SLP-76 Sterile α Motif (SAM) and Individual H5 α Helix Mediate Oligomer Formation for Microclusters and T-cell Activation*

Background: SLP-76 possesses an N-terminal sterile α motif (SAM) domain of unknown function. Results: SLP-76 SAM and its isolated H5 domain self-associates for microclusters and NFAT transcription. Conclusion: SLP-76 self-associates in response to T-cell receptor (TCR) ligation as mediated by the SAM domain. Significance: SAM-mediated SLP-76 dimerization is crucial to understanding how SLP-76 forms complexes for T-cell activation.

SAM domains are found in numerous surface receptors, signaling proteins, and transcription factors (40). To date, Ͻ25% of SAM domains have been reported, or predicted, to form dimers/oligomers either between themselves or with other proteins (41). Examples include transcription factors such as the ETS transcription factor TEL, and polyhomeotic, as well as cell surface receptors ephrin B (EphB) and LAR (leukocyte common antigen-related) (42)(43)(44)(45). Their versatility in binding has implicated them in an array of biological processes that includes signal transduction, protein translation, and gene transcription (46,47). Structurally, the SAM regions are generally comprised of multiple ␣ helices (H1-H5). The crystal structure of the self-associating EphB2 SAM domain has shown that presence of two binding interfaces, one formed by adjacent monomer exchange of amino-terminal peptides that insert into a hydrophobic groove on each neighbor and a second composed of the carboxyl-terminal H5 helix and a nearby loop (44,48). The SLP-76 SAM region is comprised of residues 1-78 with five conserved ␣ helices (H1-5) (6,48). Previous work has shown that the partial loss (i.e. residues 12-78) of the SLP-76 SAM region can impair positive and negative thymic selection (49).
Despite the importance of SLP-76, it has been unclear whether the adaptor can directly self-associate in response to T-cell receptor ligation and whether this event is needed for the activation of T-cells. Although complexes comprised of SLP-76 associated with adaptors such as Nck and Vav-1 have been described (50), the direct binding of SLP-76 to SLP-76 has not been reported. Here, we report that anti-CD3 induces SLP-76 self-association mediated by the SAM domain, and this event was needed for SLP-76 microcluster formation and T-cell activation. Furthermore, different regions in the SAM domain contributed to this self-association with the H5 helix alone supporting co-precipitation of SLP-76 at reduced levels, smaller microclusters, and enhanced T-cell activation. Our data identified for the first time that anti-CD3 ligation induces SLP-76 self-association as mediated by its N-terminal SAM domain.
Fluorescence Microscale Therophoresis (MST)-MST experiments were performed using a Monolith NT.115 instrument (NanoTemper) as reviewed (55). Temperature was controlled at 25°C in the following buffer: 10 mM HEPES, pH 7.5, 500 mM NaCl, 0.5 mM tris(2-carboxyethyl)phosphine, and 0.05% Tween 20. Standard glass capillaries were used. Fluorescence labeling of SLP-76 SAM domain was performed using primary amide coupling of NT-647 dye (NanoTemper) using the manufacturer's instructions. A labeling efficiency of one label per one protein molecule was verified by spectrophotometric analysis using the following molar extinction coefficients (⌺ 280 SLP-76 SAM ϭ 9,970 M Ϫ1 cm Ϫ1 ; ⌺ 650 NT-647 ϭ 250,000 M Ϫ1 cm Ϫ1 ). Individual titrations of 10 nM NT-647 labeled SLP-76 SAM domain and unlabeled SLP-76 SAM domain (0 -90.5 M) were made via 1:1 dilution from stock protein. The reported monomer-dimer K D value was calculated using Origin software from the averages of two separate experimental setups and 11 full titration series, including a range of thermal gradients (from ϳ3-12°C).
Circular Dichroism (CD)-The native far-UV CD spectrum of SLP-76 SAM domain was obtained using a Jasco J-810 instrument with temperature control (25°C; Julabo AWC 100) in the following buffer: 10 mM HEPES, pH 7.5, 150 mM NaCl, and 0.5 mM tris(2-carboxyethyl)phosphine. The reported spectrum is the average of four individual spectra using a 1-mm path length cuvette.
Dynamic Light Scattering (DLS)-DLS experiments were performed using a temperature-controlled (25°C) DynaPro instrument (Protein Solutions). Raw data were analyzed using manufacturer provided Dynamics software (version 6; Wyatt Technologies), and each data point was the result of at least three averages of 20 individual scans. An estimation of SLP-76 SAM domain monomer hydrodynamic radius was performed using the deposited Protein Data Bank structure of the NMR solution structure of SLP-76 SAM Domain (Protein Data Bank code 2EAP), including flanking residues to mimic the expression construct and submitted to HYDROPRO (56). The approximate dimer (and tetramer) hydrodynamic radius was calculated using HYDROPRO with a dimer model constructed by GRAMM-X (57, 58) using the 2EAP structure.

SAM Domain of SLP-76 Mediates Dimer/Oligomer Formation-
A minority of SAM domains have been reported to undergo complex formation with themselves or other proteins (41,42). Given the importance of complexes to signal transduction, we assessed whether SLP-76 could self-associate and whether the SLP-76 SAM domain could form oligomers. We therefore first examined the in vitro binding of a recombinant, purified SLP-76 SAM domain (amino acids 1-78). HIS-tagged human protein corresponding to the SLP-76 SAM domain region (1-78 amino acids) was expressed in E. coli followed by purification using Ni 2ϩ affinity column chromatography. This procedure yielded a single major protein at ϳ10 kDa. (Fig. 1A, left  panel). Purified SLP-76 SAM domain was then analyzed by far-UV CD to verify the ␣ helical secondary content that is typical of natively folded SAM domains (Fig. 1A, right panel). Two CD bands observed at ϳ210 and 225 nm are characteristic of high helical content protein structures, as described for other SAM domains (59). MST performed by titrating increasing concentrations of unmodified SLP-76 SAM domain into fluorescently labeled SLP-76 SAM domain suggested a monomerdimer K D of 2.5 Ϯ 0.9 M (Fig. 1B). In addition, DLS corroborated the oligomerization of the SLP-76 SAM domain (Fig. 1C). Analysis of DLS data (see "Experimental Procedures") indicated that the SLP-76 SAM domain formed dimers as well as higher order oligomers. Estimated hydrodynamic radii for SLP-76 SAM domain are shown as a monomer (solid line), dimer (large dashed line), and tetramer (small dashed line) as determined by the programs HYDROPRO and GRAMM-X. The R H showed a monomer radius of ϳ2 nm that increased to a 3-nm species at a concentration transition of 10 to 20 M that correlated with a size shift from 10 to 20 kDa. Tetramers were also observed at concentrations Ͼ50 M as well as possible higher order complexes Ͼ150 M. These findings indicated that the SLP-76 SAM domain is capable of self-associating in the formation of dimers and higher order oligomers in solution.
SLP-76 Self-associates in Response to CD3 Ligation-Next, to assess whether SLP-76 could bind to itself in T-cells, two tagged versions of SLP-76 were generated, one with an YFP and another with a His 6 tag ( Fig. 2A, left panel). EYFP-tagged full- length SLP-76 (i.e. WT) was then co-expressed with His-tagged SLP-76 in SLP-76-deficient J14 Jurkat cells. Transfected cells were stimulated with anti-CD3 (right panel, lanes 2 and 4), or an isotype control (lanes 1 and 3) for 5 min, followed by lysis and precipitation of His-tagged SLP-76 with anti-His and blotting with anti-SLP-76. Significantly, anti-His precipitation of SLP-76-His co-precipitated SLP-76 EYFP from anti-CD3 ligated cells ( Fig. 2A, lane 4). A weaker co-precipitated band was occasionally seen in resting cells (lane 3); however, in all experiments, the level of co-precipitation was markedly increased with anti-CD3 ligation. As a negative control, no SLP-76 was seen in the IgG control precipitates ( Fig. 2A, lanes 1 and 2). SLP-76 binding to SLP-76 occurred as early as 2 min post-ligation and peaked at 5 min, followed by a slight decrease at 15 min post-anti-CD3 ligation (Fig. 2B, lanes 2-4). Concentrations of anti-CD3 as low as 0.5 g/ml induced binding and this increased slightly with higher concentrations of 2 and 5 g/ml (Fig. 1C, lanes 2-4). Importantly, the deletion of the SAM domain eliminated the ability of SLP-76 to co-precipitate SLP-76 (Fig. 3A). Although His-tagged WT SLP-76 co-precipitated EYFP-SLP-76 in response to anti-CD3 ligation (Fig. 3A,  left lower panel, lanes 1 and 2), His-tagged dN78 (lacking residues 1-78) failed to co-precipitate WT EYFP-SLP-76 (lanes 3 and 4) or EYFP-dN78 (lane 5). No co-precipitation was seen at either 2 or 5 min post-anti-CD3 ligation (Fig. 3A, right lower panel, lanes [3][4][5][6] contrary to the wild type SAM domain (lanes 1 and 2). Consistent with SAM domain binding to itself to form higher order complexes, these observations showed that anti-CD3 induces self-association of SLP-76, which is dependent on the SAM domain.
SLP-76 forms microclusters in response to anti-CD3 ligation (35,36). To assess this in the context of the SAM domain, J14 cells expressing EYFP-tagged WT or dN78 SLP-76 were imaged on anti-CD3-coated slides, as described (35,37,54). WT SLP-76 formed microclusters that migrated to the inner contact region over time (35,54) (Fig. 3B, also right panel). By contrast, EYFP-tagged dN78 SLP-76 completely failed to generate microclusters and, instead, showed a diffuse pattern of membrane localization (Fig. 3B, lower panel). This occurred despite the fact that dN78 could still co-precipitate GADS and ADAP (Fig. 3C). This showed that the SAM domain was essential for microcluster formation, to a greater extent than previously observed with the partial SAM domain mutant (49).
SAM H5 ␣ Helix alone Can Support Self-association and Microcluster Formation-The crystal structure of the SLP-76 SAM domain has been solved recently 4 (Protein Data Bank code 2EAP). The structure has similarities and differences to other solved SAM structures (42)(43)(44)(45). The orientation of the individual N-terminal ␣ H1-4 helices differs among SLP-76, EphB2 receptor, and polyhomeotic SAM domains, whereas the position of the larger H5 ␣ helix is similar in each case (Fig. 4A).
In this context, a previous study had shown that the self-association of the EphB2 SAM domain is mediated by two distinct interfaces: one by a hydrophobic interaction between aminoterminal H1-H4 helices and a second by the binding between J14 T-cells were co-transfected and subjected to precipitation as described in Fig. 1 (n ϭ 3). Lower left panel: SLPWT His and WT EYFP (lanes 1 and 2); SLPdN78 His and WT EYFP (lanes 3 and 4); SLPdN78 His and dN78 EYFP (lane 5). Lanes 1 and 3, isotype IgG control; lanes 2, 4, and 5, anti-CD3. The arrows indicate the EYFP-SLP-76 (higher band) and His 6 -SLP-76 or His 6 -SLPdN78 (lower band). Lower right panel: anti-CD3 time course of ligation for 2 and 5 min as was indicated. SLPWT His and WT EYFP (lanes 1 and 2); SLPdN78 His and WT YFP (lanes 3 and 4); SLPdN78 His and dN78 EYFP (lanes 5 and 6). adjacent H5 helices and a nearby loop (44,48). This H5 interaction shows pseudodyadic symmetry in the packing of the monomer against the same region in another molecule (44).
To test whether the SLP-76 H5 domain could independently mediate SLP-76 self-association, the first four of the five SAM ␣ helices (i.e. residues 1-57) were deleted leaving the single H5 helix attached to the rest of the SLP-76 protein (termed dN57). EYFP-and His-tagged versions were expressed in J14 cells, either alone or with WT SLP-76 (Fig. 4B). Remarkably, anti-His readily co-precipitated dN57 EYFP from lysates of cells co-transfected with dN57 HIS (Fig. 4B, left panel, lane 5). dN57 also coprecipitated SLP-76 WT His when co-expressed in J14 cells (Fig.   4B, lanes 3 and 4). In both cases, the association was anti-CD3dependent. As a further positive control, anti-His co-precipitated SLP-76 WT EYFP from cells co-transfected with SLP-76 WT His and SLP-76 WT EYFP (Fig. 4B, lane 2). Densiometric readings showed that dN57 co-precipitated less dN57 than WT SLP-76 (i.e. 40% less). dN57 co-precipitation of WT SLP-76 was in turn less than WT SLP-76 co-precipitation of WT SLP-76 (right histogram). These observations showed that the SLP-76 SAM H5 helix was sufficient to bind an H5 helix in an adjacent SLP-76 molecule; however, this self-association was less efficient than to WT SLP-76 or between WT SLP-76 molecules with full-length SAM domains. With the ephrin receptor (EphB2) SAM domain, self-association is mediated by a hydrophobic interaction between amino-terminal H1-H4 helices and distinct binding between adjacent H5 helices and a nearby loop (44,48). B, left panel: dN57 lacking H1-H4 domains but retaining H5 supports SLP-76 binding to itself. J14 T-cells were co-transfected with two tagged versions of SLP-76, one with an EYFP and another with a His 6 tag and precipitated using anti-His monoclonal antibody as described in Fig. 1. Lanes 1 and 3 Intriguingly, dN57 SLP-76 also supported the formation of microclusters (Fig. 4, C-E). Furthermore, consistent with the reduced level of co-precipitated SLP-76, the dN57 microclusters were significantly smaller than SLP-76 WT clusters (Fig.  4C, upper versus lower image). Clusters form initially in the peripheral contact region followed by migration to the central contact region (35,37,54). Once the clusters migrated to the interior of the cell contact region, they coalesced to form larger clusters. Although the average size of the full length WT clusters in the peripheral contact region was 1.1 m 2, the mean size of dN57 clusters was 0.62 m 2. (Fig. 4D, middle panel). Interestingly, this reduced size was accompanied by an increase in the numbers of dN57 clusters (i.e. 70 clusters/cell to 47 for WT SLP-76 clusters) (left panel) and by a slight increase in the motility of clusters (0.096 versus 0.075 m/s) (right panel). Kymograph analysis confirmed the more rapid movement of dN57 clusters (Fig. 4E). For 0 -125 s, the dN57 clusters moved more rapidly to the central contact region than did the WT clusters. These data showed that the single SAM H5 helix selfassociation was sufficient to support SLP-76 microcluster formation.
SLP-76 SAM H5 ␣ Helix Supports Enhanced T-cell Proliferation-dN57 SLP-76 also supported anti-CD3 induced NFAT-mediated transcription in J14 cells, as well as the production of IL-2 or proliferation of primary T-cells. Surprisingly, dN57 supported significantly higher levels of activation relative to WT SLP-76 (Fig. 5). J14 cells transfected with dN57 or WT SLP-76 with a 3ϫ-NFAT-promoter construct were ligated with anti-CD3 followed by a measurement of luciferase activity. dN57 reconstituted the promoter activity at 2-3-fold higher levels than WT SLP-76 (p ϭ 0.017). In contrast, dN78 SLP-76 failed to support increased transcription. As a control, the blotting of cell lysates confirmed expression of transfected SLP-76 constructs (Fig. 5A, right panel). Furthermore, the same effect of dN57 was observed in primary mouse and human primary T-cells (Fig. 5B). CD4-positive mouse T-cells were transfected and stimulated with anti-CD3 for 12 h followed by a measure of IL-2 by an ELISA assay (Fig. 5, left panel). Each transfected SLP-76 was expressed at similar levels (Fig. 5B, middle panel). dN57 enhanced IL-2 production relative to that seen with WT SLP-76, whereas the N78 mutant (a deletion mutant lacking N-terminal amino acids 1-78) failed to support IL-2 production. Similarly, transfection of primary human T-cells showed that dN57 greatly enhanced proliferation as measured by [ 3 H]thymidine incorporation relative to WT SLP-76 and the dN78 mutant (Fig. 5B, right panel) (p ϭ 0.043). Similar levels of expression of transfected constructs were observed in primary human and mouse cells. These data showed that the H5 SAM helix effectively supported the activation of T-cells.
To further assess the importance of the H5 motif in inter-SLP-76 binding, a deletion mutant form of SLP-76 lacking the H5 domain (⌬H5) was generated and expressed in J14 Jurkat T-cells (Fig. 6). SLP-76-WT-EYFP was expressed with HA-SLP-76 WT, or SLP-76-⌬H5-EYFP was expressed with HA-SLP-76-⌬H5 followed by anti-CD3 ligation for 5 min and then precipitation with anti-GFP followed by blotting with anti-GFP or anti-HA. As shown previously, anti-GFP precipitated SLP-76-WT-EYFP from resting and activated cells (Fig.  6A, upper panel, lanes 1 and 2) and co-precipitated HA-SLP-76 WT from stimulated cells (lower panel, lane 3 versus 2). Anti-GFP also precipitated SLP-76-⌬H5-EYFP from resting and activated cells (Fig. 6A, upper panel, lanes 4 and 5). However, it failed to co-precipitate HA-SLP-76-⌬H5 from resting or activated T-cells (Fig. 6A, lower panel, lanes 4 and 5). A faint amount of material in the HA-SLP-76-⌬H5 M r range was occasionally seen; however, this observation was not reproducible. In agreement with the impaired self-association between SLP-76 lacking the H5 helix, we also examined in vitro selfassociation of the SAM domain lacking the H5 domain (i.e. SLP-76 residues 1-61) by MST (Fig. 6B). This assay showed that isolated protein fragment failed to show specific binding as seen by the absence of a sigmoidal shaped curve. Four different laser powers were used with a triplicate measurement at each power. Last, expression of the SLP-76-⌬H5-EYFP mutant failed  4). B, SLP-76 SAM lacking the H5 helix (residues 1-61) failed to show higher order complexes as monitored by MST. Analysis of SLP-76 protein residues 1-61 was analyzed by MST as described in Fig. 1 and under "Experimental Procedures." C, SLP-76 ⌬H5-EGFP failed to support anti-CD3-induced NF-AT/AP1 promoter activity in J14 Jurkat cells. J14 cells were co-transfected with SR␣ vector (mock), SLP-76 WT or SLP-76 ⌬H5-EYFP plus a NF-AT/AP1 luciferase promoter. Eighteen hours after transfection, cells were stimulated with anti-CD3 for 6 h, followed by a measure of luciferase activity. Right panel: anti-SLP-76 blotting of lysates from transfected J14 cells.
to support an increase in anti-CD3-induced AP-1/NFAT transcription in T-cells (Fig. 6C). These data indicated that the H5 ␣ helix is needed for the binding of the SAM domain to itself, and the ability of SLP-76 to generate signals from the T-cell receptor needed for NFAT-AP1 transcription.

DISCUSSION
SLP-76 integrates signals from the antigen-receptor for the activation of T-cells. Despite this, it had been unclear whether the SLP-76 can directly self-assemble to form dimers and higher order oligomers in the generation of intracellular signals. Furthermore, although SLP-76 can form microclusters, it had been unclear whether self-assembly is needed for the formation of these large assemblies of proteins (35,36,50). In this work, we have shown for the first time that SLP-76 can selfassociate in response to T-cell receptor ligation as mediated by its own N-terminal SAM domain. The purified SAM domain can form dimers, tetramers, and possibly higher ordered complexes, as detected by MST and DLS analysis. Furthermore, deletion of the SAM domain prevented SLP-76 self-association, whereas the retention of the single H5 helix sufficed to mediate self-association, albeit to a lesser degree than wild-type SLP-76. The H5 helix supported for formation of smaller clusters and enhanced T-cell activation. Overall, these observations show that SLP-76 SAM domain can self-associate for the formation of complexes for T-cell activation.
Our first observation was that the purified SLP-76 SAM domain self-associated to form dimers and other higher order oligomers as determined by MST and DLS analysis. We observed dimers as well as tetramers and possible higher order complexes. Dimer formation occurred at protein concentrations similar to, or lower than, those seen for other SAMs such in the EphB2 SAM domain (44,48). The SLP-76 SAM domain therefore is a member of the minority of SAM domains (Ͻ25%) that have been reported or predicted to self-associate (41). We also observed that SLP-76 employed the SAM domain to selfassociate in T-cells as seen by co-precipitation where anti-His co-precipitated SLP-76 EYFP with SLP-76 His . This effect was lost with the dN78 mutant where the SAM domain has been deleted. Although some co-precipitation was occasionally observed in resting cells, the formation of the SAM-dependent complex was largely dependent on anti-CD3-induced signals. The nature of the T-cell receptor signals that facilitate this process is not known, but at a minimum, is likely to involve the increased plasma membrane localization for increased SLP-76-SLP-76 interactions. Despite the binding of SLP-76 to other proteins, GADs and ADAP, no SLP-76 co-precipitation of SLP-76 was noted with the loss of the SAM domain. This indicates that self-association depends on the SAM domain for assembly and cannot occur independently by the binding of other proteins to SLP-76. Instead, a more likely scenario would involve initial SAM mediated binding followed by the participation of other binding proteins, possibly for the assembly of even larger multiprotein complexes. Complexes involving two NCK and VAV1 molecules binding to SLP-76 have been described (50), whereas LAT can cluster independently due to the dimerization of GRB2 by SOS1 (61). SAM-mediated dimerization has also been shown to activate associated pro-teins such as in the ETS transcription factor TEL (TEL-SAM) (42). A similar activation event could possibly occur in the case of SLP-76 associated proteins.
Our second finding was that a subdomain of the SAM region, the C-terminal H5 helix alone, sufficed to support self-association. The observation was consistent with the conserved orientation of the H5 helix in different SAM domains, and the fact that the H5 helix in the EphB2 SAM domain serves as a second binding site between SAM domains (44). However, to our knowledge, the demonstration that the SLP-76 H5 helix alone can mediate SLP-76 dimer formation is the first example of autonomous SAM subregion mediated binding between SAM domains. At the same time, a major difference was seen in the efficiency of co-precipitation, where dN57 co-precipitated dN57 at lower levels than observed with full-length SLP-76. The importance of the H5 domain was further underscored by impaired ability of the intact SLP-76 protein with the deleted H5 helix (SLP-76⌬H5) to support co-precipitation in response to anti-CD3 ligation. MST analysis of the SAM domain lacking H5 helix also failed to show an evidence of self-association. Whether the H1-H4 region can form a second interface that depends on the presence of the H5 helix will require further structural analysis.
In the case of the dN57 versus WT SLP-76 proteins, confocal imaging showed remarkably that the H5 helix alone could support the formation of anti-CD3-induced clusters. This indicated that the observed H5 helix self-association was sufficient to mediate the formation of microclusters. However, the dN57 clusters were considerably smaller that the WT clusters as most evident in the peripheral region where clusters are known to arise. It is tempting to speculate that these smaller clusters represent dN57-dN57 dimers, whereas the larger WT clusters includes additional interactions that form larger oligomers. As well, upon migration to the interior of the cell contact region, the smaller dN57 clusters were observed to coalesce to form larger clusters, an event that we would speculate may involve the subsequent recruitment of other associated proteins such as VAV and NCK, which may also contribute to the formation of higher order complex structures. Although the complete loss of SAM completely prevented cluster formation, the partial deletion of residues 12-78 has been described to partially affect the longevity of cluster formation (49).
Last, the smaller sized oligomers and clusters of SLP-76 H5 supported IL-2 transcription in J14 cells at significantly higher levels than WT SLP-76 (Fig. 5). This effect was also seen in the activation of IL-2 production and proliferation in primary mouse and human T-cells. The basis for this gain-of-function is not clear but may involve the presence of a greater number and speed of microclusters for more interactions with other signaling proteins. Alternatively, although dimer formation is needed for the propagation of signals as shown by the loss of function with the dN78 and ⌬H5 mutants, additional higher order complex formation may be inhibitory. Preliminary data showed that dN57 clusters have greater co-localization with LAT clusters (data not shown). The basis for the presence of a full-length SAM domain that limits the activation potential of the adaptor relative to the individual H5 helix remains to the uncovered. Further studies will be needed to uncover the role of individual