This work shows for the first time that HPK1 modulates TCR-proximal signaling and T cell activation by regulating the stability of critical protein complexes at the immunological synapse. We found that the persistence of SLP76 microclusters induced by TCR stimulation is dependent on HPK1 activity. HPK1 incorporation into SLP76 microclusters coincides with their rapid dissipation caused by a 14-3-3–mediated uncoupling of SLP76–GADS complexes from LAT. Persistence of phospho-LAT microclusters appears also affected by HPK1 but with some delay compared with SLP76 microclusters. These kinetic differences suggest that the effect on phospho-LAT clustering is consequent to the release of the SLP76–GADS complex, possibly because of the loss of stabilizing protein–protein interactions dependent on SLP76 and/or GADS. In support of the hypothesis that HPK1 does not target LAT directly, phospho-LAT microclusters are increased when 14-3-3 binding to SLP76 and GADS is impaired (Fig. S5 A). Collectively, these results show that a major function of HPK1 is to uncouple the SLP76–GADS from phosphorylated LAT, leading to negative regulation of T cell activation.
The results described here reveal that the mechanism of HPK1-dependent tuning of TCR-proximal signaling is more complex than initially reported because it involves a previously unidentified 14-3-3 binding site in GADS in addition to Ser376 of SLP76 (Di Bartolo et al., 2007
). Thr254 in murine GADS (corresponding to Thr262 in the human protein) was necessary for 14-3-3 binding and for negative regulation of microcluster stability and T cell activation. Moreover, disruption of this 14-3-3 binding site had a stronger impact on SLP76 microcluster persistence than mutation of Ser376, possibly because binding of 14-3-3 to GADS disturbs the interaction of the SH2 domain of GADS with phospho-LAT, either by steric hindrance or by inducing a conformational change. However, simultaneous mutation of both Ser376 and Thr254 had additive effects on both persistence of SLP76 microclusters and NFAT transcriptional activity upon T cell stimulation, suggesting a cooperative stabilization of the interaction of 14-3-3 with the SLP76–GADS complex. This finding is consistent with 14-3-3 proteins forming homo- and heterodimers that usually require interaction with two phosphorylation sites for stable binding (Tzivion and Avruch, 2002
). Based on these results, we propose a model whereby recruitment of HPK1 in SLP76-containing microclusters leads to phosphorylation of both SLP76 and GADS on Ser376 and Thr262, respectively. These posttranslational modifications enable recruitment of a 14-3-3 protein dimer, which in turn enforces dissociation of the SLP76–GADS–14-3-3 complex from phospho-LAT and consequently down-regulates TCR-induced signal transduction ().
Figure 8. Model of regulation of microcluster persistence and signaling by HPK1. (A) SLP76–GADS complexes (only one is depicted for clarity) are recruited to the phosphorylated transmembrane adaptor LAT to form signaling-competent microclusters. (B and (more ...)
The subsequent fate of the pool of SLP76 (and GADS) released from microclusters is unknown yet. Although a fraction of it could still be signaling competent because of its interaction with other effectors (Bunnell et al., 2006
), the correlation between removal from microclusters and reduced T cell activation reported here rather hints at an inactivation process. Hence, 14-3-3–bound SLP76 might be sorted for degradation or recycling. We could not detect significant changes in the total amount of SLP76 during the stimulation time analyzed (10 min), but we cannot exclude that degradation occurs later. Reincorporation of SLP76 into new microclusters has been previously described by others (Barr et al., 2006
), but according to our model, it would imply release of 14-3-3 and dephosphorylation of Ser376. However, we found that both Ser376 phosphorylation and 14-3-3 binding to SLP76 were long lasting (i.e., 45–60 min; Di Bartolo et al., 2007
) and that no additional microclusters were generated after the extinction of the first wave accompanying cell spreading on anti-CD3 coverslips in cells overexpressing HPK1 ( and Video 1). Both observations argue against a recycling of released SLP76; hence, further studies are required to address these issues.
Intriguingly, HPK1 knockdown more potently impaired GST–14-3-3ζ binding to SLP76 than to GADS in the overlay and in situ PLA assays, whereas direct mutation of the 14-3-3 binding site in GADS had stronger effects on 14-3-3ζ coprecipitation and on microcluster stability than mutation in SLP76. The reason for this difference is unknown and may depend, in part, on the different assay used. However, although HPK1 phosphorylates GADS in vitro, they do not allow us to exclude the existence of redundant kinases that may replace HPK1 in phosphorylating GADS in vivo.
The effect of HPK1 on microcluster persistence is reminiscent of that previously described for the ubiquitin E3 ligase c-Cbl (Balagopalan et al., 2007
). Indeed, c-Cbl incorporation into LAT- and SLP76-containing microclusters induces their dissipation, although the mechanisms allowing c-Cbl–dependent control of microcluster stability remain to be identified (Balagopalan et al., 2007
). Therefore, HPK1 and c-Cbl appear to modulate microcluster persistence, likely by different mechanisms, suggesting that multiple negative feedback loops operate to control protein clustering at immunological synapses. This also implies that the dissipation of signaling protein complexes is not only dictated by “passive” mechanisms, e.g., stochastic release of SH2-mediated interactions followed by dephosphorylation of critical tyrosine residues, but also by specific mechanisms “actively” driving the dissociation of key components.
Tuning of T cell signaling by HPK1 might be regulated in an activation- and/or differentiation-dependent fashion. Indeed, it has been shown that the expression of HPK1 in stimulated T cells is first up-regulated and then decreased because of caspase-dependent cleavage. Such modulation appears to control the activation of the NFκB pathway, thus inducing a change in T cell sensitivity to activation-induced apoptosis (Brenner et al., 2005
), but it may also affect upstream signaling events. If this is the case, the regulatory mechanism described here would be active in naive but not in previously activated cells.
TCR-independent stimuli may also tune HPK1 activity and, consequently, T cell responsiveness. For instance, it has been proposed that secretion of prostaglandin E2 by cancer cells activates HPK1 and impairs CD8+
T cell–dependent antitumor responses (Alzabin et al., 2010
). Interestingly, anti-CD3–stimulated binding of GST–14-3-3ζ to GADS in vitro is reinforced by pretreating Jurkat cells with PGE2 (unpublished data). Hence, it will be interesting to address the involvement of HPK1-dependent control of microclusters in altering T cell responsiveness under pathological conditions.
In conclusion, our data demonstrate that negative feedback triggered by HPK1 plays an important role in regulating the stability of critical signaling complexes at immunological synapses. This mechanism may represent a flexible device adapting T cell responsiveness according to cell differentiation and/or external cues.