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的细胞间信号中起着重要作用。它 在许多关键过程中发挥作用，如器官构型、生长控制、细胞极性和干细胞 维修。当放松管制时，它与一系列疾病有关，从先天性心脏病和畸形 血管生成与癌症的关系。我实验室最近的研究表明，鞭毛内转运复合体A(IFT-A) 蛋白质调节典型的Wnt/Wg信号，独立于IFTS的纤毛作用。我们证明了这一点 它们与Kinesin2一起起作用(就像它们在睫毛功能中所做的那样)，并促进β-2的核转位。 连环蛋白作用于Wnt途径的激活，并作用于β-连环蛋白稳定的下游。激动素-2和IFT-A 在果蝇和哺乳动物中，蛋白质在Wg信号传递过程中扮演着一个复合体的角色。两者的突变体，激动素2和 IFT-A以相同的方式影响Wg/Wnt信号，它们在遗传和物理上相互作用。Kap3，a 动蛋白相关蛋白在睫状体功能中作为动蛋白2和IFT-A之间的桥梁因子 也以同样的方式需要，并参与形成运动蛋白2-Kap3-的物理复合体- 如果是这样。然后，IFT-A蛋白IFT140直接与β-连环蛋白结合，在果蝇中称为Armadillo/Arm。vt.在.的基础上 Wg/Wnt对通路的刺激和由此产生的通路激活，所有这些因素都是相互共定位的 和β-连环蛋白，并与微管(MT)结合。Kinesin-2、IFT-AS或 Kap3不能正确激活果蝇和MEF中的Wg/Wnt信号靶点。此外，Axin加倍 带有ift-A或kinesin-2的突变背景显示细胞质中高水平的ARM/β-连环蛋白，但不能激活 WG/WNT靶，由于核β-连环蛋白定位减少。这些数据表明，Kinesin-2/IFT-A 复合体通过保护ARM/β-连环蛋白免受细胞质锚定/抑制物的影响而促进其核定位。我们 从而确定了KINEIN-2/IFT-A复合体在WNT信号转导中的机械功能，不依赖于 它们在纤毛中的作用。由于相关机制(S)在哺乳动物细胞中是保守的，这些观察到 有可能接受药物治疗。然而，还有几个问题仍然存在：(I)Kinesin-2/IFT-A如何 促进核局部化的复合体；我们的数据表明它是通过沿着微管移动的，但这需要 与IFT140结合的ARM/β-连环蛋白竞争的因素是什么？ ARM/β-连环蛋白核定位的“抑制”功能。我们将在目标1中解决这一问题的细胞生物学 机制使用了一个优雅的体外Wnt信号系统，Aim 2是为识别拮抗剂而量身定做的 与IFT140结合竞争的因子(S)。我们的初步数据表明，这种知识可以作为一种 新药开发的切入点，以抑制过度活跃的Wnt/β-连环蛋白信号。因此，所获得的信息 这一应用将促进我们对WNT/β-连环蛋白信号的机械理解，并有可能还 导致后续研究，以开发新药，从而有利于疾病相关背景下的治疗。