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 line.
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 FcepsilonRI aggregation, suggesting that the scFv do not affect the recruitment of Syk to the receptor. Nevertheless, FcepsilonRI-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-gamma2 tyrosine phosphorylation and activation. Interestingly, FcepsilonRI-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-gamma2 activation.
Institut de Génétique Moléculaire de Montpellier, Unité Mixte de Recherche 5535 Centre National de la Recherche Scientifique, Montpellier, France.
Stéphanie
S
Dauvillier
Peggy
P
Mérida
Michela
M
Visintin
Antonino
A
Cattaneo
Christian
C
Bonnerot
Piona
P
Dariavach
D000077329:Agammaglobulinaemia Tyrosine Kinase; D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001666:Binding Sites, Antibody; D002118:Calcium / Q000037:antagonists & inhibitors / Q000378:metabolism; D004789:Enzyme Activation / Q000235:genetics / Q000276:immunology; D004792:Enzyme Precursors / Q000276:immunology* / Q000378:metabolism; D006801:Humans; D007135:Immunoglobulin Variable Region / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D007424:Intracellular Fluid / Q000276:immunology* / Q000378:metabolism; D047908:Intracellular Signaling Peptides and Proteins; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000378:metabolism; D008969:Molecular Sequence Data; D051966:Phospholipase C gamma; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors / Q000276:immunology* / Q000378:metabolism; D051381:Rats; D017455:Receptors, IgE / Q000037:antagonists & inhibitors* / Q000502:physiology*; D015398:Signal Transduction / Q000235:genetics / Q000276:immunology*; D000072377:Syk Kinase; D014162:Transfection; D014407:Tumor Cells, Cultured / Q000276:immunology* / Q000378:metabolism; D010738:Type C Phospholipases / Q000037:antagonists & inhibitors / Q000378:metabolism; D014443:Tyrosine / Q000037:antagonists & inhibitors / Q000378:metabolism; D018909:src Homology Domains / Q000276:immunology*; D019061:src-Family Kinases / Q000378:metabolism
D000911:Antibodies, Monoclonal; D004792:Enzyme Precursors; D007135:Immunoglobulin Variable Region; D047908:Intracellular Signaling Peptides and Proteins; D007527:Isoenzymes; D017455:Receptors, IgE; D014443:Tyrosine; D011505:Protein-Tyrosine Kinases; D000077329:Agammaglobulinaemia Tyrosine Kinase; C000606896:SYK protein, human; D000072377:Syk Kinase; C000606898:Syk protein, rat; C060902:lyn protein-tyrosine kinase; D019061:src-Family Kinases; D010738:Type C Phospholipases; D051966:Phospholipase C gamma; D002118:Calcium