Molecular Mechanisms of Wnt Signal Transduction

 说明(申请人提供)：WNT生长因子是一种脂类修饰的分泌型蛋白质，通过确定胚胎发育过程中的细胞命运和成人组织的更新，对多细胞生物体的发育至关重要。在“规范”途径中，WNTS通过转录共激活因子�-连环蛋白激活控制细胞增殖的靶基因。我们的目标是确定WNT与其细胞表面辅助受体FrizzledFZD和LRP5/6结合从而稳定β-连环蛋白的分子机制。在没有WNTS的情况下，β-连环蛋白结合在一个“破坏复合体”中，该“破坏复合体”包括支架蛋白Axin、激酶GSK-3和CK1；β-连环蛋白被Ck1和GSK-3磷酸化，导致其泛素化，并被蛋白酶体破坏。WNT与FZD和LRP5/6的结合使FZD能够招募胞浆蛋白DVL，DVL进而与轴蛋白结合，从而将破坏复合体招募到激活的受体复合体中。这导致LRP5/6胞内域的磷酸化，从而抑制β-连环蛋白的破坏。了解这一过程是一个重要的生物医学问题，因为这一途径的组件在大量癌症中发生突变，导致β-连环蛋白不适当稳定和细胞增殖失控。战略。我们使用生化和生物物理方法对纯化的途径成分进行分析，解决了两个尚未解决的基本机制问题：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β-连环蛋白介导的信号通路的特异性FZD4配体Norrin，研究配体和DVL与FZD的偶联作用。定量亲和力测量将用于测试与FZD(Aim 1a)结合的配体和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的相互作用(Aim 1c)。目的2检测LRP6ICD的磷酸化机制及其对β-连环蛋白磷酸化的抑制作用。目的2a明确LRP6 ICD被GSK-3和CK1磷酸化的机制，以及磷酸化如何影响LRP6 ICD与轴蛋白的亲和力。目的2b测试最近描述的Axin自身抑制模型，在该模型中，分子内相互作用抑制Axin与LRP6和β-连环蛋白的结合，并被GSK-3磷酸化解除。这个子目标还测试Axin和β-catenin是否竞争磷酸化的Axin。目的2c检测LRP6对GSK-3的抑制是否足以使β-连环蛋白从破坏复合体中逃逸。结果。从这些研究中获得的机制洞察力有望为控制癌症和其他疾病的途径所需的治疗方法的发展提供信息。

 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.