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Regulation of Ca²⁺ Signaling in Mast Cells by Tyrosine-Phosphorylated and Unphosphorylated Non-T Cell Activation Linker¹

Lubica Dráberová^*, Gouse Mohiddin Shaik^*, Petra Volná^*, Petr Heneberg^*,, Magda Tmová^*, Pavel Lebduka^*, Jan Korb and Petr Dráber^2,*

^* Department of Signal Transduction, Department of Micromorphology of Biopolymers, Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, and Center for Research in Diabetes, Metabolism and Nutrition, 3rd Medical Faculty, Charles University, Prague, Czech Republic

	Abstract

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Engagement of the FcRI in mast cells and basophils leads to a rapid tyrosine phosphorylation of the transmembrane adaptors LAT (linker for activation of T cells) and NTAL (non-T cell activation linker, also called LAB or LAT2). NTAL regulates activation of mast cells by a mechanism, which is incompletely understood. Here we report properties of rat basophilic leukemia cells with enhanced or reduced NTAL expression. Overexpression of NTAL led to changes in cell morphology, enhanced formation of actin filaments and inhibition of the FcRI-induced tyrosine phosphorylation of the FcRI subunits, Syk kinase and LAT and all downstream activation events, including calcium and secretory responses. In contrast, reduced expression of NTAL had little effect on early FcRI-induced signaling events but inhibited calcium mobilization and secretory response. Calcium response was also repressed in Ag-activated cells defective in Grb2, a major target of phosphorylated NTAL. Unexpectedly, in cells stimulated with thapsigargin, an inhibitor of the endoplasmic reticulum Ca²⁺ ATPase, the amount of cellular NTAL directly correlated with the uptake of extracellular calcium even though no enhanced tyrosine phosphorylation of NTAL was observed. The combined data indicate that NTAL regulates FcRI-mediated signaling at multiple steps and by different mechanisms. At early stages NTAL interferes with tyrosine phosphorylation of several substrates and formation of signaling assemblies, whereas at later stages it regulates the activity of store-operated calcium channels through a distinct mechanism independent of enhanced NTAL tyrosine phosphorylation.

	Introduction

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Aggregation of FcRI in mast cells and basophils triggers numerous signaling steps, which eventually lead to degranulation and cytokine production. Early signaling events involve sequential activation of Src family protein tyrosine kinases Lyn and Fyn, and Syk/Zap family kinase Syk (1, 2, 3, 4). The kinases phosphorylate several substrates, including and subunits of the FcRI and transmembrane adaptor protein linker for activation of T cells (LAT).³ Phosphorylated LAT becomes a docking site for phospholipase C (PLC)1 and PLC2 and some other Src homology 2 (SH2) domain containing signaling proteins, namely Grb2 adaptor (5, 6). Recently two groups have identified in mast cells another transmembrane adaptor protein called NTAL (non-T cell activation linker) or LAB (linker for activation of B cells) (7, 8), a product of the Williams-Beuren syndrome gene, Wbscr5. This protein, also expressed in B cells and NK cells but not in resting T cells, resembles LAT in possessing a short extracellular domain, a single transmembrane region, and a cytoplasmic tail with two palmitoylation cysteine residues and evolutionary conserved motifs containing tyrosine residues. Five of these motifs are of the YXN type (where X is any amino acid), and thus are potential binding sites for the SH2 domain of the cytosolic adaptor protein Grb2. However, unlike LAT, NTAL does not possess a consensus binding motif for PLC1 and PLC2 (7, 8, 9).

An important role of NTAL in immunoreceptor signaling was inferred from experiments in which diminution of NTAL expression by silencing RNA oligonucleotides resulted in reduced BCR-mediated activation of MAPK in A20 cell line (8), as well as impaired degranulation in FcRI-activated human mast cells (9). Unexpectedly, bone marrow-derived mast cells (BMMCs) isolated from NTAL-deficient mice were hyperresponsive to stimulation via the FcRI, as evidenced by enhanced tyrosine phosphorylation of several substrates, calcium response, degranulation, and cytokine production. However, BMMCs obtained from mice lacking both LAT and NTAL had a more severe block in FcRI-mediated signaling than BMMCs deficient in LAT alone (10, 11), suggesting that under certain circumstances NTAL may exert a positive signaling role even in BMMC.

Positive regulatory role of NTAL in immunoreceptor signaling was also observed in studies with immature chicken B cell line, DT40 (12). In these cells, Grb2 negatively regulates the Ca²⁺ response through its binding to so far unidentified suppressor. It has been shown that SH2-mediated binding of Grb2 to tyrosine phosphorylated NTAL resulted in sequestering of the Grb2 inhibitory complex away from the cytosol, enhancing thus the calcium response. However, the role of NTAL in Ca²⁺ signaling is more complex as indicated by previous studies describing enhanced calcium responses in NTAL-deficient BMMCs (10, 11).

To enlighten the role of NTAL in FcRI signaling, we investigated by genetic and biochemical approaches the properties of rat basophilic leukemia (RBL) cells with enhanced or reduced expression of NTAL, and cells defective in Grb2 alone or in combination with NTAL. Our data indicate multiple regulatory roles of NTAL in FcRI signaling in mast cells and document for the first time that activity of the store-operated Ca²⁺ (SOC) channels could be regulated by NTAL even in the absence of its enhanced tyrosine phosphorylation.

	Materials and Methods

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Abs, reagents, and cell cultures

The following mAbs were used: anti-Syk (13), anti-Lyn (14), anti-LAT (15), anti-FcRI subunit (JRK) (16), trinitrophenyl (TNP)-specific IgE mAb (IGEL b4 1) (17), DNP-specific IgE (18), and anti-NTAL (NAP-07; Exbio). Phospho-Tyr-specific mAb (PY-20), conjugated to HRP, was purchased from Transduction Laboratories. Rabbit polyclonal Abs specific for Syk, Lyn, LAT, and NTAL were prepared by immunization with recombinant fragments of Syk (13), Lyn (14), LAT (15) or rat NTAL (aa 30–196; GenBank accession no. Q8CGL2), respectively. Rabbit anti-IgE was prepared by immunization with whole IGEL b4 1. Polyclonal Abs specific for PLC1, PLC2, Erk1, phospho-Erk (specific for phosphorylated Tyr²⁰⁴), Grb2, Akt1, phospho-Akt1 (specific for phosphorylated Ser⁴⁷³), and HRP-conjugated donkey anti-goat IgG, goat anti-mouse IgG and goat anti-rabbit IgG, were obtained from Santa Cruz Biotechnology. Rabbit anti-PI3K p85 subunit Ab (a mixture of equal amounts of antisera against the intact p85 subunit and the N-SH2 region of PI3K) was obtained from Upstate Biotechnology. Goat anti-mouse IgG and anti-rabbit IgG conjugated to colloidal gold particles of 10- or 5-nm were obtained from Amersham Biosciences. Fura-2/AM and ⁴⁵Ca (sp. act. 566 MBq/mg Ca²⁺) were purchased, respectively, from Molecular Probes and MP Biomedicals. Origin of RBL cells (clone 2H3) and their culture conditions have been described (19).

Cloning of rat NTAL cDNA and its sequencing

Based on the nucleotide sequence of human Wbscr5 (GenBank accession no. AF045555) and mouse Wbscr5 (AF139987) we used 5' primer 5'-AAAGAATTCGTCAGTGGTGTTGGCATCAGC-3' (EcoRI site underlined) and 3' primer 5'-AAAAAGCTTGGGCTTCCAGTCAGCACAGTC-3' (HindIII site underlined) to amplify the NTAL cDNA from RBL cells by RT-PCR as described (20). The PCR product was digested with EcoRI and HindIII and ligated into pGEM3Z vector (Promega). The plasmid was amplified and the sequence of the insert was verified by DNA sequencing. All primers used in this study were obtained from Generi Biotech.

Construction of plasmid vectors and isolation of cell lines with changes in expression of NTAL and/or Grb2

Mouse NTAL cDNA was obtained from V. Hoejí and cloned into EcoRI site of pcDNA3.1/Zeo vector (Invitrogen). The plasmid, pZeo-NTAL-1, was isolated and its sequence confirmed by sequencing. The plasmid or empty pcDNA3.1/Zeo vector (negative control) were transfected into RBL cells by electroporation (250 V and 750 µF) using Gene Pulser (Bio-Rad). Colonies resistant to zeocin (300 µg/ml) were then isolated, and clones with enhanced expression of NTAL were selected.

For production of NTAL– and Grb2-specific RNA silencing vectors, two sets of oligonucleotides, 5'-TTTGAACTCCTACGAGAATGTGCTCGGAAGCTTGCGAGCACATTCTCGTAGGAGTTTTTTT-3' and 5'-CTAGAAAAAAACTCCTACGAGAATGTGCTCGCAAGCTTCCGAGCACATTCTCGTAGGAGTT-3' (for NTAL), and 5'-TTTGAATAGATTACCACAGATCAACATAAGCTTTTGTTGATCTGTGGTAATCTATTTTTTT-3' and 5'-CTAGAAAAAAATAGATTACCACAGATCAACAAAAGCTTATGTTGATCTGTGGTAATCTATT-3' (for Grb2) were annealed and cloned into mU6pro vector as described (21). These sequences upon expression form hairpins using the loops in the middle of the sequences (underlined). The plasmids, pU6/siNTAL and pU6/siGrb2 were amplified, the sequences of the inserts were verified by DNA sequencing, and cotransfected at a ratio 10:1 with pstNeoB vector (22) into RBL cells by electroporation. In some experiments RBL cells were transfected with a mix of plasmids, pU6/siGrb2, pU6/siNTAL and pSTneoB at a ratio 5:5:1. Negative controls included empty mU6pro vector or mU6pro vector with the annealed NTAL insert as above except that two mismatches were introduced at positions 30 and 50 (pU6/NTAL-30/50). Clones resistant to antibiotic G418 (0.4 mg/ml) were isolated and analyzed by immunoblotting for NTAL and/or Grb2 expression.

Cell activation, immunoprecipitation, and immunoblotting

Cells were harvested, resuspended in culture medium at a concentration 10 x 10⁶ cells/ml and sensitized with IgE (IGEL b4 1; ascites diluted 1/1000). After 30 min at 37°C the cells were washed in buffered saline solution (BSS) containing 20 mM HEPES (pH 7.4), 135 mM NaCl, 5 mM KCl, 1.8 mM CaCl₂, 1 mM MgCl₂, 5.6 mM glucose, and 0.1% BSA, and challenged with Ag (TNP-BSA) for different time intervals. When the cells were activated with thapsigargin, the sensitization step was omitted. Toward the end of the activation period the cells were briefly centrifuged, and -glucuronidase released into supernatant was determined as described (23) using 4-methylumbelliferyl -D-glucuronide (Sigma-Aldrich) as a substrate. The cell pellets were lysed in an ice-cold lysis buffer containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 2 mM EDTA, 10 mM -glycerophosphate, 1 mM Na₃VO₄, 1 mM PMSF, 1 µg/ml aprotinin, 1 µg/ml leupeptin, and supplemented with 1% Nonidet P-40 (NP-40) (for Lyn, Syk, Erk), 0.2% Brij 96 (for FcRI) or 1% NP40 plus 1% n-dodecyl -D-maltoside (for NTAL and LAT). In experiments analyzing the association of proteins with large signaling assemblies, the activated or nonactivated cells were resuspended in ice-cold PBS supplemented with 0.1% saponin, 5 mM MgCl₂ and 1 mM Na₃VO₄ (permeabilization buffer). After 5 min of incubation on ice, the cells were spun down and extracted for 15 min in a lysis buffer containing 1% Triton X-100. Postnuclear supernatants were immunoprecipitated with corresponding Abs prebound to UltraLink-immobilized protein A or G (Pierce), size fractionated by SDS-PAGE and immunoblotted with PY-20-HRP conjugate or with protein-specific Abs followed by an appropriate second stage HRP-conjugated anti-mouse or anti-rabbit IgG. HRP signal was detected by the ECL reagent (Amersham Biosciences). Tyrosine-phosphorylated Erk and Akt were determined by direct immunoblotting with phosphospecific Abs. Immunoblots were quantified by Luminescent Image Analyzer LAS 3000 (Fuji Photo Film) and further analyzed by AIDA image analyzer software (Raytest). The amount of tyrosine-phosphorylated proteins was corrected for the amount of proteins immunoprecipitated as determined by densitometry of immunoblots after stripping of the membranes, followed by development with the corresponding Abs.

Flow cytofluorometry analysis of FcRI and F-actin

To determine the surface FcRI, cells were exposed to 1 µg/ml anti-TNP IgE followed by FITC-conjugated anti-mouse IgG cross-reacting with mouse IgE, and probed by flow cytofluorometry using a FACSCalibur (BD Biosciences). The total amount of polymeric actin was measured as previously described (24, 25). In brief, 10⁶ cells in 200 µl of BSS-BSA were sensitized with IgE and stimulated or not with Ag for various time intervals. The reaction was terminated by adding 300 µl of PBS containing 50 µg of lysophosphatidylcholine, 6% formaldehyde, and 0.125 µg/ml FITC-phalloidin (Sigma-Aldrich). After 10 min of incubation at 37°C, the cells were centrifuged and resuspended in 1 ml of PBS before flow cytofluorometry analysis. The geometric mean fluorescence intensity was determined for each sample, and data points were plotted relative to the mean fluorescence intensity of nonactivated control cells.

Cytokine detection

Quantitative measurements of rat TNF- was performed using murine TNF- ELISA development kit (cross-reacting with rat TNF-; PeproTech) according to the manufacturer’s instructions.

Lyn kinase assay

In vitro Lyn kinase assay was performed as previously described (26). In brief, Lyn was immunoprecipitated from cells lysed by sequential treatment with 0.1% saponin and 1% Triton X-100. Saponin/Triton X-100-extracted material was incubated with rabbit anti-Lyn Ab and the immunocomplexes were collected on protein A beads. The kinase reaction was conducted for 30 min at 37°C in kinase buffer (25 mM HEPES (pH 7.2) 3 mM MnCl₂, 0.1% NP-40, 100 mM Na₃VO₄, 20 mM MgCl₂) containing 1 µCi [-³²P]ATP (Amersham Biosciences), 100 µM cold ATP, and 0.5 µg/µl denatured enolase as exogenous substrate. The kinase reaction products were resolved by SDS-PAGE, transferred to nitrocellulose, visualized by autoradiography, and quantified by Fuji Bio-Imaging Analyzer Bas 5000.

Electron microscopy

Plasma membrane sheets were prepared from nonactivated or activated cells and examined by electron microscopy as described (27) with some modifications (28).

Immune complex PI3K and PLC assay

PI3K and PLC enzymatic activity was measured as previously described (25). In brief, FcRI-activated or control cells (2 x 10⁶) were solubilized in lysis buffer supplemented with 1% Triton X-100. PI3K in postnuclear supernatant was immunoprecipitated with anti-PI3K p85 subunit Ab and immunocomplexes were collected on UltraLink-immobilized protein A. PI3K assay was initiated by addition of 25 µl of kinase buffer (20 mM HEPES (pH 7.4), 20 mM MgCl₂, and 0.25 mM EGTA) containing 10 µg of sonicated phosphatidylinositol (Sigma-Aldrich) and 37 kBq [-³²P]ATP. After 30 min at 25°C, the reaction was terminated and lipids were separated on TLC Silica gel-60 plate (Merck) in a mixture of chloroform/methanol/4 M ammonium hydroxide (9:7:2, v/v/v) for 1 h. ³²P-labeled materials were visualized by autoradiography and quantified by Fuji Bio-Imaging analyzer Bas 5000.

To determine the PLC enzymatic activity, postnuclear supernatants from nonactivated or activated cells were immunoprecipitated with anti-PLC1 and immunocomplexes were collected on beads of UltraLink-immobilized protein A. The beads were washed and resuspended in 25 µl of reaction buffer followed by addition of 10 µl substrate solution (25 mM sodium phosphate (pH 6.8), 50 mM KCl, 2.5% Triton X-100, 6 µg of phosphatidylinositol 4,5-bisphosphate (PIP₂)) supplemented with 1.1 kBq of P[³H]IP₂ (PerkinElmer Life Sciences). After 30 min at 37°C, the reaction was stopped by adding 300 µl of ice-cold 0.5% BSA in PBS. The samples were centrifuged and 300-µl aliquots of the supernatant were mixed with 100 µl of ice-cold 25% (w/v) TCA. Precipitates were removed by centrifugation, and supernatants were collected for quantification of released [³H]inositol 1,4,5-trisphosphate (IP₃) by liquid scintillation counting.

IP₃ determination

The procedure used a commercially available [³H]IP₃ radioreceptor assay kit and followed the manufacturer’s protocol (PerkinElmer Life Sciences). In brief, IgE-sensitized cells (6 x 10⁶) were stimulated or not with TNP-BSA (500 ng/ml) in 500 µl BSS-BSA. At various time intervals the reactions were terminated by adding 100 µl of ice-cold 100% TCA and the tubes were incubated on ice for 15 min. After centrifugation, supernatants were incubated for 15 min at room temperature and then mixed with a mixture of 1,1,2-trichloro-1,2,2,-trifluoroethane-trioctylamin (3:1). The tubes were vortexed, centrifuged, and water phase was used for determination the radioactivity bound to IP₃-binding protein.

Measurement of intracellular Ca²⁺ concentrations

Changes in the concentration of free intracellular Ca²⁺ [Ca²⁺]_i were determined using fura-2/AM as a probe as previously described (23). IgE-sensitized and control cells were resuspended in BSS-BSA supplemented with 2.5 mM probenecid and 2 µM fura-2/AM. After 40 min at 37°C, the cells were washed with BSS-BSA-probenecid and immediately before measurement briefly centrifuged and resuspended in BSS-BSA. The levels of [Ca²⁺]_i were monitored using luminescence spectrometer LS-50B (PerkinElmer Life Sciences) with excitation wavelengths 340 and 380 nm, and with constant emission at 510 nm. The values were calculated using ICBC Calibration PerkinElmer Fluorescence WinLab software (PerkinElmer Life Sciences).

Uptake of extracellular calcium

Calcium uptake was determined by a modified previously described procedure (29). Briefly, the cells (2 x 10⁶) were resuspended in 100 µl of BSS-BSA with 1 mM Ca²⁺, mixed with 100 µl of BSS-BSA supplemented with ⁴⁵Ca²⁺and various concentrations of thapsigargin, and incubated for 5 min at 37°C. The reaction was terminated by placing the tubes on ice followed by suspending 100-µl aliquots on the wall of the microtest tube separated by air space from the 12% BSA in PBS (300 µl) at the bottom. Cells with bound ⁴⁵Ca were separated from free ⁴⁵Ca²⁺ by centrifugation at 1200 x g for 15 min at 4°C through 12% BSA. The cell pellets were recovered by freezing the tubes, slicing off the tube bottom, and solubilized with 1 ml of 1% Triton X-100. The radioactivity was measured in 10 ml of scintillation liquid (EcoLite; ICN Biomedicals) in a scintillation counter with QuantaSmart software (PerkinElmer). The efflux of calcium was determined in cells loaded with ⁴⁵Ca²⁺ after 2 µM thapsigargin-induced ⁴⁵Ca²⁺ influx for 15 min at 37°C. The cells were then washed and incubated in BSS-BSA containing 1 mM Ca²⁺ for different time intervals. The amount of cell-associated ⁴⁵Ca²⁺ was determined after removing free extracellular ⁴⁵Ca²⁺ as described above.

	Results

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Generation and initial characterization of mast cell lines with enhanced or reduced expression of NTAL

To prepare RNA silencing probes specific for rat NTAL, we first cloned rat NTAL (Wbscr5) cDNA (GenBank accession no. AY170849). As shown in Fig. 1A, the predicted amino acid sequence of rat NTAL (204 aa) is one amino acid longer than its mouse ortholog (GenBank accession no. NM_020044). All tyrosine motifs as well as a potential palmitoylation site (CxxC motif) are conserved in rat and mouse NTAL sequence, suggesting identical functions.

View larger version (34K): [in this window] [in a new window]   FIGURE 1. Rat NTAL cDNA sequence and initial characterization of RBL-derived cell lines with either enhanced or reduced NTAL expression. A, Comparison of predicted amino acid sequence of rat NTAL (top line) and mouse NTAL (bottom line; only the different amino acids are shown). In rat NTAL, the putative transmembrane region is boxed, and potential palmitoylation sequence (CVQC) and tyrosine-containing motifs are underlined and in bold. B, RBL cells were transfected with vector pZeo-NTAL-1 or pU6/siNTAL-1, and stable clones with, respectively, enhanced or reduced NTAL expression were selected. Total cell lysates were analyzed by immunoblotting (IB) with anti-NTAL or anti-Lyn mAbs and amounts of the corresponding proteins were quantified by densitometry. NTAL expression was normalized to control RBL cells and to the amount of Lyn in individual samples (Fold). C, IgE-sensitized control RBL cells or individual transfectants with enhanced expression of NTAL were activated for 5 min by Ag (TNP-BSA, 100 ng/ml; +) or incubated with BSS-BSA alone (–). Lysates from 107 cells were immunoprecipitated (IP) with anti-NTAL mAb, and analyzed by immunoblotting with anti-pTyr-HRP conjugate (PY-20), anti-Grb2 and anti-NTAL Abs. D and E, IgE-sensitized control RBL cells or cells with enhanced (D) or decreased (E) expression of NTAL were stimulated for the indicated time intervals (0–30 min) with Ag (100 ng/ml) and release of -glucuronidase was determined in individual clones. F, IgE-sensitized control RBL cells, or cells with enhanced [clone 2A/4 (NTAL+)] or decreased [clone C4 cells (NTAL–)] expression of NTAL were stimulated for 30 min with different concentrations of Ag and release of -glucuronidase was determined. Data in D–F represent the average of three to four separate experiments, and are expressed as the mean ± SD.

After stimulation of RBL cells with Ag, NTAL was phosphorylated on tyrosine residues as detected by immunoblotting with PY-20-HRP conjugate (Fig. 1C, top). We confirmed previous data (7, 8, 10, 11, 12) that phosphorylated NTAL bound the Grb2 adaptor (Fig. 1C, middle); the amount of bound Grb2 correlated with that of phosphorylated NTAL present.

The relationship between the amount of NTAL and FcRI-mediated degranulation was estimated by the production of -glucuronidase in individual cell lines at various time intervals after triggering with Ag (Fig. 1, D and E). In cells expressing high levels of NTAL (3B/18, 3B/17 and 2A/4), the secretory response was reduced to 50% of that found in control RBL cells or cells transfected with empty vector (not shown). In the cell line with lower level of exogenous NTAL expression (1A/1), the secretory response was inhibited less, indicating a correlation between the inhibitory effect and the extent of NTAL overexpression. The secretory response of 2A/4 cells was reduced at all concentrations of Ag used; two other clones with high NTAL levels (3B/18 and 3B/17) exhibited similar properties. To simplify the presentation, only data from clone 2A/4 (NTAL+) are included in Fig. 1F and other figures. The secretory response was also inhibited in all cell lines with decreased amount of NTAL (Fig. 1E) at all concentrations of Ag used; only data from clone C4 (NTAL–) are shown in Fig. 1F and other figures. The finding that FcRI-mediated secretory response was inhibited in both NTAL+ and NTAL– cells was unexpected and induced additional experiments.

Using IgE-sensitized cells and fluorescently labeled anti-IgE we found that all cell lines differing in NTAL expression exhibited comparable amount of FcRI as detected by flow cytometry (Fig. 2A). Light microscopy of cultured NTAL+ cells (Fig. 2B) and other NTAL overexpressors (not shown) revealed their decreased adhesion to tissue culture plastic surface, a more rounded morphology and less developed processes when compared with RBL cells. In contrast, NTAL– cells had fewer but more developed processes (Fig. 2B). The observed changes in morphology in NTAL– cells were probably related to an enhanced amount of F-actin observed in nonactivated cells (Fig. 2C). After FcRI-triggering, the amount of F-actin rose as described before (30, 31) and remained higher in NTAL– cells than RBL cells. In contrast, in NTAL+ cells activation-induced increase in F-actin was less pronounced (Fig. 2C).

View larger version (56K): [in this window] [in a new window]   FIGURE 2. Expression of FcRI, cell morphology, actin polymerization, and topography of NTAL on plasma membrane sheets. A, The cells were stained for surface FcRI by sequential exposure to TNP-specific IgE (thick line; 1 µg/ml) or PBS alone (negative control; thin line) followed by anti-mouse IgE-FITC conjugate. The samples were analyzed by flow cytometry. B, Phase contrast images of cells cultured for 48 h under standard conditions. Bar, 10 µm. C, Actin polymerization in nonactivated or Ag-activated cells. The cells were sensitized with TNP-specific IgE (1 µg/ml) and then activated by Ag (TNP-BSA; 100 ng/ml) for the indicated time intervals. The amount of F-actin was determined by flow cytometry. Means ± SD were calculated from three independent experiments. D, Membrane sheets were prepared from resting cells (a–c) or from Ag-activated cells (DNP-BSA, 1 µg/ml, 2 min; d–f), and double-labeled from the cytoplasmic side of the plasma membrane for FcRI subunit (10-nm gold particles, arrows) and NTAL (5-nm gold particles, arrowheads). Bar, 100 nm.

NTAL overexpression inhibits the FcRI-induced tyrosine phosphorylation of FcRI subunits, Syk, and LAT

Next we assessed the tyrosine phosphorylation of several proteins known to be pivotal for initial phases of FcRI signaling. When total cell lysates from nonactivated or Ag-activated cells were analyzed by SDS-PAGE and immunoblotting with phosphotyrosine-specific mAb, NTAL+ cells, compared with the control RBL cells, exhibited significantly reduced tyrosine phosphorylation of several proteins (38, 55, 70, 97, and 115 kDa) and enhanced phosphorylation of some other proteins, including a protein of 30 kDa, presumably NTAL (Fig. 3A). In contrast, NTAL– cells showed a phosphorylation pattern more similar to that in control RBL cells; some proteins (e.g., 40 and 50 kDa) showed an increase, other (30 kDa (presumably NTAL) and 70 kDa) a decrease in phosphorylation.

View larger version (42K): [in this window] [in a new window]   FIGURE 3. Overexpression of NTAL inhibits tyrosine phosphorylation of FcRI, Syk, and LAT but enhances enzymatic activity of Lyn kinase. IgE-sensitized cells were stimulated with Ag (TNP-BSA, 500 ng/ml) for the indicated time intervals. A, Cells were lysed in 1% NP-40-containing lysis buffer and total cell lysates were analyzed for protein tyrosine phosphorylation by immunoblotting with PY-20-HRP conjugate. Numbers on the right indicate positions of molecular mass standards (in kDa). B–D, Cells were solubilized in 0.2% Brij 96 (B), 1% NP-40 (C) or a mix of 1% NP-40 and 1% n-dodecyl -D-maltoside (D) and the target proteins were immunoprecipitated with Abs specific for IgE (B), Syk (C), or LAT (D) and analyzed by immunoblotting with PY-20-HRP conjugate. After stripping, the membranes were reblotted with protein-specific Abs as indicated. E–G, Densitometry analysis of phosphotyrosine immunoblots (as in B–D) normalized to the amount of the proteins immunoprecipitated and to their phosphorylation in nonactivated RBL cells. H, Lyn was immunoprecipitated and its enzymatic activity was determined by in vitro kinase assay using 32P-ATP and enolase as a substrate. The kinase reaction products were quantified by autoradiography and after stripping off the membranes the amount of Lyn was determined by immunoblotting. Kinase activity of Lyn, as determined by autoradiography of 32P-labeled Lyn and enolase, normalized to the parameters in nonactivated RBL cells and corrected for the amount of Lyn in each immunoprecipitate is also indicated. Means ± SD in E–H were calculated from three experiments.

Properties of signaling assemblies depend on NTAL expression levels

To examine signaling assemblies during FcRI-induced activation, the cells were first permeabilized with cholesterol-sequestering reagent saponin to release free cytoplasmic components. All membrane components, including those residing in lipid rafts and otherwise insoluble in nonionic detergents, were then efficiently solubilized with Triton X-100. In our previous study, we have found that this two-step solubilization procedure allows better estimation of formation of signaling assemblies in the course of cell activation (25). Using this two-step solubilization procedure we compared the signaling assemblies formed by Grb2, which is the major adaptor protein bound to tyrosine phosphorylated NTAL (7, 8). Immunoblotting analyses of Grb2 immunoprecipitates with PY-20 mAb showed that the amount of Grb2-associated and tyrosine phosphorylated proteins increased during Ag-mediated activation (Fig. 4A). One of the participating tyrosine phosphorylated proteins was LAT, as determined by its molecular mass (38 kDa) and immunoblotting with LAT-specific Abs (Fig. 4, A, B, and D). Consistent with previous data (Fig. 3), the amount of phosphorylated LAT bound to Grb2 was reduced in NTAL+ cells and enhanced in NTAL– cells. There was only an insignificant decrease in the amount of LAT in Grb2 immunocomplexes from NTAL+ cells, compared with RBL cells (Fig. 4D), suggesting that reduced LAT phosphorylation did not remove all Grb2 binding sites. Another of the tyrosine phosphorylated proteins was NTAL, as determined by its molecular mass (30 kDa) and immunoblotting with NTAL-specific Abs (Fig. 4, A, C, and E). As expected, the amount of Grb2-associated NTAL was enhanced in NTAL+ cells and undetectable in NTAL– cells.

View larger version (44K): [in this window] [in a new window]   FIGURE 4. Changes in Grb2 signaling assemblies, PI3K activity and tyrosine phosphorylation of Akt. Cells were activated as in Fig. 3, solubilized with saponin/Triton X-100 procedure, and Grb2 immunocomplexes were isolated by precipitation with anti-Grb2 Ab. A, Grb2 immunocomplexes were analyzed by immunoblotting for the presence of total tyrosine-phosphorylated proteins (PY-20), LAT, NTAL and Grb2. B–E, Densitometry analysis of tyrosine-phosphorylated (PY) LAT (B) and NTAL (C), and total amount of LAT (D) and NTAL (E) in Grb2 immunocomplexes. Means ± SD in B–E were calculated from three to four experiments. F, PI3K activity associated with Grb2 immunocomplexes was estimated using [32P-]ATP and phosphatidylinositol as a substrate in PI3K assay (PI3K a.). Positions of [32P]PI (PIP) and start (S) are indicated by arrows. G, PI3K immunoprecipitates were analyzed in parallel for PI3K enzymatic activity by PI3K assay and for the amount of immunoprecipitated PI3K by immunoblotting with anti-p85 subunit of PI3K. Enzymatic activity of PI3K normalized to its levels in nonactivated RBL cells and corrected for the amount of PI3K-p85 subunit precipitated is also indicated. H, The cells were solubilized with the saponin/Triton X-100 procedure and postnuclear supernatants were analyzed by immunoblotting with anti-phospho-Akt, followed by stripping and immunoblotting with Akt-specific Ab. Relative amounts of the proteins were normalized to nonactivated RBL cells. Representative data from three experiments performed are shown.

Enzymatic activity of PI3K results in the production of phosphatidylinositol 3,4,5-trisphosphate (PIP3). PIP3 and phosphatidylinositol 3,4-bisphosphate recruit Akt to the plasma membrane, where it is phosphorylated and activated. Phosphorylation of Akt was enhanced after FcRI triggering in RBL cells (Fig. 4H). In nonactivated NTAL+ cells the amount of Akt and phospho-Akt associated with saponin-permeabilized cells was higher, but the changes faded out 2 and 5 min after FcRI triggering. In accordance with the enhanced activity of PI3K in NTAL– cells (Fig. 4G), the amount of membrane-bound Akt and its phosphorylation was also enhanced. These data support the concept that NTAL regulates the formation of signaling assemblies containing PI3K and Akt.

Cytokine TNF- production in NTAL⁺ and NTAL^– cells

Previous experiments showed that production of several inflammatory mediators, including TNF-, is enhanced in FcRI-activated BMMC from NTAL-deficient mice (11). Central to regulation of cytokine gene transcription is Ras/Raf/MEK/Erk signaling pathway (35, 36, 37). As expected, enhanced tyrosine phosphorylation of Erk was indeed observed in NTAL^–/– BBMC (10). In additional experiments we therefore examined phosphorylation of Erk and secretion of TNF- in NTAL+ and NTAL– RBL cells. Immunoblotting experiments showed that the amount of tyrosine phosphorylated Erk in Ag-activated RBL cells was increased, reaching the peak 2 min after triggering (Fig. 5A). NTAL overexpression resulted in an impaired phosphorylation of Erk. In contrast, the onset of Erk phosphorylation in NTAL– cells was faster and remained higher at all time intervals tested. Inhibition of Erk phosphorylation in NTAL+ cells correlated with an inhibition of TNF- secretion from the cells activated by two different doses of Ag (Fig. 5B). In NTAL– cells, Ag-induced secretion of TNF- was, surprisingly, also reduced, but less dramatically than in NTAL+ cells and only at lower Ag concentration (100 ng/ml); no inhibition was observed at 500 ng/ml. These data suggest that NTAL in NTAL+ cells negatively regulates cytokine production through inhibition of Ras/Raf/MEK/Erk signaling pathway. In NTAL– cells this pathway is potentiated; the observed inhibition of TNF- production in NTAL– cells seems therefore to reflect some positive effects of NTAL on later stages of mast cell signaling (see below).

View larger version (31K): [in this window] [in a new window]   FIGURE 5. Changes in tyrosine phosphorylation of Erk and TNF- production. A, Cells were activated as in Fig. 3 and then solubilized with 1% Triton X-100. Postnuclear supernatants were analyzed by immunoblotting with anti-phospho-Erk, followed by stripping and immunoblotting with Erk-specific Ab. Relative amounts of the proteins were normalized to nonactivated RBL cells. A typical result from three experiments is shown. B, IgE-sensitized cells were activated with different concentrations of Ag for 3 h and the amount of TNF- released into supernatant was determined by ELISA. Data represent means ± SD from three experiments performed in triplicates.

Expression levels of NTAL modulate activity of PLC

Immunoblotting analyses of PLC1 and PLC2 immunoprecipitates from saponin/Triton X-100 solubilized cells showed that the amount of tyrosine phosphorylated PLC1 associated with signaling assemblies decreased in both NTAL+ cells and NTAL– activated cells (Fig. 6A). Recruitment of PLC2 and degree of its phosphorylation were comparable in all cell lines (Fig. 6B). Enzymatic activity of PLC was detected by immunocomplex PLC assay, determining the production of [³H]IP₃ from P[³H]IP₂ substrate. In nonstimulated cells the activity of PLC was comparable in RBL, NTAL+ and NTAL– cells (Fig. 6C). After FcRI triggering, PLC activity rapidly increased in all cell lines; however, in NTAL– cells, and especially in NTAL+ cells the increase was lower than in RBL cells. These findings were corroborated by direct measurements of IP₃ levels (Fig. 6D). In nonactivated cells IP₃ concentrations were similar in all cell lines under study, but in FcRI-activated cells IP₃ reached higher levels in RBL cells compared with NTAL– cells, and especially to NTAL+ cells.

View larger version (44K): [in this window] [in a new window]   FIGURE 6. Changes in the properties of PLC1 and PLC2. A and B, Cells were activated as in Fig. 3, solubilized with saponin/Triton X-100 procedure, and PLC1 (A) and PLC2 (B) were immunoprecipitated and analyzed by immunoblotting with PY-20-HRP conjugate. After stripping the same membrane was reblotted with protein-specific Abs. Relative amounts of proteins were normalized to nonactivated cells. Data from representative experiments from at least three performed are shown. C, Cells were lysed in 1% Triton X-100, and enzymatic activity of the immunoprecipitated PLC1 was measured by immune complex PLC assay. D, Cellular IP3 levels were determined by 3H-labeled radioreceptor assay kit. Data in C and D represent means ± SD from three to four experiments.

Enhanced levels of IP3 induce a release of calcium from intracellular stores, followed by calcium influx through SOC channels in the plasma membrane (38). To determine the role of NTAL in both these steps we followed the calcium response in cells under different conditions. NTAL– cells activated with Ag in the presence of extracellular Ca²⁺showed a lower calcium response than control cells, but identical initial kinetics. In contrast, NTAL+ cells exhibited a delay in calcium response and a slower decline to baseline levels (Fig. 7A). Activation of the cells by Ag in the absence of extracellular calcium resulted in 30% inhibition of the calcium response in NTAL– cells relative to RBL cells, whereas in NTAL+ cells this response was dramatically inhibited and delayed (Fig. 7B). After increasing the concentration of extracellular Ca²⁺, both RBL and NTAL– cells showed rapid increase in [Ca²⁺]_i, with faster return to initial levels in NTAL– cells, whereas NTAL+ cells showed only a week response (Fig. 7B).

View larger version (27K): [in this window] [in a new window]   FIGURE 7. Intracellular Ca2+ mobilization, extracellular 45Ca uptake and calcium release from the cells. A–C, Calcium responses in fura-2 loaded RBL, NTAL+, and NTAL– cells. A, The cells were sensitized with IgE and stimulated in the presence of 1 mM extracellular calcium with TNP-BSA (500 ng/ml; arrow, Ag). Calcium levels were determined by spectrophotometry. B, Cells were stimulated with Ag in the absence of extracellular calcium (arrow, Ag) and 1 mM Ca2+ was added after Ca2+ calcium levels returned to original values (arrow, Ca2+). C, Cells were exposed to thapsigargin (1 µM; arrow, Th) in the absence of extracellular calcium and 1 mM Ca2+ was added later on (arrow, Ca2+). D, The cells were activated with various concentrations of thapsigargin in the presence of extracellular 45Ca2+ (1 mM). After 5 min at 37°C, the reaction was terminated and the cells were centrifuged through 12% BSA in BSS and cell-bound radioactivity was determined. E, The cells were loaded with 45Ca2+ during 15-min activation with thapsigargin, unbound 45Ca2+ was washed out and calcium efflux was determined at different time intervals. F, The cells were activated with thapsigargin (2 µM) for different time intervals and solubilized in lysis buffer containing 1% NP-40 plus 1% n-dodecyl -D-maltoside. NTAL was immunoprecipitated from postnuclear supernatant and analyzed by immunoblotting with PY-20-HRP conjugate. After stripping, the membrane was reblotted with NTAL-specific Ab. Data in A–C and F are representative experiments from, respectively, three and two performed. Data in D and E represent means ± SD from four experiments performed in duplicates or triplicates.

These data suggested that NTAL could regulate the transport of extracellular Ca²⁺ through the plasma membrane. To test this we measured the ⁴⁵Ca uptake in thapsigargin-stimulated cells. A direct correlation was found between the amount of NTAL expressed and calcium uptake; it was high in NTAL+ cells, medium in RBL cells and low in NTAL– cells (Fig. 7D). The observed correlation could imply not just an important role of NTAL in regulating Ca²⁺ uptake but also an inhibitory effect of NTAL on the release of Ca²⁺ from the cells. Next we therefore labeled the cells with ⁴⁵Ca²⁺, washed them and measured the radioactivity retained within the cells at different time intervals. Results presented in Fig. 7E show that all cell lines exhibited the same kinetics of ⁴⁵Ca²⁺ efflux. Together with previous findings on Ca²⁺ uptake, these data indicate that NTAL positively regulates Ca²⁺ uptake rather than Ca²⁺ efflux.

Experiments with DT40 chicken B cells suggested that phosphorylated NTAL could regulate the Ca²⁺ uptake by a mechanism involving its binding with Grb2, a negative regulator of Ca²⁺ signaling (12). We therefore attempted to find out whether or not NTAL is phosphorylated in thapsigargin-activated cells. NTAL was immunoprecipitated from nonactivated or thapsigargin-activated RBL or NTAL+ cells and its phosphorylation was assessed by immunoblotting with PY-20-HRP. Data presented in Fig. 7F indicate that thapsigargin had no effect on NTAL tyrosine phosphorylation in either RBL or NTAL+ cells. To determine whether Grb2 could function as a negative regulator of Ca²⁺ response in RBL cells, we transfected the cells with pU6/siGrb2 plus pSTneoB and selected G418-resistant clones, G1 and G9, with decreased amount of Grb2 (Fig. 8A). We also isolated RBL-derived cells with decreased amounts of both NTAL and Grb2 after simultaneous transfection with pU6/siNTAL, pU6/siGrb2 and pSTneoB (Fig. 8B, clones NG2 and NG10). It should be noted that the amount of LAT was not affected in any transfectant (Fig. 8, A and B), confirming the specificity of the knock-down procedure. Detailed analysis showed that Ag-activated RBL cells with decreased amount of Grb2 exhibited lower increase in [Ca²⁺]_i than RBL cells transfected with empty vector (Fig. 8C). When the expression of both NTAL and Grb2 was reduced, an even deeper decrease in [Ca²⁺]_i was observed (Fig. 8D). These data indicate that Grb2 functions as a positive regulator of Ca²⁺ response in RBL cells.

View larger version (41K): [in this window] [in a new window]   FIGURE 8. Positive regulatory role of Grb2 in Ag-induced Ca2+ signaling. A and B, Immunoblotting analysis of Grb2- or Grb2/NTAL-deficient cells. RBL cells transfected with empty vector (RBL-C), pU6/siGrb2 vector (clones G1 and G9) or both pU6/siNTAL-1 and pU6/siGrb2 (clones NG2 and NG10) were lysed and postnuclear supernatants were analyzed by immunoblotting for the presence of Grb2, LAT, and NTAL. Relative amounts of Grb2 and NTAL were normalized to the amount of LAT in each sample. C and D, Calcium response in fura-2-loaded control and transfected cells. The cells were sensitized with IgE and stimulated in the presence of 1 mM extracellular Ca2+ with TNP-BSA (500 ng/ml, arrow, Ag). Calcium levels were determined by spectrophotometry. Data are representative of at least two experiments performed.

	Discussion

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Initial characterization revealed that cells with increased or decreased amount of NTAL exhibited restricted secretory response after FcRI triggering, unrelated to differences in surface expression of FcRI and evident at all concentrations of Ag used. NTAL levels also affected morphology of the cells; NTAL+ cells being less adherent to tissue culture plastic surface and NTAL– cells having longer processes. The observed changes in cell morphology could be related to the amount of F-actin and its formation during FcRI signaling. Thus, NTAL– cells had more F-actin than parental RBL or NTAL+ cells, and only a weak increase in F-actin formation was induced by activation of both NTAL+ and NTAL– cells.

Previously we have examined the distribution of NTAL and LAT by electron microscopy of membrane sheets. NTAL in resting RBL cells was localized in small clusters, topographically separated from clusters of LAT and FcRI (10), but not Thy-1 (40). When membrane sheets were isolated from NTAL overexpressors, no difference in the distribution of FcRI and NTAL was observed except that the average cluster size and density of NTAL-bound gold particles was higher in NTAL+ than control cells. Importantly, the absence of NTAL from the FcRI aggregates is not confined just to control RBL cells, as reported previously (10), but applies also to NTAL+ cells (this study). Together with our finding of normal redistribution of aggregated FcRI into osmiophilic regions in NTAL+ cells, these data suggest that the initial FcRI aggregation step induced by multivalent Ag is not changed in NTAL overexpressors.

Immunochemical studies showed that overexpression of NTAL inhibited the tyrosine phosphorylation of FcRI and subunits, Syk and LAT in Ag-activated cells. Consequently, recruitment of PLC to signaling assemblies and its tyrosine phosphorylation and activation were inhibited. Reduced production of PLC metabolite IP3 led to a reduction in both the release of Ca²⁺ from intracellular stores and the uptake of extracellular Ca²⁺ through SOC channels. This could explain the inhibition of the secretory response in Ag-activated cells. The MAP kinase signaling pathway was also inhibited in NTAL+ cells as reflected in the impaired tyrosine phosphorylation of Erk and subsequent low secretion of TNF-.

Decreased tyrosine phosphorylation of FcRI subunits in NTAL+ cells suggested that the activity of Lyn kinase is inhibited. However, immunocomplex kinase assays showed Lyn kinase activity in NTAL+ cells is undiminished, implying that NTAL interferes with the accessibility of Lyn to FcRI. This possibility is strengthened by data indicating that the amount of Lyn coprecipitated with FcRI was higher in activated RBL cells than in NTAL+ cells. Because Lyn, like NTAL, seems to be localized in lipid rafts (7, 41), it is possible that direct or indirect interactions of Lyn with NTAL preclude the interaction between Lyn and FcRI subunits. Although immunoprecipitation and immunocomplex kinase assays failed to show NTAL-Lyn interactions (L. Dráberová, unpublished data), it is possible that procedures used to isolate NTAL immunocomplexes destroyed these interactions.

Our results indicate that the signaling assemblies formed in NTAL+ cells are different from those formed in control or NTAL– cells. Indeed, in Ag-activated NTAL+ cells, more NTAL and less LAT is bound to Grb2, supporting the concept of competition between NTAL and LAT for Grb2 as a substrate (10). In contrast, in activated NTAL– cells phosphorylation of FcRI subunits, Syk and LAT was not inhibited. However, association of PLC with insoluble complexes, its phosphorylation and its enzymatic activity were less efficient and resulted in a partial inhibition of downstream events. The impaired function of PLC could reflect involvement of NTAL in the formation of signaling assemblies required for PLC enzymatic activity (42, 43, 44). As previously shown (10, 45), Grb2 forms complexes with SHP-2 phosphatase, and these complexes could bind to NTAL to regulate early signaling events. Alternatively, if PLC somehow interacts with NTAL, its enzymatic activity would be inhibited in NTAL– cells even though the early FcRI-activation events proceed normally. Although we were unable to coprecipitate PLC with NTAL (L. Dráberová, unpublished data), it remains possible that these interaction are sensitive to the solubilization and immunoprecipitation procedures.

If IP3 signal generated by FcRI aggregation is bypassed by thapsigargin, NTAL+ cells show a higher uptake of extracellular Ca²⁺ than control RBL cells. This suggests that NTAL could have a positive regulatory role in Ca²⁺ uptake. This is corroborated by complementary studies in which thapsigargin-activated NTAL– cells showed a lower Ca²⁺ uptake. The role of NTAL in uptake of extracellular Ca²⁺ is unclear but could be related to NTAL-dependent Ca²⁺-regulating signal circuit recently described in DT40 B lymphocytes (12). In these cells, Grb2 plays a negative regulatory role in Ca²⁺ uptake, which appears to be eliminated upon binding to NTAL. However, several pieces of evidence indicate that in rodent mast cells NTAL plays a different role in Ca²⁺ response. First, no inhibition in [Ca²⁺]_i was observed in Ag-activated BMMC from NTAL^–/– mice; rather, there was an enhancement of the Ca²⁺ response (10, 11). Second, Ag-activated NTAL^–/– BMMC showed no decrease, but rather an increase in the uptake of extracellular ⁴⁵Ca²⁺; this could be related to enhanced activity of PLC (10, 11). Third, there was a higher Ca²⁺ response to FcRI triggering in NTAL– cells than in NTAL+ cells. It should be emphasized that the highest Ca²⁺ response was observed in control RBL cells, suggesting that an optimal concentration and/or topography of NTAL is required for maximum Ca²⁺ response. Fourth, after activation by Ag in the absence of extracellular Ca²⁺ and restoration of Ca²⁺ level later on, there was no dramatic difference between control and NTAL– cells in initial Ca²⁺ uptake. In contrast, the response in NTAL+ was markedly inhibited. This inhibition reflected low levels of Ca²⁺ released from intracellular stores and consequently low Ca²⁺ influx through the SOC channels. Finally, [Ca²⁺]_i in cells activated with thapsigargin in the absence of extracellular Ca²⁺ was not dependent on NTAL. However, when Ca²⁺ was replenished, a significant increase in [Ca²⁺]_i occurred in the following sequence: NTAL+ cells > control RBL cells > NTAL– cells. These data suggest that NTAL could play a positive role in regulating the Ca²⁺ uptake at the level of SOC channels. Our finding of correlation between ⁴⁵Ca²⁺ uptake and the amount of NTAL in thapsigargin-activated cells supports the notion. The possibility that NTAL modulated Ca²⁺ efflux rather then its influx was excluded by experiments measuring the kinetics of calcium release from activated ⁴⁵Ca²⁺-labeled cells. Interestingly, no increase in tyrosine phosphorylation of NTAL was observed in thapsigargin-triggered cells. Thus, our data indicate that NTAL could have a novel regulatory role in Ca²⁺ uptake independent of de novo NTAL tyrosine phosphorylation.

Possible regulatory functions of NTAL at different phases of FcRI-mediated Ca²⁺ signaling are shown in Fig. 9. At early stages of activation (phase I) NTAL serves as a substrate for protein tyrosine kinases and thus could interfere with phosphorylation of FcRI and LAT by a competitive mechanism. Furthermore, phosphorylated NTAL binds Grb2 and other signaling molecules, which could modulate the activity of various enzymes, including PI3K and PLC. Enhanced activity of PLC leads to increased production of IP3 and consequently to elevated levels of cytoplasmic Ca²⁺. At later stages of activation (phase II), NTAL could affect the function of SOC channels, reflecting its direct or indirect interactions with channel-forming proteins and/or their regulators such as Orai1 and/or Stim1 (46, 47, 48, 49). This function of NTAL is not dependent on its enhanced tyrosine phosphorylation.

View larger version (29K): [in this window] [in a new window]   FIGURE 9. A model of NTAL function in FcRI-mediated Ca2+ signaling. At early stages after FcRI aggregation (phase I), NTAL is rapidly tyrosine phosphorylated, competing with phosphorylation of FcRI and LAT. Phosphorylated NTAL binds Grb2 complexes and interferes with the activity of PI3K and several other signaling molecules. NTAL affects activity of PLC and in this way the generation of IP3 followed by release of Ca2+ from internal stores. At later stages of activation (phase II), extracellular Ca2+ flows into the cytoplasm through SOC channels. Activity of these channels could be modulated by direct or indirect interaction of NTAL with SOC channel proteins and/or regulators of their activity. However, this effect is independent of enhanced NTAL tyrosine phosphorylation.

In summary, NTAL overexpression suppresses early FcRI-induced activation events, but has a positive effect on the uptake of extracellular Ca²⁺ in thapsigargin-stimulated cells. Accordingly, inhibition of NTAL expression has no inhibitory effect on early signaling pathways but does suppress the late ones, including the uptake of extracellular Ca²⁺. Expression levels of NTAL may thus regulate mast cells activation at multiple steps. Differences in interaction of NTAL with various regulatory proteins and signaling pathways (50) could explain why down-regulation of NTAL expression either inhibited (9) or enhanced (10, 11) FcRI-induced secretory response in different mast cell types.

	Acknowledgments

 
We thank H. Mrázová, R. Budoviová, D. Loreníková, M. Dráber, I. Liková, and J. Musilová for technical assistance; and A. Koffer for critical reading of the manuscript.

	Disclosure

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The authors have no financial conflict of interest.

	Footnotes

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This work was supported by projects 1M6837805001 (Center of Molecular and Cellular Immunology) and LC-545 from Ministry of Education, Youth and Sports of the Czech Republic; Grants 204/05/H023 and 301/06/0361 from the Grant Agency of the Czech Republic; Grants A5052310 and 1QS500520551 from the Grant Agency of the Academy of Sciences of the Czech Republic; and Institutional project AVOZ50520514. The research of P.D. and P.H. was supported, respectively, by an International Research Scholar’s award from Howard Hughes Medical Institute and Research goal MSM0021620814 from the 3rd Faculty of Medicine, Charles University, Prague.

² Address correspondence and reprint requests to Dr. Petr Dráber, Department of Signal Transduction, Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, Videská 1083, Prague, Czech Republic. E-mail address: draberpe{at}biomed.cas.cz

³ Abbreviations used in this paper: LAT, linker for activation of T cells; PLC, phospholipase C; SH2, Src homology 2; NTAL, non-T cell activation linker; BMMC, bone marrow-derived mast cell; RBL, rat basophilic leukemia; SOC, store-operated Ca²⁺; BSS, buffered saline solution; NP-40, Nonidet P-40; PIP₂, phosphatidylinositol 4,5-bisphosphate; IP₃, inositol 1,4,5-trisphosphate; [Ca²⁺]_i, concentration of free intracellular Ca²⁺; PIP3, phosphatidylinositol 3,4,5-trisphosphate; TNP, trinitrophenyl.

Received for publication March 23, 2007. Accepted for publication August 7, 2007.

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