Molecular Mechanisms of Wnt Signal Transduction

Wnt信号转导的分子机制

• 批准号: 9245707
• 负责人: William I Weis
• 金额: $ 31.09万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-04-01 至 2020-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9245707
• 关键词: Address; Adenomatous Polyposis Coli Protein; Adult; Affect; Affinity; Agonist; Binding; Biochemical; Biological Assay; Biophysics; CSNK1A1 gene; Cell Proliferation; Cell membrane; Cell surface; Cells; Complex; Coupling; Cytoplasmic Protein; Cytoplasmic Tail; Development; Disease; Dsh protein; Embryonic Development; Family member; Foundations; G-Protein-Coupled Receptors; Gene Targeting; Glycogen Synthase Kinase 3; Goals; Growth Factor; Kinetics; Ligand Binding; Ligands; Lipids; Malignant Neoplasms; Measurement; Measures; Mediating; Modeling; Molecular; Molecular Conformation; Mutate; Organism; Outcome; Pathway interactions; Peptides; Phosphorylation; Phosphotransferases; Process; Proteins; Recruitment Activity; Retinal; Role; Scaffolding Protein; Signal Pathway; Signal Transduction; Specific qualifier value; Tail; Testing; Tissues; Transcription Coactivator; WNT Signaling Pathway; Wnt proteins; beta catenin; biophysical techniques; experimental study; inhibitor/antagonist; insight; member; multicatalytic endopeptidase complex; prevent; programs; public health relevance; receptor; seven-transmembrane G-protein-coupled receptor; therapeutic development

 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.

 描述（由适用提供）：Wnt生长因子是脂质修饰的，分泌的蛋白质，对于在胚胎发生过程中指定细胞命运和成人组织的更新，对于多细胞生物的发展至关重要。在“规范”途径中，WNT激活了通过转录共激活蛋白控制细胞增殖的靶基因。我们的目标是将Wnt与其细胞表面共受体（FZD）和LRP5/6的结合定义分子机制，以稳定β-catenin。理由在没有WNT的情况下，β-catenin与“破坏复合物”结合，其中包括脚手架蛋白Axin和激酶GSK-3和CK1。 CK1和GSK-3对β-catenin的磷酸化导致蛋白质组的泛素化和破坏。 Wnt与FZD和LRP5/6的结合使FZD能够募集细胞质蛋白质染色（DVL），这又与Axin结合，从而将破坏复合物收集到激活的接收器配合物中。这导致LRP5/6细胞内结构域（ICD）的磷酸化，从而抑制β-catenin的破坏。理解这一过程是一个重要的生物医学问题，因为该途径的成分在许多癌症中被突变，这会导致不适当地稳定β-catenin和不受控制的细胞增殖。战略。我们使用具有纯化途径成分的生化和生物物理测定法解决了两个未解决的基本机械问题：1）WNT如何促进FZD-DVL相互作用？ 2）LRP6尾的磷酸化如何抑制β-catenin的破坏？ AIM 1检查配体和DVL与FZD的偶联，使用Norrin，Norrin，Norrin是特定于β-catenin介导的信号通路的FZD4配体。定量亲和力测量目标2A定义了LRP6 ICD磷酸化的机制及其抑制β-catenin磷酸化的机制。 AIM 2A定义了LRP6 ICD磷酸化的机制及其如何抑制β-catenin磷酸化。由GSK-3和CK1，以及磷酸化如何影响LRP6 ICD对Axin的亲和力。 AIM 2B测试最近描述了Axin自抑制的模型，其中分子内相互作用抑制了Axin与LRP6和β-catenin的结合，并通过GSK-3磷酸化来缓解。该子AIM还测试了Axin和β-catenin是否竞争磷酸化的Axin。 AIM 2C测试LRP6抑制GSK-3是否足以允许β-catenin从破坏复合物中逃脱。结果。这些研究得出的机械洞察力有望为控制癌症和其他疾病中的途径所需的治疗发展提供信息。