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Molecular and Cellular Biology, August 2006, p. 5559-5568, Vol. 26, No. 15
0270-7306/06/$08.00+0 doi:10.1128/MCB.00357-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.
Laboratory of Molecular Oncology, Clinical Research Institute of Montreal, Montréal, Québec, Canada,1 Unité INSERM U768, Hôpital Necker Enfants-Malades, Paris, France,2 Department of Medicine, University of Montréal, Montréal, Québec, Canada,3 Department of Medicine, McGill University, Montréal, Québec, Canada4
Received 27 February 2006/ Returned for modification 12 April 2006/ Accepted 22 May 2006
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ABSTRACT |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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INTRODUCTION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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SAP regulates immunity as a result of its capacity to interact with the SLAM family of receptors (11, 17, 23, 31). This family includes SLAM; 2B4; natural killer, T- and B-cell antigen (NTB-A); Ly-9; CD84; and CD2-like receptor activating cytotoxic cells (CRACC). Most SLAM-related receptors are involved in homotypic interactions and, therefore, are self ligands. The lone exception is 2B4, which interacts with CD48, another receptor expressed on hemopoietic cells. SAP interacts with SLAM family receptors by way of an association involving its SH2 domain and the tyrosine-based motif TIYXXV/I (where T is threonine, I is isoleucine, Y is tyrosine, V is valine, and X is any residue), which is found in the cytoplasmic region of all SLAM-related receptors except CRACC.
Crystallographic and nuclear magnetic resonance analyses revealed that the interaction of the SAP SH2 domain with the TIYXXV/I motif from SLAM is quite unusual in comparison to other associations involving SH2 domains (13, 18, 26). First, contrary to most other SH2 domains (24), the SAP SH2 domain makes three rather than two contacts with the tyrosine-based motif of SLAM. In addition to classical interactions with the tyrosine residue and the amino acids located C-terminally to the tyrosine, the SAP SH2 domain contacts residues located N-terminally to the tyrosine. This feature is likely to stabilize the association of SAP with SLAM. Second, the SAP SH2 domain can interact with the tyrosine-based motif of SLAM even in the absence of tyrosine phosphorylation. This results in a ligand-independent association of SAP with SLAM in immune cells, a property that may facilitate the initiation of SLAM signaling. The affinity of the SLAM-SAP association is, however, augmented (approximately fivefold) when SLAM becomes tyrosine phosphorylated (18, 26).
The capacity of SAP to regulate the function of SLAM family receptors appears to reflect its ability to promote protein tyrosine phosphorylation signals (6, 15, 29). This is due to the aptitude of SAP to bind and activate FynT, a Src-related protein tyrosine kinase expressed in immune cells (3, 5, 10, 15, 16). Biochemical and crystallographic studies demonstrated that SAP directly associates with FynT by way of a second binding surface in the SAP SH2 domain that directly interacts with the FynT Src homology 3 (SH3) domain (5, 16). This surface is located between the sixth ß sheet and the second 
helix of the SAP SH2 domain and involves the motif RF/YFR78 (where R is arginine, F is phenylalanine, Y is tyrosine, and R78 represents arginine 78). It is centered on arginine 78 of SAP, and contrary to most other motifs interacting with SH3 domains, it does not contain any proline.
In the present report, we wanted to further understand the mechanism by which SAP couples SLAM to FynT. We found that, contrary to most other associations mediated by SH3 domains, the interaction of the SH3 domain of FynT with the SLAM-SAP complex is strictly inducible. It is absolutely dependent on engagement of the extracellular domain of SLAM by ligands. This property does not appear to reflect the need of a SLAM-dependent biochemical signal to evoke binding of the FynT SH3 domain to SLAM-SAP, but rather the requirement of a conformational change in SLAM-associated SAP to allow stable recognition of SAP by the FynT SH3 domain.
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MATERIALS AND METHODS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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chain of the human interleukin-2 receptor (named Tac-SLAM) in the absence or in the presence of SAP were described elsewhere (15). Transfectants expressing mutant Tac-SLAM molecules, in which one or more tyrosines in the cytoplasmic domain were replaced by phenylalanines, or full-length SLAM in combination with an arginine 78-to-alanine 78 mutant of SAP (SAP R78A) were also reported previously (15). Thymocytes were isolated from 5- to 6-week-old wild-type, fyn –/– or sap –/– mice (8, 30). The fyn –/– and sap –/– mice were backcrossed at least five generations to the C57BL/6 background. Antibodies. Polyclonal rabbit antisera or rat monoclonal antibodies (MAbs) directed against SLAM, SAP, FynT, and phosphotyrosine were produced in our laboratory (9, 15, 27). A rabbit antiserum against the extracellular domain of Tac was purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, Calif.). Rat anti-mouse SLAM MAb 12F12 was kindly provided by A. O'Gara, DNAX Research Institute of Molecular and Cellular Biology, Palo Alto, Calif. (4). Mouse anti-Tac MAb 7G7 and rat anti-mouse CD4 MAb GK1.5 were described elsewhere (15).
Immunoprecipitation and immunoblotting. Cells were lysed in TNE buffer (50 mM Tris [pH 8.0], 1% Nonidet P-40, and 2 mM EDTA) containing protease and phosphatase inhibitors, as detailed elsewhere (9). After preclearing, lysates were immunoprecipitated for 1.5 h with the indicated antibodies. Immune complexes were then captured with formalin-fixed Staphylococcus aureus (Calbiochem-Novabiochem, San Diego, Calif.), coupled in some cases to rabbit anti-mouse or rabbit anti-rat immunoglobulin G (IgG) (Jackson Immunoresearch Laboratories, West Grove, Pa.). After being washed, proteins were eluted in sodium dodecyl sulfate (SDS)-containing sample buffer, boiled, and separated by gel electrophoresis. Immunoblotting was performed as previously described (32). Immunoreactive products were detected using 125I-protein A (Amersham Pharmacia Biotech) or 125I-goat anti-mouse IgG (ICN Biomedicals, Aurora, Ohio), or protein A-horseradish peroxidase (HRP) or goat anti-rat antibody-HRP plus enhanced chemiluminescence reagents (Amersham Pharmacia Biotech). Radioactivity was quantitated with a Storm Phosphorimager (Molecular Dynamics, General Electrics Canada, Mississauga, Ontario, Canada).
Cell stimulation. BI-141 cells (1 x 107 cells/ml) were incubated for 30 min on ice with mouse anti-Tac MAb 7G7 (6 µg/ml). After the unbound antibodies were removed, the cells were stimulated for the indicated time at 37°C or 4°C with either sheep anti-mouse or rabbit anti-mouse IgG. For inhibition by tyrosine phosphatase (PTP), the cells were treated for 10 min at 37°C with pervanadate (100 µM), as outlined elsewhere (12). After being stimulated, the cells were lysed in 2x TNE lysis buffer. The lysates were subsequently processed for immunoprecipitation, in vitro binding studies, or immunoblotting.
In vitro binding assays.
Glutathione S-transferase (GST) proteins alone or encompassing the FynT SH3 and/or SH2 domains were produced in bacteria and purified on agarose-glutathione beads, as previously described (25). In vitro binding assays were performed using 1.5 mg of cell lysates and 
20 µg of fusion proteins. After being washed, the proteins were detected by immunoblotting with anti-SLAM or anti-Tac antibodies.
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RESULTS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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chain of the human interleukin-2 receptor (Tac) (15). Engagement of Tac-SLAM on these cells was not constitutive and required the addition of anti-Tac MAb 7G7 to the culture medium. Using this reagent, we were able to show that the SH2 domain-mediated association of SLAM with SAP was ligand independent. This finding was consistent with work showing that the SAP SH2 domain can interact with the TIYXXV/I motif from SLAM even in the absence of tyrosine phosphorylation (18, 26). In the present study, we decided to use this system to elucidate the mechanisms controlling the association of the preformed SLAM-SAP complex with the third partner, FynT. Hence, BI-141 T cells expressing Tac-SLAM in the presence or in the absence of SAP were stimulated or not with anti-Tac MAb 7G7. After the cells were lysed, Tac-SLAM was recovered by immunoprecipitation and probed by immunoblotting with a variety of antibodies (Fig. 1, lanes 3 to 6). Cells expressing full-length SLAM with or without SAP were analyzed as controls (Fig. 1, lanes 1 and 2).
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To ensure that the association of the FynT SH3 domain with SLAM in these in vitro assays was mediated by SAP, we examined the impact of a mutation of arginine 78 of SAP (Fig. 2C). Mutation of this residue was previously shown to prevent the binding of SAP to the FynT SH3 domain, without interfering with the capacity of SAP to associate with SLAM (5, 16). Thus, BI-141 T cells expressing SLAM alone or in the presence of wild-type SAP or arginine 78-to-alanine 78 SAP (SAP R78A) were tested in the assays described for Fig. 1 and 2A. In keeping with earlier results (16), we observed that SLAM was associated with SAP R78A (Fig. 2C, first panel, lane 3) to the same extent as with wild-type SAP (Fig. 2C, lane 2). Despite this, the FynT SH3 domain failed to recover SLAM in cells expressing SAP R78A (Fig. 2C, second panel, lane 3), whereas a strong association was noted in cells containing wild-type SAP (Fig. 2C, lane 2). These data firmly supported the idea that the interaction between the FynT SH3 domain and SLAM in the in vitro assays was dependent on the ability of the FynT SH3 domain to bind SAP.
SLAM-SAP signaling is insufficient for binding of the SLAM-SAP complex to the FynT SH3 domain. These findings suggested two potential explanations. One was that SLAM engagement triggered an active biochemical signal that modified SLAM and/or SAP and that was required for the arginine 78-based motif of SAP to bind the FynT SH3 domain. An alternative was that SLAM engagement induced a signal-independent change in the conformation of SLAM and/or SAP that was necessary for the arginine 78-based surface of SAP to interact with the FynT SH3 domain.
To help distinguish between these possibilities, we assessed whether SLAM-SAP signaling in the absence of SLAM engagement was able to trigger binding to the FynT SH3 domain (Fig. 3). For this purpose, Tac-SLAM tyrosine phosphorylation was induced by treatment of cells with pervanadate, a potent protein tyrosine phosphatase inhibitor. We had previously reported that this compound was able to stimulate SAP-dependent Tac-SLAM tyrosine phosphorylation in the absence of Tac stimulation (15). After cell lysis, the association of the FynT SH3 domain with Tac-SLAM was ascertained as for Fig. 2A. Pervanadate treatment (Fig. 3, lane 2) induced a pronounced increase in Tac-SLAM tyrosine phosphorylation (Fig. 3, first panel), as well as a marked augmentation of the association of Tac-SLAM with SAP (Fig. 3, second panel), compared to no stimulation (Fig. 3, lane 1). These changes were, in fact, more extensive than those observed in cells stimulated with anti-Tac (Fig. 3, lane 4). Despite this, PTP inhibition (Fig. 3, lane 2) allowed only a minimal increase in binding of the FynT SH3 domain to Tac-SLAM-SAP (Fig. 3, fourth panel). This was in striking contrast to the more-robust increase noted after anti-Tac stimulation (Fig. 3, lane 4). These findings implied that SLAM-SAP signaling alone was insufficient to trigger binding of the SLAM-SAP complex to the FynT SH3 domain.
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Second, we tested the importance of the tyrosine residues in the cytoplasmic domain of SLAM (Fig. 5). Mouse SLAM possesses three tyrosines in its cytoplasmic domain, tyrosine 288 (Y288), Y315, and Y335, which play important roles in SLAM signaling (Fig. 5A) (4, 15). Y288 is responsible for the phospho-independent association with SAP, while Y315 and Y335 undergo ligand-induced phosphorylation in the presence of SAP and enable recruitment of downstream signaling effectors such as 5' inositol phosphatase SHIP-1 and the adaptors Dok-1 and Dok-2 (15). To address the importance of these residues for binding of SLAM-SAP to the FynT SH3 domain, we used T cells expressing SAP in addition to Tac-SLAM variants in which either Y288 ("Y1F") or Y315 and Y335 ("Y2,3F") were mutated to phenylalanines. The cells were stimulated with anti-Tac, and binding of Tac-SLAM-SAP to the FynT SH3 domain was revealed as detailed for Fig. 1 and 2A.
Unlike cells expressing wild-type Tac-SLAM (Fig. 5B, fourth panel, lanes 1 and 2), cells expressing Tac-SLAM Y1F (Fig. 5B, fourth panel, lanes 3 and 4) exhibited no binding of Tac-SLAM-SAP to the FynT SH3 domain. This was not surprising, given that the Y1F mutation abolished the ability of Tac-SLAM to bind SAP (Fig. 5B, second panel) (15). However, cells containing Tac-SLAM Y2,3F (Fig. 5B, second panel, lanes 5 and 6) showed normal association of Tac-SLAM-SAP to the FynT SH3 domain (Fig. 5B, fourth panel). While this mutation had no appreciable effect on the capacity of Tac-SLAM to bind SAP (Fig. 5B, second panel), it completely abolished the ability of Tac-SLAM to undergo tyrosine phosphorylation (Fig. 5B, first panel, lane 6) and promote tyrosine phosphorylation of intracellular substrates like SHIP-1, Dok-1, and Dok-2 (data not shown) (15). These findings gave further credence to the notion that binding of SLAM-SAP to the FynT SH3 domain required SLAM engagement but not an active SLAM biochemical signal.
Third, we ascertained the effect of ablating expression of FynT, the protein tyrasine kinase responsible for SLAM-SAP signaling (Fig. 6) (15, 29). To this end, thymocytes were obtained from wild-type or fyn–/– mice, and the ability of GST-FynT SH3 domains to capture SLAM-SAP complexes was determined as for Fig. 2A (Fig. 6A, first panel). Thymocytes from sap–/– mice were studied as controls. As expected, recombinant FynT SH3 domains were able to recover SLAM-SAP complexes from lysates of wild-type (Fig. 6, first panel, lane 2), but not sap–/– (Fig. 6, first panel, lane 6) animals. Interestingly, they were also able to bind SLAM-SAP from lysates of FynT-deficient mice (Fig. 6, first panel, lane 4). While the extent of SLAM-SAP binding was partially reduced in fyn–/– mice (Fig. 6, first panel, lane 4) compared to wild-type mice (Fig. 6, first panel, lane 2), this diminution likely reflected the decreased SLAM-SAP association in FynT-deficient T cells (Fig. 6B, compare lanes 2 and 4). The diminished association of SLAM with SAP in FynT-deficient T cells probably mirrored the observation that, although SLAM tyrosine phosphorylation is not required for binding to the SAP SH2 domain, the affinity of this interaction is increased approximately fivefold when SLAM is tyrosine phosphorylated (18, 26). Since expression of FynT is required for SLAM-triggered protein tyrosine phosphorylation (15, 29), these results provided additional evidence that the interaction of SLAM-SAP with the FynT SH3 domain did not necessitate active SLAM-SAP-derived biochemical signals.
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DISCUSSION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Several experiments were performed to elucidate the mechanism of this inducible SH3 domain-mediated association. Above all, we were interested in determining whether a SLAM-dependent biochemical signal, a conformational modification, or both were implicated in this phenomenon. We found that treatment of cells with the protein tyrosine phosphatase inhibitor pervanadate was able to trigger ligand-independent tyrosine phosphorylation of Tac-SLAM in the presence of SAP. Nonetheless, this compound provoked only a small increase in the binding of Tac-SLAM-SAP to the FynT SH3 domain. This observation suggested that SLAM signaling was not sufficient to trigger the association of the FynT SH3 domain with SLAM-SAP.
Subsequent analyses provided compelling indication that SLAM-dependent biochemical signals were in fact not involved in the ligand-induced association of the FynT SH3 domain with SLAM-SAP. First, it was observed that incubation of cells at 4°C, a condition that significantly delayed the induction of Tac-SLAM tyrosine phosphorylation in response to anti-Tac, did not interfere with binding of the FynT SH3 domain to Tac-SLAM-SAP. Second, we determined that mutation of the two distal tyrosines in the cytoplasmic domain of SLAM, required for SLAM signaling but dispensable for SAP binding (15), did not prevent the ligand-induced association of the SH3 domain of FynT with Tac-SLAM-SAP. And third, it was revealed that ablation of FynT expression in the mouse, previously shown to abrogate SLAM-triggered protein tyrosine phosphorylation (15, 29), did not hinder the interaction between SLAM-SAP and the FynT SH3 domain. Although we cannot exclude the possibility that these various conditions did not block all SLAM-derived signals, these data strongly supported the hypothesis that SLAM signaling is not necessary for binding of the SLAM-SAP complex to the FynT SH3 domain. They also lent credence to the idea that a signal-independent conformational modification in the SLAM-SAP complex, brought about by SLAM engagement, is responsible for the induced SH3 domain-mediated association.
One prospect that deserves consideration is that signals other than protein tyrosine phosphorylation that are triggered by SLAM engagement are responsible for the inducible binding of SLAM-associated SAP to the FynT SH3 domain. However, this is an unlikely possibility for several reasons. First, there is no indication that SLAM-SAP signaling implicates pathways occurring independently of protein tyrosine phosphorylation. Second, the rapidity of binding of SAP to the FynT SH3 domain was not influenced in any way by incubation at 4°C, which inhibits not only protein tyrosine phosphorylation but also serine/threonine kinase reactions. And third, we have performed experiments in which we tested the impact of the serine/threonine phosphatase inhibitor calyculin, the serine/threonine kinase inhibitor staurosporine, and the serine/threonine kinase activator PMA on the binding of SLAM-associated SAP to the FynT SH3 domain in the T-cell line BI-141 (our unpublished results). We observed that these compounds had no effect on the ability of SAP to interact inducibly with the FynT SH3 domain. Hence, it seems unlikely that alternative signaling pathways such as serine/threonine phosphorylation are involved.
Based on these findings, we propose that SLAM engagement catalyzes a change in the conformation of SAP that allows the arginine 78-based surface of the SAP SH2 domain to recognize the FynT SH3 domain (Fig. 7). In the absence of SLAM engagement (Fig. 7A), the arginine 78-based motif may be structured in such a way that it is not capable of binding with sufficient affinity to the FynT SH3 domain. Some of the critical residues in the arginine 78-based motif may be inaccessible or misaligned at the surface of the SAP SH2 domain, thereby preventing binding to the FynT SH3 domain. Upon SLAM engagement (Fig. 7B), a change of conformation in the cytoplasmic domain of SLAM may be transmitted to associated SAP, thereby altering the arginine 78-based surface and allowing recognition by the FynT SH3 domain. Binding of SAP to the FynT SH3 domain would then activate FynT, which proceeds to phosphorylate SLAM and its downstream effectors (Fig. 7C). A variant of this model is that, prior to SLAM engagement, the arginine 78-based motif of SAP may be masked by binding to another protein. This protein may dissociate from SAP following SLAM engagement, thus freeing the arginine 78-based motif and allowing binding to the FynT SH3 domain. Nevertheless, this possibility seems much less likely, as the recognition of Tac-SLAM-SAP by the FynT SH3 domain was induced promptly (within 2 min) after anti-Tac stimulation. Furthermore, there are no known bona fide binding partners for SAP other than SLAM family receptors and FynT (31).
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A formal proof that SAP undergoes conformational changes in response to SLAM binding with or without SLAM engagement will necessitate solving the molecular structure of intracellular SAP in the absence or presence of SLAM ligation. Unfortunately, the currently available technologies do not allow this type of analysis. Alternative ways to probe the conformation of SAP would be to study its sensitivity to various proteases or its reactivity with various anti-SAP antibodies in the absence or in the presence of SLAM engagement. We did attempt these experiments, but they were sadly inconclusive (data not shown). Hence, a definitive demonstration of our model will have to await technological improvements in atomic structure determination.
Whereas the interaction of the SAP SH2 domain with SLAM is phospho independent, it should be pointed out that the association of the SAP SH2 domain with other SLAM family receptors such as 2B4 is phospho dependent (31). We believe that, in the latter cases, a variation of the model that we are proposing for SLAM is likely to apply. Receptor engagement by ligand would first induce inefficient SAP-independent tyrosine phosphorylation of the receptor, possibly by surrounding membrane-associated Src family kinases. This may be similar to the mechanism implicated in the tyrosine phosphorylation of immunoreceptors by Src-related kinases. This initial phosphorylation of SLAM family receptors would trigger binding of SAP, which would rapidly adopt the necessary conformation for binding and subsequent activation of FynT. Associated FynT would further increase the tyrosine phosphorylation of the SLAM family receptor, which would recruit more SAP and more FynT. This amplification loop would ultimately lead to a full-blown SAP-FynT-dependent tyrosine phosphorylation signal.
Our results also bring up several interesting points about SH3 domains. First of all, they highlight the plasticity of the SH3 module. Typically, SH3 domains, including that of FynT, bind canonical proline-rich ligands forming a left-handed polyproline II helix (24). However, in the case of the interaction between the FynT SH3 domain and SAP, the ligand is an arginine-based motif that lacks any proline. Interestingly, the subdomains of the FynT SH3 domain that make contact with the arginine 78-based motif of SAP appear to be very similar to those utilized to bind proline-rich ligands (5). Therefore, the SH3 domain of FynT is able to bind two very different ligands using the same binding surface. A somewhat distinct phenomenon was documented for the GADS SH3 domain, which binds an arginine-based motif in SLP-76 (2, 19). In this case, the SH3 domain is binding the arginine-based ligand using subdomains that are partly different from those utilized by other SH3 domains to interact with proline-rich ligands. This variation adds further breadth to the ligand-binding modalities used by SH3 domains. There are also other types of ligands for SH3 domains that further exemplify the plasticity of SH3 domains. One example is the RKXXYXXY motif in the adaptor SKAP-55 that interacts with the FynT and ADAP SH3 domains (14).
What is the purpose of having this wide an array of binding motifs for SH3 domains? One possibility is that alternative ligands such as arginine-based motifs may confer a different affinity or avidity to the SH3 domain-mediated association. In support of this, it was shown that the affinity of the GADS SH3 domain for the arginine-rich ligand from SLP-76 was greater than that of other SH3 domains for proline-rich ligands (Kd = 0.24 µM versus 
1 to 10 µM) (19). However, this may not always be the case, as the affinity of the FynT SH3 domain for SAP was 3.45 µM, in keeping with the affinity of traditional SH3 domain-mediated associations (5). Nonetheless, it should be pointed out that, in this last study, the SAP SH2 domain was not complexed to SLAM. In light of the results reported herein, it is plausible that binding of SAP to ligand-stimulated SLAM would further augment the affinity of SAP for the FynT SH3 domain. Another possibility is that the structure of alternative ligands may be more flexible than that of proline-rich ligands. As a result, they may be more susceptible to being regulated, as described here for the inducible binding of the arginine 78-based motif of SAP to the FynT SH3 domain. Furthermore, they may be easier to "insert" in other structural determinants of a molecule, while preserving the global structure and function of this molecule. This may be especially important in the case of SAP, where the arginine-based motif is inserted in the middle of an SH2 domain.
Lastly, our findings uncovered the fact that associations mediated by SH3 domains can be highly regulated. Typically, SH3 domain-mediated interactions are viewed as constitutive (24). In contrast, the association of the FynT SH3 domain with the SLAM-SAP complex is strictly inducible. Whereas the precise structural basis for this phenomenon remains to be established, we propose that a change in the conformation of SAP, induced upon SLAM engagement, is required to enable binding of the FynT SH3 domain to SLAM-associated SAP. Whereas these results constitute a description of a purely inducible association mediated by an SH3 domain, it appears likely that other similar associations exist. One likely example is the association between GADS and hemopoietic progenitor kinase 1 (HPK-1). This interaction was reported to be inducible and to be mediated at least in part by one of the SH3 domains of GADS and a proline-based motif in HPK-1 (20). Perhaps these inducible interactions will be primarily found in situations where SH3 domains interact with ligands other than proline-rich motifs.
In addition to the interaction between the FynT SH3 domain and SAP, other modifications probably contribute to stabilizing the association between SLAM and FynT in T cells. In support of this, we found that mutations of Y315 and Y335 of SLAM (collectively the Y2,3F mutation) dramatically reduced the association of SLAM-SAP with full-length FynT in T cells. However, as demonstrated herein, this mutation had no effect on the ability of SLAM-SAP to bind the SH3 domain of FynT in vitro (15; this report). One scenario is that other interactions exist between the two phosphorylated tyrosines of SLAM and the FynT molecule, perhaps involving the FynT SH2 domain. Against this idea, though, is the finding that the FynT SH2 domain did not enhance the capacity of the FynT SH3 domain to bind SLAM-SAP complexes in vitro. An alternative prospect is that phosphorylation of Y315 and Y335 may change the conformation of SLAM and/or SLAM-associated SAP, thereby stabilizing the SH3 domain-mediated interaction of FynT with SAP in intact cells. Obviously, further studies will be necessary to elucidate this additional level of regulation of the SLAM-SAP-FynT interaction.
The overall mechanism of action of SAP, a molecule composed almost exclusively of an SH2 domain, is quite unusual. First of all, the SAP SH2 domain makes three contacts with tyrosine-based ligands (13, 18, 26). Usually, SH2 domains make only two contacts with their ligands. Second, the SAP SH2 domain interacts in a phosphorylation-independent manner with the tyrosine-based motif from the SLAM receptor (13, 18, 26). As a consequence, SLAM-SAP exists as a preformed complex in T cells (15). Third, the SH2 region of SAP contains a second binding surface, distinct from the phosphotyrosine-binding fold, that makes contact with the SH3 domain of the FynT kinase (5, 16). This feature enables SAP to link SLAM to FynT. Fourth, the SH3 domain-binding site of SAP is not a proline-based motif but is an arginine-based motif (5, 16). And fifth, the interaction of SAP with the FynT SH3 domain is inducible and dependent on engagement of SLAM by extracellular ligands (this report). This is contrary to most other SH3 domain-mediated associations, which are constitutive. Hence, studies of a small adaptor molecule composed only of an SH2 domain have highlighted well the plasticity of modular interactions in cell signaling.
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ACKNOWLEDGMENTS |
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This work was supported by grants from the Canadian Institutes of Health Research (to A.V.), the National Cancer Institute of Canada (to A.V.), and the CANVAC National Centre of Excellence (to A.V.). S.L. held a fellowship from the Canadian Institutes of Health Research and is currently a scientist of the Centre National de la Recherche Scientifique (France). A.V. holds the Canada Research Chair in Signaling in the Immune System.
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FOOTNOTES |
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