Supramolecular assembly of the beta-catenin destruction complex and the effect of Wnt signaling on its localization, molecular size, and activity in vivo.
Supramolecular assembly of the beta-catenin destruction complex and the effect of Wnt signaling on its localization, molecular size, and activity in vivo.
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DOI：
10.1371/journal.pgen.1007339
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发表时间：
2018-04
期刊：
影响因子：
4.5
通讯作者：
Peifer M
中科院分区：
文献类型：
--
作者：
Schaefer KN;Bonello TT;Zhang S;Williams CE;Roberts DM;McKay DJ;Peifer M
Wnt信号提供了一种细胞-细胞信号的范例，这种信号调节胚胎发育和干细胞稳态，并且在癌症中被不适当地激活。肿瘤抑制因子APC和Axin形成多蛋白破坏复合物的核心，其靶向Wnt效应子β-连环蛋白进行磷酸化、泛素化和破坏。基于早期的工作，我们假设破坏复合物是一个超分子实体，自组装的轴蛋白和APC聚合，并调节组装和稳定性的破坏复合物的基础上，其功能。我们在果蝇胚胎中测试了这一假设，这是Wnt信号传导的首要模型。结合生物化学、操纵Axin和APC 2水平的遗传工具、先进的成像和分子计数，我们定义了破坏复合物组装、化学计量和体内定位，以及其响应于Wnt信号传导的下调。我们的发现挑战和修订了目前的破坏复杂功能的模型。内源性Axin和APC 2蛋白及其拮抗剂Dishevelled以大致相似的水平积累，表明结合竞争可能是至关重要的。通过以接近内源水平表达Axin：GFP，我们发现在不存在Wnt信号的情况下，Axin和APC 2共组装成含有数十至数百个Axin蛋白的大细胞质复合物。Wnt信号触发这些细胞向膜的募集，而细胞质Axin水平增加，表明组装/分解改变。糖原合成酶激酶3调节破坏复合物向膜的募集和从破坏复合物释放Armadillo/β-连环蛋白。当Wnt信号不存在时，操纵Axin或APC 2水平对破坏复合物活性没有影响，但令人惊讶的是，当Wnt信号存在时，对破坏复合物具有相反的影响。升高Axin使复合物对失活更具抵抗力，而升高APC 2水平则增强失活。我们的数据表明，这两种核心成分的绝对水平和比例都会影响破坏复合物的功能，支持Axin合作伙伴之间的竞争决定破坏复合物活性的模型。细胞间的通讯对于细胞在胚胎发育过程中选择命运至关重要，并且在癌症等疾病中经常出错。Wnt细胞信号通路提供了一个极好的例子。APC和Axin等负调控蛋白的丢失会阻止细胞增殖，从而导致结肠癌。我们研究APC，Axin及其合作伙伴如何保持细胞信号关闭，以及细胞间Wnt信号如何逆转这一点。我们使用果蝇胚胎，结合生化和遗传工具与先进的显微镜。我们发现破坏复合物蛋白APC 2，Axin及其拮抗剂Dishevelled以相似的水平存在，使它们能够有效地相互竞争。我们进一步发现，Wnt信号传导关闭负调节破坏复合体机器的能力受到Axin和APC 2水平及其水平比例的影响。我们可视化动物体内的主动破坏复合物，并计算该复合物中Axin蛋白的数量。最后，我们发现Wnt信号对破坏复合物有两种影响-将其招募到质膜并改变其组装/拆卸。然后，我们提出了一个新的模型，如何调节这一重要的信号通路。
Wnt signaling provides a paradigm for cell-cell signals that regulate embryonic development and stem cell homeostasis and are inappropriately activated in cancers. The tumor suppressors APC and Axin form the core of the multiprotein destruction complex, which targets the Wnt-effector beta-catenin for phosphorylation, ubiquitination and destruction. Based on earlier work, we hypothesize that the destruction complex is a supramolecular entity that self-assembles by Axin and APC polymerization, and that regulating assembly and stability of the destruction complex underlie its function. We tested this hypothesis in Drosophila embryos, a premier model of Wnt signaling. Combining biochemistry, genetic tools to manipulate Axin and APC2 levels, advanced imaging and molecule counting, we defined destruction complex assembly, stoichiometry, and localization in vivo, and its downregulation in response to Wnt signaling. Our findings challenge and revise current models of destruction complex function. Endogenous Axin and APC2 proteins and their antagonist Dishevelled accumulate at roughly similar levels, suggesting competition for binding may be critical. By expressing Axin:GFP at near endogenous levels we found that in the absence of Wnt signals, Axin and APC2 co-assemble into large cytoplasmic complexes containing tens to hundreds of Axin proteins. Wnt signals trigger recruitment of these to the membrane, while cytoplasmic Axin levels increase, suggesting altered assembly/disassembly. Glycogen synthase kinase3 regulates destruction complex recruitment to the membrane and release of Armadillo/beta-catenin from the destruction complex. Manipulating Axin or APC2 levels had no effect on destruction complex activity when Wnt signals were absent, but, surprisingly, had opposite effects on the destruction complex when Wnt signals were present. Elevating Axin made the complex more resistant to inactivation, while elevating APC2 levels enhanced inactivation. Our data suggest both absolute levels and the ratio of these two core components affect destruction complex function, supporting models in which competition among Axin partners determines destruction complex activity. Cell-cell communication is critical for cells to choose fates during embryonic development and often goes wrong in diseases like cancer. The Wnt cell signaling pathway provides a superb example. Loss of negative regulatory proteins like APC and Axin takes the brakes off cell proliferation and thus contributes to colon cancer. We study how APC, Axin and their partners keep cell signaling off, and how cell-to-cell Wnt signals reverse this. We use the fruit fly embryo, combining biochemical and genetic tools with advanced microscopy. We found that the destruction complex proteins APC2, Axin, and their antagonist Dishevelled are present at similar levels, allowing them to effectively compete with one another. We further find that the ability of Wnt signaling to turn off the negative regulatory destruction complex machine is influenced both by the levels of Axin and APC2 and by the ratio of their levels. We visualize the active destruction complex in the animal, and count the number of Axin proteins in this complex. Finally, we find that Wnt signals have two effects on the destruction complex—recruiting it to the plasma membrane and altering its assembly/disassembly. We then propose a new model for how this important signaling pathway is regulated.
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4
作者：
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9.2
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