SLP-76 (Src homology (SH) 2-domain-containing leukocyte protein of 76 kDa) and FYB/SLAP (FYN-T-binding protein/SLP-76-associated protein) are two hemopoietic cell-specific adaptor proteins downstream of TCR-activated protein tyrosine kinases. SLP-76 has been implicated as an essential component in T cell signaling. FYB is selectively phosphorylated by FYN-T, providing a template for the recruitment of FYN-T and SLP-76 SH2 domains. Coexpression of FYN-T, FYB, and SLP-76 can synergistically up-regulate IL-2 production in T cells upon TCR ligation. In this report, we show that two tyrosines, Tyr595 and Tyr651, of FYB are major sites of phosphorylation by FYN-T and mediate binding to SLP-76 in Jurkat T cells. Furthermore, the synergistic up-regulation of IL-2 promoter activity in the FYN-T-FYB-SLP-76 pathway is contingent upon the interaction between FYB and SLP-76, but not the interaction between FYB and FYN-T. These observations define a pathway by which SLP-76 interacts with downstream components in the up-regulation of T cell cytokine production.
Engagement of the TCR by peptide/MHC complexes results in the activation of a number of protein-tyrosine kinases (1, 2, 3), including members of the src family, LCK and FYN-T, and syk family, ZAP-70 and SYK. Activated protein-tyrosine kinases can then phosphorylate a variety of substrates that act as adaptor proteins to activate other signaling pathways, including the phosphatidylinositol and Ras/extracellular signal-related kinase second messenger pathways, leading to T cell proliferation and cytokine production.
Among the substrates identified are the hemopoietic cell-specific adaptor proteins, SLP-76 (Src homology (SH) 2-domain-containing leukocyte protein of 76 kDa)2 and FYB/SLAP (Fyn T-binding protein/SLP-76-associated protein) (4, 5, 6). SLP-76 is an adaptor protein that is comprised of three domains allowing for protein-protein interactions: an amino-terminal acidic region containing tyrosine phosphorylation sites (7), a central proline-rich region that binds to the SH3 domain of Grb2 family members (8, 9), and a carboxyl-terminal SH2 domain that associates with FYB/SLAP (5, 6). The importance of SLP-76 in T cell signaling has been demonstrated in experiments in the Jurkat human T cell leukemia line and in SLP-76-deficient mice. SLP-76 overexpression in Jurkat cells augments TCR-mediated activation of the IL-2 promoter (9, 10). Furthermore, Jurkat T cells lacking SLP-76 show defects in TCR-mediated signals (11). Studies of SLP-76−/− mice also indicate that SLP-76 is required for pre-TCR signaling, as these mice exhibit arrest of thymocyte development at the double negative CD25+ CD44− stage (12, 13).
FYB/SLAP was independently cloned on the basis of its ability to bind to the src kinase FYN-T and SLP-76 (5, 6). It also has the hallmarks of an adaptor protein with several proline-rich regions, two putative nuclear localization sequences, a carboxyl-terminal SH3 domain, and multiple tyrosine-containing motifs. Two forms of FYB at 120 and 130 kDa exits that differ due to an insertion of 46 aa toward the C terminus (14). FYB-120 corresponds to the version of SLAP termed SLAP-130 (5). FYB undergoes tyrosine phosphorylation in response to TCR ligation, an event diminished in FYN-T-deficient T cells (15). However, unlike with SLP-76, transfection studies with FYB/SLAP have yielded conflicting results on the role of FYB/SLAP in the regulation of IL-2 production. Recent studies from our laboratory demonstrated that FYB is selectively phosphorylated between residue 585 and 670 by FYN-T (16), providing a template for the recruitment of the SH2 domains of FYN-T and SLP-76. The interaction between FYN-T, FYB, and SLP-76 is unusual in its distinct cytoplasmic localization and its stable kinetics of phosphorylation. Furthermore, coexpression of all three components of the FYN-T-FYB-SLP-76 matrix can cooperatively up-regulate anti-CD3-mediated IL-2 transcription in Jurkat T cells (16). However, previous studies have not addressed whether direct association between these components is required for their synergy.
In this paper, we demonstrate that two YDDV sites on FYB are major sites of phosphorylation by FYN-T and serve as major sites for SLP-76 SH2 domain binding. We further demonstrate that the loss of SLP-76 binding by mutation of these sites markedly reduced the ability of FYN-T-FYB-SLP-76 to up-regulate IL-2 transcription. By contrast, mutation of the FYN-T SH2 domain binding site had no consistent effect. Overall, these results indicate a role for SLP-76/FYB complex formation in the regulation of TCR-driven IL-2 production.
Materials and Methods
Monoclonal Ab to SLP-76 was kindly provided by Dr. Paul R. Findell (Syntex, Palo Alto, CA). Anti-phosphotyrosine mAb 4G10 was kindly provided by Dr. Tom Roberts (Dana-Farber Cancer Institute, Boston, MA). Anti-human CD3 (OKT3) was obtained from American Type Culture Collection (Manassas, VA).
Wild-type FYB were cloned into the EcoRI and SalI sites of pSRα mammalian expression vector containing a sequence encoding the influenza hemaaglutinin (HA) epitope tag at the N terminal. The FYB deletion mutations have been described. The tyrosine mutants were created with the Quickchange Mutagenesis kit (Stratagene, La Jolla, CA) using oligonucleotides (Life Technologies, Gaithersburg, MD) encoding the appropriate mutations plus silent mutations to provide novel restriction sites for diagnosis. The NF-AT luciferase reporter construct was provided by Dr. Burakoff (Dana-Farber Cancer Institute).
Transfections, immunoprecipitation, and immunoblotting
Transfections and immunoprecipitations were conducted as previously described (17, 18). Briefly, 20 × 106 SV40 large tumor Ag-transfected Jurkat cells were electroporated and allowed to sit for 20 h. Cells were harvested and lysed with 200 μl lysis buffer (20 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1% (v/v) Triton X-100, 1 mM sodium vanadate, 1 mM PMSF, 1 mM leupeptin). Immunoprecipitation was conducted by incubation of the lysate with the Ab for 1 h at 4°C, followed by incubation with 50 μl of protein A-Sepharose beads (10% w/v) for 1 h at 4°C. Immunoprecipitates were washed three times with ice-cold lysis buffer and subjected to SDS-PAGE. For immunoblotting, the immunoprecipitates were separated by SDS-PAGE and transferred onto nitrocellulose filters (Schleicher and Schuell, Keene, NH). Filters were blocked with 5% (w/v) skim milk for 1 h in TBS, pH 8.0, and then probed with the indicated Ab. Bound Ab was revealed with HRP-conjugated rabbit anti-mouse or donkey anti-rabbit Abs using enhanced chemiluminescence (Amersham, Arlington Heights, IL).
A total of 5 × 105 transfected Jurkat cells were stimulated with 1 μg/ml OKT3 and 2 μg/ml rabbit anti-mouse Ab at 37°C for 6 h and subsequently assayed for luciferase activity following manufacturer suggested protocols (dual luciferase system kit; Promega, Madison, WI). Luciferase activity was determined using the luminometer (MicroLumat, EG&G Berthold Bad WildBad, Germany). Luciferase units of the experimental vector were normalized to the level of the control vector in each sample.
Results and Discussion
Tyrosines 595 and 651 are major phosphorylation sites by FYN-T
Recent studies in our laboratory demonstrated that the region between residues 585 and 670 on FYB was selectively phosphorylated by FYN-T, but not LCK and ZAP-70, and mediates binding to SH2 domains of FYN-T and SLP-76 (16). Fig. 1,A shows the amino acid sequence of a portion of this region highlighting the three potential tyrosine phosphorylation motifs that would be suited for SH2 domain binding (YDDV, residues 595–598; YDGI, residues 625–628; YDDV, residues 651–654). The YDGI site has previously been identified as the predominant FYN-T-binding site in vitro by peptide binding assays and in vivo using mutant FYB in which the tyrosine of the YDGI site was replaced by phenylalanine (16). The two Y595DDV and Y651DDV motifs are similar, each containing conserved EV residues preceding the YDDV sequence (Fig. 1 A). By in vitro peptide binding assay, the C-terminal YDDV motif was shown to be able to bind to SLP-76 SH2 domain (16). However, in vivo studies are necessary to confirm this result and to test whether the N-terminal YDDV motif or other tyrosine-containing motifs in FYB can bind to SLP-76 as well.
To address this, mutant human FYB-120 cDNA constructs encoding substitutions at one or both tyrosines of the YDDV motifs with FDDV were generated (Fig. 1,A). Each construct included an epitope tag (HA) for detection of the transfected molecule. The FYB-120 isoform was used in this study and is referred to as FYB in the rest of the paper. To determine whether the YDDV motifs are sites of FYN-T phosphorylation, wild-type FYB (WT FYB) and FYB mutants were coexpressed with FYN-T in Jurkat T cells and were assessed for phosphorylation by anti-phosphotyrosine blotting (Fig. 1,B, upper panel). As previously reported, WT FYB was tyrosine-phosphorylated by FYN-T (lane 2 vs 1). The Y595F and Y651F single mutants were significantly less phosphorylated when compared with WT FYB (lane 3 and 4 vs 2). Phosphorylation was reduced even further (generally <30% of WT) in the Y595/651F double mutant (lane 5; also see histogram in Fig. 1,C). As an internal control for expression, anti-HA Ab detected similar levels of expression for FYB and the mutants (Fig. 1,B, lower panel). These data demonstrate that Y595 and Y651 are major sites of phosphorylation by FYN-T. Much longer exposure revealed a slight FYB band in lane 1 (Fig. 1 B), indicating a basal level of FYB phosphorylation in resting Jurkat, as previously reported in other studies (5, 15). The remaining 30% phosphorylation in the Y595/651F double mutant can be explained by protein tyrosine kinase phosphorylation of other tyrosines in the sequence of FYB, in particular Y625 of the YDGI motif that has been demonstrated to mediate direct binding to FYN-T (16).
Both YDDV sites are necessary for optimal SLP-76 binding
To assess whether the two pYDDV sites are responsible for binding to SLP-76, the three FYB mutants (Y595F, Y651F, and Y595/651F) were coexpressed with FYN-T and HA-tagged SLP-76 in Jurkat T cells followed by immunoprecipitation with anti-SLP-76 mAb and anti-HA blotting. The expression levels of FYB and the mutants and SLP-76 were similar as shown in the whole-cell lysates (Fig. 2,A, right upper and lower panels, respectively). As expected, SLP-76 coprecipitated WT FYB (left panel, lane 1), whereas the double mutant Y595/651F fail to coprecipitate with SLP-76. The single mutations resulted in significant reductions in SLP-76 binding, with Y651F having a greater effect (55% reduction) than Y595F (20% reduction) (left panel, lanes 2 and 3; also see Fig. 2 B: densiometric readings in histogram).
From these observations, it appears that in T cells both Tyr595 and Tyr651 are essential for optimal binding of SLP-76. Both Tyr595 and Tyr651 are found in similar sequence motifs (EVYDDV), either of which would be predicted to bind the SLP-76 SH2 domain. Indeed, we previously showed that a phosphorylated peptide corresponding to the C-terminal EVpYDDV motif precipitated SLP-76 from cell lysates (16). The involvement of two sites is reminiscent of the interaction between SLP-76 and the VAV SH2 domain, where both DYESP motifs are required for VAV SH2 domain binding to SLP-76 (17, 19). Although the basis for this observation is not clear, it may simply be related to the fact that both sites are used for SLP-76 binding, perhaps with Y651F being the preferred site.
FYB and SLP-76 interaction is required to up-regulate NF-AT activity in the FYN-T-FYB-SLP-76 pathway
To address the role of SLP-76 binding to FYB in T cell function, Jurkat T cells were transiently transfected with FYB mutants or a control SRα vector together with FYN-T and SLP-76 and a luciferase reporter construct driven by an IL-2 NF-AT/AP-1 promoter. The mutants used include the YDDV mutants, a C-terminal deletion mutant D585 (deletion from amino acid 585 to C terminus), and a FYB mutant with the Tyr625 in the YDGI motif substituted by phenylalanine (Y625F). This latter site has previously been shown to bind to FYN-T, but had not been examined for an effect on IL-2 transcription (16). As reported, the combined expression of FYN-T/FYB/SLP-76 potentiated IL-2 transcription by about 100-fold beyond vector-transfected control (Fig. 3, A and B). Deletion mutant D585, which lacks the region of FYN-T and SLP-76 binding, effectively eliminated the potentiating effect (Fig. 3 A, left panel). By contrast, the Y625F mutant had little effect on FYB synergy with FYN and SLP-76 (left panel). Several experiments showed no efect, while a slight degree of inhibition. This result can be explained by two scenarios. One possibility is that FYN-T complex formation with FYB and SLP-76 is not necessary for the FYB-FYN-SLP-76 pathway. FYN-T may only be required to phosphorylate FYB, which can then be accessed by a third anchor protein such as SLP-76. Alternatively, the Y625F mutant retains some 20% WT FYB binding to FYN-T, indicating a site other than YDGI can mediate the FYB-FYN-T interaction, albeit much less efficiently. This limited interaction may be sufficient to mediate the up-regulation of TCR-stimulated NF-AT/AP-1 activity
Analysis of the single and double YDDV mutants indicate that FYB-SLP-76 interaction is crucial in the FYN-T-FYB-SLP-76 pathway, as a marked reduction of NF-AT/AP-1 activity was observed in transfectants expressing the mutants compared with the WT FYB (Fig. 3,A, right panel). The reduction was greatest with the Y651F single mutant and the double Y595/651F mutant, reducing the NF-AT activity by 50–70% compared with the wild type (Fig. 3,B and data not shown). The different effect of the two single mutants correlates with the differences in the relative importance of each site for SLP-76 binding (Fig. 2 B, lower left panel), further confirming the functional importance of the two sites. Indeed, often the effect of the single Y651 site was found as effective as Y595/651F mutant in reducing IL-2 transcription. In several other experiments, the reduction mediated by the Y651 was midway between the Y595F and Y595/651F double mutant. In both cases, these data confirm the importance of the SLP-76 binding to the FYB scaffold at sites Y595/651F in the optimal up-regulation of TCR-driven IL-2 production.
Recent studies have demonstrated that the importance of the SLP-76 (and LAT) adaptors in T cell signaling (9, 10, 11, 17, 20, 21, 22). Further, the combined expression of SLP-76 with the FYB adaptor and FYN-T has demonstrated potent increases in TCR-driven IL-2 production (14, 16). Whether this cooperativity was related to separate effects of each component on different pathways or was genuinely related to binding between the components had been unclear. Our mapping of the SLP-76 binding sites and our finding that binding is needed for the potentiation of IL-2 transcription demonstrates that FYB’s role as a scaffold for SLP-76 binding contributes to optimal activation. The functional significance of the FYB-SLP-76 interaction in SLP-76 function is also consistent with the report that the SLP-76 SH2 domain is needed for its ability to augment IL-2 transcription (9). FYB/SLAP is the principal protein that has been found to bind to the SLP-76 SH2 domain (5, 15). The residual stimulatory effects of the Y595/651F double mutant further argue that other signaling components (albeit less potent than SLP-76) can also use other sites on the FYB scaffold in the mediation of signals leading to IL-2 transcription (Fig. 3 B). We are now in the process of determining the identity of the second messenger pathways downstream of the interaction.
Abbreviations used in this paper: SLP-76, SH2-domain-containing leukocyte protein of 76 kDa; SH, Src homology; FYB, Fyn T-binding protein; SLAP, SLP-76-associated protein; HA, hemagglutinin.