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Signaling in the RBL-2H3 Cell Line RI Ε Domains of Syk Partially Inhibit Fc

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Signaling in the RBL-2H3 Cell Line RI Ε Domains of Syk Partially Inhibit Fc Intracellular Single-Chain Variable Fragments Directed to the Src Homology 2 Domains of Syk Partially Inhibit FcεRI Signaling in the RBL-2H3 Cell Line This information is current as of September 27, 2021. Stéphanie Dauvillier, Peggy Mérida, Michela Visintin, Antonino Cattaneo, Christian Bonnerot and Piona Dariavach J Immunol 2002; 169:2274-2283; ; doi: 10.4049/jimmunol.169.5.2274 http://www.jimmunol.org/content/169/5/2274 Downloaded from References This article cites 65 articles, 37 of which you can access for free at: http://www.jimmunol.org/content/169/5/2274.full#ref-list-1 http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication by guest on September 27, 2021 *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2002 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology Intracellular Single-Chain Variable Fragments Directed to the Src Homology 2 Domains of Syk Partially Inhibit Fc⑀RI Signaling in the RBL-2H3 Cell Line1 Ste´phanie Dauvillier,* Peggy Me´rida,* Michela Visintin,† Antonino Cattaneo,† Christian Bonnerot,‡ and Piona Dariavach2* 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 Downloaded from 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 intra- cellular G4G11 and G4E4 bind to Syk, but do not inhibit the activation of Syk following Fc⑀RI aggregation, suggesting that the scFv do not affect the recruitment of Syk to the receptor. Nevertheless, Fc⑀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-␥2 tyrosine phosphorylation and activation. Interestingly, Fc⑀RI-induced mitogen-activated protein kinase phosphorylation is not http://www.jimmunol.org/ 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-␥2 activation. The Journal of Immunology, 2002, 169: 2274–2283. n basophils and mast cells, cross-linking the high affinity IgE of tyrosine residues in their activation loops and by conformational receptor, Fc⑀RI, results in a number of biochemical events changes in the case of Syk (9, 10). Active Lyn and Syk phosphor- I leading to the release of a panel of proinflammatory media- ylate themselves and other protein substrates such as phospho- tors. Fc⑀RI signal transduction is mediated by three distinct fam- lipase C (PLC)-␥ and Btk (11–15). Hydrolysis of phosphatidyl- ilies of cytoplasmic protein tyrosine kinase (PTK)3: the Src family inositol 4,5-biphosphate by PLC-␥ generates two second by guest on September 27, 2021 PTK Lyn, the Syk family PTK Syk, and Bruton’s tyrosine kinase messengers, inositol 1,4,5-triphosphate (IP3) and diacylglycerol. (Btk)/Tec. The PTK Lyn is activated by transphosphorylation upon 2ϩ IP3 mobilizes calcium (Ca ) from intracellular storage sites, and Fc⑀RI cross-linking (1). Activated Lyn phosphorylates the tyrosine diacylglycerol together with Ca2ϩ activates protein kinase C. Both residues in the immunoreceptor tyrosine-based activation motifs Ca2ϩ and protein kinase C are required for optimal mast cell ⑀ ␤ ⑀ ␥ (ITAM) (2) of the cytoplasmic regions of Fc RI- and Fc RI- degranulation. subunits (3–5), enabling the recruitment of Lyn and Syk through The Syk family of cytoplasmic PTKs comprises two known Src homology (SH)2 domain-phosphotyrosine interactions (3, members termed Syk and Zap-70. Syk is present in most hemo- 6–8). Newly recruited PTKs are activated by transphosphorylation poietic cell types, including B cells and mast cells. The importance of Syk to receptor-mediated signaling in hemopoietic cells is un- derscored by the signaling defects observed in Syk-deficient vari- *Institut de Ge´ne´tique Mole´culaire de Montpellier, Unite´Mixte de Recherche 5535 ants of the chicken B cell line DT-40 and the rat basophilic leu- Centre National de la Recherche Scientifique, Montpellier, France; †International School for Advanced Studies, Neuroscience Program, Trieste, Italy; and ‡Institut kemia cell line RBL-2H3 (13, 16). The creation of Syk-deficient Curie, Unite´520, Institut National de la Sante´et de la Recherche Me´dicale, Paris, mice by homologous recombination has also highlighted the im- France portance of Syk in developmental processes (17, 18). The structure Received for publication August 13, 2001. Accepted for publication June 20, 2002. of Syk includes from the N to the C terminus: 1) two SH2 domains, 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 which bind doubly phosphorylated ITAMs (19); 2) a linker region, with 18 U.S.C. Section 1734 solely to indicate this fact. containing sites of tyrosine phosphorylation that are direct binding 1 This work was supported by the EEC BIOTECH Grant BIO4-CT97-2285, the As- sites for SH2 domains and phosphotyrosine-binding domains of sociation pour la Recherche contre le Cancer Grant 9232, Center National pour la signaling molecules, including PLC-␥, Vav, and Cbl (20–22); 3) a Recherche Scientifique, Ministe`re de l’Enseignement Supe´rieur et de la Recherche, and Universite´Montpellier II. catalytic domain, including sites for ATP-binding and tyrosine 2 Address correspondence and reprint requests to Dr. Piona Dariavach, CRLC Val phosphorylation; and 4) a short C-terminal extension of yet unde- d’Aurelle, Baˆt. de Recherche, Unite´Mixte de Recherche 5094, Centre National de la termined function. Recherche Scientifique, 35 rue de la Croix Verte, 34298 Montpellier Cedex 5, France. In this study, we report the use of intracellular Ab technology to E-mail address: [email protected] target Syk. We established stable transfectants of RBL-2H3 cell 3 Abbreviations used in this paper: PTK, protein tyrosine kinase; ␤-gal, ␤-galactosi- line that express in their cytoplasm single-chain variable fragment dase; Btk, Bruton’s tyrosine kinase; ER, endoplasmic reticulum; IP3, inositol 1,4,5- triphosphate; ITAM, immunoreceptor tyrosine-based activation motif; LAT, linker (scFv) Abs directed against the SH2 domains of Syk. We studied for activation of T cells; MAPK, mitogen-activated protein kinase; PI 3-kinase, phos- phatidylinositol 3-kinase; PKB, protein kinase B; PLC, phospholipase C; scFv, single- the biological effects of the binding of intracellular scFv to Syk, chain variable fragment; SH2, Src homology 2; TNP, trinitrophenyl; WT, wild type. and we found that despite an intact kinase activity of Syk, the cells Copyright © 2002 by The American Association of Immunologists, Inc. 0022-1767/02/$02.00 The Journal of Immunology 2275 that expressed the scFv exhibited a defect in the Fc⑀RI-mediated enzymes, and they were cloned into the pscFvexp-cyt vector (26). This signal transduction as visualized by an impaired calcium mobili- vector directs the expression of cytosolic Ab fragments, and contains the zation, and the inhibition of the secretion of allergic mediators. neomycin phosphotransferase gene (neo). All scFv fragments contain at their COOH terminal end a c-myc tag that permits their detection with the The analysis of the proteins that are implicated in that signaling mAb 9E10. For stable transfection, 50 ␮g recombinant vectors were trans- pathway revealed an inhibition in the tyrosine phosphorylation and fected into 2 ϫ 106 RBL-2H3 cells by electroporation (960 ␮F, 260 V). activation of Btk and PLC-␥2. Nevertheless, Fc⑀RI-induced mito- Neomycin-resistant transfectant cells were grown in the presence of 2 gen-activated protein kinase (MAPK) phosphorylation was not al- mg/ml G418 (Life Technologies). Monoclonal cell lines expressing the scFv were produced by limiting dilution and identified by immunofluores- tered, suggesting that the scFv inhibited selectively the link be- cence with 9E10 mAb. All RBL-2H3 clones used in our experiments ex- tween Syk and Btk and PLC-␥2 for their subsequent tyrosine pressed unaltered levels of Fc⑀RI (data not shown). phosphorylation and activation. Immunoprecipitations and immunoblots Materials and Methods Cells were seeded in petri dishes and, for activation, they were cultured Materials and Abs overnight with anti-trinitrophenyl (TNP) IgE. After 12–16 h, the excess IgE was removed by washing twice with RPMI without additives, and cells All reagents, unless otherwise mentioned, were from Sigma-Aldrich (St. were stimulated at 37°C in RPMI containing 100 ng/ml Ag DNP-BSA. Louis, MO). The mAb 9E10 directed against the amino acid sequence After 3 min, the supernatant was harvested, and the cell monolayers were EQKLISEEDLN of human c-myc was kindly provided by G.
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