beta-Galactosidase enzyme fragment complementation for the measurement of Wnt/beta-catenin signaling.
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.
Verkaar
Folkert
F
Schering-Plough Research Institute, P.O. Box 20, 5340 BH Oss, The Netherlands.
Blankesteijn
W Matthijs
WM
Smits
Jos F M
JF
Zaman
Guido J R
GJ
D000818:Animals; D016764:Cell Polarity; D049109:Cell Proliferation; D015536:Down-Regulation / Q000187:drug effects* / Q000502:physiology; D005816:Genetic Complementation Test / Q000379:methods*; D038362:Glycogen Synthase Kinase 3 / Q000037:antagonists & inhibitors / Q000378:metabolism; D006801:Humans; D019169:Jurkat Cells; D051379:Mice; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D011758:Pyrroles / Q000494:pharmacology; D011799:Quinazolines / Q000494:pharmacology; D015398:Signal Transduction / Q000187:drug effects* / Q000502:physiology; D051820:TCF Transcription Factors; D014158:Transcription, Genetic; D019076:Transgenes / Q000502:physiology; D051153:Wnt Proteins / Q000494:pharmacology*; D000071818:Wnt-5a Protein; D060508:Wnt3 Protein; D060509:Wnt3A Protein; D051176:beta Catenin / Q000235:genetics / Q000378:metabolism*; D001616:beta-Galactosidase / Q000096:biosynthesis* / Q000235:genetics
C495267:CTNNB1 protein, mouse; D047428:Protein Kinase Inhibitors; D011758:Pyrroles; D011799:Quinazolines; D051820:TCF Transcription Factors; C555510:WNT3A protein, human; D051153:Wnt Proteins; D000071818:Wnt-5a Protein; D060508:Wnt3 Protein; D060509:Wnt3A Protein; C555511:Wnt3a protein, mouse; C438615:Wnt5a protein, mouse; D051176:beta Catenin; C543528:sotrastaurin; D011493:Protein Kinase C; D038362:Glycogen Synthase Kinase 3; D001616:beta-Galactosidase