In this report, we have examined the function and regulation of the lipid raft-associated transmembrane adaptor PAG in T cells. First, as previously reported for human T cells (2
), we observed that PAG was extensively tyrosine phosphorylated and associated with Csk in ex vivo mouse thymocytes. Furthermore, following antigen receptor stimulation on these cells, PAG underwent rapid dephosphorylation and became dissociated from Csk. In time-course analyses, PAG dephosphorylation temporally coincided with, or perhaps even preceded, the overall intracellular protein tyrosine phosphorylation signal induced by TCR engagement. Taken together, these findings supported the earlier idea that PAG dephosphorylation and dissociation from Csk are early events of T-cell activation and that they might be required for TCR signaling to proceed normally.
To address the mechanism of action of PAG, wild-type PAG and phosphorylation-defective PAG mutants were expressed in normal mouse T cells via transgenesis. Our analyses of these mice revealed that overexpression of wild-type PAG caused a striking inhibition of TCR-induced proliferation and IL-2 production. This effect was observed in several T-cell populations, namely, CD4+ splenic T cells, CD8+ splenic T cells, CD4+ thymocytes, and CD4+ lymph node T cells. In contrast to wild-type PAG, the phosphorylation-defective PAG mutants PAG Y314F and PAG 9Y→F caused an enhancement of these TCR-triggered responses. In addition to demonstrating the importance of tyrosine phosphorylation for the inhibitory function of PAG, the dominant-negative effect of these mutants implied that the inhibitory impact of wild-type PAG was not a spurious effect of overexpression. Rather, it reflected the true function of endogenous PAG molecules.
Several lines of evidence indicated that PAG inhibits T-cell activation primarily by recruiting Csk and inactivating Src kinases. First, we found that the inhibitory influence of PAG was eliminated by mutation of Y314, the major Csk-binding site of PAG (20
). Obviously, the possibility that this site was also implicated in recruiting other SH2 domain-containing molecules to PAG cannot be excluded. Second, it was noted that augmented PAG expression resulted in an inhibition of TCR-induced protein tyrosine phosphorylation, an effect analogous to that observed upon overexpression of Csk (8
). And lastly, PAG-mediated inhibition was rescued by expression of a Src kinase mutant that is refractory to the effect of Csk (FynT Y528F). While this last finding is in keeping with our model, it is worth mentioning that the activated FynT might also function by causing enhanced phosphorylation of proteins other than PAG.
While PAG overexpression inhibited TCR-induced proliferation and IL-2 secretion, it is noteworthy that it had no impact on the production of IL-4 and IFN-γ. This finding suggested that the intensity and/or nature of the TCR signals required for release of IL-2 and proliferation might be distinct from those needed for production of IL-4 and IFN-γ. Interestingly, a similar alteration in the profile of cytokine production was reported for anergic T cells. Like PAG-overexpressing cells, these cells have severe defects in TCR-induced proliferation and IL-2 secretion but tend to exhibit normal secretion of IL-4 or IFN-γ (1
). This qualitative difference was proposed to reflect a hierarchy in the TCR signaling thresholds required for production of the various cytokines (18
). It is possible that a similar phenomenon explains the differential effects of PAG on cytokine production. Given the similarities between anergic and PAG-overexpressing T cells, it is also tempting to speculate that PAG is involved in the pathophysiology of T-cell anergy.
A surprising finding in our studies was that expression of the dominant-negative PAG molecules had no appreciable effect on thymocyte development. This is in striking contrast to the previously described severe effects of Csk deficiency on T-cell maturation (29
). A possible explanation for this distinction is that PAG-independent mechanisms exist for membrane recruitment of Csk. Along these lines, it was reported that the Csk SH2 domain can interact with other molecules such as Dok-related adaptors, paxillin, and focal adhesion kinase (35
). Alternatively, the expression levels of the phosphorylation-defective PAG polypeptides might have been insufficient to obliterate fully the physiological function of endogenous PAG molecules. While the creation of PAG-deficient mouse T cells should help distinguish between these possibilities, it seems probable, based on the available evidence, that additional mechanisms of Csk recruitment exist.
Considering the importance of PAG tyrosine phosphorylation for its inhibitory function, we attempted to identify the PTPs regulating this process. By analyzing T cells lacking various PTPs, evidence was adduced that PEP and SHP-1 were not involved in controlling PAG tyrosine phosphorylation. The lack of effect of PEP on PAG tyrosine phosphorylation was also confirmed by analyses of transgenic mice overexpressing wild-type PEP or phosphatase-inactive versions of PEP (our unpublished results). The observation that PEP had no apparent effect on PAG tyrosine phosphorylation was unexpected, given that PEP associates with Csk by way of the Csk SH3 domain (10
). Nonetheless, we recently obtained indications that the pool of Csk molecules associated with PEP does not interact simultaneously with PAG (our unpublished results). Therefore, PAG might not be accessible to PEP-mediated dephosphorylation.
However, our results provided an indication that CD45 is involved in inhibiting PAG tyrosine phosphorylation in T cells. In support of this idea, CD45, but not PTPs like PEP and SHP-1, partially colocalized with PAG in lipid raft fractions. Moreover, we found that the phosphotyrosine content of PAG was increased in lipid raft fractions of CD45-deficient thymocytes as well as in a CD45-negative variant of the mouse T-cell line YAC-1. While it is impossible with the currently available technologies to prove that CD45 was acting directly on PAG, this notion was suggested by the finding that a substrate-trapping mutant of CD45 can interact with tyrosine-phosphorylated PAG in transiently transfected Cos-1 cells. On the other hand, it is also plausible that CD45 regulated PAG phosphorylation by an indirect mechanism, for instance by inactivating Src kinases through dephosphorylation of their activating tyrosine (31
). The development of new methodologies capable of identifying enzyme-substrate interactions in vivo is needed to resolve these issues. Lastly, it should be pointed out that, in addition to CD45, other PTPs are likely to be involved in regulating PAG tyrosine phosphorylation. This is definitely true for nonhemopoietic cells, which express PAG but lack CD45.
The finding that CD45 is involved, directly or indirectly, in regulating PAG tyrosine phosphorylation is likely to be important. It suggests that CD45 sets the threshold of TCR signaling by at least two mechanisms. First, as documented in the past, CD45 dephosphorylates the inhibitory tyrosine of Src kinases (31
). And second, as reported herein, it promotes the dephosphorylation of PAG, thereby diminishing the amount of Csk located in lipid rafts. Both effects converge on increasing the catalytic activity of Src kinases, and their combination might be critical to the generation of sufficient Src kinase activation to allow productive TCR signaling to occur.
In summary, the data reported in this work provide compelling evidence that PAG is a negative regulator of T-cell activation in normal T cells as a result of its capacity to recruit Csk and inactivate Src kinases. They also support the idea that the dephosphorylation of PAG is a pivotal event during the initiation of T-cell activation. In the light of these results, additional studies are warranted to elucidate the mechanism responsible for PAG dephosphorylation upon TCR engagement. One possibility is that TCR stimulation activates or alters the cellular localization of PTPs like CD45 and others. Alternatively, triggering of the TCR might inactivate or sequester the PTK(s) responsible for PAG phosphorylation. An understanding of this phenomenon would undoubtedly provide valuable insights into the molecular changes responsible for initiating T-cell activation.