The adapter molecule Src homology 2 (SH2) domain-containing leukocyte phosphoprotein of 76 kDa (SLP-76) is essential for FcεRI-mediated signaling, degranulation and IL-6 production in mast cells. To test the structural requirements of SLP-76 in mast cell signaling and function, we have studied the functional responses of murine bone marrow-derived mast cells (BMMCs) expressing mutant forms of SLP-76. We found that the N-terminal tyrosines as well as the central proline-rich region of SLP-76 are required for participation of SLP-76 in FcεRI-mediated signaling and function. The C-terminal SH2 domain of SLP-76 also contributes to optimal function of SLP-76 in mast cells. Another adapter molecule, adhesion- and degranulation-promoting adapter protein (ADAP), is known to bind the SH2 domain of SLP-76, and cell line studies have implicated ADAP in mast cell adhesion and FcεRI-induced degranulation. Surprisingly, we found that mast cells lacking ADAP expression demonstrate no defects in FcεRI-induced adhesion, granule release, or IL-6 production, and that ADAP-deficient mice produce a normal passive systemic anaphylactic response. Thus, failure to bind ADAP does not underlie the functional defects exhibited by SLP-76 SH2 domain mutant-expressing mast cells.
The high affinity IgE receptor (FcεRI) is an immunoreceptor tyrosine-based activation motif (ITAM)4-bearing multimolecular complex expressed on the surface of mast cells. Ag cross-linking of IgE-engaged FcεRI leads to activation of Src family kinases Lyn and Fyn and subsequent phosphorylation of the protein tyrosine kinase (PTK) Syk. Syk then associates with the ITAMs of the FcεRI and cooperates in the phosphorylation of multiple substrates. In striking analogy to the signaling cascades initiated by TCR engagement, FcεRI stimulation induces formation of a signaling complex assembled by the adapter proteins Src homology 2 (SH2) domain-containing leukocyte phosphoprotein of 76 kDa (SLP-76), Gads, Nck, adhesion- and degranulation-promoting adapter protein (ADAP), and membrane-anchored linker for activation of T cells (LAT) and containing the effector molecules phospholipase Cγ1 (PLCγ1), PLCγ2, Vav, and Btk (for reviews, see Refs. 1 and 2).
The SH2 domain-containing leukocyte phosphoprotein of 76 kDa (SLP-76) is expressed in nearly all hemopoietic cells and has been shown to be critical for signaling through a variety of ITAM-bearing receptors as well as integrins expressed on T cells (3, 4), platelets (5), neutrophils (6), and mast cells (7). Structure/function analyses conducted in T cells have identified several motifs critical for SLP-76-dependent signaling (8, 9, 10). Three tyrosines near the N terminus of the protein (Y112, Y128, and Y145) are phosphorylated and mediate inducible interactions with Vav (11, 12, 13), Nck (14), and Itk (15, 16), respectively. The central, proline-rich region of SLP-76 mediates a constitutive interaction with the adapter protein Gads (17, 18) and the enzyme PLCγ1 (19), whereas the C-terminal SH2 domain can bind tyrosine-phosphorylated ADAP (20, 21) and hemopoietic progenitor kinase-1 (HPK1) (22).
Despite having normal numbers of mature, granule-containing dermal mast cells, SLP-76-deficient mice have a severely blunted passive systemic anaphylactic response, as measured by heart rate elevation and serum histamine concentration. Bone marrow from these mice produces normal numbers of bone marrow-derived mast cells (BMMCs) when cultured in vitro, and these cells express normal levels of c-Kit and FcεRI. Consistent with their in vivo activation defect, these cells fail to degranulate or secrete IL-6 upon FcεRI stimulation (7).
Recently, two parallel, but intersecting, signaling pathways downstream of the FcεRI have been described: the canonical Lyn/Syk/LAT/SLP-76 pathway and a novel Fyn/Gab2/phosphotidylinositol 3-kinase (PI3K) pathway (23). Parravicini et al. (23) have demonstrated enhanced Fyn/Gab2/PI3K signaling and degranulation in the absence of Lyn, whereas Lyn/Syk/LAT/SLP-76 signaling is normal, but degranulation is impaired, in the absence of Fyn. Two molecules were hypothesized to mediate the apparent cross-talk/convergence of these two pathways: 1) the Tec family kinase Btk, which can be activated downstream of both PLCγ and PI3K; and 2) ADAP, which can bind both SLP-76 and Fyn. Mast cells derived from mice lacking Btk have been extensively studied and demonstrate a variety of defects in FcεRI-induced signaling and function (24, 25). To date, however, studies of ADAP in mast cells have been limited to overexpression in the RBL-2H3 cell line and have indicated that ADAP can enhance basal mast cell adhesion to fibronectin as well as FcεRI-induced degranulation (26, 27).
To further elucidate the structural requirements of SLP-76 in mast cell signaling and function, we have studied the functional responses of BMMCs expressing mutant forms of SLP-76. These experiments demonstrate that the N-terminal tyrosines as well as the central proline-rich region of SLP-76 are required for participation of SLP-76 in FcεRI-mediated signaling and function. The C-terminal SH2 domain of SLP-76 also contributes to optimal function of SLP-76 in mast cells. To test whether the functional defects demonstrated by BMMCs expressing the SH2 domain mutant of SLP-76 are attributable to the lack of ADAP binding and to examine the postulated role of ADAP in integrating Lyn- and Fyn-mediated signals regulating adhesion and degranulation, we have also studied ADAP-deficient BMMCs. We found that ADAP-deficient mast cells demonstrate no defects in FcεRI-induced adhesion, granule release, cytokine production, or passive systemic anaphylaxis. Thus, failure to bind ADAP does not underlie the functional defects exhibited by SLP-76 SH2 domain mutant-expressing mast cells, and ADAP is unlikely to mediate the cross-talk between Lyn- and Fyn-initiated FcεRI signaling.
Materials and Methods
cDNA constructs and production of retrovirus
SLP-76 mutants were generated from mouse SLP-76 cDNA and subcloned into the murine stem cell virus-based retroviral MiGR vector (28) as previously described (29). High titer retroviral supernatants were produced via cotransfection of 293-T cells with retroviral constructs (30) and the Helper Virus packaging construct (Imgenex, San Diego, CA).
Retroviral infection of BMMCs
2, and retroviral spin infection was repeated the following day. After overnight incubation, cells were cultured as described above to generate mast cells. After 3–4 wk, green fluorescence protein-expressing cells were sorted using a FACSVantage SE flow cytometer (BD Biosciences, Mountain View, CA).
Flow cytometric analysis
Cells were stained according to standard protocols using the following labeled Abs: mouse anti-DNP IgE (Sigma-Aldrich), anti-mouse IgE-biotin (BD PharMingen, San Diego, CA), streptavidin-allophycocyanin (BD PharMingen), and anti-mouse SLP-76-PE (31). Two-color flow cytometry was performed on a FACSCalibur (BD Biosciences).
β-Hexosaminidase release assay
BMMCs (1 × 106/ml) were starved of SCF overnight, then sensitized at 1 × 107/ml in complete RPMI 1640 without cytokines with 1 μg/ml anti-DNP IgE (clone SPE-7; Sigma-Aldrich) for 4 h at 37°C in 5% CO2. Cells were then washed once in Tyrode’s buffer (130 mM NaCl, 10 mM HEPES, 1 mM MgCl2, 5 mM KCl, 1.4 mM CaCl2, 5.6 mM glucose, and 1 mg/ml BSA, pH 7.4) and resuspended at 2 × 106/ml in Tyrode’s buffer. Cells (200 μl) were then stimulated with varying amounts of DNP-human serum albumin (HSA) (0–1000 ng/ml) for 1 h at 37°C. Cells were spun down, and 30 μl of supernatant was transferred to a 96-well, flat-bottom plate. Thirty microliters of 1 mM p-nitrophenyl-N-acetyl-β-d-glucosamide was then added to each supernatant and mixed before incubating for 1 h at 37°C. The reaction was terminated by the addition of 200 μl of 0.1 M Na2CO3-NAHCO3 buffer, and OD was read on a plate reader at a wavelength of 405 nm.
IL-6 production assay
BMMCs (1 × 106/ml) were starved of SCF overnight, then sensitized at 1 × 107/ml in complete RPMI 1640 without cytokines with 1 μg/ml anti-DNP IgE (clone SPE-7; Sigma-Aldrich) for 4 h at 37°C in 5% CO2. Cells were washed once and resuspended at 1 × 106/ml in complete RPMI 1640. Cells (5 × 104) in complete RPMI 1640 were then incubated with varying concentrations of DNP-HSA overnight at 37°C in 5% CO2 in a total volume of 100 μl in a 96-well, flat-bottom plate. Each sample was assayed in triplicate. The following day, the plate was removed from the incubator and frozen at −20°C. An ELISA was performed on thawed supernatants using a murine IL-6 ELISA kit (Pierce/Endogen, Rockford, IL).
Lysate preparation and immunoblotting
BMMCs (1 × 106/ml) were starved of SCF overnight, then sensitized at 1 × 107/ml in complete RPMI 1640 without cytokines with 1 μg/ml anti-DNP IgE (clone SPE-7; Sigma-Aldrich) for 4 h at 37°C in 5% CO2. Cells were washed and resuspended at 2 × 107/ml in Tyrode’s buffer. Cells were stimulated for various times with 100 ng/ml DNP-HSA. Cells were then pelleted, supernatant was aspirated, and cells were lysed in ice-cold 1% Nonidet P-40 containing proteinase (50 μg/ml aprotinin, 10 μg/ml leupeptin, 50 μg/ml pepstatin A, and 1 mM Pefablock) and phosphatase (400 μM sodium vanadate, 10 mM sodium fluoride, and 10 mM sodium pyrophosphate) inhibitors. Western blotting was performed using the following Abs: anti-PLCγ1, anti-PLCγ2, 4G10, anti-phospho-PLCγ2, anti-phospho-extracellular signal-regulated kinase 1/2 (anti-phospho-Erk1/2; Upstate Biotechnology, Lake Placid, NY), and anti-Erk1/2 (Zymed Laboratories, San Francisco, CA).
Calcium flux assay
BMMCs were sensitized with anti-DNP-IgE as described in the above section. Cells were then washed once in Tyrode’s buffer and resuspended at 1 × 107 in Tyrode’s buffer containing 25 mM Probenecid (Sigma-Aldrich) and 2 mg/ml Indo-1 (Molecular Probes, Eugene, OR). Cells were protected from the light and incubated at 37°C for 30 min. Indo-1-loaded cells were washed twice and resuspended in warm Tyrode’s buffer. Data were collected using an LSR flow cytometer (BD Biosciences). Baseline Ca2+ levels were measured for 30 s before addition of DNP-HSA (100 ng/ml). The sample was collected for a total of 7–8 min, collecting ∼500–700 events/s. Ionomycin was added 30 s before the end of the assay. Data were analyzed using FlowJo software (TreeStar, Ashland, OR) and are represented as the mean Ca2+ flux of cells over time.
Mast cell adhesion assay
BMMCs were sensitized with anti-DNP IgE as described above. Cells were washed once in PBS and resuspended at 1 × 107 cells/ml in PBS containing 5 μg/ml calcein-AM (Molecular Probes, Eugene, OR). Cells were incubated at 37°C for 15 min for labeling, then washed twice with PBS and resuspended at 2 × 106 cells/ml in Tyrode’s buffer. Wells of a 96-well tissue culture plate were coated with varying concentrations of fibronectin (0, 0.1, 1, and 10 μg/ml) and washed once with PBS. Fifty microliters of Tyrode’s buffer containing 200 ng/ml DNP-HSA, 80 ng/ml PMA, or no stimulus was added to triplicate wells. Labeled BMMCs (1 × 105) in 50 μl of Tyrode’s buffer were then added, and the plate was incubated at 37°C for 1 h. Wells were washed five times with 200 μl of Tyrode’s buffer, and remaining cells were quantitated by calcein fluorescence on a SpectraMax 190E microplate reader (Molecular Devices, Sunnyvale, CA). The percent maximal adhesion was calculated relative to the adhesion induced by PMA stimulation. Assays were performed in triplicate for each stimulation condition and concentration of fibronectin.
Passive systemic anaphylaxis assay
Mice were anesthetized by i.p. injection of 300 μl of 2.5% 2,2,2-tribromoethanol in tert-amyl alcohol/PBS (1/40; Sigma-Aldrich). In vivo mast cells were then sensitized with 3 μg of anti-DNP IgE in 200 μl of PBS by i.v. retro-orbital injection. Twenty-four hours later mice were again anesthetized and challenged with 100 μg of DNP-HSA in 200 μl of PBS by i.v. retro-orbital injection. Ninety seconds after challenge, mice were cervically dislocated, and blood was collected by cardiac puncture. Plasma was separated from blood by centrifuging samples for 10 min at 8000 rpm at 4°C. The plasma histamine concentration was determined by competitive histamine immunoassay (Immunotech, Marseilles, France).
Reconstitution of SLP-76-deficient BMMCs with SLP-76 mutants
To determine the requirements for the different domains of SLP-76 for FcεRI-mediated mast cell function, we expressed wild-type SLP-76 or one of three mutant forms in BMMCs by retroviral transduction. For the Y3F mutant of SLP-76, the three N-terminal tyrosines shown to mediate phosphorylation-inducible binding of Vav, Nck, and Itk have been mutated to phenylalanine (32). The Δ20 mutant of SLP-76 lacks aa 224–244, which are known to mediate a constitutive association with the adapter Gads (18, 33). The R448K mutant of SLP-76 has a point mutation abolishing the arginine residue known to be critical for SH2 domain binding of phosphorylated tyrosines in ADAP (20) and HPK-1 (22) (Fig. 1⇓A). After reconstitution of SLP-76-deficient BMMCs with retrovirally expressed wild-type or mutant SLP-76, BMMCs were sorted for equivalent expression of green fluorescence protein (expressed by MIGR1 retroviral plasmid) by flow cytometry. Cells expressed comparable levels of FcεRI and SLP-76, as determined by cell surface and intracellular FACS, respectively (Fig. 1⇓B).
N-terminal tyrosines and the proline-rich region are required for SLP-76 function
SLP-76 has been shown to be required for several FcεRI-induced mast cell functions in vitro, including granule release and cytokine production (7). We therefore investigated the ability of mast cells harboring mutant forms of SLP-76 to release the granule component hexosaminidase and to secrete IL-6. SLP-76-deficient BMMCs do not release hexosaminidase upon FcεRI cross-linking, whereas BMMCs reconstituted with wild-type SLP-76 respond robustly (Fig. 2⇓A). Neither the Y3F nor the Δ20 mutant of SLP-76 restores significant granule release, whereas the R448K mutant rescues ∼50% of wild-type function. A similar pattern of function is seen for IL-6 production, with Y3F and Δ20 mutants providing negligible augmentation of cytokine production over SLP-76-deficient BMMCs, and the R448K mutant restoring ∼50% of wild-type function (Fig. 2⇓B).
In an effort to elucidate the signal transduction events underlying the functional deficits apparent in BMMCs bearing mutant forms of SLP-76, we assessed several biochemical markers of mast cell activation. SLP-76-deficient BMMCs demonstrate markedly decreased calcium flux upon FcεRI cross-linking, whereas restoration of wild-type SLP-76 expression restores normal flux. Here again expression of the Y3F and Δ20 mutants of SLP-76 restored calcium flux minimally, with the Δ20 mutant consistently demonstrating a marginally better calcium response than the Y3F mutant. As with hexosaminidase and cytokine production, expression of the R448K mutant resulted in an intermediate response (Fig. 3⇓A). As mast cells express two isoforms of PLCγ, PLCγ1 and PLCγ2, we investigated phosphorylation of these two enzymes in SLP-76 mutant BMMCs. SLP-76-deficient as well as Y3F and Δ20 mutant BMMCs show significantly diminished inducible phosphorylation of PLCγ2. The R448K mutant again demonstrates an intermediate phenotype, partially restoring phosphorylation of PLCγ2 (Fig. 3⇓B). In contrast, SLP-76-deficient BMMCs show only slight defects in inducible phosphorylation of PLCγ1 (Fig. 3⇓B). We further investigated activation of the mitogen-activated protein kinase Erk in SLP-76-deficient mast cells, finding only mildly decreased Erk phosphorylation in these cells (Fig. 3⇓C).
ADAP-deficient BMMCs degranulate and produce cytokines normally
Given the partial defects in calcium flux, degranulation, and cytokine production seen in mast cells expressing the R448K mutant of SLP-76, and the augmentation of mast cell adhesion and degranulation reported upon overexpression of ADAP in the RBL-2H3 mast cell line (27), we investigated FcεRI signaling and function in ADAP-deficient BMMCs. ADAP is tyrosine-phosphorylated after FcεRI stimulation in wild-type cells (Fig. 4⇓A). ADAP-deficient cells demonstrate normal surface expression of c-Kit and FcεRI and develop normally in vitro (data not shown). In addition, ADAP-deficient BMMCs show no defects in hexosaminidase release (Fig. 4⇓B) or IL-6 production (Fig. 4⇓C) after FcεRI stimulation.
ADAP-deficient BMMCs have normal FcεRI-induced adhesion to fibronectin, but SLP-76-deficient BMMCs do not
In T cells, ADAP deficiency has been shown to impair TCR-induced clustering of the integrin LFA-1 (αLβ2) as well as adhesion to integrin substrates ICAM-1 and VCAM-1 (34, 35). Recently, others reported that overexpression of ADAP in RBL-2H3 cells results in increased VLA-4 (α4β1) clustering and enhanced basal adhesion to fibronectin (26, 27). To test whether ADAP deficiency impairs the ability of BMMCs to inducibly adhere, we performed static adhesion assays. Mast cells were stimulated through the FcεRI, via phorbol ester, or were left unstimulated, and adhesion to fibronectin-coated plates was assessed. Surprisingly, ADAP-deficient BMMCs demonstrate a robust FcεRI-induced adhesion to fibronectin, similar to wild-type BMMCs (Fig. 4⇑D). We then questioned whether SLP-76 was required for mast cell adhesion. BMMCs from SLP-76-deficient mice show decreased adhesion to fibronectin upon FcεRI stimulation, although some inducible adhesion can be detected at the highest dose of fibronectin (10 μg/ml). However, BMMCs expressing the R448K mutant of SLP-76 adhere normally to fibronectin (Fig. 4⇑D). This result indicates that inside-out activation of β1 integrins on BMMCs requires SLP-76, but is independent of both ADAP and the SH2 domain of SLP-76, and may therefore be mechanistically distinct from TCR-induced integrin activation.
Passive systemic anaphylactic response is intact in ADAP-deficient mice
Finally, to extend these results in vivo, we examined the passive systemic anaphylactic response in ADAP-deficient and wild-type mice. Mast cells were sensitized in vivo by i.v. injection of monoclonal α-DNP-IgE and later challenged by i.v. injection of DNP. Mice were sacrificed 90 s after Ag challenge and blood was collected for analysis of histamine levels. As shown in Fig. 5⇓, ADAP-deficient and wild-type mice developed comparable levels of histamine in the blood after Ag administration, whereas SLP-76-deficient mice demonstrated very low histamine response, as previously reported (7). Thus, we have detected no defects in FcεRI-mediated mast cell function in vitro or in vivo in the absence of ADAP.
We have demonstrated in this study that the N-terminal tyrosines and the central proline-rich region of SLP-76 are required for its function downstream of the FcεRI in mast cells. BMMCs expressing SLP-76 that lacks either of these domains flux calcium, degranulate, and produce IL-6 only marginally better than cells lacking expression of SLP-76 entirely (7). The SH2 domain of SLP-76 is also required for optimal mast cell function, and BMMCs expressing this mutant have ∼50% the functional response of wild-type cells. We have further shown that the phenotype of this SH2 domain mutant of SLP-76 is probably not due to an inability to associate with the adapter protein ADAP. ADAP-deficient BMMCs show no functional defects in FcεRI-induced adhesion, degranulation, or cytokine production, and ADAP-deficient mice have an intact passive systemic anaphylactic response. Taken together, these data suggest that the formation of a multimolecular signaling complex anchored by SLP-76, Gads, and LAT and recruiting Vav, Nck, and Btk is an important element of signaling downstream of the FcεRI in mast cells. In contrast, ADAP is dispensable for these FcεRI-induced mast cell functions and is unlikely to mediate its postulated role as an integrator of Lyn- and Fyn-mediated signaling.
A structure/function analysis of SLP-76 in mast cells has recently been reported (36). Although the results reported in this study are largely in accord with those findings, there are some notable differences. Most strikingly, Kettner et al. (36) have reported that BMMCs expressing a Y3F mutant of SLP-76 retain the ability to produce normal amounts of IL-6 and to flux calcium and degranulate significantly better than SLP-76-deficient mast cells. In contrast, the expression of a mutant of SLP-76 lacking the entire N-terminal acidic region, including all three phosphorylated tyrosines, fails to restore significant FcεRI function in mast cells. Although we have not tested an N-terminal truncation mutant, we observed minimal cytokine production, degranulation, and calcium flux for the Y3F mutant of SLP-76. We do not believe that these discrepancies are due to differences in the degree of FcεRI cross-linking, because the Y3F mutant of SLP-76 functions poorly across a wide range of Ag doses in our experiments (Fig. 2⇑ and data not shown). Notably, our studies were performed with the mouse monoclonal anti-DNP IgE SPE7, whereas Kettner et al. (36) used polyclonal rat IgE. SPE7 has been reported to induce mast cell signaling, survival, and cytokine production in the absence of Ag cross-linking (37, 38). This reagent and other methodologic differences may account for the divergent results. Thus, our results indicate that if there exists an additional domain in the N-terminal region of SLP-76 contributing to mast cell function, it is unable to compensate for the absence of the tyrosine phosphorylation motifs in our experimental approach.
The R448K point mutant of SLP-76 is able to only partially restore each of the aberrant functional responses we measured in SLP-76-deficient BMMCs, suggesting that recruitment of a binding partner by the SH2 domain of SLP-76 is necessary for optimal FcεRI signaling. ADAP is known to bind this region of SLP-76 (20), is phosphorylated after FcεRI stimulation in BMMCs (Fig. 4⇑A), and has been reported to modulate adhesion and FcεRI-induced degranulation in cell lines (26, 27). However, ADAP-deficient BMMCs have no demonstrable defects in adhesion, degranulation, or cytokine production, and ADAP-deficient mice produce a robust passive systemic anaphylactic response. Thus, we postulate that a protein other than ADAP is responsible for SLP-76 SH2 domain-dependent FcεRI function. One possible explanation for the intact FcεRI-induced functions of ADAP-deficient BMMCs is the presence of a homologous protein that may compensate for the absence of ADAP. We were unable to detect mRNA or protein expression of the only known homologue of ADAP, PRAM-1 (39), in mast cells (data not shown). A more likely candidate is HPK-1 (22), a known binder of the SLP-76 SH2 domain in T cells. Furthermore, we submit that ADAP is unlikely to mediate the cross-talk between the Lyn and Fyn pathways described by Parravicini et al. (23). Nonetheless, confirmation of this will require studying degranulation and calcium flux in mast cells doubly deficient for Lyn and ADAP or for Fyn and ADAP.
Two main pathways for activation of Erk have been described in mast cells and T cells: a PLCγ/1,2-diacylglycerol/Ras-guanine nucleotide-releasing protein (40) cascade and a LAT/Grb2/Sos (41) pathway. Consistent with the previous report (36), we found that Erk is significantly inducibly phosphorylated in SLP-76-deficient mast cells. Although assessment of Erk activation in SLP-76-deficient T cells is not possible, varying levels of Erk phosphorylation can be observed in the SLP-76-deficient Jurkat T cell line, J14 (our unpublished observation). Thus, in both Jurkat and mast cells, partial Erk activation is possible in the absence of SLP-76. In contrast, Erk phosphorylation is absent in LAT-deficient BMMCs and in two models of LAT-deficient Jurkat cells, J.CaM2 and ANJ3 (42, 43). Given that SLP-76-deficient mast cells, much like LAT-deficient mast cells, retain some residual ability to inducibly phosphorylate PLCγ1, these data suggest the possibility that Erk activation in primary mast cells is largely dependent on the LAT/Grb2/Sos pathway.
We also report that SLP-76-deficient BMMCs have a more pronounced defect in phosphorylation of PLCγ2 than of PLCγ1. Studies have suggested that these two isoforms of PLCγ are differentially localized and activated in RBL-2H3 cells upon FcεRI stimulation (44, 45). Barker et al. (45) reported that inhibition of PI3K activity by wortmannin suppresses phosphorylation and lipase activity of PLCγ1, but not PLCγ2. Furthermore, Wilson et al. (44) describe the formation of distinct signaling domains in FcεRI-stimulated RBL-2H3 cells: primary signaling domains containing FcεRI, Syk, PLCγ2, Vav, and a variety of other proteins; and secondary domains characterized by the presence of LAT and PLCγ1. Preferential localization of SLP-76 to either of these domains has not yet been described, but our data suggest that SLP-76 may be more important for recruitment or stabilization of PLCγ2 in close proximity to its activating kinase within primary signaling domains.
SLP-76-deficient BMMCs have been reported to retain the ability to inducibly phosphorylate a number of substrates, including LAT, PLCγ, Vav, Btk, and Erk (7, 36), suggesting that, as in LAT-deficient BMMCs, several components of FcεRI signaling, perhaps along the Fyn/Gab2/PI3K pathway, are intact. Future studies will continue to address the integrity of these pathways and their relevance to in vitro and in vivo mast cell functions.
↵1 This work was supported by the National Institutes of Health (to G.A.K., E.J.P., J.N.W., and M.A.S.), the Sandler Foundation for Asthma Research (to G.A.K.), the Arthritis Foundation (to E.J.P.), the Cancer Research Institute (to M.S.J.), and the Abramson Family Cancer Research Institute.
↵2 J.N.W. and M.S.J. contributed equally to this work.
↵3 Address correspondence and reprint requests to Dr. Gary A. Koretzky, 415 BRB II/III, 421 Curie Boulevard, Philadelphia, PA 19104. E-mail address:
↵4 Abbreviations used in this paper: ITAM, immunoreceptor tyrosine-based activation motif; ADAP, adhesion- and degranulation-promoting adapter protein; BMMC, bone marrow-derived mast cell; Erk, extracellular signal-regulated kinase; HSA, human serum albumin; LAT, linker for activation of T cell; PI3K, phosphotidylinositol 3-kinase; PLCγ1, phospholipase Cγ1; PTK, protein tyrosine kinase; SCF, stem cell factor; SH2, Src homology 2; SLP-76, Src homology 2 domain-containing leukocyte phosphoprotein of 76 kDa.
- Received January 8, 2004.
- Accepted March 18, 2004.
- Copyright © 2004 by The American Association of Immunologists