Molecular mechanisms of Wnt and mechanical signaling through β-catenin

Wnt 的分子机制和通过 β-catenin 的机械信号传导

• 批准号: 10382116
• 负责人: William I Weis
• 金额: $ 2.21万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-06-07 至 2024-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10382116
• 关键词: APC gene; Actins; Address; Adherens Junction; Adult; Affect; Area; Binding; Biochemical; Cadherins; Cell Fate Control; Cell Nucleus; Cell-Cell Adhesion; Cells; Complex; Cysteine-Rich Domain; Cytoplasmic Protein; Defect; Development; Dsh protein; E-Cadherin; Embryonic Development; Epithelial; Family; Frizzled Domain; Genes; Goals; Growth Factor; Homeostasis; Intercellular Junctions; Knowledge; Ligands; Link; Maintenance; Malignant Neoplasms; Mechanics; Microfilaments; Molecular; Molecular Conformation; Neoplasms; Pathway interactions; Phosphorylation; Phosphotransferases; Process; Property; Proteins; Role; Scaffolding Protein; Signal Transduction; Signaling Protein; Solid; Specific qualifier value; Structure; Tight Junctions; Tissues; Vinculin; WNT Signaling Pathway; Wnt proteins; afadin; alpha catenin; base; beta catenin; biophysical techniques; extracellular; mechanical force; multicatalytic endopeptidase complex; novel strategies; protein B; receptor; recruit; tissue regeneration; transmission process; zonula occludens-1 protein

The development and homeostasis of solid tissues depends upon biochemical and mechanical signals that control cell fate and the resulting organization of cells in the tissue. The conserved protein β-catenin is a key effector of signals from both the Wnt family of secreted growth factors that specify cell fate during embryogenesis and tissue renewal in the adult, and mechanical force transmitted through cell-cell junctions in multicellular tissues. We hypothesize that mechanical force transmitted through β-catenin links Wnt signaling and cell-cell adhesion, and our overall goal is to understand the molecular mechanisms underlying these dual roles of β-catenin. Our strategy is to use biochemical, structural and biophysical methods to address critical knowledge gaps in these areas. In the absence of Wnts, the β-catenin is bound in a “destruction complex” that includes the proteins Axin and Adenomatous Polyposis Coli (APC), and kinases that phosphorylate β-catenin; phosphorylation leads to ubiquitylation and destruction of β-catenin by the proteasome. Wnt binding to the receptors Frizzled (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, which inhibits β-catenin destruction; the stabilized β-catenin enters the nucleus and activates target genes. We will address critical mechanistic aspects of this pathway that are not understood: 1) how secreted ligands “activate” the Fzd-Dvl interaction needed for β-catenin stabilization through interaction with the extracellular cysteine-rich domain of Fzd and LRP5/6; 2) how activated Dvl recruits Axin to turn off β-catenin destruction; 3) how the β-catenin destruction complex forms and interacts with the ubiquitylation/proteosomal machinery; 4) the essential role of APC in β-catenin destruction. Force transmission through cell-cell adherens junctions (AJ) requires a complex of E-cadherin, β-catenin, and α-catenin, which binds to actin filaments and forms a minimal force-sensing unit. Tension on cadherins can release β-catenin and cause its translocation to the nucleus independent of, but synergized by, Wnt signaling. Understanding such tension-triggered release of β-catenin requires understanding how force is transmitted through the AJ complex. α-Catenin additionally has a central role in organizing epithelial tissues based on its interactions with vinculin, Epithelial Protein Lost in Neoplasm (EPLIN), the tight junction (TJ) protein Zonula Occludens (ZO)-1, and afadin, all of which bind actin and recruit other scaffolding and signaling proteins. We will study: 1) The force-dependent conformational landscape and force responsivness of α-catenin alone and bound to its partners, including how β-catenin modifies α-catenin force responsiveness; 2) How αE-catenin conformation and force transmission properties are affected by its binding to its other junctional partners.

实体组织的发育和体内平衡取决于生物化学和机械信号， 控制细胞命运和组织中细胞的最终组织。保守蛋白β-catenin是一个关键 来自分泌生长因子的Wnt家族的信号的效应物，其在细胞生长期间指定细胞命运。 胚胎发生和组织更新，以及通过细胞-细胞连接传递的机械力 在多细胞组织中。我们假设通过β-连环蛋白连接Wnt传递的机械力 我们的总体目标是了解潜在的分子机制， β-catenin的双重作用。我们的策略是使用生物化学、结构和生物物理方法， 解决这些领域的关键知识差距。 在缺乏Wnt的情况下，β-连环蛋白结合在包括Axin蛋白的“破坏复合物”中， 和腺瘤性结肠息肉病（APC），以及磷酸化β-连环蛋白的激酶;磷酸化导致 蛋白酶体对β-catenin的泛素化和破坏。Wnt与卷曲受体（Fzd）的结合 而LRP 5/6使Fzd能够募集细胞质蛋白Dishevelled（Dvl），Dvl又与Axin结合， 从而将破坏复合物募集到活化的受体复合物上。这导致了磷酸化， LRP 5/6细胞内结构域，其抑制β-连环蛋白破坏;稳定的β-连环蛋白进入细胞内， 细胞核并激活靶基因。我们将解决这一途径的关键机制方面， 理解：1）分泌的配体如何“激活”β-连环蛋白稳定化所需的Fzd-Dvl相互作用 通过与Fzd和LRP 5/6的胞外富含半胱氨酸结构域的相互作用; 2）如何激活Dvl 募集Axin以关闭β-连环蛋白破坏; 3）β-连环蛋白破坏复合物如何形成和相互作用 与泛素化/蛋白体机制; 4）APC在β-catenin破坏中的重要作用。 通过细胞-细胞粘附连接（AJ）的力传递需要E-钙粘蛋白，β-连环蛋白， 和α-连环蛋白，它与肌动蛋白丝结合，形成一个最小的力感应单位。钙粘蛋白张力 可释放β-连环蛋白并使其不依赖于Wnt而移位至细胞核，但与Wnt协同作用 信号理解这种张力触发的β-连环蛋白释放需要理解力是如何 通过AJ综合体传播此外，α-连环蛋白在组织上皮组织中具有重要作用。 基于其与黏着斑蛋白、肿瘤中的上皮蛋白丢失（EPLIN）、紧密连接（TJ） 蛋白质Zonula Occludens（ZO）-1和afadin，所有这些都结合肌动蛋白并招募其他支架， 信号蛋白我们将研究：1）力依赖的构象景观和力 α-catenin单独和与其伴侣结合的反应性，包括β-catenin如何修饰α-catenin 力响应性; 2）α E-连环蛋白的构象和力传递特性如何受到其 与其他连接性伴侣结合。