Yokoyama Noriko, Markova Nelli G, Wang Hsien-Yu, Malbon Craig C
Departments of Pharmacology, School of Medicine, State University of New York at Stony Brook, Stony Brook, NY, 11794-8651, USA.
J Mol Signal. 2012 Jun 29;7(1):8. doi: 10.1186/1750-2187-7-8.
Dishevelled-3 (Dvl3) is a multivalent scaffold essential to cell signaling in development. Dsh/Dvls enable a myriad of protein-protein interactions in Wnt signaling. In the canonical Wnt/β-catenin pathway specifically, Dvl3 polymerizes to form dynamic protein aggregates, so-called "signalsomes", which propagate signals from the Wnt receptor Frizzled to downstream elements.
Very large Dvl3-based supermolecular complexes form in response to Wnt3a. These complexes are identified by steric-exclusion chromatography, affinity pull-downs, proteomics, and fluorescence correlation microscopy (fcs). In the current work, the roles of Dvl3 phosphorylation and of Axin in the assembly of Dvl3-based supermolecular complexes in response to Wnt3a are probed in totipotent mouse F9 teratocarcinoma cells. Point mutations of phosphorylation sites of Dvl3 which interfere with Lef/Tcf-sensitive transcriptional activation by Wnt3a are shown to interfere more proximally with the assembly of Dvl3-based supermolecular complexes. Axin, a Dvl-interacting protein, plays a central role in organizing the beta-catenin destruction complex. The assembly of Dvl3-based supermolecular complexes is blocked either by depletion of Axin or by mutation of Axin sites necessary for polymerization in response to Wnt3a.
These data demonstrate that Wnt3a activation of the canonical pathway requires specific phosphorylation events as well as Axin to assemble very large, Dvl3-based supermolecular complexes; these complexes are a prerequisite to activation of Lef/Tcf-sensitive transcription.
Dishevelled-3（Dvl3）是发育过程中细胞信号传导所必需的多价支架蛋白。Dsh/Dvls在Wnt信号通路中促成了大量的蛋白质-蛋白质相互作用。具体而言，在经典的Wnt/β-连环蛋白通路中，Dvl3聚合形成动态的蛋白质聚集体，即所谓的“信号小体”，它将信号从Wnt受体卷曲蛋白传递至下游元件。
响应Wnt3a会形成基于Dvl3的非常大的超分子复合物。这些复合物通过空间排阻色谱法、亲和下拉法、蛋白质组学和荧光相关显微镜技术（fcs）得以鉴定。在当前研究中，在全能性小鼠F9畸胎瘤细胞中探究了Dvl3磷酸化和Axin在响应Wnt3a时基于Dvl3的超分子复合物组装中的作用。Dvl3磷酸化位点的点突变会干扰Wnt3a对Lef/Tcf敏感的转录激活，结果表明这些突变更直接地干扰了基于Dvl3的超分子复合物的组装。Axin是一种与Dvl相互作用的蛋白质，在组织β-连环蛋白破坏复合物中起核心作用。响应Wnt3a时，基于Dvl3的超分子复合物的组装会因Axin的缺失或因Axin聚合所需位点的突变而受阻。
这些数据表明，经典通路的Wnt3a激活需要特定的磷酸化事件以及Axin来组装非常大的、基于Dvl3的超分子复合物；这些复合物是激活Lef/Tcf敏感转录的先决条件。
