Molecular Mechanisms of Wnt Signal Transduction

 描述（由申请人提供）：Wnt生长因子是脂质修饰的分泌蛋白，通过在胚胎发生和成体组织更新期间指定细胞命运，对多细胞生物体的发育至关重要。在"经典"途径中，Wnt通过转录辅激活因子β-连环蛋白激活控制细胞增殖的靶基因。我们的目标是确定将Wnt与其细胞表面共受体Frizzled（Fzd）和LRP 5/6的结合转化为β-连环蛋白稳定化的分子机制。在缺乏Wnt的情况下，β-连环蛋白结合在"破坏复合物"中，该"破坏复合物"包括支架蛋白Axin和激酶GSK-3和CK 1; CK 1和GSK-3对β-连环蛋白的磷酸化导致其泛素化和被蛋白酶体破坏。Wnt与Fzd和LRP 5/6的结合使得Fzd能够募集细胞质蛋白Dishevelled（Dvl），其进而与Axin结合，从而将破坏复合物募集至活化的受体复合物。这导致LRP 5/6胞内结构域（ICD）的磷酸化，其抑制β-连环蛋白破坏。理解这一过程是一个重要的生物医学问题，因为该途径的组分在大量癌症中发生突变，这导致不适当地稳定的β-连环蛋白和不受控制的细胞增殖。战略我们解决了两个未解决的基本机制问题，使用纯化的途径组分的生物化学和生物物理测定：1）Wnt如何促进Fzd-Dvl相互作用？2)LRP6尾部的磷酸化如何抑制β-连环蛋白的破坏？目的1使用Norrin（一种对β-连环蛋白介导的信号传导途径特异的Fzd4配体）检查配体和Dvl与Fzd结合的偶联。定量亲和力测量将用于测试配体和Dvl与Fzd结合之间是否存在变构偶联（Aim 1a），LRP 6（Aim 1b）或Dvl磷酸化（Aim 1c）是否促进该偶联，以及Dvl磷酸化是否促进其与Axin的相互作用（Aim 1c）。目的2测试LRP6 ICD磷酸化的机制以及它如何抑制β-catenin磷酸化。目的2a定义GSK-3和CK1对LRP 6 ICD磷酸化的机制，以及磷酸化如何影响LRP 6 ICD对Axin的亲和力。目的2b测试最近描述了Axin自身抑制的模型，其中分子内相互作用抑制Axin与LRP 6和β-连环蛋白的结合，并且通过GSK-3磷酸化来缓解。该子目标还测试了Axin和β-catenin是否竞争磷酸化Axin。目的2c测试GSK-3的LRP 6抑制是否在动力学上足以允许β-连环蛋白从破坏复合物中逃逸。结果。从这些研究中获得的机制见解有望为控制癌症和其他疾病途径所需的治疗方法的开发提供信息。

 DESCRIPTION (provided by applicant): Wnt growth factors are lipid-modified, secreted proteins that are essential for the development of multicellular organisms by specifying cell fate during embryogenesis and the renewal of tissues in the adult. In the "canonical" pathway, Wnts activate target genes that control cellular proliferation through the transcriptional co-activator�-catenin. Our goal is to define the molecular mechanisms that transduce the binding of a Wnt to its cell surface co-receptors Frizzled (Fzd) and LRP5/6 into stabilization of β-catenin. Rationale In the absence of Wnts, β-catenin is bound in a "destruction complex" that includes the scaffolding protein Axin and the kinases GSK-3 and CK1; phosphorylation of β-catenin by CK1 and GSK-3 leads to its ubiquitylation and destruction by the proteasome. Wnt binding to Fzd and LRP5/6 enables Fzd to recruit the cytoplasmic protein Dishevelled (Dvl), which in turn binds to Axin and thereby recruits the destruction complex to the activated receptor complex. This leads to phosphorylation of the LRP5/6 intracellular domain (ICD), which inhibits β-catenin destruction. Understanding this process is an important biomedical problem, as components of this pathway are mutated in a large number of cancers, which leads to inappropriately stabilized β-catenin and uncontrolled cellular proliferation. Strategy. We address two unsolved, fundamental mechanistic questions, using biochemical and biophysical assays with purified pathway components: 1) How do Wnts promote the Fzd-Dvl interaction? 2) How does phosphorylation of the LRP6 tail inhibit β-catenin destruction? Aim 1 examines the coupling of ligand and Dvl binding to Fzd, using Norrin, a Fzd4 ligand specific to a β-catenin-mediated signaling pathway. Quantitative affinity measurements will be used to test whether there is allosteric coupling between ligand and Dvl binding to Fzd (Aim 1a), whether LRP6 (Aim 1b) or Dvl phosphorylation (Aim 1c) promotes this coupling, and whether Dvl phosphorylation promotes its interactions with Axin (Aim 1c). Aim 2 tests proposed mechanisms of LRP6 ICD phosphorylation and how it inhibits β-catenin phosphorylation. Aim 2a defines the mechanism of LRP6 ICD phosphorylation by GSK-3 and CK1, and how phosphorylation affects the affinity of the LRP6 ICD for Axin. Aim 2b tests recently described models of Axin autoinhibition, in which intramolecular interactions inhibit binding of Axin to LRP6 and to β-catenin, and are relieved by GSK-3 phosphoryation. This sub-Aim also tests whether Axin and β-catenin compete for phosphorylated Axin. Aim 2c tests whether LRP6 inhibition of GSK-3 is kinetically sufficient to allow β-catenin to escape from the destruction complex. Outcomes. The mechanistic insights derived from these studies promise to inform development of therapeutics needed to control the pathway in cancers and other diseases.