_-Catenin is an important protein for cancer research as it influences numerous events in the cell that lead to the development of cancer when gone awry. At the adherens junctions, _-catenin... Show more_-Catenin is an important protein for cancer research as it influences numerous events in the cell that lead to the development of cancer when gone awry. At the adherens junctions, _-catenin functions in cell-cell adhesion to maintain epithelial organisation. As an effector of Wnt signaling, _-catenin controls numerous developmental processes as well as homeostatic self-renewal. The effector function of _-catenin is to form a transcriptional complex in the nucleus with TCF/Lef transcription factors to regulate target gene expression. Due to the dual function of _-catenin in cell adhesion and signaling, there are different pools of the protein. The research described in this thesis focuses on the role of _-catenin in the Wnt signaling pathway. What is the pool of _-catenin that is active in signaling? Where is active _-catenin localized? Where and how is _-catenin activated and how is its nuclear export regulated to terminate Wnt signaling. Chapter 1 provides a general introduction about the different aspects of nuclear transport and the Wnt signaling cascade, putting it into the context of cancer development. Chapter 2 describes the identification of Ran-binding protein 3 (RanBP3) as a novel regulator of the active signaling form of _-catenin. We initiated this study to investigate the nuclear translocation of _-catenin and found that RanBP3 directly inhibits _-catenin signaling by stimulating nuclear export of the transcriptionally active form of _-catenin. The active form of _-catenin is unphosphorylated on its N-terminus, and covers only a small fraction of the total amount of _-catenin in the cell. We therefore continued to study the localization of this pool of _-catenin in Chapter 3. We describe that a relative large pool of unphosphorylated _-catenin resides at the adherens junctions, where it most likely functions in cell-cell adhesion. As Wnt treatment induces recruitment of unphosphorylated _-catenin to the plasma membrane, it is impossible to distinguish the resident junctional pool of unphosphorylated _-catenin from the signaling pool. We emphasize the importance of an E-cadherin null background in studying signaling competent unphosphorylated _-catenin. In Chapter 4, we study the unphosphorylated _-catenin pool at the plasma membrane upon Wnt signal induction in E-cadherin knock out cells. Plasma membrane recruitment of _-catenin in the early steps of the Wnt signaling cascade fits with recent new insights, which suggest recruitment of Axin and Dvl to the activated Wnt receptor LRP5/6. We expand these insights by showing that active _-catenin, Axin, APC and activated LRP6 receptor all localize to the plasma membrane upon Wnt stimulation. Moreover, we find that Wnt induced _-catenin is transcriptionally more active than overexpressed _-catenin. We suggest a model in which plasma membrane recruitment of _-catenin represents an important step in _-catenin processing and Wnt signal transduction. In Chapter 5, we determine the nuclear export kinetics of _-catenin in human cells and show that _-catenin exits the nucleus very fast, independently of the CRM1 export pathway and that _-catenin can enhance export of the small molecule GFP (green fluorescent protein). These observations fit into a model in which _-catenin can translocate quickly into and out of the nucleus independently of nuclear transport receptors. Therefore, the activity and localization of _-catenin are likely to be regulated by retention of the protein in the nucleus, cytoplasm and plasma membrane. Finally, in Chapter 6 we reconcile our findings with current knowledge of the Wnt signaling cascade. Show less
