Paclíková Petra, Radaszkiewicz Tomasz Witold, Potěšil David, Harnoš Jakub, Zdráhal Zbyněk, Bryja Vítězslav
Department of Experimental Biology, Faculty of Science, Masaryk University, Brno, Czech Republic.
Central European Institute of Technology, Masaryk University, Brno, Czech Republic.
Cell Signal. 2021 Sep;85:110058. doi: 10.1016/j.cellsig.2021.110058. Epub 2021 May 31.
Dishevelled (DVL) proteins are key mediators of most Wnt pathways. In all vertebrates, three DVL paralogs are present (DVL1, DVL2 and DVL3) but it is poorly defined to what extent they are functionally redundant. Here, we generated T-REx HEK 293 cells with only one DVL paralog (i.e., DVL1-only, DVL2-only, and DVL3-only) and compared their response to Wnt-3a and Wnt-5a ligands with wild type and DVL triple knockout cells. We show that DVL is essential, in addition to the previously shown Wnt-3a-induced phosphorylation of LRP6 and transcriptional activation of TCF/LEF-dependent reporter, also for Wnt-3a-induced degradation of AXIN1 and Wnt-5a-induced phosphorylation of ROR1. We have quantified the molar ratios of DVL1:DVL2:DVL3 in our model to be approximately 4:80:16. Interestingly, DVL-only cells do not compensate for the lack of other paralogs and are still fully functional in all analyzed readouts with the exception of Wnt-3a-induced transcription assessed by TopFlash assay. In this assay, the DVL1-only cell line was the most potent; on the contrary, the DVL3-only cell line exhibited only the negligible capacity to mediate Wnt signals. Using a novel model system - complementation assays in T-REx HEK 293 with amplified Wnt signal response (RNF43/ZNRF3/DVL1/DVL2/DVL3 penta KO cells) we demonstrate that it is not the total amount of DVL but ratio of individual paralogs what decides the signal strength. In sum, this study contributes to our better understanding of the role of individual human DVL paralogs in the Wnt pathway.
散乱蛋白（DVL）是大多数Wnt信号通路的关键介质。在所有脊椎动物中，存在三种DVL旁系同源物（DVL1、DVL2和DVL3），但它们在功能上的冗余程度尚不清楚。在这里，我们构建了仅含有一种DVL旁系同源物的T-REx HEK 293细胞（即仅含DVL1、仅含DVL2和仅含DVL3），并将它们对Wnt-3a和Wnt-5a配体的反应与野生型和DVL三基因敲除细胞进行了比较。我们发现，除了先前显示的Wnt-3a诱导的LRP6磷酸化和TCF/LEF依赖性报告基因的转录激活外，DVL对于Wnt-3a诱导的AXIN1降解和Wnt-5a诱导的ROR1磷酸化也是必不可少的。我们在模型中量化了DVL1:DVL2:DVL3的摩尔比约为4:80:16。有趣的是，仅含一种DVL的细胞不能弥补其他旁系同源物的缺失，除了通过TopFlash试验评估的Wnt-3a诱导的转录外，在所有分析的读数中仍然完全功能正常。在该试验中，仅含DVL1的细胞系最有效；相反，仅含DVL3的细胞系介导Wnt信号的能力微不足道。使用一种新型模型系统——在具有放大Wnt信号反应的T-REx HEK 293中进行互补试验（RNF43/ZNRF3/DVL1/DVL2/DVL3五基因敲除细胞），我们证明决定信号强度的不是DVL的总量，而是各个旁系同源物的比例。总之，这项研究有助于我们更好地理解人类个体DVL旁系同源物在Wnt信号通路中的作用。
