Wnt/beta-catenin signaling: phosphorylation regulation of Wnt receptor-Axin inter

描述（由申请人提供）：Wnt家族分泌蛋白的信号调节胚胎发生和体内平衡的许多方面。不受管制的Wnt信号与癌症、骨质疏松症和退行性疾病等人类疾病有关。因此，研究Wnt信号对于理解基础生物学和人类健康具有至关重要的意义。在调节转录辅激活因子2-连环蛋白稳定性的典型Wnt通路中，涉及两种类型的跨膜受体，它们构成Wnt受体复合物。一个是卷曲（Fz）蛇形受体家族的成员，另一个是低密度脂蛋白受体相关蛋白（LRP）家族的单遍跨膜受体LRP5或LRP6。我们已经证明Wnt诱导LRP6磷酸化/激活，从而募集轴蛋白支架蛋白，这是调节2-连环蛋白稳定性所必需的。但是LRP6磷酸化和激活以及轴蛋白的募集如何导致2-连环蛋白的稳定一直知之甚少，这是本应用的重点。目的1：研究LRP6的磷酸化和信号转导。我们生成了Lrp5-/-；Lrp6-/-双敲除（LrpDKO）小鼠胚胎成纤维细胞。这些LrpDKO细胞为深入研究LRP6磷酸化和信号传导提供了理想的遗传加生化系统。我们将在Lrp6DKO细胞中重新表达野生型和各种LRP6突变体，以确定LRP6磷酸化的起始步骤，不同磷酸化基序之间的功能和关系，以及它们在LRP6信号传导和所谓的LRP6“信号体”形成中的潜在作用。目的2研究Wnt对轴蛋白去磷酸化的调控作用。Axin是2-catenin磷酸化/降解和LRP6磷酸化/活化的关键支架蛋白，Axin与磷酸化的LRP6相互作用是2-catenin稳定的关键步骤。我们已经生成了磷酸化轴蛋白的特异性抗体，并表明Wnt通过蛋白磷酸酶1 （PP1）促进轴蛋白去磷酸化。我们发现这可能是通过PP1调节蛋白的磷酸化介导的，PP1调节蛋白是Wnt信号传导中一个以前未被怀疑的成分。我们将研究轴蛋白去磷酸化及其在介导wnt调控的爪蟾胚胎模式中的作用。目的3：研究Axin-LRP6的相互作用。我们将在Axin和LRP6之间建立一种新的磷酸化调节蛋白相互作用，并揭示Axin磷酸化如何通过一种新的分子内和自调节机制控制其与其他伙伴（如LRP6）的相互作用。我们将提出一个内聚的工作模型，通过调节轴蛋白磷酸化-去磷酸化将LRP6激活与2-连环蛋白稳定联系起来。目的4：研究Wnt激活轴蛋白PP1去磷酸化的机制。我们将研究LRP6如何使用LrpDKO细胞调节PP1的激活，我们对鉴定介导PP1 Wnt激活的激酶（或激酶）特别感兴趣。这些研究可能会提供从Wnt受体到2-连环蛋白稳定的Wnt信号的全面理解。

DESCRIPTION (provided by applicant): Signaling by the Wnt family of secreted proteins regulates many aspects of embryogenesis and homeostasis. Deregulated Wnt signaling is implicated in human diseases such as cancer, osteoporosis and degenerative disorders. The study of Wnt signaling thus has critical importance for understanding basic biology and human health. In the canonical Wnt pathway that regulates the stability of the transcriptional coactivator 2-catenin, two types of transmembrane receptors that constitute the Wnt receptor complex are involved. One is a member of the Frizzled (Fz) family of serpentine receptors, and the other is a single-pass transmembrane receptor of the LDL receptor related protein (LRP) family, LRP5 or LRP6. We have shown that Wnt induces LRP6 phosphorylation/activation, which recruits the Axin scaffolding protein that is essential for the regulation of 2-catenin stability. But how LRP6 phoshorylation and activation and the recruitment of Axin lead to 2- catenin stabilization has been poorly understood, and is the focus of this application. Aim 1 is to study LRP6 phosphorylation and signaling. We have generated Lrp5-/-; Lrp6-/- double knockout (LrpDKO) mouse embryonic fibroblast cells. These LrpDKO cells provide an ideal genetic plus biochemical system for in-depth investigation of LRP6 phosphorylation and signaling. We will re-express the wild type and various LRP6 mutants in Lrp6DKO cells to define the initiating step in LRP6 phosphorylation, the function and relationship among distinct phosphorylation motifs, and their potential roles in LRP6 signaling and in the so-called LRP6 "signalosome" formation. Aim 2 is to study Wnt regulation of Axin dephosphorylation. Axin is a pivotal scaffolding protein for 2-catenin phosphorylation/degradation and LRP6 phosphorylation/activation, and Axin interaction with phosphorylated LRP6 is a key step in 2-catenin stabilization. We have generated specific antibodies for phoshorylated Axin, and show that Wnt promotes Axin dephosphorylation via protein phosphatase 1 (PP1). We found that this is likely mediated through phosphorylation of a PP1 regulatory protein, a previously unsuspected component in Wnt signaling. We will study Axin dephosphorylation and its role in mediating Wnt-regulated Xenopus embryonic patterning. Aim 3 is to study Axin-LRP6 interaction. We will establish a new type of phosphorylation-regulated protein interaction between Axin and LRP6 and uncover how Axin phosphorylation controls its interactions with other partners such as LRP6 via a novel intra- molecular and auto-regulatory mechanism. We will present a cohesive working model that links LRP6 activation to 2-catenin stabilization through regulation of Axin phosphorylation-dephosphorylation. Aim 4 is to study the mechanism by which Wnt activates PP1 dephosphorylation of Axin. We will study how LRP6 regulates PP1 activation using LrpDKO cells, and we are particularly interested in the identification of the kinase (or kinases) that mediates Wnt activation of PP1. These studies will likely provide a comprehensive understanding of Wnt signaling from the Wnt receptor to 2-catenin stabilization. PUBLIC HEALTH RELEVANCE: Cell-to-cell communication is vital for human embryonic development and tissue homeostasis, and is mediated by key "signal transduction" pathways. Defects in these pathways cause human cancer. This proposal aims to understand the molecular nature of one of these signaling pathways, the so-called Wnt pathway, in animal development and human cells. Our study will provide better understanding of human embryonic development and pathogenesis of cancer.