The role of beta-Arrestin in achieving Wnt-signaling specificity

分泌型形态发生素的Wnt家族激活不同的信号级联，其传统上分为一个经典Wnt/β-连环蛋白途径和多个非经典β-连环蛋白非依赖性途径。不同的Wnt通路通过相互作用、串扰和拮抗调节而紧密联系在一起，这导致了Wnt信号通路可能是一个信号网络而不是一组不同的通路的观点。我们对第一个资助期的联合提案是基于这样的假设，即β-Arrestin 2（Arrb 2）是不同Wnt途径中的中心蛋白，与不同的结合伴侣相互作用，从而有助于信号特异性和信号整合。事实上，我们观察到β-Arrestin 2与Dishevelled形成了信号转导所需的核心复合物，并由G（β/γ）调节，并连接了由Wnt/Frizzled受体复合物激活的主要细胞内模块。通过定量功能蛋白质组学，我们已经表征了这种β-Arrestin/ Dishevelled核心复合物的蛋白质-蛋白质相互作用，并鉴定了新的相互作用蛋白，以及与已知Wnt信号转导调节剂的新的物理相互作用。此外，在对CamKII和Dishevelled 2（Dvl 2）之间的新型物理相互作用的后续实验过程中，我们观察到CamKII与Dvl 1、Dvl 2和Dvl 3的差异结合。引人注目的是，这些差异物理相互作用与Wnt/Ca 2+信号传导中三种Dvl旁系同源物的非冗余、部分拮抗的生物学功能有关。在这个更新的应用程序中，我建议分析一组新的激酶，磷酸酶和支架蛋白，我们已经确定为相互作用的Arrb 2/Dvl 2核心复合物的Wnt信号网络的调制器和潜在的分子机制方面的假定作用。此外，三个Dvl旁系同源物可以形成不同的蛋白质复合物的显著迹象提出了不同的蛋白质复合物组合物如何与功能分歧相关的问题，我打算更详细地研究。为了实现这一目标，我们建议除了建立一个体内读出系统的同步，多路复用的Wnt途径中的激酶活性的定量质谱监测。

The Wnt family of secreted morphogens activates divergent signaling cascades, which are traditionally divided into one canonical Wnt/beta-Catenin pathway and multiple non-canonical beta-Catenin-independent pathways. The different Wnt pathways are intimately linked by interactions, cross-talk and antagonistic regulation, which led to the emerging opinion that Wnt signaling might be a signaling network rather than a group of distinct pathways. Our joint proposal for the first funding period was based on the hypothesis that beta-Arrestin 2 (Arrb2) is a central protein in different Wnt pathways that interacts with different binding partners and thereby contributes to signal specificity and signal integration. Indeed, we observed that beta-Arrestin 2 forms a core complex with Dishevelled that is required for signal transduction and that is modulated by G (beta/gamma) and links the major intracellular modules activated by Wnt/Frizzled receptor complexes. By quantitative functional proteomics we have characterized the protein-protein interactions of this beta-Arrestin / Dishevelled core complex and identified new interacting proteins and also new physical interactions with known modulators of Wnt signaling. Further, in the course of follow-up experiments on the novel physical interaction between CamKII and Dishevelled 2 (Dvl2), we observed differential binding of CamKII to Dvl1, Dvl2 and Dvl3. Strikingly, these differential physical interactions were related to non-redundant, partially antagonistic biological functions of the three Dvl paralogs in Wnt/Ca2+ signaling. In this renewal application, I propose to analyze a set of novel kinases, phosphatases and scaffold proteins that we have identified as interactors of the Arrb2/Dvl2 core complex with respect to their putative role as modulators of the Wnt signaling network and the underlying molecular mechanisms. In addition, the striking indication that the three Dvl paralogs may form distinct protein complexes poses the question how different protein complex compositions relate to functional divergence and I intend to investigate in more detail. To achieve this aim we propose in addition to establish an in vivo read-out system for synchronous, multiplexed monitoring of kinase activity in Wnt pathways by quantitative mass spectrometry.