Dishevelled enables casein kinase 1–mediated phosphorylation of Frizzled 6 required for cell membrane localization
Dishevelled enables casein kinase 1–mediated phosphorylation of Frizzled 6 required for cell membrane localization
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DOI：
10.1074/jbc.ra118.004656
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发表时间：
2018-10
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通讯作者：
Kateřina Straková;Maria Kowalski-Jahn;Tomáš Gybeľ;J. Valnohova;V. Dhople;J. Harnoš;Ondřej Bernatík;R. Ganji;Z. Zdráhal;J. Mulder;C. Lindskog;V. Bryja;G. Schulte
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作者：
Kateřina Straková;Maria Kowalski-Jahn;Tomáš Gybeľ;J. Valnohova;V. Dhople;J. Harnoš;Ondřej Bernatík;R. Ganji;Z. Zdráhal;J. Mulder;C. Lindskog;V. Bryja;G. Schulte
Frizzleds（FZD）是Wingless/Int-1（WNT）家族分泌脂糖蛋白的受体，在多细胞生物中启动重要的信号转导网络。FZD是G蛋白偶联受体（GPCR），众所周知其通过磷酸化调节，导致特异性下游信号传导或受体脱敏。FZD磷酸化的作用和潜在机制在很大程度上尚未探索。在这里，我们研究了人FZD 6的磷酸化。使用MS分析和磷酸化状态和位点特异性抗体，我们发现位于FZD 6 C末端的Ser-648被酪蛋白激酶1 α（CK 1 β）有效地磷酸化，并且这种磷酸化需要支架蛋白Dishevelled（DVL）。在过表达系统中，DVL 1、DVL-2和DVL-3促进CK 1 β介导的FZD 6在Ser-648上的磷酸化。该DVL活性需要完整的DEP结构域和FZD介导的该结构域向细胞膜的募集。CK 1 β靶向磷酸化酶的取代降低了FZD 6表面表达，表明Ser-648磷酸化控制FZD 6的膜运输。与总FZD 6蛋白表达相比，人输卵管上皮中的磷酸化-Ser-648 FZD 6免疫反应性主要为顶端，与上皮细胞亚群中的纤毛相关，表明FZD 6磷酸化有助于细胞内受体功能的不对称定位和上皮极性。鉴于FZD 6在平面细胞极性中的关键作用，我们的研究结果提出了FZD 6的不对称磷酸化而不是不对称蛋白质分布解释极化受体信号传导的可能性。
Frizzleds (FZDs) are receptors for secreted lipoglycoproteins of the Wingless/Int-1 (WNT) family, initiating an important signal transduction network in multicellular organisms. FZDs are G protein–coupled receptors (GPCRs), which are well known to be regulated by phosphorylation, leading to specific downstream signaling or receptor desensitization. The role and underlying mechanisms of FZD phosphorylation remain largely unexplored. Here, we investigated the phosphorylation of human FZD6. Using MS analysis and a phospho-state– and -site–specific antibody, we found that Ser-648, located in the FZD6 C terminus, is efficiently phosphorylated by casein kinase 1 ϵ (CK1ϵ) and that this phosphorylation requires the scaffolding protein Dishevelled (DVL). In an overexpression system, DVL1, -2, and -3 promoted CK1ϵ-mediated FZD6 phosphorylation on Ser-648. This DVL activity required an intact DEP domain and FZD-mediated recruitment of this domain to the cell membrane. Substitution of the CK1ϵ-targeted phosphomotif reduced FZD6 surface expression, suggesting that Ser-648 phosphorylation controls membrane trafficking of FZD6. Phospho-Ser-648 FZD6 immunoreactivity in human fallopian tube epithelium was predominantly apical, associated with cilia in a subset of epithelial cells, compared with the total FZD6 protein expression, suggesting that FZD6 phosphorylation contributes to asymmetric localization of receptor function within the cell and to epithelial polarity. Given the key role of FZD6 in planar cell polarity, our results raise the possibility that asymmetric phosphorylation of FZD6 rather than asymmetric protein distribution accounts for polarized receptor signaling.