Department of Pharmacology, Health Sciences Center, State University of New York at Stony Brook, Stony Brook, NY 11794-8651, USA.
J Cell Sci. 2009 Dec 15;122(Pt 24):4439-51. doi: 10.1242/jcs.051847. Epub 2009 Nov 17.
Wnt3a activates the ;canonical' signaling pathway, stimulating the nuclear accumulation of beta-catenin and activation of Lef/Tcf-sensitive transcription of developmentally important genes. Using totipotent mouse F9 teratocarcinoma cells expressing frizzled-1 (Fz1), we investigated roles of tyrosine kinase activity in Wnt/beta-catenin signaling. Treatment with either genistein or Src family kinase inhibitor PP2 attenuates Wnt3a-stimulated Lef/Tcf transcription activation and primitive endoderm formation. siRNA-induced knockdown of Src likewise attenuates Lef/Tcf transcription and primitive endoderm formation in response to Wnt3a, implicating Src as a positive regulator of Wnt/beta-catenin signaling. We discovered that Src binds dishevelled-2 (Dvl2), a key phosphoprotein in Wnt signaling, at two positions: an SH3-binding domain and a C-terminal domain. The Y18F mutant of Dvl2 attenuates the Wnt3a-stimulated Lef/Tcf-sensitive transcriptional response. Wnt3a stimulates Src docking to Dvl2 and activation of this tyrosine kinase. Activated Src, in turn, enhances Wnt activation of the canonical pathway. We show that Dvl2 and beta-catenin are crucially important substrates for tyrosine phosphorylation in the canonical Wnt/beta-catenin pathway.
Wnt3a 激活“经典”信号通路，刺激β-连环蛋白的核积累，并激活 Lef/Tcf 敏感的发育重要基因转录。我们使用表达 frizzled-1（Fz1）的全能性小鼠 F9 畸胎瘤细胞，研究了酪氨酸激酶活性在 Wnt/β-连环蛋白信号通路中的作用。用金雀异黄素或 Src 家族激酶抑制剂 PP2 处理，可减弱 Wnt3a 刺激的 Lef/Tcf 转录激活和原始内胚层形成。Src 的 siRNA 诱导敲低同样减弱了 Wnt3a 诱导的 Lef/Tcf 转录和原始内胚层形成，表明 Src 是 Wnt/β-连环蛋白信号的正调节剂。我们发现 Src 在两个位置与盘状结构域受体 2（Dvl2）结合，Dvl2 是 Wnt 信号中的关键磷酸化蛋白：一个 SH3 结合结构域和一个 C 端结构域。Dvl2 的 Y18F 突变体减弱了 Wnt3a 刺激的 Lef/Tcf 敏感的转录反应。Wnt3a 刺激 Src 与 Dvl2 对接，并激活这种酪氨酸激酶。激活的 Src 反过来增强了 Wnt 对经典途径的激活。我们表明 Dvl2 和β-连环蛋白是经典 Wnt/β-连环蛋白途径中酪氨酸磷酸化的关键底物。
