Destabilization of heterologous proteins mediated by the GSK3β phosphorylation domain of the β-catenin protein.

Background and Aims: Wnt/beta-catenin signaling plays important roles in development and cellular processes. The hallmark of canonical Wnt signaling activation is the stabilization of beta-catenin protein in cytoplasm and/or nucleus. The stability of beta-catenin is the key to its biological functions and is controlled by the phosphorylation of its amino-terminal degradation domain. Aberrant activation of beta-catenin signaling has been implicated in the development of human cancers. It has been recently suggested that GSK3 beta may play an essential role in regulating global protein turnover. Here, we investigate if the GSK3 beta phosphorylation site-containing degradation domain of beta-catenin is sufficient to destabilize heterologous proteins. Methods and Results: We engineer chimeric proteins by fusing beta-catenin degradation domain at the N- and/or C-termini of the enhanced green fluorescent protein (eGFP). In both transient and stable expression experiments, the chimeric GFP proteins exhibit a significantly decreased stability, which can be effectively antagonized by lithium and Wnt1. An activating mutation in the destruction domain significantly stabilizes the fusion protein. Furthermore, GSK3 inhibitor SB-216763 effectively increases the GFP signal of the fusion protein. Conversely, the inhibition of Wnt signaling with tankyrase inhibitor XAV939 results in a decrease in GFP signal of the fusion proteins, while these small molecules have no significant effects on the mutant destruction domain-GFP fusion protein. Conclusion: Our findings strongly suggest that the beta-catenin degradation domain may be sufficient to destabilize heterologous proteins in Wnt signaling-dependent manner. It is conceivable that the chimeric GFP proteins may be used as a functional reporter to measure the dynamic status of beta-catenin signaling, and to identify potential anticancer drugs that target beta-catenin signaling. Copyright (C) 2013 S. Karger AG, Basel