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

Wnt家族分泌的形态因子激活分化的信号级联，传统上分为一个典型的Wnt/ β - catenin通路和多个非典型的β - catenin独立通路。不同的Wnt通路通过相互作用、串扰和拮抗调控紧密相连，这导致了Wnt信号可能是一个信号网络而不是一组不同的信号通路的新观点。我们第一个资助期的联合提案是基于β -阻滞蛋白2 （Arrb2）是不同Wnt通路中的中心蛋白，与不同的结合伙伴相互作用，从而有助于信号特异性和信号整合的假设。事实上，我们观察到β - arrestin 2与disheveled形成一个核心复合物，这是信号转导所必需的，由G （β / γ）调节，并连接由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，我们表征了这种β -阻滞蛋白/ dishevelded核心复合物的蛋白-蛋白相互作用，并鉴定了新的相互作用蛋白以及与已知Wnt信号调节因子的新的物理相互作用。此外，在CamKII与disheveled2 （Dvl2）之间新型物理相互作用的后续实验中，我们观察到CamKII与Dvl1、Dvl2和Dvl3的差异结合。引人注目的是，这些不同的物理相互作用与Wnt/Ca2+信号传导中三个Dvl相似物的非冗余，部分拮抗的生物学功能有关。在这个更新的申请中，我建议分析一组新的激酶、磷酸酶和支架蛋白，我们已经确定它们是Arrb2/Dvl2核心复合物的相互作用物，关于它们作为Wnt信号网络调节剂的假设作用和潜在的分子机制。此外，三种Dvl相似物可能形成不同的蛋白质复合物的惊人迹象提出了不同的蛋白质复合物组成如何与功能分化相关的问题，我打算更详细地研究。为了实现这一目标，我们建议建立一个体内读出系统，通过定量质谱法同步、多路监测Wnt通路中的激酶活性。

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.