Molecular Mechanisms of Wnt Signal Transduction

Wnt信号转导的分子机制

• 批准号: 9106024
• 负责人: William I Weis
• 金额: $ 31.04万
• 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/9106024
• 关键词: Address; Adenomatous Polyposis Coli Protein; Adult; Affect; Affinity; Agonist; Binding; Biochemical; Biological Assay; CSNK1A1 gene; Cell Proliferation; Cell membrane; Cell surface; Cells; Complex; Coupling; Cytoplasmic Protein; Cytoplasmic Tail; Development; Disease; Embryonic Development; Family member; Foundations; G-Protein-Coupled Receptors; Gene Targeting; Glycogen Synthase Kinase 3; Goals; Growth; 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; Therapeutic Agents; Tissues; Transcription Coactivator; Wnt proteins; beta catenin; biophysical techniques; inhibitor/antagonist; insight; member; multicatalytic endopeptidase complex; prevent; programs; public health relevance; receptor; research study; 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人组织更新过程中指定细胞命运，对于多细胞生物的发育至关重要。在“规范”途径中，Wnt 激活。通过转录共激活因子β-连环蛋白控制细胞增殖的靶基因我们的目标是定义转导 Wnt 与其细胞表面共受体 Frizzled 结合的分子机制。 (Fzd) 和 LRP5/6 稳定 β-连环蛋白 基本原理 在没有 Wnt 的情况下，β-连环蛋白结合在“破坏复合物”中，该复合物包括支架蛋白 Axin 以及 β 磷酸化激酶 GSK-3 和 CK1。 CK1 和 GSK-3 的连环蛋白导致其泛素化，并被蛋白酶体破坏，Wnt 与 Fzd 和 LRP5/6 结合使其成为可能。 Fzd 招募细胞质蛋白 Disheveled (Dvl)，后者又与 Axin 结合，从而将破坏复合物招募到激活的受体复合物上，这导致 LRP5/6 胞内结构域 (ICD) 磷酸化，从而抑制 β-连环蛋白破坏。了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大量癌症中发生突变，从而导致β-连环蛋白的不适当稳定和不受控制的细胞增殖策略。使用纯化的途径成分进行生化和生物物理测定，解决两个未解决的基本机制问题：1) Wnts 如何促进 Fzd-Dvl 相互作用？2) LRP6 尾部磷酸化如何抑制 β-catenin 破坏？配体和 Dvl 与 Fzd 的结合，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）进行定量亲和力测量。测试配体和与 Fzd (Aim 1a) 结合的 Dvl 之间是否存在变构偶联，LRP6 (Aim 1b) 或 Dvl 磷酸化 (Aim 1c) 是否促进这种偶联，以及 Dvl 磷酸化是否促进其与 Axin (Aim Aim Aim Aim) 的相互作用。 2 测试提出的 LRP6 ICD 磷酸化机制及其如何抑制 β-连环蛋白磷酸化 Aim 2a。定义了 GSK-3 和 CK1 磷酸化 LRP6 ICD 的机制，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 自抑制模型，其中分子内相互作用抑制 Axin 与 LRP6 和 β 的结合。 -连环蛋白，并通过 GSK-3 磷酸化而缓解。该子目标还测试 Axin 和 β-连环蛋白是否竞争磷酸化。 Axin.Aim 2c 测试 LRP6 对 GSK-3 的抑制是否足以在动力学上使 β-catenin 逃离破坏复合物。这些研究得出的机制见解有望为控制癌症和癌症通路所需的治疗方法的开发提供信息。其他疾病。