Src Family Kinase Regulation in Allergy

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Secretion of inflammatory mediators after allergen-induced aggregation of the high affinity IgE receptor (FceRI) on mast cells is a critical component of the pathogenesis of asthma and other allergic disorders. The Src family tyrosine kinases, Lyn and Fyn, both positively and negatively regulate FceRI signaling and secretion in mast cells. We hypothesize that the functional differences between Lyn and Fyn in regulating mast cell secretion arise from the ability of their unique and Src homology (SH) domains to differentially associate with positive or negative regulators of FceRI signaling. Furthermore, we hypothesize that the compartmentalization of Lyn into membrane rafts facilitates its participation in negatively regulating activation of Fyn kinase. The specific aims are 1) to express dominant negative (DN) Lyn mutants in bone marrow derived mast cells (BMMC) and measure antigen-induced mediator secretion, signaling changes and association between Lyn and the FceRI. To determine the site of Lyn association on the FceRI beta with unaggregated receptors. To reintroduce either Lyn A or Lyn B into Lyn -/- BMMC to investigate the specific contribution of each isoform to FceRI signaling, chemotaxis and secretion. 2) To express DN Fyn mutants in BMMC and measure antigen-induced mediator secretion, signaling changes and association between Fyn and the FceRI. To determine the site of Fyn association on the FceRI beta with unaggregated receptors. 3) To directly measure the ability of Lyn to associate with FceRI beta and gamma subunits in living cells using fluorescence resonance energy transfer. To compare the activity and functions of raft-localized and raftexcluded Lyn kinase on FceRI signaling and secretion. Relevance to public health: The incidence of allergic disease in Westernized countries is increasing and treatment costs worldwide now total billion of dollars. Understanding the molecular details of the interaction of Src family kinases with FceRI may allow the development of drugs that will limit allergic reactions rather than mitigating symptoms.