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Laboratory of Molecular Oncology, Clinical Research Institute of Montreal, Montréal, Québec, Canada,1 Institute of Immunology, Otto von Guericke University, 39120 Magdeburg, Germany,2 Department of Medicine, University of Montreal, Montréal, Québec, Canada,3 Department of Medicine, McGill University, Montréal, Québec, Canada4
Received 21 October 2006/ Returned for modification 16 November 2006/ Accepted 14 December 2006
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ABSTRACT |
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INTRODUCTION |
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So far, the major protein shown to associate with PAG/Cbp (hereafter named PAG) is Csk, a cytoplasmic protein tyrosine kinase (PTK) implicated in the inhibition of Src-related PTKs (4, 16, 34). Peptide binding and site-directed mutagenesis studies indicated that this association is mediated by phosphorylation of PAG at tyrosine 314 (Y314) in mice (Y317 in humans) and by the SH2 domain of Csk (4, 6, 16, 28). This interaction was shown to enhance the catalytic activity of Csk and to favor inactivation of Src family kinases in lipid rafts (4, 16, 28). It was proposed that PAG is implicated in the negative regulation of Src kinases in lipid rafts and, thus, is likely to be a negative regulator of cellular processes mediated by Src kinases.
Direct support for this idea was provided by the finding that enforced expression of PAG in COS-7 cells caused pronounced reductions of the enzymatic activities of Src-related kinases (16). This effect was dependent on Y314 of PAG. Furthermore, it was reported that overexpression of PAG in normal T cells, the T-cell line Jurkat, or the basophil leukemia cell line RBL-2H3 suppressed cellular responses mediated by Src-related kinases (4, 6, 21). The inhibitory impact of PAG in normal T cells also required Y314 (6). Finally, it was shown that diminution of PAG expression in mouse embryo fibroblasts by RNA interference caused changes in cell spreading and adhesion similar to those observed in Csk-deficient fibroblasts (25). While these various findings provided a compelling argument that PAG-Csk plays a role in the negative regulation of Src family kinases, it is noteworthy that two groups recently reported that mice lacking PAG exhibited little or no phenotype (8, 36). Given that a severe phenotype was observed in Csk-deficient mice (11, 19), it was postulated that other, PAG-independent mechanisms of Csk recruitment may exist and that these mechanisms compensate for the absence of PAG expression.
Characterizations of the interaction between PAG and Csk have been mostly performed with T cells. In resting T cells, PAG was shown to be constitutively tyrosine phosphorylated and associated with Csk (4, 6, 29). These modifications are rapidly lost upon engagement of the T-cell antigen receptor (TCR) complex. In this light, it was proposed that the PAG-Csk complex may prevent activation of resting T cells and that relief of this inhibition upon TCR engagement may be instrumental in allowing T-cell activation. In agreement with this, we reported that augmentation of expression of wild-type PAG in T cells by transgenesis caused inhibitions of TCR-triggered protein tyrosine phosphorylation, cellular proliferation, and interleukin-2 (IL-2) secretion (6). Opposite effects were seen in transgenic mice expressing a PAG mutant in which Y314 was replaced by phenylalanine (PAG Y314F), supporting the idea that these effects were mediated by Csk.
In addition to Csk, other proteins interact with PAG. In T cells, PAG is associated with the Src-related protein tyrosine kinase FynT (4). While the structural basis of this association is not known, this complex has been reported not to be modulated by TCR stimulation (4), suggesting that it is not mediated by PAG tyrosine phosphorylation. The interaction between PAG and FynT is probably critical for the function of PAG, as PAG tyrosine phosphorylation and binding of PAG to Csk were noted to be dramatically reduced in FynT-deficient T cells (37). A similar association of PAG with Lyn, another member of the Src family, was described for mast cells (20). Moreover, PAG was observed to interact by way of its carboxyl-terminal tail with EBP-50, a PDZ domain-containing protein associated with the cytoskeleton (12). This interaction seemingly links lipid raft-associated PAG to the cytoskeleton.
In this paper, we examined in greater detail the regulation and function of the interaction between PAG and FynT. Our data showed that, as is the case for the PAG-Csk interaction, the PAG-FynT association was rapidly lost in response to TCR stimulation. Dissociation of PAG-FynT complexes preceded disappearance of PAG-Csk complexes, suggesting that the former may trigger the loss of the PAG-Csk interaction in activated T cells. In other experiments, we noted that the extents of the interactions between PAG and FynT, but not PAG and Csk, were augmented in anergic T cells. Moreover, enforced expression in normal T cells of a PAG mutant capable of interacting with FynT, but not Csk, resulted in a selective enhancement of TCR-triggered calcium fluxes and promoted T-cell anergy. Together, these data provide a better understanding of the role of the PAG-FynT interaction in T cells. Furthermore, they further implicate FynT in the induction of T-cell anergy.
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MATERIALS AND METHODS |
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B
, or phospho-IB were purchased from Cell Signaling Technology Inc. (Beverly, MA), Santa Cruz Biotechnology Inc. (Santa Cruz, CA), or Upstate (Charlottesville, VA). Biotinylated anti-CD3 MAb 145-2C11, biotinylated anti-TCR MAb H57-597, and anti-CD28 MAb 37-51 were purchased from eBiosciences (San Diego, CA) or BD Biosciences (Mississauga, Ontario, Canada). Avidin was purchased from Calbiochem-Novabiochem Corporation, San Diego, CA. Mice. Transgenic mice expressing wild-type PAG, a PAG mutant in which tyrosine 314 was mutated to phenylalanine (PAG Y314F), or a PAG mutant in which all nine cytoplasmic tyrosines were mutated to phenylalanines (PAG 9YF), under the control of the human CD2 promoter, were reported elsewhere (6). A similar approach was used to create transgenic mice expressing a PAG mutant in which all cytoplasmic tyrosines except Y314 were mutated to phenylalanines (PAG 8YF). Transgenic mice were produced by the IRCM Transgenic Service, according to established protocols. At least two independent founders of each transgenic type were used. The levels of the exogenous PAG proteins were 6- to 12-fold greater than those of endogenous PAG (data not shown). Mice lacking expression of Fyn (fyn–/–) (27) were obtained from Jackson Laboratory, Bar Harbor, ME. Transgenic mice expressing the class I major histocompatibility complex-restricted, lymphocytic choriomeningitis virus (LCMV) gp33-specific TCR P14 (22) were obtained from Taconic, Hudson, NY. All animals were backcrossed for at least five generations to the C57BL/6 background.
Cell stimulation. Thymocytes were obtained by making cell suspensions from thymus tissue. CD4+ lymph node or spleen T cells, depleted of natural killer T cells, were purified from mice by using a Stem Cell purification kit (Stem Cell Technology Inc., Vancouver, British Columbia, Canada) or T-cell enrichment columns (R&D Systems, Minneapolis, MN). Cell purity was consistently greater than 90% (data not shown). Effector CD4+ T cells were generated by stimulating purified CD4+ T cells with coated anti-CD3 (3 µg per ml) and soluble anti-CD28 (1 µg per ml) for 2 to 3 days and then expanding them in IL-2 (50 units per ml)-containing medium for 3 days generally. Cells (at a concentration of 30 x 106 per ml) were stimulated for the indicated periods of time at 37°C or room temperature (21°C) with biotinylated anti-CD3 (10 µg) or anti-TCR (10 µg) and avidin (14 µg) in a volume of 200 µl. Unstimulated controls were incubated with avidin alone. After lysis in buffer containing maltoside supplemented with protease and phosphatase inhibitors (6), lysates were subjected to immunoprecipitation or immunoblot analysis.
Cell fractionation. Cells were lysed in 1 ml of Brij-58-containing buffer supplemented with protease and phosphatase inhibitors, as detailed elsewhere (6). Lysates were then mixed with 80% sucrose (made in the same buffer without detergent) and overlaid sequentially with 30% sucrose and 5% sucrose. After centrifugation at 200,000 x g, 0.5-ml fractions were collected from the top of the gradient. Fractions 2 and 3 contained most of the lipid rafts, while fractions 8 and 9 contained the soluble proteins (data not shown) (6). Individual fractions were subjected to immunoblot analysis after solubilization using 1% maltoside.
Immunoprecipitations and immunoblot analyses. To extract PAG and PAG-associated proteins from lipid rafts, cells were lysed in a buffer containing maltoside (1% n-dodecyl-ß-D-maltoside, 50 mM Tris [pH 7.6], 150 mM NaCl, and 2 mM EDTA) supplemented with protease and phosphatase inhibitors (6). Immunoprecipitations and immunoblot analyses were performed as previously described (7, 33). Radioactivity was quantitated with a Storm PhosphorImager (Molecular Dynamics, General Electrics Canada, Mississauga, Ontario, Canada).
Intracellular-calcium fluxes. Thymocytes or splenic and lymph node T cells were loaded with Indo-1 (10 µM; Molecular Probes, Eugene, OR) for 45 min at 37°C and stained with phycoerythrin-coupled anti-CD4 MAb L3T4 and fluorescein isothiocyanate-coupled anti-CD8 MAb Ly-2 (BD Biosciences, Mississauga, Ontario, Canada), as described previously (6). After the cells were washed, they were stimulated at 37°C with biotinylated anti-TCR or anti-CD3 and avidin. Changes in intracellular calcium over time were monitored using the FL4 and FL5 channels of a BD LSR cell analyzer (BD Biosciences, Mississauga, Ontario, Canada). CD4+ single-positive cells were selectively analyzed by gating on CD4+ CD8– cells. As a control, cells were stimulated with the calcium ionophore ionomycin (100 ng per ml).
Anergy induction. For induction of anergy in vitro using ionomycin or anti-CD3, CD4+ T cells were first activated for 48 h with anti-CD3 (3 µg per ml; on plastic) and anti-CD28 (1 µg per ml; soluble) (14, 15, 17). They were then expanded in IL-2 (50 U per ml) for 3 days. After this, anergy was induced by treating cells with ionomycin (1 µM) or coated anti-CD3 alone (1 µg per ml) for 16 h. Controls were with no addition. For anergy induced by ionomycin, cells were subsequently processed immediately, either for biochemical studies (immunoprecipitation or immunoblot analysis) or for functional studies (proliferation induced by anti-CD3 plus anti-CD28). For anergy triggered with anti-CD3, cells were first washed and rested in growth medium for 30 h. They were then restimulated with anti-CD3 (0.11 µg per ml) and anti-CD28 (1 µg per ml) for 48 h. Thymidine incorporation and IL-2 secretion were measured (6). For induction of anergy in vivo, mice were injected intraperitoneally with the superantigen Staphylococcus aureus enterotoxin B (SEB; 100 µg in 100 µl of phosphate-buffered saline [PBS]; Sigma-Aldrich Canada Ltd., Oakville, Ontario, Canada) or with PBS alone (18). After 10 days, CD4+ T cells were purified from spleen and stimulated for 48 h with various concentrations of SEB (0.013 to 10 µg per ml) in the presence of irradiated splenocytes from C57BL/6 mice. Thymidine incorporation and IL-2 secretion were measured (6). The percentages of Vß8.1/8.2-positive cells in purified CD4+ T cells were determined by staining with TCR Vß8.1/8.2-specific MAb MR5-2 (BD Biosciences, Mississauga, Ontario, Canada). To induce anergy in vivo in transgenic mice expressing the class I major histocompatibility complex-restricted, LCMV gp33-specific TCR P14, mice (12 in each group) were injected intravenously with gp33 peptide (300 µg in PBS) or PBS alone on day 0 and day 3. On day 4, CD8+ T cells were purified by negative selection, using a Stem Cell purification kit. In all cases, flow cytometry analyses confirmed that more than 90% of purified CD8+ T cells expressed the transgenic anti-gp33 TCR (data not shown). Anergy induction was verified by stimulating purified CD8+ T cells with gp33 peptide (lysine-alanine-valine-tyrosine-asparagine-phenylalanine-alanine-threonine-methionine; 0.1 µM) and irradiated syngeneic splenocytes and measuring proliferation and IL-2 secretion. Purified CD8+ T cells were then processed for biochemical analyses as detailed above.
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RESULTS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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80%) and rapid decrease in the association of PAG with FynT (Fig. 1A, first panel), in comparison to no stimulation (Fig. 1A, first panel, lane 1). This effect was seen within 30 seconds of TCR ligation (Fig. 1A, first panel, lane 2) and was maximal at 1 minute (Fig. 1A, first panel, lane 3). Interestingly, the loss of the PAG-FynT association preceded the disappearance of the interaction between PAG and Csk (Fig. 1A, second panel). In the case of PAG-Csk, little or no change was occurring at 30 seconds (Fig. 1A, second panel, lane 2) and maximal dissociation required 2 minutes of stimulation (Fig. 1A, second panel, lane 4). These results are represented graphically in Fig. 1C. The dissociation of PAG-FynT also preceded the induction of overall protein tyrosine phosphorylation in T cells, as revealed by immunoblotting total cell lysates with antiphosphotyrosine antibodies (Fig. 1B).
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To confirm the differences in the kinetics of dissociation of PAG-FynT, dissociation of PAG-Csk, and dephosphorylation of Y314, this experiment was also conducted by stimulating cells at room temperature rather than 37°C (Fig. 1D). This is known to slow down the onset of TCR-triggered signaling events and to delay dephosphorylation reactions. Under these conditions, we found that dissociation of the PAG-FynT complex was maximal after 1 to 2 min of TCR stimulation. In contrast, loss of the PAG-Csk association and Y314 dephosphorylation were delayed, reaching their maxima after 6 min. These observations firmly supported the notion that loss of the PAG-FynT interaction in response to TCR stimulation preceded dephosphorylation of PAG Y314 and disappearance of the PAG-Csk association.
One interpretation of these results is that dissociation of PAG-FynT may lead to dephosphorylation of Y314 and, consequently, dissociation of PAG-Csk. To address further this possibility, the impact of FynT expression on phosphorylation of Y314 was directly examined by immunoblotting total cell lysates from fyn+/+ and fyn–/– T cells with the phospho-specific anti-pY314 antibody (Fig. 2A, first panel). We found that cells lacking FynT, both thymocytes (Fig. 2A, lanes 3 and 4) and lymph node T cells (Fig. 2A, lane 6), showed marked decreases in Y314 phosphorylation, in comparison to cells from wild-type mice (Fig. 2A, lanes 1, 2, and 5). It is noteworthy, however, that some degree of PAG tyrosine phosphorylation remained in unstimulated FynT-deficient T cells (Fig. 2A, lanes 3 and 6). This residual tyrosine phosphorylation was partially affected by TCR stimulation (Fig. 2A, lane 4) and was equivalent to the extent of Y314 tyrosine phosphorylation remaining in TCR-stimulated normal T cells (Fig. 2A, lane 2). It also correlated with a residual binding of PAG to Csk in FynT-deficient T cells (Fig. 2A, fourth panel, compare lane 3 with lane 1). While the precise basis of these findings is not known, they likely reflect the existence of a FynT-independent, TCR-regulated pool of tyrosine-phosphorylated PAG in T cells. Thus, combined with the data shown in Fig. 1 and previously published results (37), those shown in Fig. 2 were consistent with the idea that dissociation of the PAG-FynT complex may be responsible for loss of the PAG-Csk interaction during T-cell activation.
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To assess whether the tyrosines other than Y314 contributed to Csk recruitment by promoting the FynT-dependent phosphorylation of Y314, the impacts of the various mutations on Y314 phosphorylation were ascertained by immunoblotting cell lysates with the phospho-specific anti-pY314 antibody (Fig. 3B, first panel). As expected, mutation of Y314 alone (Fig. 3B, first panel, lane 3) or replacement of all cytoplasmic tyrosines of PAG, including Y314 (Fig. 3B, first panel, lane 4), abolished the increased phosphorylation at Y314. Surprisingly, this reactivity was also eliminated when all cytoplasmic tyrosines except Y314 (Fig. 3B, first panel, lane 5) were mutated. Coupled with the observation that FynT was needed for Y314 phosphorylation, this result provided a compelling indication that binding of PAG to FynT is required for phosphorylation of PAG at Y314 and the subsequent association of PAG with Csk.
To ensure that the differences in the associations of the various PAG polypeptides with FynT and Csk were not due to variations in their associations with lipid rafts, cell fractionation experiments using T cells from the various mice were performed, in order to separate lipid raft and soluble proteins (Fig. 3C). In these studies, fractions 2 and 3 contain most lipid raft-associated proteins, while fractions 8 and 9 bear most non-lipid raft-associated proteins (data not shown). The distribution of PAG in these fractions was examined by immunoblotting with anti-PAG. This analysis showed that, for all the various PAG polypeptides, >80% were located in lipid raft fractions (Fig. 3C, lanes 1 to 4). This is consistent with previous assessments of the extents of the associations of endogenous PAG molecules in T cells with lipid raft fractions (4, 6). Thus, it is improbable that variations in the extents of targeting of the various PAG polypeptides to lipid rafts explained the differences in their associations with FynT and Csk.
Evidence that PAG-associated FynT selectively promotes antigen receptor-triggered calcium fluxes in T cells. These findings showed that one of the roles of PAG-associated FynT is to regulate the recruitment of Csk to PAG. To evaluate whether this pool of FynT molecules may have additional functions, we studied TCR-mediated responses in T cells from transgenic mice expressing wild-type PAG (which binds FynT and Csk), PAG Y314F (which binds FynT but not Csk), or PAG 9YF (which binds neither FynT nor Csk). Along these lines, it is noteworthy that we had already reported that TCR-triggered T-cell proliferation and IL-2 secretion were reduced in T cells expressing wild-type PAG (6). In contrast, these responses were enhanced in T cells containing PAG Y314F or PAG 9YF. These data had implied that wild-type PAG had an inhibitory effect on TCR signaling and that this effect was largely dependent on the capacity of PAG to recruit Csk.
We extended these findings by studying the impacts of the various PAG polypeptides on TCR-triggered calcium fluxes (Fig. 4). Purified splenic and lymph node T cells were loaded with the calcium indicator dye Indo-1, and T cells were stimulated at 37°C with biotinylated anti-TCR and avidin. Changes in intracellular calcium over time were monitored using flow cytometry, by gating on single-positive CD4+ T cells. In agreement with our earlier results (6), cells with augmented levels of expression of wild-type PAG (Fig. 4B) exhibited a striking inhibition of TCR-triggered calcium fluxes, in comparison to control nontransgenic cells (Fig. 4A). In contrast, cells containing PAG Y314F (Fig. 4C) had an enhancement of TCR-initiated calcium fluxes, compared to control cells (Fig. 4A). Unexpectedly, however, we found that cells bearing PAG 9YF (Fig. 4D) had normal TCR-mediated calcium changes. These differences were not due to variations between pools of intracellular calcium in the various T cells, since all cells responded equally well to ionomycin, as reported elsewhere (6) (data not shown). Similar results were obtained when calcium responses in thymocytes or CD8+ T cells were studied (data not shown).
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B (Fig. 6, fourth panel) and total IB (Fig. 6, fifth panel) showed no convincing difference in IB activation and IB degradation. Since IB degradation predicates the activation of NF-B during T-cell activation (35), this suggested that PAG Y314F did not affect NF-B activation. Thus, together with the results shown in Fig. 4 and 5, those shown in Fig. 6 supported the idea that augmented levels of PAG-associated FynT, in the absence of elevated PAG-Csk, caused an increase in TCR-triggered protein tyrosine phosphorylation and calcium fluxes, coupled with a decrease in Erk activation and no effect on NF-B.
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B and AP-1, can lead to a state of T-cell unresponsiveness known as anergy (or adaptive tolerance) (15, 17). In a variety of systems, it was observed that T-cell anergy is accompanied by an increase in the expression levels of FynT, raising the possibility that FynT may be an effector of anergy (3, 9, 23, 30, 31). To ascertain whether a selective increase in the association of PAG with FynT may favor anergy, we first examined whether anergy was accompanied by a change in the stoichiometry of this association, using the ionomycin-induced T-cell anergy model (Fig. 7A and B) (15, 17). Ionomycin is a very efficient inducer of T-cell anergy, seemingly because it causes strong calcium-dependent signals in the absence of parallel activation of other signaling pathways, such as NF-B (10, 17).
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We also studied whether similar changes occurred in an in vivo model of T-cell anergy (Fig. 7C and D). For this purpose, transgenic mice expressing the class I-restricted, LCMV gp33-specific TCR P14 were injected or not intravenously on day 0 and day 3 with soluble gp33 peptide. On day 4, CD8+ T cells were purified, and the extents of the associations of PAG with FynT and Csk were examined (Fig. 7C). We observed that the amount of PAG-associated FynT was increased approximately twofold in CD8+ T cells from peptide-treated mice (Fig. 7C, first panel, lane 2), compared to that in CD8+ T cells from control PBS-injected mice (Fig. 7C, first panel, lane 1). In contrast, the amount of PAG-associated Csk was dramatically reduced (Fig. 7C, second panel). The increase in PAG-associated FynT in CD8+ T cells from peptide-injected mice correlated with an increment in the abundance of FynT in total cell lysates (Fig. 7C, fourth panel), despite the fact that, as was the case for the ionomycin-triggered anergy (Fig. 7A), there was a pronounced reduction of the abundance of PAG in T cells from peptide-injected mice (Fig. 7C, third panel, lane 2). However, there was no change in the abundance of Csk in these lysates (Fig. 7C, fifth panel). To verify the efficiency of the protocol used for anergy induction in vivo, CD8+ T cells from the treated mice were also stimulated in vitro with gp33 peptide plus irradiated syngeneic splenocytes, and T-cell proliferation and IL-2 secretion were determined (Fig. 7D). This experiment demonstrated that, compared to CD8+ T cells from control mice, CD8+ T cells from peptide-injected mice exhibited a reduction of T-cell proliferation and, to a greater extent, IL-2 secretion.
Evidence that PAG-associated FynT can promote T-cell anergy. Considering these observations, we addressed the effect of PAG-associated FynT on T-cell anergy by evaluating the impacts of PAG Y314F and the other PAG polypeptides in two different models of anergy (Fig. 8 and 9). First, we examined the influence of these PAG molecules in an in vitro model of T-cell anergy (Fig. 8) (14). CD4+ T cells from the various mice were first activated in vitro for 2 days by a combination of anti-CD3 and anti-CD28, subsequently expanded for 3 days in the presence of IL-2, and then stimulated or not for 16 h by anti-CD3 alone to induce anergy. After an additional rest period of 30 h, cells were restimulated for 48 h with anti-TCR and anti-CD28 and assayed for proliferation or IL-2 secretion.
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These data showed that PAG Y314F, but not wild-type PAG or PAG 9YF, markedly stimulated the induction of anergy in response to anti-CD3. To examine whether this effect was mediated by FynT, similar experiments were conducted with mice expressing PAG Y314F that were bred or not with fyn–/– mice (Fig. 8C and D). In PAG Y314F-expressing T cells, a lack of FynT expression caused a reduction of the anergy index from 79 to 1.4 in proliferation assays (Fig. 8C). The index became essentially identical to that seen in T cells from normal mice (1.7). These results indicated that, as was the case for the calcium fluxes, the augmented anergy caused by PAG Y314F was mediated by FynT. Equivalent results were obtained when IL-2 secretion was measured (Fig. 8D).
We also studied the impact of PAG Y314F, in the presence or in the absence of FynT, on anergy induction in vivo (Fig. 9) (18). Mice were injected with the superantigen SEB or with PBS, and after 10 days, CD4+ T cells were restimulated in vitro with various concentrations of SEB. They were then assayed for proliferation (Fig. 9A) or IL-2 secretion (Fig. 9B). Importantly, staining of purified CD4+ T cells from the diverse animals indicated that there was no marked difference between the relative abundances of Vß8.1/8.2-positive T cells in mice injected with PBS or those injected with SEB (data not shown). This T-cell subset is known to mediate responses to SEB.
According to our expectation, injection of control mice with SEB caused a reproducible inhibition of superantigen-induced T-cell proliferation (Fig. 9A) or IL-2 release (Fig. 9B) in the restimulation assay, in comparison to injection with PBS alone. Here, the anergy index corresponds to the ratio between the thymidine incorporation or IL-2 secretion levels of PBS-injected and SEB-injected mice at a given concentration of SEB. In normal mice and at 0.12 µg per ml of SEB, these values were 1.7 for proliferation (Fig. 9A) and 4.0 for IL-2 production (Fig. 9B). They were enhanced to 4.0 and 10.0, respectively, in mice expressing PAG Y314F. Similar differences were seen when higher concentrations of SEB were used for restimulation (data not shown). As was the case for the anti-CD3-triggered anergy, the impact of PAG Y314F on SEB-induced anergy was eliminated when mice were crossed with fyn–/– mice. The values of the anergy index for these mice were 1.0 for proliferation (Fig. 9A) and 3.4 for IL-2 secretion (Fig. 9B). It is noteworthy that SEB-mediated anergy was also abrogated in FynT-deficient mice in the absence of PAG Y314F, supporting a role for FynT in anergy induction even in the absence of PAG Y314F. Therefore, the results shown in Fig. 8 and 9 were consistent with the hypothesis that increased PAG-associated FynT, in the absence of augmented PAG-associated Csk, can promote T-cell anergy.
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DISCUSSION |
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In the present report, we wanted to understand better the role and regulation of the PAG-FynT interaction. Our data showed that, like Csk, FynT was constitutively associated with PAG in unstimulated T cells. Moreover, as was the case for Csk, FynT became rapidly dissociated from PAG following TCR engagement. Interestingly, time course analyses indicated that dissociation of the PAG-FynT complex preceded PAG dephosphorylation, cleavage of the PAG-Csk interaction, and induction of overall protein tyrosine phosphorylation following TCR stimulation. Using an antibody specifically recognizing phosphorylated Y314 of PAG, it was also established that phosphorylation of Y314 was reduced in FynT-deficient T cells, extending previous findings that FynT was required for global PAG tyrosine phosphorylation (37). Lastly, we found that a PAG mutant (PAG 8YF) that cannot bind FynT but has a preserved Csk-binding site (Y314) was no longer phosphorylated at Y314 and was unable to interact with Csk in T cells. Presumably, the lack of phosphorylation of PAG 8YF at Y314 was due to the inability of this mutant to associate with FynT.
Based on these findings and previously published results (4, 6, 29), we propose that constitutive binding of PAG to FynT in resting T cells is critical for phosphorylation of PAG at Y314 and subsequent binding of PAG to Csk. We also postulate that, following TCR stimulation, dissociation of the PAG-FynT complex is instrumental in causing loss of PAG phosphorylation at Y314, disruption of the PAG-Csk association and, ultimately, induction of TCR-triggered protein tyrosine phosphorylation. Although the mechanism of PAG-FynT dissociation in response to TCR engagement is not known, possibilities include changes in the conformation of PAG or FynT, dephosphorylation of PAG at the tyrosines other than Y314, or posttranslational modifications of FynT, such as serine/threonine phosphorylation. Obviously, future studies are warranted to elucidate the mechanism of this dissociation.
The precise mechanism by which FynT interacts with PAG also remains to be clarified. Since this association required tyrosines in the cytoplasmic domain of PAG, it may be mediated, at least in part, by direct binding of the FynT SH2 domain to sites of tyrosine phosphorylation in PAG. In keeping with this, we have observed that recombinant FynT SH2 domains can interact with PAG in vitro and that mutation of all tyrosines of PAG other than Y314 (PAG 8YF mutation) abrogates this association (our unpublished results). It seems, however, that more than one tyrosine would be implicated, as mutation of the first five or the last three tyrosines of PAG does not abolish the association of PAG with FynT SH2 domains in vitro. It is possible that additional mechanisms, including binding of the FynT SH3 domain to proline-rich sequences in PAG or participation of intermediary proteins, are also implicated. Future studies are needed to address exhaustively these various possibilities.
We were interested in examining whether PAG-associated FynT may mediate other functions, in addition to allowing the recruitment of Csk via phosphorylation of Y314. To this end, the functional impact in normal T cells of the expression of wild-type PAG, a PAG mutant that can bind FynT but not Csk (PAG Y314F), or a PAG mutant that can bind neither FynT nor Csk (PAG 9YF) was examined. We observed that PAG Y314F caused a pronounced increase in TCR-triggered calcium fluxes. Importantly, this effect was not seen in T cells expressing PAG 9YF. Furthermore, it was eliminated when mice expressing PAG Y314F were crossed with fyn–/– mice. Hence, it is likely that PAG-associated FynT was responsible for this augmentation of TCR-triggered calcium fluxes. In contrast to the enhanced calcium response, PAG Y314F had no effect on phosphorylation of IB, a marker of NF-B activation, and caused a reduction of Erk activation. Therefore, the stimulatory effect of PAG Y314F on calcium fluxes was rather specific. Although the basis for the concomitant inhibition of Erk activation remains to be established, it is possible that this effect was due to inhibition of Ras activation by calcium-triggered recruitment of CAPRI, a calcium-regulated Ras-GTPase-activating protein (2).
The mechanism by which PAG-associated FynT caused an increase in TCR-triggered calcium fluxes needs to be determined. Surprisingly, we did not detect any increase in TCR-induced tyrosine phosphorylation of phospholipase C-
1 in T cells expressing PAG Y314F (our unpublished results). We were also not able to notice any change in the tyrosine phosphorylation of the inositol triphosphate receptor, which was previously reported to be a target of FynT (13). Moreover, we did not observe any impact on the increase of intracellular inositol triphosphate in response to TCR stimulation. Therefore, it is likely that another mechanism is involved, and future studies are warranted to resolve this issue.
In the light of the selective impact of PAG Y314F on TCR-triggered calcium fluxes, we examined the possibility that PAG-associated FynT may be implicated in the induction of T-cell anergy. This suggestion was prompted by earlier work indicating that TCR-mediated activation of calcium signaling in the absence of sufficient parallel activation of other pathways, such as NF-B, can promote T-cell anergy (17). In a physiological setting, this is presumably occurring when the TCR is engaged by antigen in the absence of adequate costimulation through CD28. Preliminary support for the idea that PAG-associated FynT may indeed be implicated in anergy induction was obtained with our observation that the abundance of PAG-associated FynT, but not PAG-associated Csk, was augmented in T cells anergized in vitro with ionomycin or in vivo with soluble peptide antigen. Furthermore, by studying two different anergy models, anergy triggered by anti-CD3 antibodies or by superantigen (SEB), we observed that PAG Y314F, but not wild-type PAG or PAG 9YF, potently augmented anergy induction in T cells. As was the case for the enhanced calcium fluxes, the effect of PAG Y314F on anergy was eliminated in T cells lacking FynT, implying that it was mediated by PAG-associated FynT. Consequently, it seems likely that PAG-associated FynT can participate in anergy induction in T cells.
One issue that arises from these results is whether PAG Y314F mimics a bona fide effect of PAG-associated FynT on anergy. We believe this to be the case for the following reasons. First, previous studies with several anergy models revealed that FynT expression was greatly enhanced in anergic T cells (3, 9, 23, 30, 31). Second, expression of FynT was shown to be required for anergy induction in an unusual population of double-negative ß T cells (30). This finding was extended by our demonstration that FynT expression was also important for superantigen-induced anergy in conventional ß T cells, even in the absence of PAG Y314F. And third, in the ionomycin-induced and the soluble-peptide-triggered models of T-cell anergy, we documented an increased abundance of endogenous PAG-FynT complexes but not PAG-Csk complexes. Hence, it appears that FynT molecules bound to endogenous PAG can also participate in the induction of T-cell anergy. While it is difficult to demonstrate this idea further at this time, one possible approach would be to introduce "knock-in" germ line mutations of all tyrosines other than Y314 in the mouse pag gene. Nevertheless, since complete ablation of PAG expression in the mouse had little or no effect on T-cell functions (presumably because of redundancy or compensation by other signaling pathways) (8, 36), this approach may not be very informative.
These observations also imply that a proper balance between PAG-associated FynT and PAG-associated Csk is crucial to avoid induction of gratuitous T-cell anergy. In the presence of adequate recruitment of Csk to PAG, PAG-associated FynT is presumably maintained relatively inactive, thereby preventing anergy induction. This notion is in agreement with our observation that, contrary to PAG Y314F, wild-type PAG did not promote T-cell anergy. In this setting, the effect of PAG-associated Csk was probably dominant. The situation would change when PAG-associated FynT dominates over PAG-associated Csk, as seen with PAG Y314F or in ionomycin-triggered anergic T cells. Under physiological circumstances, this may occur because of a change in the relative abundances or activities of FynT and Csk or an alteration of their affinities for PAG.
Initial reports suggested that the exclusive role of PAG was to recruit the negative regulator of Src family kinases, Csk, to lipid rafts (4, 6, 16, 29). According to these studies, PAG-associated FynT was simply implicated in the phosphorylation of PAG at Y314, an event that enables binding of PAG to Csk. While the data presented here further supported the importance of PAG-associated FynT for Csk recruitment, they also provided evidence that PAG-associated FynT can transduce other biochemical signals during TCR signaling. These signals lead to a marked increase in TCR-induced calcium fluxes and can, under certain circumstances, promote T-cell anergy. In this manner, PAG is reminiscent of other lipid raft-associated adaptors, such as NTAL/LAB, SIT, and LAT, which can have dual, and at times opposing, functions in immune cells (26).
Our finding that PAG can generate multiple signals may also explain in part why little or no phenotype was observed in PAG-deficient mice (8, 36). Removal of opposing signals, as a result of PAG deficiency, may cause no appreciable net effect on cell signaling. The generation of knock-in mice in which PAG can bind only FynT or Csk may help address this intriguing idea. However, as alluded to already (8, 36), it is likely that other mechanisms of recruitment of Csk and FynT can compensate in the absence of PAG. Indeed, Csk can be recruited by several other molecules, including Dok-related adaptors, LIME, SIT, and paxillin (34). Likewise, FynT can be recruited by the TCR, the adaptor SAP, and others (24, 32). Clearly, more work remains to be done to elucidate the cooperative importance of these various mechanisms in the regulation and functions of Csk and FynT, not only in T cells but also in other cell types.
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ACKNOWLEDGMENTS |
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This work was supported by grants from the National Cancer Institute of Canada and the Canadian Institutes of Health Research (to A.V.) and the Deutsche Forschungsgemeinschaft (to B.S.). A.V. holds the Canada Research Chair in Signaling in the Immune System.
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FOOTNOTES |
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Published ahead of print on 1 January 2007.
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REFERENCES |
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