Schering-Plough Research Institute, P.O. Box 20, 5340 BH Oss, The Netherlands.
FASEB J. 2010 Apr;24(4):1205-17. doi: 10.1096/fj.09-141671. Epub 2009 Nov 25.
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.
Wnt/β-连环蛋白信号通路是细胞极性、增殖和成年期干细胞维持的重要调节因子。Wnt 蛋白诱导β-连环蛋白的核积累，通过与 TCF/LEF 转录因子结合来调节 Wnt 反应基因的表达。异常的 Wnt/β-连环蛋白信号通路与多种病理学有关，尤其是在几种癌症的起始和扩张中起着基础作用。在这里，我们应用酶片段互补来测量β-连环蛋白的核积累。β-连环蛋白被β-半乳糖苷酶的肽段标记，并转染到仅在核中表达酶相应缺失突变体的细胞中。用重组 Wnt-3a 刺激细胞，以纳米摩尔效力剂量依赖性地恢复β-半乳糖苷酶活性。使用该测定法，我们证实 Wnt-5a 抑制β-连环蛋白进入细胞核后驱动的报告基因活性。此外，我们测试了 >2000 种合成化学化合物文库，以确定它们诱导β-连环蛋白核积累的能力。免疫抑制蛋白激酶 C 抑制剂 sotrastaurin（AEB-071）在微摩尔浓度下被鉴定为 Wnt/β-连环蛋白信号的激活剂。已证实该化合物稳定内源性β-连环蛋白蛋白并能诱导 TCF/LEF 依赖性基因转录。随后对 >200 种激酶的生化分析揭示了糖原合成酶激酶 3 的两种同工型，这是 sotrastaurin 以前未被重视的靶标。我们表明，β-连环蛋白核积累测定法有助于我们了解 Wnt/β-连环蛋白途径中的分子相互作用，并可用于寻找针对 Wnt/β-连环蛋白信号的新治疗方法。-Verkaar，F.，Blankesteijn，W. M.，Smits，J. F. M.，Zaman，G. J. R.用于测量 Wnt/β-连环蛋白信号通路的β-半乳糖苷酶酶片段互补。
