Essential role of the Dishevelled DEP domain in a Wnt-dependent human-cell-based complementation assay.
Essential role of the Dishevelled DEP domain in a Wnt-dependent human-cell-based complementation assay.
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DOI：
10.1242/jcs.195685
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发表时间：
2016-10-15
影响因子：
4
通讯作者：
Bienz M
中科院分区：
文献类型：
--
作者：
Gammons MV;Rutherford TJ;Steinhart Z;Angers S;Bienz M
Disheveled (DVL) 借助其 DIX 结构域通过动态头尾聚合来组装 Wnt 信号体。因此，它将 Wnt 信号转导至包括 β-连环蛋白在内的细胞质效应器，以控制正常发育、组织稳态以及癌症过程中的细胞命运。迄今为止，Disheveled 的大多数功能研究都依赖于其因过度表达而产生的不依赖于 Wnt 的信号活动，这足以触发聚合，绕过了对 Wnt 信号的要求。在这里，我们生成了一种不含内源性 Disheveled 的人类细胞系 (DVL1-DVL3)，该细胞系缺乏 β-连环蛋白的 Wnt 信号转导。然而，Wnt 反应可以通过以接近内源水平稳定地重新表达的 DVL2 来恢复。使用该测定法测试突变体 DVL2，我们发现其 DEP 结构域对于 β-连环蛋白的信号传导是必需的，而其 PDZ 结构域是可有可无的。我们的结果表明 DEP 结构域在 Wnt 信号转导中具有两个相互排斥的功能：与 Frizzled 结合以将 Disheveled 招募到受体复合物中，并二聚化以交联 DIX 结构域聚合物以进行信号小体组装。我们的检测避免了与过度表达 Dishevelled 相关的警告，并为这种关键的人类信号蛋白的严格功能测试提供了强大的工具。摘要：在 Disheveled 无效突变人类细胞中进行的生理互补测定确定了 Disheveled 的 DEP 结构域在与 Frizzled 结合以将信号转导至 β-连环蛋白方面的基本功能。
Dishevelled (DVL) assembles Wnt signalosomes through dynamic head-to-tail polymerisation by means of its DIX domain. It thus transduces Wnt signals to cytoplasmic effectors including β-catenin, to control cell fates during normal development, tissue homeostasis and also in cancer. To date, most functional studies of Dishevelled relied on its Wnt-independent signalling activity resulting from overexpression, which is sufficient to trigger polymerisation, bypassing the requirement for Wnt signals. Here, we generate a human cell line devoid of endogenous Dishevelled (DVL1– DVL3), which lacks Wnt signal transduction to β-catenin. However, Wnt responses can be restored by DVL2 stably re-expressed at near-endogenous levels. Using this assay to test mutant DVL2, we show that its DEP domain is essential, whereas its PDZ domain is dispensable, for signalling to β-catenin. Our results imply two mutually exclusive functions of the DEP domain in Wnt signal transduction – binding to Frizzled to recruit Dishevelled to the receptor complex, and dimerising to cross-link DIX domain polymers for signalosome assembly. Our assay avoids the caveats associated with overexpressing Dishevelled, and provides a powerful tool for rigorous functional tests of this pivotal human signalling protein. Summary: A physiological complementation assay in Dishevelled null-mutant human cells establishes an essential function of the DEP domain of Dishevelled in its binding to Frizzled for signal transduction to β-catenin.
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