Department of Developmental and Regenerative Biology, Mount Sinai School of Medicine, New York, New York 10029, USA.
J Biol Chem. 2011 Apr 8;286(14):12093-100. doi: 10.1074/jbc.M110.185280. Epub 2011 Feb 1.
Wnt pathways play essential roles in cell proliferation, morphogenesis, and cell fate specification during embryonic development. According to the consensus view, the Wnt pathway prevents the degradation of the key signaling component β-catenin by the protein complex containing the negative regulators Axin and glycogen synthase kinase 3 (GSK3). Stabilized β-catenin associates with TCF proteins and enters the nucleus to promote target gene expression. This study examines the involvement of HIPK2 (homeodomain-interacting protein kinase 2) in the regulation of different TCF proteins in Xenopus embryos in vivo. We show that the TCF family members LEF1, TCF4, and TCF3 are phosphorylated in embryonic ectoderm after Wnt8 stimulation and HIPK2 overexpression. We also find that TCF3 phosphorylation is triggered by canonical Wnt ligands, LRP6, and dominant negative mutants for Axin and GSK3, indicating that this process shares the same upstream regulators with β-catenin stabilization. HIPK2-dependent phosphorylation caused the dissociation of LEF1, TCF4, and TCF3 from a target promoter in vivo. This result provides a mechanistic explanation for the context-dependent function of HIPK2 in Wnt signaling; HIPK2 up-regulates transcription by phosphorylating TCF3, a transcriptional repressor, but inhibits transcription by phosphorylating LEF1, a transcriptional activator. Finally, we show that upon HIPK2-mediated phosphorylation, TCF3 is replaced with positively acting TCF1 at a target promoter. These observations emphasize a critical role for Wnt/HIPK2-dependent TCF phosphorylation and suggest that TCF switching is an important mechanism of Wnt target gene activation in vertebrate embryos.
Wnt 通路在胚胎发育过程中对细胞增殖、形态发生和细胞命运特化起着至关重要的作用。根据共识观点，Wnt 通路通过包含负调节因子 Axin 和糖原合成酶激酶 3 (GSK3) 的蛋白复合物来防止关键信号成分β-catenin 的降解。稳定的β-catenin与 TCF 蛋白结合并进入细胞核，促进靶基因表达。本研究在体内检查了 HIPK2（同源域相互作用蛋白激酶 2）在调控 Xenopus 胚胎中不同 TCF 蛋白方面的作用。我们表明，在 Wnt8 刺激和 HIPK2 过表达后，胚胎外胚层中的 TCF 家族成员 LEF1、TCF4 和 TCF3 被磷酸化。我们还发现，TCF3 磷酸化是由经典 Wnt 配体 LRP6 和 Axin 和 GSK3 的显性负突变体触发的，表明这一过程与β-catenin 稳定具有相同的上游调节因子。HIPK2 依赖性磷酸化导致 LEF1、TCF4 和 TCF3 在体内从靶启动子解离。这一结果为 HIPK2 在 Wnt 信号中的上下文相关功能提供了一个机制解释；HIPK2 通过磷酸化转录抑制因子 TCF3 而上调转录，但通过磷酸化转录激活因子 LEF1 抑制转录。最后，我们表明，在 HIPK2 介导的磷酸化作用下，TCF3 在靶启动子处被正向作用的 TCF1 取代。这些观察结果强调了 Wnt/HIPK2 依赖性 TCF 磷酸化的关键作用，并表明 TCF 转换是脊椎动物胚胎中 Wnt 靶基因激活的重要机制。
