title: Syk and Lyn mediate distinct Syk phosphorylation events in FcɛRI-signal transduction: implications for regulation of IgE-mediated degranulation.
abstract: Spleen tyrosine kinase (Syk) is a key regulatory factor in the IgE-mediated allergic signal transduction pathway in mast cells and basophils. Syk is phosphorylated on a number of tyrosines following the binding of IgE/allergen complexes to FcɛRI receptors leading to initiation of inflammatory signaling via downstream enzymes and scaffolding proteins. We examined the kinases responsible for the phosphorylation of key Syk tyrosines in rat RBL-2H3 basophilic cells and bone marrow-derived mast cells (BMMCs). The phosphorylation of Syk tyrosine 346 was completely blocked by the novel Src family kinase inhibitor BIRA766, suggesting this tyrosine is a pure substrate for Src family kinases. This was supported by the findings that kinase-dead (KD) Syk was efficiently phosphorylated on this tyrosine and that a specific Syk inhibitor BAY61-3606 was without effect. The phosphorylation of other Syk tyrosines 317, 342, 519 and 520 was reduced by Syk and Src family inhibitors, suggesting a role for auto- and trans-phosphorylation. Lyn was the predominant Src family kinase expressed and activated in RBL-2H3 cells, meanwhile Lyn knockdown with a specific siRNA interfered with the phosphorylation of all Syk tyrosines and the Syk substrates SLP-76 and LAT. Pharmacological inhibition of Syk completely blocked the degranulation of RBL-2H3 and BMMCs. However, Lyn knockdown sensitized RBL-2H3 cells to FcɛRI-induced degranulation. We showed that whilst interference with Lyn expression disrupts FcɛRI proximal signaling via Syk and its direct substrates including SLP-76 and LAT, distal activation of downstream proteins including Erk is enhanced. This study identifies the responsible kinases for the phosphorylation of key Syk tyrosines and the propagation of FcɛRI receptor mediated signal transduction in allergic responses.
authors.affiliation: Department of Pulmonary Diseases Research, Boehringer-Ingelheim Pharma GmbH & Co. KG, Biberach an der Riss, Germany. michael.sanderson@boehringer-ingelheim.com
authors.forename: Michael P
authors.initials: MP
authors.lastname: Sanderson
authors.forename: Eva
authors.initials: E
authors.lastname: Wex
authors.forename: Takeshi
authors.initials: T
authors.lastname: Kono
authors.forename: Katsuhiro
authors.initials: K
authors.lastname: Uto
authors.forename: Andreas
authors.initials: A
authors.lastname: Schnapp
mesh_terms: D000818:Animals; D001491:Basophils / Q000276:immunology / Q000378:metabolism; D015550:Cell Degranulation / Q000276:immunology*; D055785:Gene Knockdown Techniques; D006801:Humans; D006967:Hypersensitivity / Q000276:immunology; D007073:Immunoglobulin E; D047908:Intracellular Signaling Peptides and Proteins / Q000276:immunology / Q000378:metabolism*; D008407:Mast Cells / Q000276:immunology / Q000378:metabolism*; D051379:Mice; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000276:immunology / Q000378:metabolism*; D051381:Rats; D017455:Receptors, IgE / Q000276:immunology / Q000378:metabolism*; D015398:Signal Transduction / Q000276:immunology*; D000072377:Syk Kinase; D014162:Transfection; D019061:src-Family Kinases / Q000276:immunology / Q000378:metabolism*
chemical_list: C553474:FCER1A protein, rat; D047908:Intracellular Signaling Peptides and Proteins; D017455:Receptors, IgE; D007073:Immunoglobulin E; D011505:Protein-Tyrosine Kinases; C000606896:SYK protein, human; D000072377:Syk Kinase; C000606897:Syk protein, mouse; C000606898:Syk protein, rat; C060902:lyn protein-tyrosine kinase; D019061:src-Family Kinases