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Address correspondence to Christopher E. Rudd, Molecular Immunology Section, Dept. of Immunology, Imperial College London, Hammersmith Campus, Du Cane Rd., London W12 ONN, England, UK. Phone: 44-20-8383-8421; Fax: 44-20-8383-8434; email: c.rudd{at}ic.ac.uk
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Abstract |
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Key Words: integrins • adaptor proteins • immunological synapse
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Introduction |
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The outer ring of the SMAC (i.e., pSMAC) is comprised of LFA-1 linked to intracellular proteins talin and RapL (regulator for cell adhesion and polarization enriched in lymphoid tissues; references 12, 13). The conversion of LFA-1 from low to higher affinity forms is a complex process that involves conformation changes combined with an increase in avidity due to clustering (13–15). TCR-CD3 ligation and chemokines promote this conversion by means of inside-out signaling. The process is influenced by various intracellular signaling proteins such as p56lck, Ras family member Rap-1, and the exchange complex Cbl–CrkL–C3G (16, 17). Rap1 binds to its effector molecule RapL (regulator of adhesion and polarization enriched in lymphoid tissues), whereas dominant negative (DN) forms of RapL can inhibit LFA-1 clustering, ICAM-1 binding, and T cell–APC conjugation (18). In addition, the head region of talin can affect conformational changes in LFA-1 (19), although it may negatively regulate LFA-1 in neutrophils (20). RhoA has also been linked to the chemokine induction of high affinity LFA-1 on lymphocytes (21, 22).
Aside from catalytic proteins, adaptor proteins or molecular scaffolds play central roles in the signaling events needed for T cell function (23, 24). These proteins lack enzymatic domains and instead carry binding domains and sites that are needed for the assembly of complexes. LAT and SLP-76 (Src homology 2 domain–containing leukocyte protein of 76 kD) couple the TCR complex to the activation of phospholipase C
1 (PLC1), Ca2+ mobilization, and NFAT activity (25–29). SLP-76 carries two NH2-terminal YESP sites that are phosphorylated by ZAP-70 (Zeta-associated protein-70) and ITK (inducible T cell kinase)/RLK (resting lymphocyte kinase; references 30–33), and that bind to the SH2 domain of the hematopoietic guanine nucleotide exchange factor VAV-1 and the adaptor NCK (34–36). The COOH-terminal Src homology 3 domain of Nck can recruit Wiskot Aldrich Syndrome protein (WASP), whereas the localization and activation of Cdc42 and WASP requires Vav1 (37). GTP-bound Cdc42 binds the WASP, which in turn regulates the Arp2/3 complex, an initiator of actin filament formation (38).
Although the NH2-terminal region of SLP-76 is needed for the activation of PLC1 and Ca2+ mobilization, the SH2 domain is not needed for these events, but is nevertheless needed, in unexplained ways, for T cell proliferation (39, 40). In this context, we and others cloned an immune cell–specific adaptor adhesion and degranulation promoting adaptor protein (ADAP; previously termed Fyn-T–binding protein [FYB] or SLP-76–associated protein [SLAP]) that binds to the SH2 domain of SLP-76 (41, 42). It is also preferentially phosphorylated by p59fyn and binds to the SH2 domain of the kinase (41, 43). Two isoforms (ADAP-120/130) exist with the 120-kD isoform preferentially expressed in the thymus and the 130-kD isoform in the peripheral T cell compartment (44). Each isoform has a proline-rich region, two putative nuclear localization sites, multiple tyrosine sites, and two SH3 domains (41–43). Each isoform also has two identical ExYDDV motifs (EVY595/Y651 DDV motifs in ADAP-120) that bind to the SH2 domain of SLP-76, and a YDGI motif that binds to same domain in p59fyn (42, 43, 45, 46). A similar DxYDDV site in hematopoetic protein kinase 1 (HPK-1) binds to the SH2 domain of BLNK/SLP-65 (47). An alternate YGYI site for SLP-76 SH2 domain binding has been proposed (48), but is the subject of some uncertainty (46, 47). Further downstream, ADAP binds to SKAP-55 (49–51), and has an EVH1 (Ena [enabled]/VASP [vasodilator-stimulated phospho protein] homology 1 domain) binding site for VASP, a regulator of actin filament elongation (52).
Although the subject of some initial debate (42), ADAP positively regulates T cell function as shown in transfection studies (44, 46) and by the phenotype of the ADAP–/– mice (53, 54). This was clearly observed in ADAP-deficient mice that show a defect in LFA-1 adhesion and clustering on T cells (53, 54). No defects in other aspects of T cell signaling were apparent. The adaptor also modulates ß1 integrin-dependent cell migration (55) as well as the clustering and adhesion of ß1 integrins on basophils (56–58). ADAP can bind to MIST (mast cell immunoreceptor signal transducer)/Clnk (for cytokine-dependent hemopoietic cell linker), leading to enhanced ß-hexosaminidase release in mast cells (56–58). A functional connection to SKAP-55 has been implied by the ability of SKAP-55 to regulate LFA-1 adhesion/clustering and T cell–APC conjugation (59). Loss of the SKAP-55 SH3 domain that binds ADAP abrogates the ability of SKAP-55 to influence these events (59).
The combined observation that the SLP-76 SH2 domain is needed for T cell function and binds to ADAP, an adaptor that in turn is needed for LFA-1 adhesion suggested that this intermolecular interaction might constitute a link in TCR-CD3–mediated activation of LFA-1 (i.e., inside-out signaling). In this work, using a mutational approach, we confirm that ADAP binding to the SLP-76 SH2 domain is needed for TCR-induced LFA-1 adhesion and T cell–APC conjugation. In addition, we further show that the ADAP–SLP-76 interaction differentially regulates pSMAC formation versus general LFA-1 clustering and conjugation. Although the loss of the SLP-76 SH2 domain binding sites on ADAP (i.e., M12) interfered with the ability of ADAP to promote conjugation and general LFA-1 clustering, it did not operate as a DN. In contrast, the same mutations converted ADAP into a potent DN in the blockade of SMAC formation, and concurrently, IL-2 production. ADAP also colocalized with LFA-1 at the immunological synapse. Our findings identify a specific intermolecular event that couples the TCR with SMAC formation in T cells, and support a role for SMAC formation in T cell function.
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Materials and Methods |
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Transfection/Infection Protocols.
ADAP, GFP, and M12 were expressed in a pSR
mammalian expression vector containing an influenza hemaglutinin (HA) epitope tag at the NH2 terminus (45, 59). The SH2 domain of SLP-76 was amplified by PCR and cloned into a pEBG mammalian expression vector containing a GST epitope tag. Transfection was conducted as described previously (45). For retroviral infection, ADAP or M12 was cloned into IRES-GFP–based pMXF5 retroviral expression vector using BamHI and NotI sites or using the Pinco-GFP–based retroviral expression vector (C. Casimir, Imperial College London, London, England) using BamHI and EcoRI sites, and T cells were infected as described using the ecotropic Phoenix retroviral producer cell line (59, 60). Primary mouse CD4+ cells were isolated using Dynabeads with mouse anti-CD4 (Dynal Biotech) and stimulated with Con A (Sigma-Aldrich) for 48 h before infection. After two washes, CD4+ T cells were cultured and washed in 10–5 M methyl--D-manno-pyranoside (Sigma-Aldrich) to remove the lectin. After three infections, T cells were collected and used in the conjugation experiments.
T Cell–APC Conjugate Assay.
Antigen-induced T cell adhesion was quantified using a colorimetric assay for measuring intracellular succinate dehydronase content with MTT (3(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl tetrazolium bromide) as described previously (59). Cleaved MTT was dissolved in DMSO, transferred into a 96-well plate, and measured for optical density at 595 nm using an ELISA reader (Bio-Rad Laboratories). The level of T8.1 cell adhesion to L625.7-presenting cells was calculated by subtracting the background signal obtained with L625.7 presenting cells alone. Alternatively, T8.1 cells were stained with PKH67 green or PKH26 red dyes (Sigma-Aldrich) and seeded on adherent L625.7 cells. After a period of culture, the supernatant containing nonadherent T8.1 cells was aspirated, and the remaining cells were observed by fluorescent microscope. Conjugates between DO11.10 Tg cells and A20 cells were measured as described previously (59). Fluorescence was analyzed using a FACSCalibur flow cytometer (Becton Dickinson) equipped with CELLQuest software. Immunoprecipitation and immunoblotting were conducted as described previously (46, 59). Levels of bound Ab were using horseradish peroxidase–conjugated rabbit anti–mouse followed by detection with enhanced chemiluminescence (Amersham Biosciences). Measurements of IL-2 were performed using GFP, ADAP-GFP, or M12-GFP–infected T8.1 cells cocultured with L625.7 cells pulsed with different concentrations of Ttox peptide for 48 h as described previously (44, 46). The supernatants were collected, and IL-2 was measured using ELISA with rat anti–mouse IL-2 monoclonal antibody (ELISA Capture) and biotinylated rat anti–mouse IL-2 monoclonal antibody (ELISA Detection).
Integrin Adhesion and Clustering Assays.
Binding to ICAM-1 or fibronectin was measured using 96-well flat-bottom plates coated with 3.4 µg of murine ICAM-1–Fc, 30 µg/ml fibronectin (Sigma-Aldrich), or 10 µg/ml BSA (Sigma-Aldrich) as described previously (59). LFA-1 capping on retroviral infected T8.1 cells was conducted as described previously (59).
Immunofluorescent and Confocal Microscopy.
Immunofluorescence microscopy was conducted as described previously (59). Cells were incubated with anti-HA antibody, followed by FITC-labeled secondary antibody and TRITC-labeled phalloidin (Sigma-Aldrich). Image acquisition was performed with an Eclipse E800 microscope (Nikon) and RT Slider digital camera (Diagnostics, Inc.). For LFA-1 confocal imaging at the T cell–APC interface, L625.7 cells were seeded on the 12-mm coverslips in 24-well plates overnight and pulsed with 2.5 µg/ml Ttox cells for 2 h. Infected T8.1 cells were added for 30 min followed by fixation with 2% paraformaldehyde for 20 min at 4°C. Cells were exposed to in a blocking solution (5% vol/vol FCS and 3% wt/vol BSA in Perm/Wash buffer; BD Biosciences) for 1 h at 4°C followed by an incubation with anti-CD11a (1 µg/250 ml) in blocking solution for 1 h at 4°C. After three washes with 0.1% Tween 20/PBS, Alexa Fluor 568–conjugated goat anti–rat antibody (1 µg/250 ml) was incubated with cells for 1 h at 4°C and washed three times in 0.1% Tween 20/PBS. Cells were viewed under a 63x oil immersion objective using a confocal laser scanning microscope (TCS SP2; Leica) equipped with argon/krypton and helium/neon lasers using excitation wavelengths of 488, 568, and 633 nm as described previously (61). Conjugates were scanned in the xy-direction every 0.3 µm throughout the z-plane. The face of the immunological synapse was reconstructed using a maximum intensity projection (Confocal Software; Leica). The percentage of fluorescence intensity at immunological synapse relative to the whole cell membranes was calculated.
Online Supplemental Material.
Conjugates and LFA-1 staining were conducted as before and scanned in the xy-direction every 0.3 µm throughout the z-plane. The face of the immunological synapse was reconstructed using a maximum intensity projection (Confocal Software; Leica). Three-dimensional movies were reconstructed using Volocity software. Online supplemental material is available at http://www.jem.org/cgi/content/full/jem.20040780/DC1.
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ADAP also localized at the interface between T cells and APCs (Fig. 1 e). Cells were transfected with HA-tagged ADAP, followed by staining with anti-HA plus FITC-conjugated goat anti–mouse. TRITC-labeled phalloidin was used to visualize F-actin. Although unconjugated cells showed ADAP throughout the cytoplasm (Fig. 1, top), the adaptor localized at the T cell–APC contact area of conjugates formed in the presence and absence of peptide (Fig. 1, middle, arrows). Often this localization was accompanied by a concurrent loss of the adaptor in other regions of the cytoplasm. Ttox-stimulated conjugates had a more compressed localization of ADAP at the interface than observed in unstimulated conjugates (Fig. 1, bottom middle vs. top middle). Furthermore, the phalloidin subcap closely overlapped with ADAP in stimulated conjugates as shown by yellow fluorescence (Fig. 1, arrows, overlay of red and green fluorescence). The overlap was less coincident in unstimulated conjugates. As a negative control, vector-transfected cells exhibited no localization (Fig. 1, bottom). Overall, these observations demonstrate that ADAP can up-regulate T cell–APC conjugate formation in a process that is accompanied by changes in the phosphorylation and localization of ADAP.
T Cell–APC Conjugation Depends on SLP-76 Binding Sites on ADAP.
We previously identified two tyrosine-based motifs (i.e., EVY595/651DDV sites) in ADAP that bind to the SH2 domains of SLP-76 (45, 46). Therefore, next we investigated whether SLP-76 binding to ADAP was needed to couple the TCR with the downstream regulation of conjugation. ADAP mutated at the tyrosine residues (i.e., Y-F) in these motifs (termed M12) was incorporated into a pMXF5-GFP–based bicistronic vector for retroviral-mediated gene transfer (60). FACS profiles showed that cells expressed moderate levels (i.e., 48–62%) of GFP, ADAP-GFP, and M12-GFP (Fig. 2). Infected T cells were generally sorted before their use in conjugation assays. Significantly, although ADAP increased conjugate formation, the M12 mutant had little if any effect (Fig. 2 b). This defect in the function of M12 was also observed in the PKH26-labeling assay (Fig. 2 c). Although ADAP expression increased T cell adhesion (middle vs. left), the M12 mutant failed to increase conjugation in the presence of Ttox. These observations clearly indicate that the two single mutations needed for SLP-76 binding interfere with ADAP enhancement of conjugation.
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SLP-76 SH2 Domain, But Not VASP Binding Mutant, Blocks Conjugation.
Although the loss of the SLP-76 binding sites abrogated conjugation, it was formally possible that another as yet identified protein binds the ADAP EVYDDV sites and acts to couple the TCR with conjugation. To address this, the SH2 domain of SLP-76 was expressed as a GST fusion protein in T cells and assessed for an effect on conjugation (Fig. 3 a). Although the expression of GST alone had no effect, expression of GST-SLP-76-SH2 reduced conjugation to nonpeptide-treated levels. GST and GST-SLP-76-SH2 were expressed at similar levels as shown in anti-GST blotting (Fig. 3 a, bottom). SLP-76 SH2 domain precipitated a major band corresponding to ADAP in addition to two weak bands (unpublished data). Although tempered by the fact that SH2 domains bind to more than one protein, the predominate binding to ADAP and its inhibition in conjugation provides further evidence in support of the importance of ADAP–SLP-76 interaction in conjugation.
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ADAP–SLP-76 Binding Regulates Integrin Adhesion and Clustering.
Given the importance of conjugation, it was of interest to determine whether this interaction was needed for integrin-mediated adhesion and aggregation (Fig. 4). Previous studies by ourselves and others have shown a requirement for ADAP expression in integrin binding (53, 54). For this, infected T cells were incubated on plates with immobilized ICAM-1 and assessed for binding as described previously (59). Anti-CD3 increased the binding of GFP-infected control cells to ICAM-1 (Fig. 4 a). ADAP infection enhanced binding by twofold relative to this control. In contrast, M12 failed to enhance adhesion. A similar analysis was conducted using purified fibronectin immobilized on plates (Fig. 4 b). In this case, the combination of immobilized fibronectin plus anti-CD3 led to a fivefold increase in binding as compared with BSA-coated plates. Similarly, ADAP-infected cells showed twofold binding increased relative to control cells. M12 infection failed to augment binding, exhibiting binding comparable to the GFP vector control. Treatment with phorbol ester served as a positive control for inside-out signaling. These data indicate that the previously documented importance of ADAP in integrin binding depends on SLP-76 SH2 binding to ADAP.
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Clustering of LFA-1 was imaged at the T cell–APC interface in conjugates formed over 30 min in the presence of Ttox. Anti-CD11a was used followed by an Alexa Fluor 568–conjugated anti–rat staining. Imaging of the immunological synapse amongst individual conjugates (>40) was conducted by laser-scanning confocal microscopy. As shown in Fig. 4 d, LFA-1 could readily be visualized at the interface of T cell–APC conjugates that had been stimulated by Ttox peptide (Fig. 4 d, top). LFA-1 at the interface was quantified as the percentage of LFA-1 located in the immunological synapse relative to the total surface of the T cell. There was considerable variation in the level of LFA-1 clustering amongst individual conjugates. Retroviral expression of ADAP-GFP increased the mean slightly, whereas M12 failed to support an increase, consistent with the observation in the capping of surface LFA-1 (Fig. 4 c). M12 also appeared to decrease the mean slightly, although this was a marginal effect relative to the overall heterogeneity amongst conjugates. Overall, these observations indicate that M12 failed to support LFA-1 clustering as determined by two independent assays.
M12 Acts as a DN in Blocking pSMAC Formation and IL-2 Production.
Importantly, so far, the importance of ADAP–SLP-76 binding was evident by the fact that M12 did not support an increased conjugation and general LFA-1 clustering observed with wild-type ADAP. However, at the same time, M12 did not act as a potent DN in interfering with the function of endogenous ADAP. In other words, M12 did not reduce in a statistically significant manner the level of conjugation or general LFA-1 clustering below the vector control. In this regard, SMAC formation is thought to occur subsequent to general LFA-1 clustering in a process that involves the segregation of LFA-1 from the TCR in the formation of the p- and c-SMAC (1, 2). Given this, next we assessed whether the characteristic ring-shaped clustering of LFA-1 at the immunological synapse (i.e., the formation of the pSMAC) was influenced by ADAP–SLP-76 binding. Imaging of the immunological synapse amongst individual conjugates (>38) was conducted by laser-scanning microscopy as described in Materials and Methods. Conjugates formed over 30 min in the presence of Ttox were stained for LFA-1 using CD11a antibody and Alexa Fluor 568–conjugated anti–rat antibody. ADAP was stained with anti-ADAP mAb and Cy5-conjugated anti–mouse. As seen in Fig. 5 a, ADAP staining generally colocalized with LFA-1 at the interface with the segregation of LFA-1 receptors that characterize a pSMAC (Fig. 5, b vs. c). Anti–LFA-1 staining was visualized perpendicular to the interfacing APCs in the context of ADAP versus M12 expression. Samples of pSMAC at the interface between T cells and APCs are shown in Fig. 5 b (top). Although 53% of conjugates of GFP-expressing cells showed the presence of a pSMAC at the immunological synapse (i.e., 21/39 conjugates), there was an increase in pSMAC formation in cells expressing ADAP-GFP, up to 71% of conjugates (i.e., 27/38 conjugates). In contrast, M12 had a marked effect as a DN, whereas only 20% of conjugates possessed a pSMAC (i.e., 8/39 conjugates; Fig. 5 a, bottom). This represents a 62% reduction in pSMAC formation relative to the GFP control cells. Three-dimensional views of the pSMAC formation are shown in Fig. S1 (available at http://www.jem.org/cgi/content/full/jem.20040780/DC1). A gallery of the patterns for the different conjugates is shown in Fig. S2 (available at http://www.jem.org/cgi/content/full/jem.20040780/DC1). It is noteworthy that, although there were significant effects of M12 on pSMAC formation, the overall intensity of LFA-1 at the immunological synapse was similar for the vector, ADAP, and M12-infected cells, a result consistent with the notion that general LFA-1 clustering was only marginally affected by M12 (i.e., not a DN). Therefore, unlike in the case of conjugation and LFA-1 clustering, M12 acted as a potent DN in abrogating the formation of pSMACs, indicating that SLP-76 binding to ADAP is needed for pSMAC formation. Thus, although other processes such as actin polymerization are known to affect the extent of accumulation of proteins at the immunological synapse, here we have identified an intracellular event that regulates assembly of the LFA-1–rich pSMAC while minimally effecting accumulation of LFA-1 at the immunological synapse itself.
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Discussion |
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ADAP joins a number of mediators such as VAV-1, WASP, WIP, Rap1/RapL, and SKAP-55 that regulate conjugation (59, 66–69). This was observed in two models with retroviral gene transfer in T8.1 cells that respond to Ttox peptide, and in DO11.10 primary T cells that respond to OVA peptide (Figs. 1 and 3). We previously identified the two ExYDDV motifs as sites of SLP-76 domain binding (45, 46), a finding confirmed with BLNK–SLP-65 SH2 binding to a related site DxYDDV in HPK-1 (47). Although our work focused on ADAP-120, the higher Mr isoform ADAP-130 also has the motifs (44), and can potentiate conjugation (unpublished data). Specificity was shown by the finding that mutations in the putative ADAP EVH1 binding sites (52) had no detrimental effect on conjugation. VASP acts to prevent the termination of growing actin filaments by competing for the binding of capping proteins (70). Therefore, VASP–ADAP binding may play other roles in conjugation, distinct from the regulation of LFA-1–ICAM-1 binding. Actin branching is generally associated with lateral extensions at the cell surface (i.e., lamellapodia) and, therefore, ADAP–VASP may extend the T cell–APC interface after LFA-1 binding, or play a supplementary nonessential role in ADAP function. In a similar vein, class II myosin modulates the cytoskeleton and motility, but not synapse formation (71). In either case, this involvement of the ADAP–VASP interaction was clearly distinct from that of the ADAP–SLP-76 interaction. One a simple level, the ADAP–SLP-76 interaction would be expected to operate downstream of TCR-p56lck-ZAP-70–SLP-76–VAV (with the possible intermediate involvement of LAT and GADS) and be integrated downstream with its other binding partner SKAP-55. We showed previously that the loss of the SKAP-55 SH3 domain that binds to ADAP abrogated the ability of SKAP-55 to enhance T cell–APC conjugation (59). The pathway is further complicated by the involvement of p59fyn. p59fyn (and not p56lck) mediates phosphorylation of ADAP on sites needed for SLP-76 SH2 domain binding (46), and p59fyn-deficient T cells show a major loss of ADAP phosphorylation (43).
Information on the intracellular process that regulates SMAC formation has been missing, as has information on the relationship between general LFA-1 clustering and the subsequent events that segregate receptors in the SMAC. TCR-CD3 clustering is controlled by WASP (72), whereas VAV-1 has been reported to regulate both TCR-CD3 and LFA-1 clustering (66). The involvement of Vav1 and RapL appears to occur at the level of general LFA-1 clustering, before the segregation of receptors leading to SMAC formation clustering (18, 66). Constitutively active Rap1 enhances LFA-1 clustering (17), whereas DN RapL blocks general clustering and conjugation (18). The proximity of SLP-76 to TCR-CD3 signaling would place ADAP–SLP-76 upstream of RapL and talin, perhaps operating in conjunction with SLP-76 binding to VAV1 and NCK during actin remodelling (36, 37). At the same time, the special DN effect of M12 on pSMAC formation and the colocalization of ADAP with LFA-1 underscores an additional close downstream connection to the SMAC (and possibly RapL and talin). Although ADAP does not coprecipitate LFA-1 (unpublished data), it must be central to this process because a single protein with two mutations essentially eliminated >50–60% of SMAC formation (Fig. 5 b). Future studies will need to be conducted to clarify the connection between ADAP and RapL/talin in the SMAC. An additional connection to SKAP-55 has also been implied by SKAP-55 binding to ADAP and its regulation of LFA-1 adhesion/clustering and T cell–APC conjugation (59).
Our findings showing a similarity in the degree of inhibition of pSMAC formation and IL-2 production suggest a role for SMAC formation in the full induction of IL-2 production in T cells (Fig. 5). Previous studies have been mixed in their support for a connection between these events, most likely due to the fact that both the clustering of receptors (i.e., a positive signal) and receptor internalization (i.e., probable termination signal) occur in the SMAC (9). SMAC formation occurs during a decrease in the optimal levels of tyrosine phosphorylation (9), and early signaling can occur outside the SMAC (4, 7, 8). T cells from CD2-associated protein-deficient mice fail to form stable SMACs and yet hyperproliferate to antigen (9). Despite this, continuous T cell receptor signaling promotes synapse formation and T cell effector function (73). The DN effect of M12 on pSMAC formation, but not conjugation, allowed for a distinction to be made between these events and IL-2 production. M12 blocked both SMAC formation and IL-2 production by >50%, and occasionally, as high as 80% (unpublished data). In this scenario, the limited numbers of agonist peptide–MHC complexes and continuous receptor degradation in in vivo responses would be offset by more effective receptor aggregation and signaling leading to enhanced cytokine production. The use of intracellular mutants and genetics may eventually be the most effective way of uncovering a role for the SMAC in T cell function.
Lastly, the connection between ADAP–SLP-76 and SMAC formation/IL-2 production provides a molecular basis to explain previous results showing that SLP-76 can exert more than one signaling function in T cells. Although NH2-terminal phosphorylation and the GADS binding sites are needed for the activation of PLC1, the SH2 domain has been found previously to be needed for proliferation, but can be dispensed for PLC1 phosphorylation (39, 40). Unlike in the case of the NH2-terminal tyrosines, the SH2 domain is also not required for progression of thymocytes beyond the pre-TCR stage (CD44+CD25+) of thymic differentiation (39, 40). The requirement for SLP-76 SH2 domain binding to ADAP in conjugation and SMAC formation provides a basis to account for its role in T cell proliferation.
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Acknowledgments |
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This work was supported by National Institutes of Health grant no. A139021 and a grant from the Wellcome Trust, London (C.E. Rudd is a Principal Research Fellow of the Wellcome Trust).
The authors have no conflicting financial interests.
Submitted: 20 April 2004
Accepted: 9 August 2004
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References |
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1 Monks, C.R., B.A. Freiburg, H. Kupher, N. Sciaky, and A. Kupfer. 1998. Three-dimensional segregation of supramolecular activation clusters in T cells. Nature. 395:82–86.[CrossRef][Medline]
2 Dustin, M.L., and A.S. Shaw. 1999. Costimulation: building an immunological synapse. Science. 283:649–650.
3 Davis, D.M. 2002. Assembly of the immunological synapse for T cells and NK cells. Trends Immunol. 23:356–363.[CrossRef][Medline]
4 Freiberg, B.A., H. Kupfer, W. Maslanik, J. Delli, J. Kappler, D.M. Zaller, and A. Kupfer. 2002. Staging and resetting T cell activation in SMACs. Nat. Immunol. 3:911–917.[CrossRef][Medline]
5 Chakraborty, A.K., M.L. Dustin, and A.S. Shaw. 2003. In silico models for cellular and molecular immunology: successes, promises and challenges. Nat. Immunol. 4:933–936.[CrossRef][Medline]
6 Davis, D.M., and M.L. Dustin. 2004. What is the importance of the immunological synapse? Trends Immunol. 25:323–327.[CrossRef][Medline]
7 Lee, K.H., A.D. Holdorf, M.L. Dustin, A.C. Chan, P.M. Allen, and A.S. Shaw. 2002. T cell receptor signaling precedes immunological synapse formation. Science. 295:1539–1542.
8 Costello, P., M. Gallagher, and D. Cantrell. 2002. Sustained and dynamic inositol lipid metabolism inside and outside the immunological synapse. Nat. Immunol. 3:1082–1089.[CrossRef][Medline]
9 Lee, K.H., A.R. Dinner, C. Tu, G. Campi, S. Raychaudhuri, R. Varma, T.N. Sims, W.R. Burack, H. Wu, J. Wang, et al. 2003. The immunological synapse balances T cell receptor signaling and degradation. Science. 302:1218–1222.
10 Igakura, T., J.C. Stinchcombe, P.K. Goon, G.P. Taylor, J.N. Weber, G.M. Griffiths, Y. Tanaka, M. Osame, and C.R. Bangham. 2003. Spread of HTLV-I between lymphocytes by virus-induced polarization of the cytoskeleton. Science. 299:1713–1716.
11 Purbhoo, M.A., D.J. Irvine, J.B. Huppa, and M.M. Davis. 2004. T cell killing does not require the formation of a stable mature immunological synapse. Nat. Immunol. 5:524–530.[CrossRef][Medline]
12 Kupfer, A., and H. Kupfer. 2003. Imaging immune cell interactions and functions: SMACs and the immunological synapse. Semin. Immunol. 15:295–300.[CrossRef][Medline]
13 Dustin, M.L., T.G. Bivona, and M.R. Philips. 2004. Membranes as messengers in T cell adhesion signaling. Nat. Immunol. 5:363–372.[CrossRef][Medline]
14 Shimaoka, M., T. Xiao, J.H. Liu, Y. Yang, Y. Dong, C.D. Jun, A. McCormack, R. Zhang, A. Joachimiak, J. Takagi, et al. 2003. Structures of the alpha L I domain and its complex with ICAM-1 reveal a shape-shifting pathway for integrin regulation. Cell. 112:99–111.[CrossRef][Medline]
15 Hogg, N., M. Laschinger, K. Giles, and A. McDowall. 2003. T-cell integrins: more than just sticking points. J. Cell Sci. 116:4695–4705.
16 Zhang, W., Y. Shao, D. Fang, J. Huang, M.S. Jeon, and Y.C. Liu. 2003. Negative regulation of T cell antigen receptor-mediated Crk-L-C3G signaling and cell adhesion by Cbl-b. J. Biol. Chem. 278:23978–23983.
17 Sebzda, E., M. Bracke, T. Tugal, N. Hogg, and D.A. Cantrell. 2002. Rap1A positively regulates T cells via integrin activation rather than inhibiting lymphocyte signaling. Nat. Immunol. 3:251–258.[CrossRef][Medline]
18 Katagiri, K., A. Maeda, M. Shimonaka, and T. Kinashi. 2003. RAPL, a Rap1-binding molecule that mediates Rap1-induced adhesion through spatial regulation of LFA-1. Nat. Immunol. 4:741–748.[CrossRef][Medline]
19 Yan, B., D.A. Calderwood, B. Yaspan, and M.H. Ginsberg. 2001. Calpain cleavage promotes talin binding to the beta 3 integrin cytoplasmic domain. J. Biol. Chem. 276:28164–28170.
20 Sampath, R., P.J. Gallagher, and F.M. Pavalko. 1998. Cytoskeletal interactions with the leukocyte integrin beta2 cytoplasmic tail. Activation-dependent regulation of associations with talin and alpha-actinin. J. Biol. Chem. 273:33588–33594.
21 Kiosses, W.B., S.J. Shattil, N. Pampori, and M.A. Schwartz. 2001. Rac recruits high-affinity integrin alphavbeta3 to lamellipodia in endothelial cell migration. Nat. Cell Biol. 3:316–320.[CrossRef][Medline]
22 Giagulli, C., E. Scarpini, L. Ottoboni, S. Narumiya, E.C. Butcher, G. Constantin, and C. Laudanna. 2004. RhoA and zeta PKC control distinct modalities of LFA-1 activation by chemokines: critical role of LFA-1 affinity triggering in lymphocyte in vivo homing. Immunity. 20:25–35.[CrossRef][Medline]
23 Wilkinson, B., H. Wang, and C.E. Rudd. 2004. Positive and negative adaptors in T-cell signalling. Immunology. 111:368–374.[CrossRef][Medline]
24 Samelson, L.E. 1999. Adaptor proteins and T-cell antigen receptor signaling. Prog. Biophys. Mol. Biol. 71:393–403.[CrossRef][Medline]
25 Zhang, W.J., J. Sloan-Lancaster, J. Kitchen, R.P. Trible, and L.E. Samelson. 1998. LAT: the ZAP-70 tyrosine kinase substrate that links T cell receptor to cellular activation. Cell. 92:83–92.[CrossRef][Medline]
26 Weber, J.R., S. Orstavik, K.M. Torgersen, N.C. Danbolt, S.F. Berg, J.C. Ryan, K. Tasken, J.B. Imboden, and J.T. Vaage. 1998. Molecular cloning of the cDNA encoding pp36, a tyrosine-phosphorylated adaptor protein selectively expressed by T cells and natural killer cells. J. Exp. Med. 187:1157–1161.
27 Jackman, J.K., D.G. Motto, Q. Sun, M. Tanemoto, C.W. Turck, G.A. Peltz, G.A. Koretzky, and P.R. Findell. 1995. Molecular cloning of SLP-76, a 76-kDa tyrosine phosphoprotein associated with Grb2 in T cells. J. Biol. Chem. 270:7029–7032.
28 Yablonski, D., M.R. Kuhne, T. Kadlecek, and A. Weiss. 1998. Uncoupling of nonreceptor tyrosine kinases from PLC-1 in a SLP-76-deficient T cell. Science. 218:413–416.
29 Zhang, W., B.J. Irvin, R.P. Trible, R.T. Abraham, and L.E. Samelson. 1999. Functional analysis of LAT in TCR-mediated signaling pathways using a LAT-deficient Jurkat cell line. Int. Immunol. 11:943–950.[Medline]
30 Raab, M., A.J. da Silva, P.R. Findell, and C.E. Rudd. 1997. Regulation of Vav-SLP-76 binding by ZAP-70 and its relevance to TcR
/CD3 induction of interleukin-2. Immunity. 6:1–11.[Medline]
31 Wardenburg, J.B., C. Fu, J.K. Jackman, H. Flotow, S.E. Wilkinson, D.H. Williams, R. Johnson, G. Kong, A.C. Chan, and P.R. Findell. 1996. Phosphorylation of SLP-76 by the ZAP-70 protein-tyrosine kinase is required for T-cell receptor function. J. Biol. Chem. 271:19641–19644.
32 Schneider, H., B. Guerette, C. Guntermann, and C.E. Rudd. 2000. Resting lymphocyte kinase (Rlk/Txk) targets lymphoid adaptor SLP-76 in the cooperative activation of interleukin-2 transcription in T-cells. J. Biol. Chem. 275:3835–3840.
33 Bunnell, S.C., M. Diehn, M.B. Yaffe, P.R. Findell, L.C. Cantley, and L.J. Berg. 2000. Biochemical interactions integrating Itk with the T cell receptor-initiated signaling cascade. J. Biol. Chem. 275:2219–2230.
34 Wu, J., D.G. Motto, G.A. Koretsky, and A. Weiss. 1996. Vav and SLP-76 interact and functionally cooperate in IL-2 gene activation. Immunity. 4:593–602.[CrossRef][Medline]
35 Onodera, H., D.G. Motto, G.A. Koretzky, and D.M. Rothstein. 1996. Differential regulation of activation-induced tyrosine phosphorylation and recruitment of SLP-76 to Vav by distinct isoforms of the CD45 protein-tyrosine phosphatase. J. Biol. Chem. 271:22225–22230.
36 Wardenburg, J.B., R. Pappu, J.Y. Bu, B. Mayer, J. Chernoff, D. Straus, and A.C. Chan. 1998. Regulation of PAK activation and the T cell cytoskeleton by the linker protein SLP-76. Immunity. 9:607–616.[CrossRef][Medline]
37 Zeng, R., J. Cannon, R. Abraham, M. Way, D. Billadeau, J. Bubeck-Wardenberg, and J. Burkhardt. 2003. SLP-76 coordinates Nck-dependent Wiskott-Aldrich syndrome protein recruitment with Vav-1-Cdc42-dependent Wiskott-Aldrich syndrome protein activation at the T cell-APC contact site. J. Immunol. 171:1360–1368.
38 Higgs, H.N., and T.D. Pollard. 2001. Regulation of actin filament network formation through ARP2/3 complex: activation by a diverse array of proteins. Annu. Rev. Biochem. 70:649–676.[CrossRef][Medline]
39 Myung, P.S., G.S. Derimanov, M.S. Jordan, J.A. Punt, Q.H. Liu, B.A. Judd, E.E. Meyers, C.D. Sigmund, B.D. Freedman, and G.A. Koretzky. 2001. Differential requirement for SLP-76 domains in T cell development and function. Immunity. 15:1011–1026.[CrossRef][Medline]
40 Kumar, L., V. Pivniouk, M.A. de la Fuente, D. Laouini, and R.S. Geha. 2002. Differential role of SLP-76 domains in T cell development and function. Proc. Natl. Acad. Sci. USA. 99:884–889.
41 da Silva, A.J., Z. Li, C. de Vera, E. Canto, P. Findell, and C.E. Rudd. 1997. Cloning of a novel T-cell protein FYB that binds FYN and SH2-domain-containing leukocyte protein 76 and modulate interleukin-2 production. Proc. Natl. Acad. Sci. USA. 94:7493–7498.
42 Musci, M.A., L.R. Hendricks-Taylor, D.G. Motto, M. Paskind, J. Kamens, C.W. Turck, and G.A. Koretzky. 1997. Molecular cloning of SLAP-130, an SLP-76-associated substrate of the T cell antigen receptor-stimulated protein tyrosine kinases. J. Biol. Chem. 272:11674–11677.
43 da Silva, A.J., J.M. Rosenfield, I. Mueller, A. Bouton, H. Hirai, and C.E. Rudd. 1997. Biochemical analysis of p120/130: a protein-tyrosine kinase substrate restricted to T and myeloid cells. J. Immunol. 158:2007–2016.[Abstract]
44 Veale, M., M. Raab, Z. Li, A.J. da Silva, S.K. Kraeft, S. Weremowicz, C.C. Morton, and C.E. Rudd. 1999. Novel isoform of lymphoid adaptor FYN-T-binding protein (FYB-130) interacts with SLP-76 and up-regulates interleukin-2 production. J. Biol. Chem. 274:28427–28435.
45 Geng, L., M. Raab, and C.E. Rudd. 1999. Cutting edge: SLP-76 cooperativity with FYB/FYN-T in the up-regulation of TCR-driven IL-2 transcription requires SLP-76 binding to FYB at Tyr595 and Tyr651. J. Immunol. 163:5753–5757.
46 Raab, M., H. Kang, A. da Silva, X. Zhu, and C.E. Rudd. 1999. FYN-FYB-SLP-76 interactions define a TcR/CD3 mediated tyrosine phosphorylation pathway that regulates interleukin-2 transcription in T-cells. J. Biol. Chem. 274:21170–21179.
47 Sauer, K., J. Liou, S. Singh, D. Yablonski, A. Weiss, and R. Perlmutter. 2001. Hematopoietic progenitor kinase 1 associates physically and functionally with the adaptor proteins B cell linker protein and SLP-76 in lymphocytes. J. Biol. Chem. 276:45207–45216.
48 Boerth, N.J., B.A. Judd, and G.A. Koretzky. 2000. Functional association between SLAP-130 and SLP-76 in Jurkat T cells. J. Biol. Chem. 275:5143–5152.
49 Liu, J., H. Kang, M. Raab, A. da Silva, S.-K. Kraeft, and C.E. Rudd. 1998. FYB (FYN binding protein) serves as a binding partner for lymphoid protein and FYN kinase substrate SKAP55 and a SKAP55-related protein in T cells. Proc. Natl. Acad. Sci. USA. 95:8779–8784.
50 Marie-Cardine, A., L.R. Hendricks-Taylor, N.J. Boerth, H. Zhao, B. Schraven, and G.A. Koretzky. 1998. Molecular interaction between the Fyn-associated protein SKAP55 and the SLP-76-associated phosphoprotein SLAP-130. J. Biol. Chem. 273:25789–25795.
51 Kang, H., C. Freund, J.S. Duke-Cohan, A. Musacchio, G. Wagner, and C.E. Rudd. 2000. SH3 domain recognition of a proline-independent tyrosine-based RKxxYxxY motif in immune cell adaptor SKAP55. EMBO J. 19:2889–2899.[CrossRef][Medline]
52 Krause, M., M.S. Sechi, M. Konradt, D. Monner, F.B. Gertler, and J. Wehland. 2000. Fyn-binding protein (Fyb)/SLP-76-associated protein, Vasodilator-stimulated phosphoprotein (VASP) proteins and the Arp2/3 complex link TCR signaling to the actin cytoskeleton. J. Cell Biol. 149:181–194.
53 Griffiths, E.K., C. Krawczyk, Y.-Y. Kong, M. Rabb, S.J. Hyduk, D. Bouchard, V.S. Chan, I. Kozieradski, A.J. Oliveira-dos-Santos, A. Wakeham, et al. 2001. Positive regulation of T cells activation and integrin adhesion by the adapter Fyb/Slap. Science. 293:2260–2263.
54 Peterson, E.J., M.L. Woods, S.A. Dmowski, G. Derimanov, M.S. Jordan, J.N. Wu, P.S. Myung, Q.-H. Liu, J.T. Pribila, B.D. Freedman, et al. 2001. Coupling of the TCR to integrin activation by SLAP-130/Fyb. Science. 293:2263–2265.
55 Hunter, A.J., N. Ottoson, N. Boerth, G.A. Koretzky, and Y. Shimizu. 2000. A novel function for the Slap-130/Fyb adapter protein in Beta-1 integrin signalling and T cell migration. J. Immunol. 164:1143–1147.
56 Geng, L., S. Pfister, S.K. Kraeft, and C.E. Rudd. 2001. Adaptor FYB (Fyn-binding protein) regulates integrin-mediated adhesion and mediator release: differential involvement of the FYB SH3 domain. Proc. Natl. Acad. Sci. USA. 98:11527–11532.
57 Geng, L., and C.E. Rudd. 2001. Adaptor ADAP (adhesion and degranulation promoting adaptor protein) regulates ß-1 integrin clustering on mast cells. Biochem. Biophys. Res. Commun. 289:2042–2050.
58 Fujii, Y., S. Wakahara, T. Nakao, T. Hara, H. Ohtake, T. Komurasaki, K. Kitamura, A. Tatsuno, N. Fujiwara, N. Hozumi, et al. 2003. Targeting of MIST to Src-family kinases via SKAP55-SLAP-130 adaptor complex in mast cells. FEBS Lett. 540:111–116.[CrossRef][Medline]
59 Wang, H., E.Y. Moon, A. Azouz, X. Wu, A. Smith, H. Schneider, N. Hogg, and C.E. Rudd. 2003. SKAP-55 regulates integrin adhesion and formation of T cell-APC conjugates. Nat. Immunol. 4:366–374.[CrossRef][Medline]
60 Pear, W.S., G.P. Nolan, M.L. Scott, and D. Baltimore. 1993. Production of high-tite helper-free retroviruses by transient transfection. Proc. Natl. Acad. Sci. USA. 90:8392–8396.
61 McCann, F.E., B. Vanherberghen, K. Eleme, L.M. Carlin, R.J. Newsam, D. Goulding, and D.M. Davis. 2003. The size of the synaptic cleft and distinct distributions of filamentous actin, ezrin, CD43, and CD45 at activating and inhibitory human NK cell immune synapses. J. Immunol. 170:2862–2870.
62 Michel, F., and O. Acuto. 1996. Induction of T cell adhesion by antigen stimulation and modulation by the coreceptor CD4. J. Immunol. 173:165–175.
63 Van Parijs, L., Y. Refaeli, A.K. Abbas, and D. Baltimore. 1999. Autoimmunity as a consequence of retrovirus-mediated expression of C-FLIP in lymphocytes. Immunity. 11:763–770.[CrossRef][Medline]
64 Prehoda, K.E., D.J. Lee, and W.A. Lim. 1999. Structure of the enabled/VASP homology 1 domain-peptide complex: a key component in the spatial control of actin assembly. Cell. 97:471–480.[CrossRef][Medline]
65 van Kooyk, Y., and C. Figdor. 2000. Avidity regulation of integrins: the driving force in leukocyte adhesion. Curr. Opin. Cell Biol. 12:542–547.[CrossRef][Medline]
66 Krawczyk, C., A. Oliveira-dos-Santos, T. Sasaki, E. Griffiths, P.S. Ohashi, S. Snapper, F. Alt, and J.M. Penninger. 2002. Vav1 controls integrin clustering and MHC/peptide-specific cell adhesion to antigen-presenting cells. Immunity. 16:331–343.[CrossRef][Medline]
67 Cannon, J.L., and J.K. Burkhardt. 2002. The regulation of actin remodeling during T-cell-APC conjugate formation. Immunol. Rev. 186:90–99.[CrossRef][Medline]
68 Katagiri, K., M. Hattori, N. Minato, and T. Kinashi. 2002. Rap1 functions as a key regulator of T-cell and antigen-presenting cell interactions and modulates T-cell responses. Mol. Cell. Biol. 22:1001–1015.
69 Anton, I.M., M. de la Fuente, T.N. Sims, S. Freeman, N. Ramesh, J.H. Hartwig, M.L. Dustin, and R.S. Geha. 2002. WIP deficiency reveals a differential role for WIP and the actin cytoskeleton in T and B cell activation. Immunity. 16:193–204.[CrossRef][Medline]
70 Bear, J.E., T.M. Svitkina, M. Krause, D.A. Schafer, J.J. Loureiro, G.A. Strasser, I.V. Maly, O.Y. Chaga, J.A. Copper, and F.B. Gertler. 2002. Antagonism between Ena/VASP proteins and actin filament capping regulates fibroblast motility. Cell. 109:509–521.[CrossRef][Medline]
71 Jacobelli, J., S.A. Chmura, D.B. Buxton, M.M. Davis, and M.F. Krummel. 2004. A single class II myosin modulates T cell motility and stopping, but not synapse formation. Nat. Immunol. 5:531–538.[CrossRef][Medline]
72 Snapper, S.B., F.S. Rosen, E. Mizoguchi, P. Cohen, W. Khan, C.H. Liu, T.L. Hagemann, S.P. Kwan, R. Ferrini, L. Davidson, et al. 1998. Wiskott-Aldrich syndrome protein-deficient mice reveal a role for WASP in T but not B cell activation. Immunity. 9:81–91.[CrossRef][Medline]
73 Huppa, J.B., and M.M. Davis. 2003. T-cell-antigen recognition and the immunological synapse. Nat. Rev. Immunol. 3:973–983.[CrossRef][Medline]
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