosphorylation and binding of PAG to Csk in T cells. Herein, we investigated the function and regulation of PAG-associated FynT. Our data showed that PAG was constitutively associated with FynT in unstimulated T cells and that this association was rapidly lost in response to TCR stimulation. Dissociation of the PAG-FynT complex preceded PAG dephosphorylation and PAG-Csk dissociation after TCR engagement. Interestingly, in anergic T cells, the association of PAG with FynT, but not Csk, was increased. Analyses of PAG mutants provided evidence that PAG interacted with FynT by way of tyrosines other than Y314. Enforced expression of a PAG variant interacting with FynT, but not Csk, caused a selective enhancement of TCR-triggered calcium fluxes in normal T cells. Furthermore, it promoted T-cell anergy. Both effects were absent in mice lacking FynT, implying that the effects were mediated by PAG-associated FynT. Hence, besides enabling PAG tyrosine phosphorylation and the PAG-Csk interaction, PAG-associated FynT can stimulate calcium signals and favor T-cell anergy. These data improve our comprehension of the function of PAG in T cells. They also further implicate FynT in T-cell anergy.

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Figures

FIG. 1.
FIG. 1.
Impact of TCR stimulation on the interactions of PAG with FynT and Csk and on PAG tyrosine phosphorylation. Thymocytes from normal mice were stimulated for the indicated periods of time at 37°C (A to C) or room temperature (D) with biotinylated anti-TCR and avidin. Lysates were then processed for immunoprecipitation or immunoblotting. (A) PAG-FynT interaction, PAG-Csk interaction, and PAG tyrosine phosphorylation. Cells were stimulated at 37°C. The associations of PAG with FynT and Csk were determined by immunoblotting anti-PAG immunoprecipitates with anti-FynT (first panel) and anti-Csk (second panel), respectively. Overall tyrosine phosphorylation of PAG was determined by immunoblotting PAG immunoprecipitates with antiphosphotyrosine (P.tyr) (third panel), whereas phosphorylation of PAG at Y314 was ascertained by probing total cell lysates with an antibody directed against phospho-Y314 of PAG (pY314) (fourth panel). The abundances of PAG, FynT, and Csk were confirmed by immunoblotting total cell lysates with anti-PAG (fifth panel), anti-FynT (sixth panel), and anti-Csk (seventh panel), respectively. (B) Overall protein tyrosine phosphorylation. Total cell lysates from the experiment whose results are depicted in panel A were immunoblotted with antiphosphotyrosine (P.tyr) antibodies. The migrations of prestained molecular mass markers (in kDa) are shown on the right. (C) Quantitation of the extents of PAG-FynT interaction, PAG-Csk interaction, and phosphorylation of Y314 of PAG (pY314) in the experiment whose results are shown in panel A. (D) Quantitation of the extents of PAG-FynT interaction, PAG-Csk interaction, and phosphorylation of Y314 of PAG (pY314). This experiment was performed as for panels A and B, except that cells were stimulated at room temperature (21°C) (data not shown).
FIG. 2.
FIG. 2.
Impact of FynT deficiency on phosphorylation of PAG at tyrosine 314. T cells from wild-type (fyn+/+) or Fyn-deficient (fyn−/−) mice were stimulated or not for 2 min with biotinylated anti-CD3 and avidin. (A) Phosphorylation of PAG at Y314. The extent of phosphorylation of PAG at Y314 was determined by immunoblotting total cell lysates with anti-pY314 (first panel). The interactions of PAG with FynT and Csk were determined by immunoblotting PAG immunoprecipitates with anti-FynT (third panel) and anti-Csk (fourth panel), respectively. The amount of PAG in these immunoprecipitates was verified by reprobing with anti-PAG (fifth panel). The abundances of PAG, FynT and Csk were verified by immunoblotting total cell lysates with anti-PAG (second panel), anti-FynT (sixth panel), and anti-Csk (seventh panel), respectively. Lanes 1 to 4, thymocytes; lanes 5 and 6, purified lymph node T cells. (B) Overall protein tyrosine phosphorylation. Total cell lysates from the experiment whose results are depicted in panel A (lanes 1 to 4) were immunoblotted with antiphosphotyrosine (P.tyr) antibodies. The positions of prestained molecular mass markers (in kDa) are shown on the right.
FIG. 3.
FIG. 3.
Associations of various PAG mutants with FynT and Csk in T cells. (A) Associations of PAG with FynT and Csk. T cells from nontransgenic (control) mice or transgenic mice expressing the indicated PAG polypeptides were lysed and processed for immunoprecipitation. The associations of PAG with FynT and Csk were determined by immunoblotting anti-PAG immunoprecipitates (lanes 1 to 5) with anti-FynT (first panel) and anti-Csk (second panel), respectively. Lane 6 shows an immunoprecipitation of lysates from control mice with normal rabbit serum. Overall phosphorylation of PAG was ascertained by immunoblotting anti-PAG immunoprecipitates with antiphosphotyrosine (P.tyr) antibodies (third panel). The abundance of PAG in the immunoprecipitates was verified by probing these immunoprecipitates with anti-PAG (fourth panel). The abundances of FynT and Csk were confirmed by immunoblotting total cell lysates with anti-FynT (fifth panel) and anti-Csk (sixth panel), respectively. PAG wt, wild-type PAG. (B) Phosphorylation of PAG polypeptides at Y314. The extents of the phosphorylations of the various PAG polypeptides at Y314 were determined by immunoblotting total cell lysates with anti-pY314 (first panel). The abundance of PAG was verified by immunoblotting total cell lysates with anti-PAG (second panel). (C) Cell fractionation. The extents of the associations of the various PAG polypeptides with lipid rafts were determined by cell fractionation studies and anti-PAG immunoblot analyses, as detailed in Materials and Methods. Fractions 2 and 3 correspond to lipid rafts, while fractions 8 and 9 contain soluble, non-lipid raft-associated proteins. The percentages of PAG in the two compartments were determined by a PhosphorImager and are shown at the bottom.
FIG. 4.
FIG. 4.
Regulation of TCR-induced calcium fluxes by various PAG polypeptides. Purified splenic and lymph node T cells from nontransgenic (control) mice or transgenic mice expressing the indicated PAG polypeptides were loaded with Indo-1 and were stimulated at 37°C with biotinylated anti-TCR and avidin. Changes in intracellular calcium were monitored using a BD LSR cell analyzer, by gating on CD4+ T cells. The ratio of bound Indo-1 (read in the FL4 channel)/free Indo-1 (read in the FL5 channel) is shown on the ordinate. The arrow corresponds to the moment at which the biotinylated anti-TCR antibody and avidin were added. Cells were observed for a total of 10 min. Similar results were obtained when thymocytes or CD8+ T cells were analyzed (data not shown). All cells exhibited equivalent calcium fluxes when they were stimulated with ionomycin, as reported elsewhere (6) (data not shown). The various transgenic mice used expressed equivalent amounts of PAG protein (data not shown).
FIG. 5.
FIG. 5.
Role of FynT in the impact of PAG Y314F on TCR-triggered calcium fluxes. The experiment was performed as explained for Fig. 4, except that mice expressing PAG Y314F in the presence or in the absence of endogenous FynT were analyzed, and biotinylated anti-CD3 was used. All cells responded equally well to ionomycin (data not shown). T cells from transgenic mice expressed equivalent amounts of PAG Y314F and contained FynT or not in agreement with their genotypes (data not shown).
FIG. 6.
FIG. 6.
Impact of PAG Y314F on other TCR-triggered signals. T cells were purified from spleen and lymph nodes of the indicated mice and stimulated for various periods of time at 37°C with biotinylated anti-CD3 and anti-CD28, followed by treatment with avidin. Global protein tyrosine phosphorylation was ascertained by immunoblotting total cell lysates with antiphosphotyrosine (P.tyr) antibodies (first panel). Activation of Erk (p42 and p44) was determined by probing total cell lysates with anti-phospho-Erk (second panel), whereas the abundance of Erk was verified by probing lysates with an antibody that recognized all forms, phosphorylated or not, of Erk (third panel). Phosphorylation of IκBα was examined by immunoblotting total cell lysates with an antibody directed against phospho-IκBα, while the abundance of IκBα was ascertained by probing lysates with an antibody recognizing all forms of IκBα. The migrations of prestained molecular mass markers (in kDa) are indicated on the right.
FIG. 7.
FIG. 7.
Influence of anergy on the associations of PAG with FynT and Csk. (A) Ionomycin-induced anergy. CD4+ T cells from normal mice were activated with anti-CD3 plus anti-CD28 and then rested in IL-2, as detailed in Materials and Methods. They were subsequently treated or not for 16 h with ionomycin (1 μM). The associations of PAG with FynT and Csk were determined by immunoblotting anti-PAG immunoprecipitates with anti-FynT (first panel) and anti-Csk (second panel), respectively. The abundance of PAG in these immunoprecipitates was verified by probing with anti-PAG (third panel). The abundances of FynT and Csk were confirmed by immunoblotting total cell lysates with anti-FynT (fourth panel) and anti-Csk (fifth panel), respectively. (B) Ionomycin-induced anergy. Cells from the experiment whose results are depicted in panel A were restimulated or not for 42 h with anti-CD3 and anti-CD28. Thymidine incorporation and IL-2 release were monitored as detailed in Materials and Methods. Standard deviations of the triplicate values in the assays are shown. (C) Soluble-peptide-induced anergy. Transgenic mice expressing the LCMV gp33-specific TCR (12 in each group) were injected intravenously with soluble gp33 peptide (300 μg in PBS) or PBS alone (control) on day 0 and day 3. On day 4, CD8+ T cells were purified by negative selection. Flow cytometry analyses confirmed that equivalent proportions (>90%) of purified CD8+ T cells from the two groups expressed the gp33-specific transgenic TCR (data not shown). The association of PAG with FynT and Csk was determined by immunoblotting anti-PAG immunoprecipitates with anti-FynT (first panel) and anti-Csk (second panel), respectively. The abundance of PAG in these immunoprecipitates was verified by probing with anti-PAG (third panel). The abundances of FynT and Csk were confirmed by immunoblotting total cell lysates with anti-FynT (fourth panel) and anti-Csk (fifth panel), respectively. (D) Soluble peptide-induced anergy. Cells from the experiment whose results are depicted in panel C were restimulated or not for 31 h with gp33 peptide (0.1 μM) and irradiated syngeneic splenocytes. Thymidine incorporation and IL-2 secretion were monitored as detailed in Materials and Methods. Standard deviations of the triplicate values in the assays are shown.
FIG. 8.
FIG. 8.
Effects of various PAG polypeptides and FynT on anti-CD3-induced T-cell anergy in vitro. CD4+ T cells from nontransgenic (control) mice or transgenic mice expressing the indicated PAG polypeptides, with or without FynT, were first activated and then rested in IL-2, as detailed in Materials and Methods. They were then treated or not with anti-CD3 (1 μg per ml), rested for an additional 30 h, and then restimulated with anti-CD3 and anti-CD28. Proliferation was monitored by assaying tritiated thymidine incorporation (A and C), while IL-2 secretion was determined by an enzyme-linked immunosorbent assay (B and D). The anergy index is the ratio between the thymidine incorporation or IL-2 secretion levels of untreated and anti-CD3-treated cells. CPM, counts per minute; PAG wt, wild-type PAG. Standard deviations for the triplicate values in the assays are shown. (A and B) Impacts of various PAG polypeptides. (C and D) Role of FynT in the effect of PAG Y314F.
FIG. 9.
FIG. 9.
Effects of PAG Y314F and FynT on superantigen-induced T-cell anergy in vivo. Nontransgenic (control) mice or transgenic mice expressing the indicated PAG polypeptides, with or without FynT, were injected intraperitoneally with SEB (100 μg) or PBS alone. After 10 days, CD4+ T cells were isolated from spleen and incubated for 48 h with SEB (0.12 μg per ml) in the presence of irradiated splenocytes from C57BL/6 mice as antigen-presenting cells. Proliferation was monitored by assaying tritiated thymidine incorporation (A), while IL-2 secretion was determined by an enzyme-linked immunosorbent assay (B). The anergy index is the ratio between the thymidine incorporation or IL-2 secretion levels of PBS-injected and SEB-injected mice. The proportions of Vβ8.1/8.2-positive cells in purified CD4+ T cells from the various mice were as follows (data not shown): for PBS-treated mice, 17.9% (control), 17.4% (PAG Y314F), 17.7% (fyn−/−), and 17.1% (PAG Y314F fyn−/−), and for SEB-treated mice, 13.8% (control), 15.3% (PAG Y314F), 15.5% (fyn−/−), and 13.0% (PAG Y314F fyn−/−). CPM, counts per minute. PAG wt, wild-type PAG. Standard deviations for the triplicate values in the assays are shown.
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