Modulation of the beta-catenin signaling pathway by the dishevelled-associated protein Hipk1.
Modulation of the beta-catenin signaling pathway by the dishevelled-associated protein Hipk1.
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DOI：
10.1371/journal.pone.0004310
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发表时间：
2009
期刊：
影响因子：
3.7
通讯作者：
Cheyette BN
中科院分区：
文献类型：
--
作者：
Louie SH;Yang XY;Conrad WH;Muster J;Angers S;Moon RT;Cheyette BN
wnt是进化上保守的配体，通过β-catenin依赖和β-catenin不依赖的途径来调节脊椎动物发育过程中细胞的命运、增殖、极性和运动。Dishevelled （Dsh/Dvl）是一种多结构域支架蛋白，几乎所有已知的Wnt信号活动都需要它，这引起了人们对Dsh相关蛋白的鉴定和功能的兴趣。我们利用Dsh的n端片段进行酵母-2杂交筛选，分离出非洲爪蟾Hipk1同源基因。通过免疫共沉淀法和质谱法证实了Dsh和Hipk1蛋白之间的相互作用，进一步的实验表明Hipk1也与转录因子Tcf3复合物。myc标记的Hipk1在X. laevis发育过程中支持核功能，主要定位于动物帽外植体的细胞核，内源性转录本在原肠和神经性阶段强烈表达。通过对人胚胎肾细胞中β-catenin报告基因的检测，以及在囊胚后期的X. laevis胚胎的背侧规范指标，Hipk1水平的实验操作表明，Hipk1可以抑制Wnt/β-catenin靶基因的激活。此外，在原肠胚形成过程中，wnt应答基因的一个子集随后需要Hipk1来激活中胚层。此外，Hipk1的过表达或敲低都会导致参与原肠胚形成和神经管闭合的趋同伸展细胞运动受到干扰。这些结果表明，在脊椎动物早期发育过程中，Hipk1以复杂的方式参与了依赖于dsh的信号活动。这包括在细胞核中调节Wnt/β-catenin靶基因的转录，可能在变化的发育环境下以抑制和激活的方式进行。这种调节是调节原肠形成所必需的基因表达和细胞运动所必需的。
Wnts are evolutionarily conserved ligands that signal through β-catenin-dependent and β-catenin–independent pathways to regulate cell fate, proliferation, polarity, and movements during vertebrate development. Dishevelled (Dsh/Dvl) is a multi-domain scaffold protein required for virtually all known Wnt signaling activities, raising interest in the identification and functions of Dsh-associated proteins. We conducted a yeast-2-hybrid screen using an N-terminal fragment of Dsh, resulting in isolation of the Xenopus laevis ortholog of Hipk1. Interaction between the Dsh and Hipk1 proteins was confirmed by co-immunoprecipitation assays and mass spectrometry, and further experiments suggest that Hipk1 also complexes with the transcription factor Tcf3. Supporting a nuclear function during X. laevis development, Myc-tagged Hipk1 localizes primarily to the nucleus in animal cap explants, and the endogenous transcript is strongly expressed during gastrula and neurula stages. Experimental manipulations of Hipk1 levels indicate that Hipk1 can repress Wnt/β-catenin target gene activation, as demonstrated by β-catenin reporter assays in human embryonic kidney cells and by indicators of dorsal specification in X. laevis embryos at the late blastula stage. In addition, a subset of Wnt-responsive genes subsequently requires Hipk1 for activation in the involuting mesoderm during gastrulation. Moreover, either over-expression or knock-down of Hipk1 leads to perturbed convergent extension cell movements involved in both gastrulation and neural tube closure. These results suggest that Hipk1 contributes in a complex fashion to Dsh-dependent signaling activities during early vertebrate development. This includes regulating the transcription of Wnt/β-catenin target genes in the nucleus, possibly in both repressive and activating ways under changing developmental contexts. This regulation is required to modulate gene expression and cell movements that are essential for gastrulation.
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