Lyn Kinase-Mediated Regulation of Allergic Inflammation

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DESCRIPTION (provided by applicant): The symptoms of an allergic reaction are generated in part by the allergen-induced release of preformed and newly synthesized mediators from basophils and mast cells bearing receptors for IgE. Initiation of signal transduction through the high affinity receptor for IgE (FceRI) requires phosphorylation of distinct tyrosine residues in the receptor's beta and gamma chains. The Src-family kinase, Lyn, is responsible for the receptor phosphorylation as well as the activation of numerous downstream signaling molecules, (e.g. Syk kinase). A small fraction of the two isoforms of Lyn (Lyn A and Lyn B) is associated with unphosphorylated IgE receptors in mast/basophil cell lines. A variety of approaches indicate that the unique domain of Lyn is associated with the C-terminus of the receptor's beta chain. We have investigated the ability of transfected Lyn unique domain to disrupt FceRI signaling and mediator secretion. The release of both histamine and TNF-alpha was partially inhibited in Rat Basophilic Leukemia (RBL) transfectants stably expressing Lyn unique domain, compared to secretion in control transfectants. This inhibition was accompanied by a partial decrease in FceRI tyrosine phosphorylation, delayed Syk activation, and reduced Gab2 tyrosine phosphorylation. Conversely, Leukotriene C4 (LTC4) secretion was upregulated three to five fold compared to control transfectants and accompanied by increased activation of the MAP kinase pathway. We propose to express Lyn unique domain in bone marrow-derived mast cells (BMMC) from Src family kinase (Lyn or Fyn) knockout mice and to monitor signaling and mediator secretion. We will investigate the role of the phosphatases PP2A, SHIP-l, and SHP-2 in LTC4 secretion in BMMC. By using in vitro kinase assays of recombinant Lyn, we have found that Lyn B has three times the specific activity towards a substrate peptide than Lyn A. We will reintroduce either Lyn A or Lyn B into Lyn knockout BMMC to investigate the specific contribution of each isoform to FceRI signaling and secretion. We also propose to prepare a transgenic Lyn unique domain mouse to investigate the ability of introduced Lyn unique domain to limit allergic inflammation, airway hyperreactivity, and BMMC mediator secretion. Understanding the molecular details of the interaction of the Lyn unique domain and FceRI beta may allow the development of drugs that will limit allergic reactions rather than mitigating symptoms.