Mechanisms for Wnt Signaling

描述(申请人提供)：Wnt家族的分泌性糖蛋白在广泛的生物学和病理生理过程中发挥重要作用，包括胚胎发育、器官发生、组织动态平衡、干细胞生物学、脂肪和葡萄糖代谢以及肿瘤发生。在这项研究中，我们计划研究WNT信号机制中仍然存在的一个主要空白，即WNT如何通过质膜传递信号。WNT3a是一个典型的典型Wnt，通过与其两个细胞表面受体FrizzledFZ和低密度脂蛋白相关蛋白5/6(LRP)结合来启动其信号转导，导致LRP的磷酸化，进而稳定b-catenin。我们先前发现，Wnt3a通过FZ和Disheveled(DVL)刺激磷脂酰肌醇(PI)激酶PI4K2和PIP5K1产生磷脂酰肌醇4，5-二磷酸(PIP2)。PIP2促进LRP簇或信号小体在细胞表面的形成，这对LRP的磷酸化和下游信号传递是重要的。我们使用包括3D-STORM(随机光学重建显微镜)在内的超分辨率荧光成像技术来直接显示LRP6和笼状蛋白簇及其在细胞表面的共定位，在本研究中，我们将把我们的研究扩展到FZ和DVL。此外，我们计划研究PI4K2和PIP5K1与DVL相互作用的分子基础，以及DVL激活这些PI激酶的机制。这项研究的具体目的是：1)用生化和先进的成像方法进一步表征WNT3a受体信号体的形成和组织。2)阐明DVL与PI4K2和PIP5K1相互作用和激活的分子机制。这些研究将为WNT3a受体信号体的形成提供新的组织和机制方面的见解，并阐明DVL是一种以前只被称为支架的蛋白质，它是如何刺激脂蛋白激酶活性的。

DESCRIPTION (provided by applicant): The Wnt family of secretory glycoproteins plays important roles in a wide range of biological and pathophysiological processes, including embryonic development, organogenesis, tissue homeostasis, stem cell biology, lipid and glucose metabolism and tumor genesis. In this study we plan to work on one of the major gaps that remains in Wnt signaling mechanisms, which is how Wnt transduces the signals cross the plasma membrane. Wnt3a, a prototypical canonical Wnt, initiates its signaling by binding to its two cell surface receptor Frizzled (Fz) and LDL- related protein 5/6 (LRP), leading to the phosphorylation of LRP and subsequently stabilization of b-catenin. We previously showed that Wnt3a, via Fz and disheveled (Dvl), stimulates phosphatidylinositol (PI) kinases PI4K2 and PIP5K1 to produce phosphatidylinositol 4, 5- bisphosphate (PIP2). PIP2 facilitates the formation of LRP clusters or signalosomes on the cell surface, which is important for LRP phosphorylation and signaling downstream. We used super resolution fluorescence imaging techniques including 3D-STORM (STochastic Optical Reconstruction Microscopy) to directly visualized LRP6 and clathrin clusters and their co-localization at cell surfaces, and in this study we will extend our study to Fz and Dvl. In addition, we plan to characterize the molecular basis for the interactions of PI4K2 and PIP5K1 with Dvl and mechanisms for the activation of these PI kinases by Dvl. The specific aims of this study are: 1) to further characterize the formation and organization of Wnt3a receptor signalosomes using both biochemical and advanced imaging approaches. 2) To elucidate the molecular mechanisms for Dvl interaction and activation of PI4K2 and PIP5K1. These studies will provide novel organizational and mechanistic insights into the formation of Wnt3a receptor signalosome and elucidate how Dvl, a protein previously only known as a scaffold, stimulates lipid kinase activity.