1. β-Galactosidase enzyme fragment complementation for the measurement of Wnt/β-catenin signaling.
- Author
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Verkaar, Folkert, Blankesteijn, W. Matthijs, Smits, Jos F. M., and Zaman, Guido J. R.
- Subjects
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ENZYMES , *WNT genes , *PROTO-oncogenes , *WNT proteins , *GLYCOPROTEINS - Abstract
Wnt/β-catenin signaling is an important regulator of cell polarity, proliferation, and stem cell maintenance during development and adulthood. Wnt proteins induce the nuclear accumulation of β-catenin, which regulates the expression of Wnt-responsive genes through association with TCF/LEF transcription factors. Aberrant Wnt/β-catenin signaling has been implicated in a plethora of pathologies and, most notably, underlies initiation and expansion of several cancers. Here, we apply enzyme fragment complementation to measure the nuclear accumulation of β-catenin. β-Catenin was tagged with a peptide fragment of β-galactosidase and transfected into cells expressing a corresponding deletion mutant of the enzyme exclusively in the nucleus. Stimulation of the cells with recombinant Wnt-3a restored β-galactosidase activity in a dose-dependent manner with nanomolar potency. Using the assay, we confirmed that Wnt-5a represses β-catenin-driven reporter gene activity downstream of nuclear entry of β-catenin. In addition, we tested a library of >2000 synthetic chemical compounds for their ability to induce β-catenin nuclear accumulation. The immunosuppressive protein kinase C inhibitor sotrastaurin (AEB-071) was identified as an activator of Wnt/β-catenin signaling at micromolar concentrations. It was confirmed that the compound stabilizes endogenous β-catenin protein and can induce TCF/LEF-dependent gene transcription. Subsequent biochemical profiling of >200 kinases revealed both isoforms of glycogen synthase kinase 3, as previously unappreciated targets of sotrastaurin. We show that the β-catenin nuclear accumulation assay contributes to our knowledge of molecular interactions within the Wnt/β-catenin pathway and can be used to find new therapeutics targeting Wnt/β-catenin signaling. [ABSTRACT FROM AUTHOR]
- Published
- 2010
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