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The Journal of Immunology, 2002, 169: 2274-2283.
Copyright © 2002 by The American Association of Immunologists

Intracellular Single-Chain Variable Fragments Directed to the Src Homology 2 Domains of Syk Partially Inhibit Fc{epsilon}RI Signaling in the RBL-2H3 Cell Line1

Stéphanie Dauvillier*, Peggy Mérida*, Michela Visintin{dagger}, Antonino Cattaneo{dagger}, Christian Bonnerot{ddagger} and Piona Dariavach2,*

* Institut de Génétique Moléculaire de Montpellier, Unité Mixte de Recherche 5535 Centre National de la Recherche Scientifique, Montpellier, France; {dagger} International School for Advanced Studies, Neuroscience Program, Trieste, Italy; and {ddagger} Institut Curie, Unité 520, Institut National de la Santé et de la Recherche Médicale, Paris, France


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Intracellular expression of Ab fragments has been efficiently used to inactivate therapeutic targets, oncogene products, and to induce viral resistance in plants. Ab fragments expressed in the appropriate cell compartment may also help to elucidate the functions of a protein of interest. We report in this study the successful targeting of the protein tyrosine kinase Syk in the RBL-2H3 rat basophilic leukemia cell line. We isolated from a phage display library human single-chain variable fragments (scFv) directed against the portion of Syk containing the Src homology 2 domains and the linker region that separates them. Among them, two scFv named G4G11 and G4E4 exhibited the best binding to Syk in vivo in a yeast two-hybrid selection system. Stable transfectants of RBL-2H3 cells expressing cytosolic G4G11 and G4E4 were established. Immunoprecipitation experiments showed that intracellular G4G11 and G4E4 bind to Syk, but do not inhibit the activation of Syk following Fc{epsilon}RI aggregation, suggesting that the scFv do not affect the recruitment of Syk to the receptor. Nevertheless, Fc{epsilon}RI-mediated calcium mobilization and the release of inflammatory mediators are inhibited, and are consistent with a defect in Bruton’s tyrosine kinase and phospholipase C-{gamma}2 tyrosine phosphorylation and activation. Interestingly, Fc{epsilon}RI-induced mitogen-activated protein kinase phosphorylation is not altered, suggesting that intracellular G4G11 and G4E4 do not prevent the coupling of Syk to the Ras pathway, but they selectively inhibit the pathway involving phospholipase C-{gamma}2 activation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
In basophils and mast cells, cross-linking the high affinity IgE receptor, Fc{epsilon}RI, results in a number of biochemical events leading to the release of a panel of proinflammatory mediators. Fc{epsilon}RI signal transduction is mediated by three distinct families of cytoplasmic protein tyrosine kinase (PTK)3: the Src family PTK Lyn, the Syk family PTK Syk, and Bruton’s tyrosine kinase (Btk)/Tec. The PTK Lyn is activated by transphosphorylation upon Fc{epsilon}RI cross-linking (1). Activated Lyn phosphorylates the tyrosine residues in the immunoreceptor tyrosine-based activation motifs (ITAM) (2) of the cytoplasmic regions of Fc{epsilon}RI-{beta} and Fc{epsilon}RI-{gamma} subunits (3, 4, 5), enabling the recruitment of Lyn and Syk through Src homology (SH)2 domain-phosphotyrosine interactions (3, 6, 7, 8). Newly recruited PTKs are activated by transphosphorylation of tyrosine residues in their activation loops and by conformational changes in the case of Syk (9, 10). Active Lyn and Syk phosphorylate themselves and other protein substrates such as phospholipase C (PLC)-{gamma} and Btk (11, 12, 13, 14, 15). Hydrolysis of phosphatidylinositol 4,5-biphosphate by PLC-{gamma} generates two second messengers, inositol 1,4,5-triphosphate (IP3) and diacylglycerol. IP3 mobilizes calcium (Ca2+) from intracellular storage sites, and diacylglycerol together with Ca2+ activates protein kinase C. Both Ca2+ and protein kinase C are required for optimal mast cell degranulation.

The Syk family of cytoplasmic PTKs comprises two known members termed Syk and Zap-70. Syk is present in most hemopoietic cell types, including B cells and mast cells. The importance of Syk to receptor-mediated signaling in hemopoietic cells is underscored by the signaling defects observed in Syk-deficient variants of the chicken B cell line DT-40 and the rat basophilic leukemia cell line RBL-2H3 (13, 16). The creation of Syk-deficient mice by homologous recombination has also highlighted the importance of Syk in developmental processes (17, 18). The structure of Syk includes from the N to the C terminus: 1) two SH2 domains, which bind doubly phosphorylated ITAMs (19); 2) a linker region, containing sites of tyrosine phosphorylation that are direct binding sites for SH2 domains and phosphotyrosine-binding domains of signaling molecules, including PLC-{gamma}, Vav, and Cbl (20, 21, 22); 3) a catalytic domain, including sites for ATP-binding and tyrosine phosphorylation; and 4) a short C-terminal extension of yet undetermined function.

In this study, we report the use of intracellular Ab technology to target Syk. We established stable transfectants of RBL-2H3 cell line that express in their cytoplasm single-chain variable fragment (scFv) Abs directed against the SH2 domains of Syk. We studied the biological effects of the binding of intracellular scFv to Syk, and we found that despite an intact kinase activity of Syk, the cells that expressed the scFv exhibited a defect in the Fc{epsilon}RI-mediated signal transduction as visualized by an impaired calcium mobilization, and the inhibition of the secretion of allergic mediators. The analysis of the proteins that are implicated in that signaling pathway revealed an inhibition in the tyrosine phosphorylation and activation of Btk and PLC-{gamma}2. Nevertheless, Fc{epsilon}RI-induced mitogen-activated protein kinase (MAPK) phosphorylation was not altered, suggesting that the scFv inhibited selectively the link between Syk and Btk and PLC-{gamma}2 for their subsequent tyrosine phosphorylation and activation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials and Abs

All reagents, unless otherwise mentioned, were from Sigma-Aldrich (St. Louis, MO). The mAb 9E10 directed against the amino acid sequence EQKLISEEDLN of human c-myc was kindly provided by G. Winter (MRC, Cambridge, U.K.). Anti-Syk, anti-Zap-70, anti-Lyn, anti-Btk, anti-PLC-{gamma}2, and anti-B cell linker protein Abs were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-phospho-p44/42 MAPK and anti-p44/42 MAPK Abs were purchased from New England Biolabs (Ozyme, France). Anti-Akt1/protein kinase B (PKB) Ab, anti-phospho-Akt1/PKB Ab, and anti-phosphotyrosine mAb 4G10 were purchased from Upstate Biotechnology (Euromedex, France). The GST-HS1 fusion protein was described elsewhere (23).

ScFv purification and BIAcore analysis

The isolation of the anti-Syk scFv fragments from the Griffin.1 human synthetic VH + VL library and their purification were described elsewhere (24). Briefly, all scFv have at their C-terminal end a c-myc tag enabling detection with the mAb 9E10, and a hexahistidine tag for immobilized metal affinity chromatography. After three rounds of selection of the library with a GST-Syk fusion protein containing the residues 6–242 of murine Syk (GST-Syk 6–242), soluble monoclonal anti-Syk scFv were isolated by ELISA tests, and the selected scFv were purified on nickel-agarose. The irrelevant scFv named dVH/dVK used in control experiments was a kind gift of I. Tomlinson (MRC Center for Protein Engineering, Cambridge, U.K.).

The BIAcore 2000 system, sensor chips CM5, and the GST kit for fusion capture used were from Biacore AB (Uppsala, Sweden). Immobilization of goat anti-GST Ab on two sensor surfaces was performed according to the manufacturer’s procedure. Recombinant GST (5 µg/ml), used as reference, were injected over one of the surfaces, whereas GST-Syk fusion protein (GST-Syk 6–242) at the same concentration was injected over the other one. The binding data of the interactions between the GST-Syk fusion protein and the scFv G4G11 and G4E4 were obtained by injecting each scFv at different concentrations (0–3.3 µM) over the surfaces at 25°C. At the end of each experiment, a regeneration procedure was performed to remove the GST fusion proteins and any binding partner, to leave anti-GST available on the surface. Association and dissociation equilibrium data were calculated using BIAevaluation 3.0 software.

Two-hybrid yeast selection system

All strains and plasmids were described elsewhere (25). Briefly, LexA fusion baits were prepared in the plasmid pBTM116. For Syk/BTM116 (pLexA-Syk), the Syk gene was amplified by PCR from murine Syk cDNA (kindly provided by A. Ziemiecki, University of Bern, Bern, Switzerland) and inserted into EcoRI-BamHI sites of pBTM116. The coding sequences of the scFv G4G11, G4E4, and G6G2 were cut from the pHEN2 recombinant vectors by using the restriction sites SfiI and NotI, and they were cloned into pVP16* vector. For the two-hybrid analysis, competent cells of Saccharomyces cerevisiae strain L40 were prepared as described (25), and positive clones were selected by using auxotrophic markers for both plasmids and for lysine and histidine prototropy. Histidine-positive colonies and controls were lysed in liquid nitrogen and assayed for {beta}-gal activity on filters, as described (25).

Cells, expression constructs, and transfection

RBL-2H3 rat basophilic leukemia cells were maintained as monolayer cultures in RPMI 1640 medium with Glutamax (Invitrogen Life Technologies, Cergy Pontoise, France) supplemented with 10% FBS (Life Technologies). For the expression of the scFv in the RBL-2H3 cell line, the cDNA encoding G4G11, G4E4, and the irrelevant scFv were isolated from the pHEN2 recombinant vectors after digestion with NcoI and NotI restriction enzymes, and they were cloned into the pscFvexp-cyt vector (26). This vector directs the expression of cytosolic Ab fragments, and contains the neomycin phosphotransferase gene (neo). All scFv fragments contain at their COOH terminal end a c-myc tag that permits their detection with the mAb 9E10. For stable transfection, 50 µg recombinant vectors were transfected into 2 x 106 RBL-2H3 cells by electroporation (960 µF, 260 V). Neomycin-resistant transfectant cells were grown in the presence of 2 mg/ml G418 (Life Technologies). Monoclonal cell lines expressing the scFv were produced by limiting dilution and identified by immunofluorescence with 9E10 mAb. All RBL-2H3 clones used in our experiments expressed unaltered levels of Fc{epsilon}RI (data not shown).

Immunoprecipitations and immunoblots

Cells were seeded in petri dishes and, for activation, they were cultured overnight with anti-trinitrophenyl (TNP) IgE. After 12–16 h, the excess IgE was removed by washing twice with RPMI without additives, and cells were stimulated at 37°C in RPMI containing 100 ng/ml Ag DNP-BSA. After 3 min, the supernatant was harvested, and the cell monolayers were rinsed twice with ice-cold PBS containing 1 mM Na3VO4, 100 mM NaF, and 5 mM {beta}-glycerophosphate. Before lysis, unstimulated cells were also rinsed twice with the same PBS buffer, and cells were solubilized in 1% Nonidet P-40 lysis buffer (1% Nonidet P-40, 25 mM Tris (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM Na3VO4, 100 mM NaF, 5 mM {beta}-glycerophosphate, 2 mM PMSF, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 10 µg/ml pepstatin A). The cells were scraped from the plates, the lysates were centrifuged for 10 min at 16,000 x g at 4°C, and the protein concentration was determined (BCA Protein Assay; Pierce, Rockford, IL). Total cell lysates were prepared by the addition of SDS sample buffer (250 mM Tris, pH 7.5, 5% SDS, 10% glycerol, 5% 2-ME, 0.01% bromphenol blue; final concentrations) to the postnuclear extracts.

For immunoprecipitations, lysates with identical protein concentration from unstimulated, and IgE/DNP-stimulated cells were incubated with preformed complexes of Abs and GammaBind G Sepharose (Amersham Pharmacia, Piscataway, NJ). To examine the in vitro kinase activity of Btk and Lyn, immunoprecipitates were suspended in kinase buffer (20 mM PIPES, pH 7.5, 10 mM MgCl2, 1 mM Na3VO4) containing [{gamma}-32P]ATP (3000 Ci/mmol; Amersham Pharmacia) and 5 µg acid-denatured enolase, and incubated at room temperature for 5 min. For the in vitro kinase assay of Syk, cells were solubilized in modified radioimmunoprecipitation assay buffer (1% Nonidet P-40, 0.25% sodium deoxycholate, 0.1% SDS in PBS buffer supplemented with phosphatase and protease inhibitors described above). Immunoprecipitates were suspended in kinase buffer (30 mM HEPES, pH 7.5, 10 mM MgCl2, 2 mM MnCl2, 1 mM Na3VO4) containing [{gamma}-32P]ATP and 5 µg GST-HS1, and incubated at room temperature for 15 min. Lysates or immunoprecipitates were separated by SDS-PAGE, transferred to nitrocellulose membrane (Schleicher & Schuell, Keene, NH), and detected by appropriate Abs and the ECL system.

Flow cytometric analysis of calcium mobilization

For the determination of intracellular free calcium concentration, wild-type (WT) and RBL-2H3 transfectants were grown for 16 h in the presence of saturating amounts of anti-TNP IgE mAb. The following day, cells were washed in RPMI 1640 medium, and 1 x 106 cells were preloaded with 5 mM Fluo-3 AM (Molecular Probes, Eugene, OR) in the presence of 0.2% Pluronic F-127 for 30 min at room temperature. Cells were washed three times in RPMI 1640 and resuspended at 1 x 106 cells/ml in complete medium; DNP-BSA was added at 100 ng/ml to initiate Ca2+ signaling, and the intracellular free calcium concentration was monitored with a BD Biosciences flow cytometer. The mean intracellular Ca2+ concentration was evaluated with FCS assistant 1.2.9 {beta} software (BD Biosciences, Le Pont de Claix, France). To detect only release of intracellular Ca2+ from endoplasmic reticulum (ER) stores, Ca2+ in the medium was buffered by adding 4 mM EGTA immediately (within 1 min) before DNP-BSA stimulation. To measure Ca2+ mobilization in response to a nonreceptor-mediated stimulation, cells were treated with 1.5 µM calcium ionophore ionomycin.

Serotonin and {beta}-hexosaminidase release

For {beta}-hexosaminidase measurements, RBL-2H3 cells and their transfectants were grown overnight in 96-well plates (2 x 105 cells/well) in the absence and in the presence of saturating amounts of anti-TNP IgE mAb. For the activation, after 12–16 h, the excess IgE was removed by washing in Tyrode buffer (10 mM HEPES, pH 7.4, 130 mM NaCl, 5 mM KCl, 1.4 mM CaCl2, 1 mM MgCl2, 5.6 mM glucose, and 0.1% BSA), and cells were stimulated at 37°C in Tyrode buffer containing 100 ng/ml Ag DNP-BSA. After 45 min, the supernatant was harvested, and the remaining cell monolayer was lysed in Tyrode buffer supplemented with 0.5% Triton X-100 and protease inhibitors. Hexosaminidase activity was measured in both supernatant and cell monolayer, using the substrate 4-nitrophenyl-2-acetamido-2-deoxy-{beta}-D-glucopyranoside (1.3 mg/ml; Sigma-Aldrich). After 40 min at 37°C, the enzymatic reaction was stopped by addition of 2 vol 0.4 M glycine, pH 10.7. Absorbance at 405 nm was read in a Dynatech (Chantilly, VA) MR5000 ELISA reader. To measure {beta}-hexosaminidase release in response to a nonreceptor-mediated stimulation, cells were treated with 1.5 µM calcium ionophore ionomycin. The percentage of hexosaminidase released from each cell line was determined by calculating: (released hexosaminidase/released + cell monolayer hexosaminidase) x 100. All experiments were done in triplicates.

For serotonin release, cells were resuspended at 1 x 106 cells/ml in RPMI 1640 medium supplemented with 10% FBS and were incubated at 37°C for 1 h with 3 µCi/ml [3H]serotonin (Amersham Pharmacia), washed, resuspended in RPMI-FBS, incubated for another hour at 37°C to remove excess [3H]serotonin, washed again, resuspended in the same medium, distributed in 96-well microculture plates at 2 x 105 cells/well, and incubated for 2 h at 37°C in the presence or absence of anti-TNP IgE in a final volume of 50 µl. Adherent cells were washed four times with 200 µl HBSS; next, 25 µl culture medium was added to each well, and cells were warmed at 37°C for 15 min before challenge. Cells were challenged for 30 min at 37°C with 100 ng/ml DNP-BSA. Reactions were stopped by the addition of 50 µl ice-cold medium and by placing plates on ice. Fifty microliters of supernatants were mixed with 200 µl Ready Protein+ scintillation fluid (Beckman Coulter, Roissy, France) and counted in a beta-plate counter (Beckman Coulter). The percentage of [3H]serotonin released was calculated using as 100%, cpm in 50 µl harvested from wells containing the same number of cells that were lysed in 100 µl 0.5% SDS and 0.5% Nonidet P-40. Determinations were done in triplicates.

Immunofluorescence

Cells were grown on glass Lab-Tek chambers (Nunc, Naperville, IL). For activation, cells were stimulated with IgE/DNP, as described above, then rinsed three times in PBS and fixed for 15 min with 4% formaldehyde in PBS. Cells were rinsed again and permeabilized for 15 min with 0.05% saponin in PBS. Cells were rinsed three times in PBS. Incubation with the mAb 9E10 was conducted at room temperature for 30 min; then a biotinylated goat anti-mouse Ab followed by a streptavidin-Texas Red complex (Amersham Pharmacia) were used for the detection of the scFv. Syk was detected with a rabbit anti-Syk Ab, followed by a FITC-conjugated donkey anti-rabbit Ab (Jackson ImmunoResearch Laboratories, West Grove, PA). Samples were routinely examined with a Leica (Deerfield, IL) DMR microscope. Confocal analysis was conducted with a Bio-Rad (Hercules, CA) 1024 CLSM beam scanning system equipped with a Nikon (Melville, NY) Optiphot II upright microscope (x60 oil immersion objective). Images were collected sequentially to avoid cross-contamination between the fluorochromes. The colocalization analysis was performed with the laser sharp 1024 software.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
In vitro and in vivo characterization of anti-Syk G4G11 and G4E4 scFv

A human synthetic scFv library was screened with a GST-Syk fusion protein containing N- and C-terminal SH2 domains of murine Syk and the linker region that separates them (24), and a panel of monoclonal scFv fragments specific to Syk was isolated and characterized. The aim of our study was the targeting of Syk in vivo, and for that purpose we needed scFv fragments that exhibit good solubility and stability in the cytoplasm of mammalian cells. To identify the potential Syk in vivo binders, we used a yeast two-hybrid selection system (25) as an intermediate selection step after the in vitro selection and before the expression in mammalian cells. This additional selection step permitted to identify two scFv, named G4G11 and G4E4, which showed the best binding to Syk in the cytoplasm of yeast cells (Fig. 1GoA, rows 1 and 2). Another scFv named G6G2 that was previously characterized for its binding to Syk in vitro (24) failed to interact with Syk in the cytoplasm of yeast cells (Fig. 1GoA, row 3). The sequence analysis of the genes encoding G4G11 and G4E4 showed that their VH domains were 100% identical and belonged to the VH4 family, and that their VL domains belonged to VL1 family and differed in 7 aa. Nucleotide sequences of the scFv G4E4 and G4G11 are available from GenBank under accession nos. AF401619 and AF401620, respectively (Fig. 1GoB). We measured kinetics and affinity of G4G11 and G4E4 binding to immobilized Syk by BIAcore analysis. The affinity (Kd) of scFv G4G11 was estimated at 50 nM with the on-rate calculated as 1.3 x 104 M-1 s-1 and the off-rate as 5.5 x 10-4 s-1; the affinity of G4E4 was estimated at 80 nM with the on-rate calculated as 5.6 x 103 M-1 s-1 and the off-rate as 4.5 x 10-4 s-1.



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  		FIGURE 1. In vivo and in vitro characterization of anti-Syk scFv fragments isolated from a phage display library. A, Interaction of Syk Ag with the anti-Syk scFv in a yeast two-hybrid selection system. In rows 1–3, three anti-Syk scFv (G4E4, G4G11, and G6G2) were cotransfected with the Syk/BTM116 bait. In rows 4–6, the same scFv were cotransfected with an irrelevant Ag bait (Lamin/BTM116). {beta}-gal activation was observed only in rows 1 and 2. B, Analysis of amino acid sequences of scFv G4G11 and G4E4. Identities are shown by dashed lines. The differences are indicated by bold letters. C, A total of 10 µg G4G11, G4E4, and the irrelevant scFv was used for the immunoprecipitation of Syk from not stimulated (-) and IgE/DNP-stimulated (+) RBL-2H3 cells. The immune complexes were analyzed by SDS-PAGE and immunoblotting with anti-Syk Abs and with 9E10 mAb. The membrane was stripped and blotted with anti-phosphotyrosine (anti-PY) Abs. D, G4G11 and G4E4 scFv were used in parallel with a polyclonal anti-Zap-70 Ab to immunoprecipitate Zap-70 from the lysates of Jurkat T cell line. The immune complexes were analyzed by SDS-PAGE and immunoblotting with anti-Zap-70 mAb for the upper section of the membrane, and with 9E10 mAb for the lower section.

 
In vitro, G4G11 and G4E4 bind specifically to Syk from human, mouse, and rat origin (Ref. 24 and unpublished data). Fig. 1GoC illustrates the immunoprecipitation of Syk from lysates of unstimulated and Fc{epsilon}RI-stimulated RBL-2H3 rat basophilic leukemia cell line, and shows that G4G11 and G4E4 bind to the unphosphorylated and tyrosine-phosphorylated Syk. The specificity of G4G11 and G4E4 for Syk was further analyzed in immunoprecipitation experiments performed on human and mouse T lymphoma cell lines and showed that neither of the scFv binds to Zap-70, the other member of the Syk family of PTKs and closely related to Syk (Fig. 1GoD and unpublished data). Consequently, based on the convincing performance of scFv G4G11 and G4E4 in binding specifically to Syk in vivo and in vitro, we selected them for the expression in mammalian cells.

Establishment of RBL-2H3 cell lines with stable expression of G4G11 and G4E4 scFv

For the intracellular targeting of Syk, we chose as a model system the RBL-2H3 cell line that has been extensively used to study the role of Syk in Fc{epsilon}RI-mediated signal transduction. The cDNA encoding the scFv were cloned into pscFvexp-cyt vector (26) and transfected into the RBL-2H3 cell line. This version of the vector directs the expression of the scFv in the cytoplasm of transfected cells, and permits their detection with the mAb 9E10 via a C-terminal c-myc tag. Cloned lines were established, and the expression of the scFv and Syk was analyzed by immunofluorescence. Our observations revealed a cytosolic distribution for G4G11 and G4E4 without any aggregates that are typically observed with insoluble scFv fragments expressed in the cytoplasm of mammalian cells (Fig. 2GoA). Next, the total lysates of the clones that exhibited a strong fluorescence staining were analyzed by SDS-PAGE and 9E10 mAb immunoblotting (Fig. 2GoB). Bands corresponding to G4G11 and G4E4 migrating at ~30 kDa were detected, and their intensities revealed that the expression level of G4E4 was higher than that of G4G11. For further analysis, at least two cell lines transfected with each cDNA were examined, although all figures present the results from only one representative clone. All the following experiments were done in triplicates.



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  		FIGURE 2. Stable expression of scFv G4G11 and G4E4 in RBL-2H3 cell line and their intracellular association with Syk. A, RBL-2H3 cell lines expressing G4G11, G4E4, and the irrelevant scFv were fixed and permeabilized, as described in Materials and Methods. The subcellular localization of the scFv and Syk was analyzed by immunofluorescence using confocal microscopy. B, Total cellular lysates of WT RBL-2H3 and clones expressing G4G11 and G4E4 scFv were analyzed by SDS-PAGE and immunoblotting with 9E10 mAb to detect the scFv. C, Immunoprecipitations were performed on lysates of WT and transfected RBL-2H3 cells using a preformed complex of GammaBind G Sepharose-9E10 mAb. The immune complexes were analyzed by SDS-PAGE and immunoblotting with anti-Syk Abs for the upper section of the membrane, and with 9E10 mAb for the lower section.

 
To examine the association of intracellular scFv with Syk, the scFv polypeptides were immunoprecipitated from lysates of RBL-2H3 cell lines using 9E10 mAb. The immune complexes were analyzed by SDS-PAGE and revealed by immunoblotting with 9E10 mAb to detect the scFv, and with a polyclonal anti-Syk Ab to check the coimmunoprecipitation of Syk with the scFv (Fig. 2GoC). A band corresponding to Syk was present in the cell lines expressing G4G11 and G4E4, indicating that Syk existed in complex with the intracellular scFv. Next, the interaction of the scFv with Syk in intact cells was analyzed using confocal microscopy. As shown in Fig. 2GoA, there is a convincing colocalization of Syk with G4G11 and G4E4 vs the irrelevant scFv.

Taken together, these observations suggested that the intracellular scFv interact with Syk in the cytoplasm of the transfected cells.

Intracellular G4G11 and G4E4 scFv inhibit the propagation of Fc{epsilon}RI signaling

To begin to examine the consequences of the intracellular association of the scFv with Syk, we studied the effector function of RBL-2H3 cell lines by examining Fc{epsilon}RI-induced degranulation, a pathway in which Syk is essential (11, 13). Cells were sensitized with IgE/DNP, and the exocytosis of inflammatory mediators was monitored by measuring serotonin and {beta}-hexosaminidase release in culture supernatants (Fig. 3GoA). In clones expressing G4G11 and G4E4 scFv, the Fc{epsilon}RI-induced serotonin and {beta}-hexosaminidase release were dramatically reduced in comparison with the control RBL-2H3 cells. However, cells still maintained the capacity to degranulate by nonreceptor-mediated stimulation with the calcium ionophore ionomycin.



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  		FIGURE 3. Inhibitory properties of intracellular G4G11 and G4E4 scFv. A, RBL-2H3 cell lines were stimulated with IgE/DNP, and {beta}-hexosaminidase and serotonin release were measured as described in Materials and Methods. Cells were treated with the calcium ionophore ionomycin to measure {beta}-hexosaminidase release in response to a nonreceptor-mediated stimulation. B, IgE-sensitized RBL-2H3 cell lines were loaded with Fluo-3 and stimulated with DNP-BSA ({uparrow}) to monitor the overall Ca2+ responses in the presence of extracellular Ca2+. To specifically measure the release of Ca2+ from ER stores, loaded cells were treated with EGTA (->) and then stimulated with DNP-BSA. To measure Ca2+ mobilization in response to a nonreceptor-mediated stimulation, cells were treated with the calcium ionophore ionomycin ({uparrow}). The figure represents the mean fluorescence of the entire population as a function of time. C, WT and transfected RBL-2H3 cell lines were either not stimulated (-) or stimulated with IgE/DNP (+). Total cellular lysates were analyzed by immunoblotting with anti-phosphotyrosine (anti-PY) Abs. The position of the proteins that exhibit reduced tyrosine phosphorylation in G4G11- and G4E4-expressing cell lines is indicated on the right.

 
Previous studies showed that Ca2+ influx is required for Ag-induced secretion in RBL-2H3 cells (27). The inhibition of degranulation observed in cell lines expressing G4G11 and G4E4 scFv suggested a defect in calcium mobilization. To test for activation-induced intracellular calcium elevation, fura 2-loaded cells were stimulated with IgE/DNP, and the rise of intracellular Ca2+ concentration was measured (Fig. 3GoB). Although all cell lines exhibited similar responses to the calcium ionophore ionomycin, the Ca2+ response in cell lines expressing G4G11 and G4E4 scFv indicated that the primary release of Ca2+ from ER stores was normal, but cells did not maintain the sustained intracellular Ca2+ elevation due to the influx of Ca2+. To confirm that the release of Ca2+ from ER stores was not altered, cells were activated in medium containing EGTA to chelate extracellular Ca2+. This experiment showed that the peak and morphology of the fluorescence ratio reflecting the initial phase of intracellular Ca2+ mobilization were unaltered, and confirmed that the defect in the calcium response of clones expressing G4G11 and G4E4 scFv was mainly due to the reduced extracellular calcium influx. FACS analysis showed that the cell surface expression of Fc{epsilon}RI in these cell lines was similar to that in the parental RBL-2H3 cells (data not shown).

Syk was shown to be primarily responsible for overall induction of tyrosine phosphorylation upon Fc{epsilon}RI stimulation (11, 13). Therefore, we next studied the pattern of tyrosine phosphorylation of cellular proteins. Cells were sensitized with IgE/DNP, and total cell lysates were analyzed by SDS-PAGE and anti-phosphotyrosine immunoblotting (Fig. 3GoC). Fc{epsilon}RI stimulation induced an increase in tyrosine protein phosphorylation in clones expressing G4G11 and G4E4 scFv to levels similar to those of parental RBL-2H3 cells, except for proteins migrating at ~67–75 kDa. These results indicated that the association of Syk with the intracellular scFv did not have a global effect on the receptor-mediated tyrosine phosphorylation of cellular proteins. Nevertheless, the defect observed in calcium mobilization suggested that the phosphorylation of a subset of proteins implicated in the regulation of calcium responses and degranulation may have been altered.

Fc{epsilon}RI-induced tyrosine phosphorylation of PLC-{gamma}2 is markedly reduced

In RBL-2H3 cells, Fc{epsilon}RI-induced tyrosine phosphorylation and activation of PLC-{gamma}2 are dependent on Syk (13), and lead to the generation of IP3 and the subsequent calcium mobilization that areessential and sufficient signals for the secretory responses to Fc{epsilon}RI aggregation. Therefore, we studied the tyrosine phosphorylation pattern of PLC-{gamma}2 after Fc{epsilon}RI aggregation. PLC-{gamma}2 was immunoprecipitated from lysates of unstimulated and IgE/DNP-stimulated cell lines, and the immune complexes were probed with anti-phosphotyrosine Abs (Fig. 4GoA). Fc{epsilon}RI stimulation still induced tyrosine phosphorylation of PLC-{gamma}2 in cells expressing anti-Syk scFv, although its extent was markedly reduced (about half of that of control cell lines). This difference was not due to differing amounts of PLC-{gamma}2 in the immunoprecipitations, as stripping the membrane and reprobing with anti-PLC-{gamma}2 Abs revealed similar amounts in PLC-{gamma}2 immunoprecipitates.



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  		FIGURE 4. Fc{epsilon}RI-mediated tyrosine phosphorylation of PLC-{gamma}2 is inhibited. Cells from WT and transfected RBL-2H3 were either not stimulated (-), or activated with IgE/DNP (+) and then solubilized with Nonidet P-40 lysis buffer. A, PLC-{gamma}2 was immunoprecipitated with anti-PLC-{gamma}2 Abs, and the immune complexes were separated by 8% SDS-PAGE and analyzed by immunoblotting with anti-phosphotyrosine (anti-PY) Abs. The membrane was stripped and reblotted with anti-PLC-{gamma}2 Abs. B, Total cellular lysates were analyzed by immunoblotting with anti-phospho-p44/42 MAPK Abs. The membrane was stripped and reprobed with anti-p44/42 MAPK Abs. C, Total cellular lysates were analyzed by immunoblotting with Abs directed against the phospho-Ser473 of the activated Akt. The membrane was stripped and reprobed with anti-Akt Abs.

 
Syk has been shown to be involved in at least two divergent signaling pathways resulting from Fc{epsilon}RI stimulation. The first is activation of PLC-{gamma}2, and the second pathway involves Ras with subsequent phosphorylation and activation of MAPK (28). Therefore, we investigated the consequences of the association of intracellular scFv with Syk on the activation of MAPK (Fig. 4GoB). Cross-linking of Fc{epsilon}RI resulted in activation of MAPK to levels comparable in WT and transfected cells, as measured by immunoblotting of cell lysates with an anti-phospho-MAPK Ab that recognizes only the activated forms of MAPK. These results indicated that G4G11 and G4E4 did not prevent the coupling of Syk to the Ras pathway, but they selectively inhibited the pathway involving PLC-{gamma} activation.

Previous studies have shown that the activation of phosphatidylinositol 3-kinase (PI 3-kinase) is dependent on Syk (29). To evaluate PI 3-kinase activity, we tested the activation of the Ser/Thr kinase Akt/PKB, a readout of the PI 3-kinase pathway (30). Using Abs that specifically recognize the P-Ser473 of the activated Akt, we found that in cell lines expressing G4G11 and G4E4, Akt phosphorylation following Fc{epsilon}RI stimulation was comparable with the control cell lines (Fig. 4GoC). A basal level of Akt phosphorylation was observed in all the unstimulated G4E4-expressing cell lines that were examined. These results indicated that intracellular G4G11 and G4E4 did not inhibit the Fc{epsilon}RI-induced activation of the PI 3-kinase.

Fc{epsilon}RI-induced tyrosine phosphorylation and activation of Syk and Lyn are not altered

It has been shown that the concerted action of Syk and Btk PTKs is required for tyrosine phosphorylation of PLC-{gamma}2 and its full activation (13, 31, 32, 33). The PTK Lyn has also been implicated in the regulation of Ag receptor-coupled Ca2+ mobilization (12, 16), and is essential for the phosphorylation of Fc{epsilon}RI-{beta} and Fc{epsilon}RI-{gamma} chains, and that of Syk and Btk, immediately after Fc{epsilon}RI stimulation (3, 10, 15, 34). Therefore, we wished to control the functional status of Lyn in the cell lines expressing G4G11 and G4E4 scFv. In vitro kinase assay was performed on Lyn immunoprecipitates, and showed that Lyn autophosphorylation, as demonstrated by the presence of two characteristic bands (p53 and p56) and the transphosphorylation of the exogenous substrate enolase, was similar in cell lines expressing G4G11 and G4E4 scFv and in the control cell lines (Fig. 5GoA). These results were consistent with normal tyrosine phosphorylation of Lyn immunoprecipitates after Fc{epsilon}RI stimulation (data not shown) and indicated that the intracellular anti-Syk scFv did not affect Fc{epsilon}RI-induced activation of Lyn.



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  		FIGURE 5. Fc{epsilon}RI-induced tyrosine phosphorylation and activation of Syk and Lyn are not inhibited. A, Cells from WT and transfected RBL-2H3 were either not stimulated (-), or activated with IgE/DNP (+) and then solubilized with Nonidet P-40 lysis buffer. Lyn was immunoprecipitated with anti-Lyn Abs. Immune complexes were subjected to in vitro kinase assays in the presence of the substrate enolase, then separated by SDS-PAGE and transferred onto nitrocellulose membrane, and radiolabeled Lyn and enolase were detected by autoradiography. The same membrane was then blotted with anti-Lyn Abs. B, Cells from WT and transfected RBL-2H3 were either not stimulated (-), or activated with IgE/DNP (+) and then solubilized with modified radioimmunoprecipitation assay lysis buffer (see Materials and Methods). Syk was immunoprecipitated with anti-Syk Abs. Immune complexes were subjected to in vitro kinase assays in the presence of the substrate GST-HS1, then separated by SDS-PAGE and transferred onto nitrocellulose membrane, and radiolabeled Syk and GST-HS1 were detected by autoradiography. The same membrane was then blotted with anti-Syk Abs, stripped, and reprobed with anti-phosphotyrosine (anti-PY) Abs.

 
Fc{epsilon}RI aggregation results in tyrosine phosphorylation and increased kinase activity of Syk. Therefore, we next analyzed the consequences of the association of intracellular scFv on Syk activation. The effect of Fc{epsilon}RI stimulation on the level of Syk tyrosine phosphorylation was examined by immunoprecipitating Syk from unstimulated and IgE/DNP-stimulated cell lines, and immunoblotting with anti-phosphotyrosine Abs (Fig. 5GoB). In cell lines expressing G4G11 and G4E4 scFv, Syk underwent an increase in tyrosine phosphorylation upon Fc{epsilon}RI stimulation to a level comparable with the Syk in control cell lines. The same blot was stripped and probed with anti-Syk Abs to show that equal amounts of Syk were present in anti-Syk immunoprecipitates. We evaluated the functional activity of Syk in an immune complex kinase assay using the GST-HS1 protein as exogenous substrate (Fig. 5GoB). We found that the enzymatic activity of Syk and the extent of its autophosphorylation in transfected cell lines were similar to the WT Syk. These data indicated that despite the association of the intracellular scFv with the SH2 domains of Syk, the recruitment of Syk to the receptor and its subsequent phosphorylation and activation were not affected.

Fc{epsilon}RI-induced tyrosine phosphorylation and activation of Btk are inhibited

The full activation of PLC-{gamma}2 requires its maximum tyrosine phosphorylation by Btk (31, 32, 33). It has been shown that Btk activation and the subsequent Btk-dependent PLC-{gamma}2 tyrosine phosphorylation require the tyrosine phosphorylation of Btk by the concerted action of Lyn and Syk kinases and by autophosphorylation (15, 34, 35, 36).

To further investigate the defect in the tyrosine phosphorylation of PLC-{gamma}2, we next examined Btk tyrosine phosphorylation and activation following Fc{epsilon}RI cross-linking. Btk was immunoprecipitated from unstimulated and IgE/DNP-stimulated cells, and analyzed by immunoblotting with anti-phosphotyrosine Abs (Fig. 6Go). In cell lines expressing scFv G4G11 and G4E4, Btk tyrosine phosphorylation following receptor stimulation was inhibited. When we evaluated the functional activity of Btk in an immune complex kinase assay using enolase as exogenous substrate, we found a decreased Btk enzymatic activity compared with the WT Btk. These results indicate that the inhibition of tyrosine phosphorylation of PLC-{gamma}2 and the subsequent reduced calcium influx were at least partly due to the inhibition of Btk enzymatic activity. Considering that the enzymatic activities of Syk and Lyn were intact, our data suggest that G4G11 and G4E4 impair the recruitment of Btk and PLC-{gamma}2 to the vicinity of Syk for their tyrosine phosphorylation and activation.



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  		FIGURE 6. Fc{epsilon}RI-induced tyrosine phosphorylation and activation of Btk are inhibited. Cells from WT and transfected RBL-2H3 were either not stimulated (-), or activated with IgE/DNP (+) and then solubilized with Nonidet P-40 lysis buffer. Btk was immunoprecipitated with anti-Btk Abs, and immune complexes were analyzed by SDS-PAGE and immunoblotting with anti-phosphotyrosine (anti-PY) Abs. The membrane was stripped and reprobed with anti-Btk Abs. To measure the enzymatic activity of Btk, the Btk immunoprecipitates were subjected to in vitro kinase assay with enolase used as exogenous substrate, then separated by SDS-PAGE and transferred onto nitrocellulose membrane, and radiolabeled enolase was detected by autoradiography.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In mast cells, the aggregation of Fc{epsilon}RI results in SH2 domain-mediated binding of Syk to the phosphorylated ITAMs of Fc{epsilon}RI and an increase in its phosphotyrosine content and its kinase activity. Recently, mutational studies showed that SH2 domain-mediated targeting of Syk to the plasma membrane is essential for Fc{epsilon}RI-mediated activation of Syk and downstream signaling events leading to secretion of allergic mediators (37).

In this study, the intracellular Ab technology (38) was used as a new approach for the dissection of the functions of Syk. Our aim was to target Syk with scFv fragments directed against its SH2 domains. As Syk is a cytosolic protein, the major problem to be encountered was the stability of the scFv in the cytoplasm of the cells. The successful expression of scFv fragments in the cell cytoplasm is influenced by various parameters (reviewed in Refs. 38 and 39). First, scFv fragments lack the disulfide bonding in the reducing environment of the cell cytoplasm (40, 41). Therefore, only the scFv that are intrinsically more stable will tolerate the loss of interchain disulfide bond, and will remain folded (42, 43). Second, other factors such as good solubility vs propensity to aggregate, cellular t1/2, and others (44) contribute also for efficient interaction with Ag in vivo. No rules or consistent predictions can yet be made about those scFv that will tolerate the cell cytoplasm conditions. Our strategy was to use an Ab-Ag two-hybrid selection system (25) to select the potential Syk in vivo binders. This experiment revealed that all the scFv that bound Syk in vitro did not necessarily bind to Syk in the yeast two-hybrid system, and only two scFv named G4G11 and G4E4 showed a very good performance in binding to Syk in the cytoplasm of yeast cells (Fig. 1GoA). Considering that in vitro, G4G11 and G4E4 were also able to bind specifically to Syk (Fig. 1Go), we selected them for the expression in mammalian cells.

Among the tyrosine residues of Syk that are phosphorylated following the receptor engagement, the tyrosine 130 is a prominent and early site of Syk autophosphorylation, and is localized within the inter-SH2 domain region. This is the only known tyrosine present in the portion of Syk that was used for the selection of the scFv, and it is implicated in the modulation of the ability of Syk to interact with the ITAMs of the Ag receptor and in the regulation of its intrinsic activity (45, 46). Therefore, in theory, the binding of the scFv to Syk may have the following consequences: 1) the inhibition of the recruitment of Syk to the ITAMs, resulting in the inhibition of Fc{epsilon}RI signaling; 2) the inhibition of the association of Syk with an effector molecule that shares the same binding site as the scFv on Syk; 3) alternatively, the creation of a steric hindrance by the scFv, preventing either the association of effector molecules or their phosphorylation by Syk. In addition to these options, all Syk molecules may not be stoichiometrically blocked by the scFv fragments, mainly due to the proportion of the active scFv molecules in the pool of the soluble cytosolic scFv.

To study the ability of G4G11 and G4E4 to impair the functions of Syk in vivo, we produced stable transfectants of RBL-2H3 cell line expressing cytosolic G4G11 and G4E4. The coimmunoprecipitation of Syk with the intracellular G4G11 and G4E4 (Fig. 2GoC), and the colocalization of Syk with the scFv in intact cells analyzed by confocal microscopy (Fig. 2GoA) strongly supported an interaction of Syk with the intracellular scFv.

Next, the activation status of Syk from the transfected cell lines was analyzed and indicated that the scFv do not affect the tyrosine phosphorylation and the activation of Syk (Fig. 5GoB), nor the overall tyrosine phosphorylation of cellular proteins known to be dependent on Syk, except for proteins migrating at ~67–75 kDa (Fig. 3GoC). Moreover, a tyrosine-phosphorylated form of Syk was coprecipitated with the intracellular G4G11 and G4E4 from lysates of IgE/DNP-activated cells (data not shown). These results suggested that the association of the scFv with Syk is independent of the phosphorylation status of Syk and/or does not affect the phosphorylation of Syk following the Fc{epsilon}RI engagement. Taken together, our data indicate that the binding of the scFv to the SH2 domains of Syk does not impair the Fc{epsilon}RI-induced recruitment of Syk to the phosphorylated ITAMs and the subsequent increase in its kinase activity.

Nevertheless, in cell lines expressing G4G11 and G4E4 scFv, Fc{epsilon}RI-mediated Ca2+ mobilization and the release of inflammatory mediators are inhibited (Fig. 3Go), and are consistent with a defect in the tyrosine phosphorylation of PLC-{gamma}2 (Fig. 4GoA) and Btk (Fig. 6Go). Both Syk and Btk are required for the full tyrosine phosphorylation of PLC-{gamma}2, with Btk phosphorylating the subset of tyrosine residues of PLC-{gamma}2 that are needed to achieve the maximal enzymatic activity for the induction of the extracellular influx (31, 32). In the cell lines expressing G4G11 and G4E4 scFv, the reduced Ca2+ influx correlates with the inhibition of Btk kinase activity, indicating that the scFv inhibit the tyrosine phosphorylation of Btk and PLC-{gamma}2 and their full activation.

The anchoring of Btk and PLC-{gamma} at the membrane at the receptor complex for these enzymes is partly due to the interaction of their pleckstrin homology and SH2 domains with PtdIns-3,4,5-P3, the product of the PI 3-kinase (47, 48, 49, 50, 51, 52). Syk controls the activation of the PI 3-kinase via the tyrosine phosphorylation of the adaptor protein Cbl (29). PtdIns-3,4,5-P3 also binds to the pleckstrin homology domain of the Ser/Thr kinase Akt/PKB and contributes to the activation of the enzyme (30). In the cell lines expressing G4G11 and G4E4, PI 3-kinase activity evaluated indirectly by examining the Akt phosphorylation was intact (Fig. 4GoC), and correlated with the normal tyrosine phosphorylation of Cbl immunoprecipitates following Fc{epsilon}RI stimulation (data not shown). These results indicate that intracellular G4G11 and G4E4 did not affect PI 3-kinase activation and the production of PtdIns-3,4,5-P3 that is necessary for Btk and PLC-{gamma} membrane localization and activation.

Considering that G4G11 and G4E4 scFv are directed against a portion of Syk that contains its SH2 domains, we wished to check their possible interaction with the SH2 domains of Btk and PLC-{gamma}2. For that purpose, we first used purified G4G11 and G4E4 scFv as reagents in immunoprecipitation experiments performed on lysates of RBL-2H3 cells. Second, the lysates of RBL-2H3 cell lines expressing G4G11 and G4E4 were subjected to immunoprecipitation experiments to detect a coprecipitation of the intracellular scFv with either Btk or PLC-{gamma}2. In both cases, neither Btk nor PLC-{gamma}2 was present in the immune complexes (data not shown), suggesting that the inhibition of Btk and PLC-{gamma}2 is not due to their interaction with the scFv. Taken together, our observations suggest that the binding of the scFv to Syk does not affect the kinase activity of Syk, but rather inhibits the link between Syk and Btk and PLC-{gamma}2 for their subsequent tyrosine phosphorylation and activation.

Studies in B cells have shown that the targeting of Btk and PLC-{gamma}2 to the proximity of Syk and Lyn is mediated by the interaction of their SH2 domains with the tyrosine-phosphorylated adaptor molecule SLP-65/B cell linker protein, a substrate of Syk that integrates the activity of Syk and Btk into downstream effectors such as PLC-{gamma}2 (53, 54, 55, 56, 57). In mast cells, the precise molecular mechanism by which Syk effectively phosphorylates Btk and PLC-{gamma}2 remains unclear. Mast cells express the adaptor molecule SLP-76, functionally and structurally related to SLP-65 and the linker for the activation of T cells (LAT), both tyrosine phosphorylated by Syk upon Fc{epsilon}RI stimulation (58, 59). It has been shown that LAT and SLP-76 are components of a macromolecular signaling complex at the plasma membrane that regulates Fc{epsilon}RI-mediated activation of PLC-{gamma} and subsequent calcium mobilization and degranulation (58, 59, 60, 61). It is possible that tyrosine-phosphorylated LAT and SLP-76 interact with SH2 domains of PLC-{gamma}2 and Btk, and recruit them to the vicinity of Syk. According to this scenario, intracellular G4G11 and G4E4 may have inhibited the phosphorylation of either LAT, SLP-76, or another adaptor molecule that bridges Syk to downstream effectors. Arguments in favor of this hypothesis are: 1) the analysis of cellular lysates of Fc{epsilon}RI-stimulated cells revealed decreased tyrosine phosphorylation of proteins migrating at 67–75 kDa in G4G11- and G4E4-expressing cell lines (Fig. 3GoC); 2) the signaling defects observed in response to IgE cross-linking in the cell lines expressing G4G11 and G4E4 are similar to those observed in the bone marrow-derived mast cells from mice deficient in SLP-76 and LAT, i.e., reduced PLC-{gamma} tyrosine phosphorylation, calcium mobilization, and granule release (61, 62). Finally, we cannot rule out that the intracellular scFv may have altered the direct binding of Syk with PLC-{gamma} and Btk for their subsequent tyrosine phosphorylation. Additional experiments, including the determination of the binding site of the scFv on Syk, are necessary to better understand the scFv inhibition mechanism.

Interestingly, the strong similarities in the signaling defects resulting from the expression of G4G11 and G4E4 suggest that both scFv recognize the same epitope on Syk. This observation is not very surprising considering the high homology of the amino acid sequences of the two scFv, and their comparable affinities for Syk. Nevertheless, although the expression level of G4E4 is higher than that of G4G11, Fc{epsilon}RI function is blocked to the same level in the cell lines expressing them, and in both cases, the defects are milder than those of Syk-deficient mast cells. These effects may be due: first, to the interaction of the scFv only with the SH2 domains of Syk and not with the entire molecule; and second, to other factors such as the folding and the stability of the cytosolic scFv that may influence the stoichiometry and the turnover of their association with Syk.

It is worth noting that we failed to establish stable transfectants of RBL-2H3 cells expressing another anti-Syk scFv named G6G2. The fact that G6G2 does not bind Syk in the yeast cells (Fig. 1GoA, row 3) suggests that the yeast two-hybrid selection system offers a valuable tool for the identification of those scFv that may be stably expressed in mammalian cells.

In conclusion, this study demonstrates that the intracellular immunization is an attractive approach to study the functions of Syk without eliminating Syk and, most of all, receptor signaling, as that was the case of all the methods used to date to study Syk functions (11, 13, 63, 64, 65, 66). More generally, the intracellular expression of scFv fragments directed against specific sequences or critical regions of proteins may be an alternative to study the functions of newly identified molecules. Finally, scFv G4G11 and G4E4 reported in this study may represent potential therapeutic intervention tools in allergy and other immune diseases.


   Acknowledgments
 
We are grateful to Drs. H. Metzger (National Institutes of Health), M. Neuberger (Medical Research Council), and P. Martineau (Unité Mixte de Recherche 5094 Centre National de la Recherche Scientifique) for critical reading of the manuscript and helpful comments. We thank Drs. P. Bruhns and M. Daëron (Institut Curie) for having taught us Ca2+ measurement techniques. We thank Drs. N. Lautredou and M. C. Guérin for their expert assistance with confocal microscopy and BIAcore analyses, respectively. We are indebted to F. Sordat for his valuable advice and support throughout this study.


   Footnotes
 
1 This work was supported by the EEC BIOTECH Grant BIO4-CT97-2285, the Association pour la Recherche contre le Cancer Grant 9232, Center National pour la Recherche Scientifique, Ministère de l’Enseignement Supérieur et de la Recherche, and Université Montpellier II. Back

2 Address correspondence and reprint requests to Dr. Piona Dariavach, CRLC Val d’Aurelle, Bât. de Recherche, Unité Mixte de Recherche 5094, Centre National de la Recherche Scientifique, 35 rue de la Croix Verte, 34298 Montpellier Cedex 5, France. E-mail address: pdariavach{at}valdorel.fnclcc.fr Back

3 Abbreviations used in this paper: PTK, protein tyrosine kinase; {beta}-gal, {beta}-galactosidase; Btk, Bruton’s tyrosine kinase; ER, endoplasmic reticulum; IP3, inositol 1,4,5-triphosphate; ITAM, immunoreceptor tyrosine-based activation motif; LAT, linker for activation of T cells; MAPK, mitogen-activated protein kinase; PI 3-kinase, phosphatidylinositol 3-kinase; PKB, protein kinase B; PLC, phospholipase C; scFv, single-chain variable fragment; SH2, Src homology 2; TNP, trinitrophenyl; WT, wild type. Back

Received for publication August 13, 2001. Accepted for publication June 20, 2002.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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S. W. Ng, J. di Capite, K. Singaravelu, and A. B. Parekh
Sustained Activation of the Tyrosine Kinase Syk by Antigen in Mast Cells Requires Local Ca2+ Influx through Ca2+ Release-activated Ca2+ Channels
J. Biol. Chem., November 14, 2008; 283(46): 31348 - 31355.
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