Asthma is characterized by eosinophilic inflammation of the airways. Interleukin-5 (IL-5) is one of the most important regulators of eosinophil differentiation and function. IL-5 activates eosinophils by stimulating a number of tyrosine kinases. However, which tyrosine kinase plays an essential role in transducing IL-5-specific signals is unknown. Based upon preliminary results we hypothesize that Lyn kinase plays an essential and non-redundant role in IL-5 -stimulated eosinophil differentiation and in the development of airway eosinophilic inflammation. In the first specific aim we will examine the physical association of Lyn with IL-5 receptor alpha subunit (IL-5Ra) and study its ability to phosphorylate the receptor. The biological relevance of Lyn for eosinophil differentiation from stem cells will be examined using Lyn knockout mice. Further, we will examine the effect of Lyn null mutation on the development of airway allergic inflammation using an ovalbumin-sensitized mouse model of asthma. The second specific aim is devoted to delineating the molecular mechanism of activation of Lyn kinase by IL-5Ra. In order to identify IL-5Ra-associated molecules that might activate Lyn kinase, we have performed yeast two-hybrid screening of a human cDNA library and cloned a novel SH2/SH3 ligand--IRIP (ImmunoReceptor-Interactive Protein). We hypothesize that IRIP activates Lyn kinase by interacting through its SH3 domain. To test the foregoing hypothesis we will map the binding sites of IRJP and Lyn kinase using recombinant Lyn domains and site directed mutagenesis. The importance of IRIP SH2/SH3 motifs for Lyn activation will be examined using motif peptides and site directed mutagenesis. Finally, the relevance of IRIP for eosinophil differentiation will be studied by overexpressing wild type and dominant negative mutants of IRIP in murine stem cells and HL-60 cells and by examining their differentiation into eosinophils. The proposed studies are designed to identify key IL-5-specific signaling molecules that are important for eosinophil differentiation and eosinophilic inflammation. The cloning and functional characterization of IRIP as a novel activator of Src-type tyrosine kinases will advance basic knowledge and will help understand the molecular mechanism of activation receptor-associated tyrosine kinases in general. Lyn and IRIP may represent excellent therapeutic targets for intervention in asthma and other allergic diseases.
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