Current models depicting the TCR signalosome may be incomplete because restricted to tyrosine-phosphorylated components and to prototypic signaling elements shared by many receptors. Thus, novel approaches may be needed to probe for yet unknown signaling elements. Moreover, understanding how TCR signals shape gene activation requires evaluating qualitative and quantitative variations within the signalosome that are likely to occur as a function of stimuli duration and/or intensity. A major hurdle toward these goals is that such a protein complex is of low abundance and highly dynamic, i.e., intrinsically labile. We tried to overcome these difficulties by combining overexpression of epitope-tagged SLP-76 and its tag-dependent isolation, followed by MS identification of associated proteins. The efficacy of this approach was demonstrated by the isolation of known SLP-76 binding partners and the identification of two novel interacting proteins, both members of the 14-3-3 protein family. This finding led us to uncover a novel SLP-76–based negative feedback mechanism that likely serves to fine tune TCR-dependent signals.
Association of 14-3-3 proteins with SLP-76 is mediated by phosphorylation of S376. This residue, which is part of a motif sharing weak homology with the mode I 14-3-3 binding motif (22
), lies in the proline-rich region of SLP-76 that mediates association with multiple partners (e.g., Gads, PLC-γ1, and inducible T cell kinase). Ser/Thr phosphorylation of SLP-76 has been reported (31
), but neither the specific residues nor the significance of these modifications was identified. Our data (see also reference 21
) reveal that four Ser residues of SLP-76 are phosphorylated either constitutively or after cell stimulation, but the role of those residues other than S376 remains to be studied. Different to the immediate induction and rapid decrease of phosphorylation of N-terminal tyrosines involved in the binding of SLP-76 to other key signaling effectors, S376 phosphorylation peaked at 10–15 min and declined slowly, matching the kinetics of 14-3-3 binding. These findings revealed a new mechanism by which SLP-76 recruits signaling partners and a rapid changing pattern of its interactions.
Our results directly implicated HPK-1 in S376 phosphorylation. Previously, this kinase was regarded as MAP4K, a positive upstream regulator of mitogen-activated protein kinases, such as JNK (32
). An implication of HPK-1 in TCR-proximal signaling has been suggested (20
), but its specific role in this context has remained elusive. We demonstrate here that HPK-1 regulates SLP-76, a major TCR signaling protein, consistent with previous data showing that HPK-1 can inducibly interact with the former (20
). We cannot exclude that once recruited to SLP-76, HPK-1 might also phosphorylate other SLP-76 partners and/or act as a MAP4K in T cells.
Data presented herein show that SLP-76 not only orchestrates positive signaling, but in combination with HPK-1 and 14-3-3, also generates a delayed negative signal regulating T cell activation. Both S376A mutation and HPK-1 knock-down correlated with increased TCR signal intensity, as demonstrated by the augmented tyrosine phosphorylation of SLP-76 and PLC-γ1. Reduction of HPK-1 expression by siRNA that increases NFAT activity (18
) or overexpression of this kinase that inhibits ERK and AP-1 activation (19
) previously suggested that HPK-1 negatively affects TCR-induced transcription. During the revision of this article, Shui et al. (34
) have reported a study of HPK-1–deficient mice confirming the role of HPK-1 as a negative regulator of T cell activation and immune response. Similar to our data, HPK-1 deficiency results in increased TCR-induced tyrosine phosphorylation of several signaling proteins, including SLP-76 and PLC-γ1, that correlates with increased calcium mobilization, ERK activation, and enhanced T cell activation. Shui et al. also showed that HPK-1 phosphorylates SLP-76 in vitro and that SLP-76 associates with 14–3-3τ. We have detected 14-3-3ζ, 14-3-3
(this work), and 14-3-3γ (unpublished data) associated with SLP-76 but not 14-3-3τ. The reason for this discrepancy is unclear, but it may be due to differences in the cells used. These results essentially corroborate our model of negative regulation of T cell activation through SLP-76, HPK-1, and 14-3-3. However, our findings that mutation of S376, a specific target of HPK-1 on SLP-76, abolished 14-3-3 binding and resulted in higher TCR-dependent IL-2 promoter induction demonstrate that 14-3-3 recruitment to SLP-76 is one of the main mechanisms by which HPK-1 negatively affects T cell activation. Finally, although Shui et al. proposed that S207 of SLP-76 may be a docking site for 14-3-3, based on conjectures about 14-3-3 consensus binding sites (34
), our work indicates that S207 does not play a major role in the 14-3-3–SLP-76 interaction (see ). Future studies of knock-in mice expressing SLP-76–S376A would address more precisely T cell abnormalities in the absence of the signal-modulating mechanism describ0ed herein because potential compensatory effects of HPK-1 on other pathways (e.g., JNK) would be avoided.
The 14-3-3ζ and τ isoforms have been implicated in negative regulation of T cell signaling through their interaction with Casitas B lineage lymphoma proto-oncogene, PKCθ, PI3K, or the adaptor 3BP2 (23
). Hence, 14-3-3 proteins appear to exert a complex control on TCR signaling, relying on multiple mechanisms. The mechanism by which S376 phosphorylation and 14-3-3 recruitment reduces Tyr phosphorylation of SLP-76 and PLC-γ1 (and perhaps other partners) is unknown. 14-3-3 binding may disrupt the interaction of SLP-76 and its partners with an upstream protein tyrosine kinase (e.g., ζ chain–associated protein of 70 kD), thus shortening the duration of tyrosine phosphorylation. Other proteins binding to the proline-rich region of SLP-76 (which contains the 14-3-3 binding site), might be released upon 14-3-3 recruitment. However, Gads association with SLP-76 does not seem to be modified by 14-3-3 binding because the S376A mutation does not affect the Gads–SLP-76 complex (Fig. S4, available at http://www.jem.org/cgi/content/full/jem.20062066/DC1
). Nonetheless, 14-3-3 proteins could compete with the binding of PLC-γ1 or inducible T cell kinase. Alternatively, 14-3-3 proteins may recruit/stabilize a PTP in the signalosome or induce dissociation of one or more proteins from the complex, leading to increased availability of phosphorylation sites to PTPs. Another intriguing possibility, not mutually exclusive of the above mechanisms, is suggested by the role of 14-3-3 proteins as regulators of trafficking and subcellular localization of their partners (35
). Upon 14-3-3 binding, SLP-76 might be released from the membrane-proximal signalosome, alone or together with some associated proteins, and translocate to a different intracellular site, as suggested by data reporting a relocation of SLP-76 to a perinuclear compartment a few minutes after receptor engagement (12
). Hence, the long-lasting phosphorylation of S376 and 14-3-3 binding might also affect potential late functions that SLP-76 would exert after leaving the membrane-proximal signalosome.
An instructive example of signal down-regulation via an adaptor is provided by insulin receptor substrate (IRS)-1, a central effector downstream of insulin receptor. Ser/Thr phosphorylation of IRS-1 counteracts its tyrosine phosphorylation, leading to termination of signaling in a physiological setting or to insulin resistance. Impairment of tyrosine phosphorylation of IRS-1, dissociation of the latter from the receptors and from the signaling complex, or its degradation has been proposed to explain this negative regulation (for review see reference 36
). Interestingly, 14-3-3 proteins bind to Ser-phosphorylated IRS-1 and regulate its function (37
). It is therefore tempting to speculate that the negative feedback loop by which SLP-76 contributes to TCR signal down-modulation occurs via one of the mechanisms described for IRS-1.
We would like to suggest that negative feedback loops such as the one described herein, acting so early on antigen receptor signals, are not required to stop the signal but rather to shape its intensity and/or duration. Indeed, in a physiological setting, T cells collect inputs from peptide/MHC over long periods of time and tuning mechanisms may be necessary to funnel the signal within a certain range of intensity and duration to orchestrate ordered and dosed gene expression.
Here, we have provided an example of how expressing an epitope-tagged central effector coupled to MS analysis can uncover previously hidden aspects of TCR signaling. Recent advances in sensitivity, mass accuracy, and resolution in MS combined with improvements in the isolation of unstable protein complexes should reveal further intricacies in the molecular mechanisms of T cell activation.