Nuclear import of beta-Catenin in Wnt-signaling

Wnt 信号转导中 β-连环蛋白的核输入

• 批准号: 10094218
• 负责人: Marek Mlodzik
• 金额: $ 21.19万
• 依托单位: ICAHN SCHOOL OF MEDICINE AT MOUNT SINAI
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-02-03 至 2022-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10094218
• 关键词: APC gene; Address; Affect; Amelia; Amino Acids; Binding; Binding Proteins; Biological; Biological Assay; Cell Maintenance; Cell Nucleus; Cell Polarity; Cells; Cellular biology; Cilia; Complex; Cytoplasm; Data; Defect; Development; Disease; Dissection; Drosophila genus; Event; Family Dasypodidae; Follow-Up Studies; Genes; Genetic; Genetic Transcription; Growth; Homeostasis; Kinesin; Knowledge; Lead; Link; Malignant Neoplasms; Mammalian Cell; Mammals; Mediating; Medical; Membrane; Microtubules; Modeling; Molecular; Movement; N-terminal; Nuclear; Nuclear Import; Nuclear Translocation; Organ; Organism; Pathway interactions; Pattern; Pharmacotherapy; Play; Process; Proteins; Proteomics; Proto-Oncogenes; Regulation; Reporting; Role; Scaffolding Protein; Signal Pathway; Signal Transduction; Stretching; System; TCF Transcription Factor; Time; Tissues; Tumor Suppressor Proteins; WNT Signaling Pathway; Work; arm; arm function; base; beta catenin; congenital heart disorder; drug development; high resolution imaging; in vivo; inhibitor/antagonist; movie; mutant; novel; novel therapeutics; osteoporosis-pseudoglioma syndrome; particle; prevent; protein complex; receptor; recruit; stem cells; vasculogenesis

Project Summary Wnt/Wingless (Wg)-signaling plays important roles in intercellular signaling in all metazoan organisms. It functions in many critical processes, such as organ patterning, growth control, cell polarity, and stem cell maintenance. When deregulated it is linked to diseases ranging from congenital heart disease and aberrant vasculogenesis to cancer. Recent studies in my lab have shown that Intraflagellar Transport complex A (IFT-A) proteins modulate canonical Wnt/Wg-signaling independently of the ciliary role of IFTs. We demonstrated that they do so together with Kinesin2 (as they do in ciliary functions) and promote nuclear translocation of β- catenin upon Wnt-pathway activation and act downstream of β-catenin stabilization. Kinesin-2 and IFT-A proteins act as a complex during Wg-signaling in Drosophila and mammals. Mutants of both, Kinesin 2 and IFT-A, affect Wg/Wnt-signaling in the same manner, and they interact genetically and physically. Kap3, a kinesin associated protein that serves as the bridging factor between Kinesin 2 and IFT-A in ciliary function is also required in the same manner and is involved in the formation of a physical complex of Kinesin 2-Kap3- IFTs. The IFT-A protein IFT140 then directly binds to β-catenin, called Armadillo/Arm in Drosophila. Upon pathway stimulation by Wg/Wnt and resulting pathway activation, all these factors co-localize with each other and β-catenin, and bind together to micro-tubules (MTs). Single or double mutant cells for Kinesin-2, IFT-As, or Kap3 fail to properly activate Wg/Wnt-signaling targets in both Drosophila and MEFs. In addition, axin double mutant backgrounds with IFT-A or Kinesin-2 reveal high levels of cytoplasmic Arm/β-catenin but fail to activate Wg/Wnt targets, due to reduced nuclear β-catenin localization. These data indicate that the Kinesin-2/IFT-A complex promotes nuclear localization of Arm/β-catenin by protecting it from a cytoplasmic tether/inhibitor. We have thus identified a mechanistic function of the Kinesin-2/IFT-A complex in Wnt-signaling, independent of their role in the cilium. As the associated mechanism(s) are conserved in mammalian cells, these observations are potentially amenable to drug treatment. However, several questions remain: (i) How is the Kinesin-2/IFT-A complex promoting nuclear localization; our data suggest it is by movement along microtubules, but this needs to be refined; and (ii) what is the factor that competes with IFT140 binding to Arm/β-catenin and serves an `inhibitory' function for Arm/β-catenin nuclear localization. We will address in Aim 1 the cell biology of this mechanism using an elegant ex vivo Wnt-signaling system, and Aim 2 is tailored to identify antagonistic factor(s) that compete with IFT140 binding. Our preliminary data suggest that this knowledge can serve as an entry-point for new drug development to inhibit overactive Wnt/β-catenin signaling. Thus, information acquired in this application will advance our mechanistic understanding of Wnt/β-catenin signaling, and potentially also lead to follow up studies to develop new drugs and thus benefit the treatment in disease associated contexts.

项目摘要 Wnt/Wingless（Wg）信号在所有后生动物生物的细胞间信号传导中起重要作用。它 在许多关键过程中发挥作用，如器官形成、生长控制、细胞极性和干细胞 上维护当放松管制时，它与从先天性心脏病和异常心脏病等疾病有关。 血管生成到癌症我实验室最近的研究表明鞭毛内转运复合物A（IFT-A） 蛋白质调节典型的Wnt/Wg信号传导，而不依赖于IFTs的纤毛作用。我们证明了 它们与驱动蛋白2一起这样做（就像它们在纤毛功能中所做的那样），并促进β- 连环蛋白在Wnt-途径活化时起作用，并在β-连环蛋白稳定化的下游起作用。驱动蛋白-2和IFT-A 在果蝇和哺乳动物中，Wg蛋白在Wg信号传导过程中作为复合物起作用。两者的突变体，驱动蛋白2和 IFT-A以相同的方式影响Wg/Wnt信号传导，并且它们在遗传和物理上相互作用。Kap3，a 在纤毛功能中作为驱动蛋白2和IFT-A之间的桥接因子的驱动蛋白相关蛋白， 也以相同的方式需要，并参与形成驱动蛋白2-Kap 3的物理复合物， IFTs。然后，IFT-A蛋白IFFT 140直接与β-连环蛋白结合，在果蝇中称为Armadillo/Arm。后 通过Wg/Wnt的通路刺激和导致的通路激活，所有这些因子彼此共定位 和β-连环蛋白，并结合在一起的微管（MT）。驱动蛋白-2、IFT-As或 Kap 3不能正确激活果蝇和MEFs中的Wg/Wnt信号靶点。此外，阿信双 具有IFT-A或驱动蛋白-2的突变背景揭示了高水平的胞质臂/β-连环蛋白，但不能激活 Wg/Wnt靶向，由于减少的核β-连环蛋白定位。这些数据表明，驱动蛋白-2/IFT-A 复合物通过保护Arm/β-连环蛋白免受细胞质系链/抑制剂的影响而促进其核定位。我们 因此，我们确定了驱动蛋白-2/IFT-A复合物在Wnt信号传导中的机制功能，独立于 它们在纤毛中的作用由于相关机制在哺乳动物细胞中是保守的，因此这些观察结果 都有可能接受药物治疗然而，仍然存在几个问题：（i）驱动蛋白-2/IFT-A是如何被激活的？ 促进核定位的复合物;我们的数据表明它是通过沿着微管运动，但这需要 （ii）与IFT 140竞争结合Arm/β-catenin并发挥作用的因子是什么？ Arm/β-连环蛋白核定位的“抑制”功能。我们将在目标1中讨论这种细胞的细胞生物学。 使用优雅的离体Wnt信号传导系统，Aim 2被定制以鉴定拮抗剂， 与IFFT 140结合竞争的因子。我们的初步数据表明，这些知识可以作为一个 抑制过度活跃的Wnt/β-catenin信号转导的新药开发的切入点。因此，获得的信息 在这个应用中，将推进我们对Wnt/β-catenin信号传导机制的理解， 导致后续研究，以开发新的药物，从而有利于疾病相关背景下的治疗。