Eosinophils play a pivotal role in the pathogenesis of asthma, various allergic and parasitic disorders. IL-5 is the principal regulatory cytokine for eosinophils. It stimulates many functions of eosinophils such as growth, differentiation, survival, secretion, adhesion, chemotaxis and cytokine production. The signal transduction mechanism of IL-5 in eosinophils is unknown, but recent evidence from the applicant indicates that the binding of IL-5 to its receptor activates lyn and Jak2 tyrosine kinases, and propagates signal through the ras-raf 1-MEK-MAP kinase and the Jak2-STAT1 signaling pathways, respectively. Further, lyn and Jak2 kinase are physically associated with the IL-5 receptor beta subunit in unstimulated eosinophils. The binding sites of lyn and Jak2 kinases on IL-5 receptor beta and alpha chains have not been investigated.
The specific aims of this research project are: 1) to map the binding sites of lyn and Jak2 kinases on the IL-5R alpha and beta subunits and to establish the structure-function relationship; 2) to determine the importance of lyn and Jak2 kinases in IL-5-induced eosinophil growth, differentiation and activation. It is hypothesized that the Jak2 kinase binds to the membrane-proximal box 1 region whereas the lyn kinase binds to the carboxyl-terminal end of the beta chain. Further, it is hypothesized that lyn and Jak2 signaling pathways are responsible for distinct cellular functions in eosinophils. The mapping for the binding sites will be performed using truncation mutants of the IL-5R alpha and beta. The truncated mutants will be constructed by inserting a stop codon at specific restriction sites. An IL-3-dependent mouse myeloid cell line will be transfected with the mutants and assessed for physical association and activation of lyn and Jak2 kinases as well as proliferative response to hIL-5. The activation of the kinases will be investigated by immunoblotting, immune-complex assay, and phosphoamino acid analysis. The physical association will be studied by co-precipitation studies. In order to determine the importance of lyn and Jak2 kinase, negative dominant mutants of the kinases will be constructed. HL-60 cells will be transfected with the lyn and jak2 mutants and their proliferation differentiation into eosinophilic and basophilic lineages will be assessed by culturing in alkaline medium in the presence of IL-5. The identification of binding sites for the kinases on IL-5R and the elucidation of the importance of the kinases in the IL-5 signaling pathway may help design novel antagonists for therapeutic interventions in allergic diseases.
