Intracellular signaling pathways transduce information from the cell surface to the nucleus, controlling the growth, division and differentiation of both normal and neoplastic cells. Protein tyrosine kinases (PTKs) are responsible for triggering the cascade of events in many of these pathways. In the case of receptor-type PTKs (rPTKs), ligand binding activates the kinase, which autophosphorylates and subsequently binds and phosphorylates substrate proteins, initiating the signaling cascade. Some non-receptor kinases form a """"""""broken"""""""" receptor complex with a cell surface molecule and function in a manner completely analogous to the rPTKs. The tyk2PTK is the prototype of a structurally distinct subfamily of non-receptor PTKs. It is activated in response to the cytokine interferon- (IFN), and it binds to and tyrosine phosphorylates one or more subunits of the IFN receptor. In addition, it is a candidate kinase for one or more members of a family of latent transcription factors, known as STATs (signal transducers and activators of transcription). Once these proteins are tyrosine phosphorylated, they translocate to the nucleus, bind an enhancer element and stimulate IFN-specific gene transcription. The resulting gene products are believed to promote an anti-viral state and/or inhibit cell growth. This latter property may account for the clinical utility of IFN in the treatment of a variety of indolent human neoplasms. Experiments are proposed to characterize, in detail, the molecular interactions between the tyk2 kinase, the IFN receptor -subunit and p113STST2 to investigate the possibility that tyk2 may be a tumor suppressor. Specifically, the regions of the tyk2 protein (p135tyk2) and the IFN receptor involved in the mutual association of these two proteins will be mapped. Next, the phosphorylation sites on both the tyk2 kinase and IFN receptor will be identified, and the role of these sites in the signaling pathway will be determined. Third, a model for STAT protein phosphorylation by p135tyk2, in which the receptor acts as a """"""""docking"""""""" protein, will be tested. Lastly, they will test the hypothesis that constitutively active derivatives of tyk2 can act as a tumor suppressor in an IFN-sensitive cell line. In summary, the research plan outlined in this proposal should increase our understanding of the molecular interactions occurring in the IFN signal transduction pathway, as well as related cytokine-mediated pathways and provide insight into the mechanisms governing the growth of human tumors, perhaps facilitating the design of improved therapies.
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