(from the application): The Wnt family of secretory glycoproteins is one of the major families of developmentally important signaling molecules and plays important roles in embryonic induction, generation of cell polarity, and specification of cell fate. Wnt pathways are also closely linked to tumorigenesis. Ectopic expression of Wnt proteins induces transformation phenotypes and tumorigenesis, and mutations in downstream signaling proteins of this pathway have been strongly linked to human colorectal cancers and melanomas. Genetic studies in Drosophila revealed the involvement of a number of proteins in Wnt signaling including dishevelled, zeste-white 3, armadillo, and transcription factor pangolin. Recent results suggest that the frizzled (Fz) family of cell surface proteins might function as the receptors for Wnt. More than 15 Wnt, 8 Fz, and 3 dishevelled homologs have been identified in mammals. Mammalian homologs to zeste-white 3, armadillo, and pangolin homologs are also identified; they are GSK3beta, beta-catenin, and TCF/LEF-1, respectively. The Wnt-signaling mechanism seems well conserved across species, because mammalian Wnt and Dishevelled (Dvl) proteins upregulate beta-catenin levels and stimulate LEF-1 activity in mammalian cells as their counterparts do in Drosophila. Despite significant advances, major gaps in the understanding of the Wnt-signaling pathway remain. Among them, the mechanisms by which Dvl receives signals from upstream regulators and transduces them to downstream effectors and the molecular identity of these upstream and downstream regulators remain unknown. In addition, it is still an open question if Fz alone can function as the receptor for Wnt. As described in this proposal, we have established cellular and biochemical assay systems to study Wnt signaling in mammalian cells. By using these assays, we have characterized two different signaling pathways led by Dvl in mammalian cells: regulation of JNK and LEF-1. We have also made significant progress in understanding the mechanism for Wnt-1- and Dvl-mediated stabilization of beta-catenin and activation of LEF-1 by discovering that Dvl binds to Axin and Frat-1. In this proposal, we will further our study using molecular, cell biological, and biochemical approaches to: 1) Continue to characterize the interactions between Dvl and Axin and between Dvl and Frat and elucidate the mechanisms by which Dvl regulates beta-catenin levels and LEF-1 activity via Axin and Frat-1. 2) Characterize the signal transduction pathway that mediates Dvl-induced JNK activation using the yeast two-hybrid system and dominant negative approach. 3) To identify upstream regulators for Dvl using the affinity chromatographic and yeast two-hybrid approaches.
