This revised proposal (l R29 AI38456-Ol) focuses on the molecular dissection of 4PS-mediated pathways in lL-4 signaling. 4PS is a 180 kDa protein that becomes tyrosine phosphorylated during IL-4 stimulation. Recently, 4PS protein was purified and its cDNA was isolated. Preliminary data directly demonstrated that 4PS plays an important role in lL-4 signaling. A full understanding of 4PS-mediated signaling pathways in IL- 4 system is scientifically and clinically important because IL-4 plays a crucial role in the regulation of protective immune responses, in limiting tissue damage by autoimmunity, and in immune elimination of certain tumor cells. IL-4 also controls the Ig class switching in B cells from IgM to IgGl (IgG4 in human) and to IgE, which is commonly associated with allergic diseases such as asthma and hay fever. Understanding the molecular mechanisms of IL-4 signaling may provide opportunities for controlling and intervening in these pathophysiological processes. IL-4 signaling system is involved in the activation of tyrosine kinases, phosphorylation of 4PS, and the activation of transcription factors; however, the kinase that phosphorylates 4PS in response to IL-4 has not been identified, and the signals linking to nuclear events have not been established. This proposal uses the recently cloned 4PS molecule to investigate the molecular linkage between the IL-4 receptor complex, 4PS kinase, phosphorylation of 4PS and activation of transcription factors in IL-4 signaling. The following specific aims will define and explore over the next five years elements in the IL-4 signaling system that are controlled by 4PS. 1. The mechanism of interaction between the IL-4 receptor complex and 4PS will be investigated by a rationally designed mutational analysis of 4PS and the alpha subunit of the IL-4 receptor. 2. The tyrosine kinases that phosphorylate 4PS (4PS kinase) in response to IL-4 will be identified by fractionation of cytoplasmic and membrane proteins, and characterized. The importance of the kinase in IL-4 signaling will be investigated. 3. The connection between phosphorylation of 4PS and the activation of transcription factors in flu signaling pathway will be established. Overexpressing 4PS in 32D cells expressing the alpha subunit of the IL-4 receptors will allow the identification of potential 4PS-mediated transcription factors (4PS-TF) that may be involved in IL-4-induced cell proliferation or differentiation.
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