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

Folkert Verkaar, W. Matthijs Blankesteijn, Jos F. M. Smits, Guido J. R. Zaman*

*Corresponding author for this work

Research output: Contribution to journalArticleAcademicpeer-review

77 Downloads (Pure)


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 identified as an activator of Wnt/beta-catenin signaling at micromolar concentrations. It was confirmed that the compound stabilizes endogenous beta-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 beta-catenin nuclear accumulation assay contributes to our knowledge of molecular interactions within the Wnt/beta-catenin pathway and can be used to find new therapeutics targeting Wnt/beta-catenin signaling.-Verkaar, F., Blankesteijn, W. M., Smits, J. F. M., Zaman, G. J. R. beta-Galactosidase enzyme fragment complementation for the measurement of Wnt/beta-catenin signaling. FASEB J. 24, 1205-1217 (2010).
Original languageEnglish
Pages (from-to)1205-1217
JournalFaseb Journal
Issue number4
Publication statusPublished - Apr 2010


  • AEB-071
  • GSK3
  • PKC
  • sotrastaurin
  • Wnt-3a
  • Wnt-5a


Dive into the research topics of 'beta-Galactosidase enzyme fragment complementation for the measurement of Wnt/beta-catenin signaling'. Together they form a unique fingerprint.

Cite this