Isolation of dishevelled-interacting proteins and their role in Wnt signal transduction

Abstract

The Dishevelled (Dvl) gene family encodes cytoplasmic proteins that are implicated in Wnt signal transduction. Wnt genes are differentially expressed throughout development and encode secreted glycoproteins that modulate a variety of processes in both invertebrate and vertebrate development, including cell growth regulation, cell fate determination, organogenesis, and oncogenesis. At present, Dvl represents the most upstream component of the Wnt signal transduction pathway in the receiving cell subsequent to receptor activation. Three murine homologues of dishevelled have been isolated, Dvl1, Dvl2, and Dvl3, which all share a region of homology known as the PDZ domain. This motif has been identified in several other proteins and mediates proteinprotein interactions; however, no ligand for the PDZ domain of Dvl has been identified. In this project, the yeast two-hybrid assay of protein-protein interaction has been used to ascertain a role for Dvl in Wnt signal transduction through the identification of Dvl-interacting proteins. Protein phosphatase 2Calpha (PP2Calpha), a serine/threonine specific phosphatase, was found to directly interact with the PDZ domains of Dvl1 and Dvl2. Immunoprecipitation studies demonstrated that PP2Calpha and Dvl also interact in vivo , and exist in a complex with beta-catenin and Axin, a negative regulator of Wnt signaling. The Wnt-1-responsive LEF-1 (lymphoid enhancer-binding factor) reporter gene assay, a recently developed system to test for the function of Wnt signaling components, was used to determine if PP2Calpha could activate or inhibit Wnt-mediated activation of LEF-1-dependent transcription. PP2Calpha expression was shown to activate transcription of the reporter gene and relieve the Axin-mediated repression of LEF-1 dependent transcription, suggesting PP2Calpha functions as a positive regulator of Wnt signaling. Investigation of the possible targets of PP2Calpha revealed that Axin serves as a substrate for PP2Calpha in vitro and in vivo. The phosphorylation of Axin by glycogen synthase kinase-3beta (GSK-3beta) has previously been shown to lead to its stabilization, therefore it appears that PP2Calpha activates Wnt signaling through the dephosphorylation and subsequent destabilization of Axin. In addition, PP2Calpha expression can rescue Dvl-mediated apoptosis. This project has identified PP2Calpha as a new component of the Wnt signal transduction pathway and provides strong evidence for a role of PP2Calpha in mediating the activation of Wnt signaling. Further studies should provide insight into the biological function of PP2Calpha with regard to the regulation of protein phosphorylation in this signal transduction pathway.