beta-Galactosidase enzyme fragment complementation for the measurement of Wnt/beta-catenin signaling

FASEB Journal : Official Publication of the Federation of American Societies for Experimental Biology
Folkert VerkaarGuido J R Zaman

Abstract

Wnt/beta-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 beta-catenin, which regulates the expression of Wnt-responsive genes through association with TCF/LEF transcription factors. Aberrant Wnt/beta-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 beta-catenin. beta-Catenin was tagged with a peptide fragment of beta-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 beta-galactosidase activity in a dose-dependent manner with nanomolar potency. Using the assay, we confirmed that Wnt-5a represses beta-catenin-driven reporter gene activity downstream of nuclear entry of beta-catenin. In addition, we tested a library of >2000 synthetic chemical compounds for their ability to induce beta-catenin nuclear accumulation. The immunosuppressive protein kinase C inhibitor sotrastaurin (AEB-071) was ...Continue Reading

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Citations

May 12, 2011·PloS One·Folkert VerkaarGuido J R Zaman
Mar 6, 2015·Protein Science : a Publication of the Protein Society·Kotaro NishiyamaTetsuya Yomo
Aug 1, 2012·Neuropharmacology·Kirsty J Martin, J Simon C Arthur

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