Functional analysis of dishevelled-3 phosphorylation identifies distinct mechanisms driven by casein kinase 1ϵ and frizzled5.
Functional analysis of dishevelled-3 phosphorylation identifies distinct mechanisms driven by casein kinase 1ϵ and frizzled5.
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DOI：
10.1074/jbc.m114.590638
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发表时间：
2014-08-22
期刊：
影响因子：
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通讯作者：
Bryja V
中科院分区：
文献类型：
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作者：
Bernatík O;Šedová K;Schille C;Ganji RS;Červenka I;Trantírek L;Schambony A;Zdráhal Z;Bryja V
背景：酪蛋白激酶1ϵ(CK1ϵ)对乱蓬蓬蛋白的磷酸化是Wnt信号转导中的关键事件。结果：用蛋白质组学方法鉴定了CK1ϵ磷酸化的Dv13残基，并进行了功能分析。结论：不同的磷酸化事件控制着DVL生物学的不同方面。意义：CK1ϵ和WNT受体FZD5通过不同的机制作用于DVL，提示CK1ϵ不直接位于Frizzled5的下游。Dvl3是Wnt信号通路的关键组成部分，作用于FrizzledFzd受体的下游，并在Wnt配体激活的通路中被大量磷酸化。酪蛋白激酶1ϵ(Casein kinase1ϵ，CK1)是Wnt诱导Dv13磷酸化的主要途径。目前还不清楚哪些DVL残基被磷酸化，以及个别磷酸化事件的后果是什么。在本研究中，我们利用质谱仪对CK1ϵ诱导的人Dv13蛋白的磷酸化进行了全面的分析。我们的分析显示，Dv13上的>；50个磷酸化位点；只有少数这些位点在CK1ϵ共表达后被动态诱导，令人惊讶的是，一簇修饰残基的磷酸化被下调。对动态磷酸化位点进行了功能分析。在非洲爪哇胚胎中，PDZ结构域的突变(S280A和S311A)降低了Dvl3激活T细胞因子/淋巴增强因子(T细胞因子/淋巴增强因子)驱动的转录和诱导次级轴的能力。相反，Dv13 C末端聚集的丝氨酸/苏氨酸突变阻止了CK1ϵ诱导Dv13的凝胶迁移率改变及其甚至亚细胞定位的能力。令人惊讶的是，在所有这些突变体中，Wnt受体Fzd5引起的迁移率变化和亚细胞定位变化与野生型Dvl3没有区别。综上所述，我们在分子水平上的数据支持了先前的假设，即CK1ϵ通过不同残基的磷酸化作为Wnt/β-连环蛋白信号的激活和关闭信号发挥作用，以及(Ii)CK1ϵ通过与FZD5不同的机制作用于DVL。
Background: Phosphorylation of Dishevelled (Dvl) by casein kinase 1ϵ (CK1ϵ) is a key event in Wnt signal transduction. Results: Dvl3 residues phosphorylated by CK1ϵ were identified by proteomics and analyzed functionally. Conclusion: Individual phosphorylation events control different aspects of Dvl biology. Significance: CK1ϵ and Fzd5, a Wnt receptor, act on Dvl via distinct mechanism, suggesting that CK1ϵ is not directly downstream of Frizzled5. Dishevelled-3 (Dvl3), a key component of the Wnt signaling pathways, acts downstream of Frizzled (Fzd) receptors and gets heavily phosphorylated in response to pathway activation by Wnt ligands. Casein kinase 1ϵ (CK1ϵ) was identified as the major kinase responsible for Wnt-induced Dvl3 phosphorylation. Currently it is not clear which Dvl residues are phosphorylated and what is the consequence of individual phosphorylation events. In the present study we employed mass spectrometry to analyze in a comprehensive way the phosphorylation of human Dvl3 induced by CK1ϵ. Our analysis revealed >50 phosphorylation sites on Dvl3; only a minority of these sites was found dynamically induced after co-expression of CK1ϵ, and surprisingly, phosphorylation of one cluster of modified residues was down-regulated. Dynamically phosphorylated sites were analyzed functionally. Mutations within PDZ domain (S280A and S311A) reduced the ability of Dvl3 to activate TCF/LEF (T-cell factor/lymphoid enhancer factor)-driven transcription and induce secondary axis in Xenopus embryos. In contrast, mutations of clustered Ser/Thr in the Dvl3 C terminus prevented ability of CK1ϵ to induce electrophoretic mobility shift of Dvl3 and its even subcellular localization. Surprisingly, mobility shift and subcellular localization changes induced by Fzd5, a Wnt receptor, were in all these mutants indistinguishable from wild type Dvl3. In summary, our data on the molecular level (i) support previous the assumption that CK1ϵ acts via phosphorylation of distinct residues as the activator as well as the shut-off signal of Wnt/β-catenin signaling and (ii) suggest that CK1ϵ acts on Dvl via different mechanism than Fzd5.