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: 9917359
• 负责人: Marek Mlodzik
• 金额: $ 25.43万
• 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/9917359
• 关键词: Address; Adenomatous Polyposis Coli; 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） - 信号在所有后生生物中都在细胞间信号传导中起重要作用。它 在许多关键过程中的功能，例如器官模式，生长控制，细胞极性和干细胞 维护。放松管制时，它与先天性心脏病和异常的疾病有关 癌症的血管生成。我的实验室的最新研究表明，flagellar运输复合物A（IFT-A） 蛋白质独立于IFT的睫状作用来调节规范的Wnt/WG信号。我们证明了这一点 他们与kinein2一起做到这一点（就像纤毛功能一样），并促进β-的核转运 Catenin在Wnt-pathway激活并在β-catenin稳定下进行作用。驱动蛋白2和IFT-A 蛋白质在果蝇和哺乳动物的WG信号期间起复合物的作用。两者的突变体，动力素2和 IFT-A，以相同的方式影响WG/WNT信号，并且它们一般和物理相互作用。 kap3，a 动力蛋白相关的蛋白质是纤毛功能中驱动蛋白2和IFT-A之间的桥接因子的蛋白质 也需要以相同的方式需要，并且参与了驱动蛋白2-kap3-物理复合物的形成 ifts。 IFT-A蛋白IFT140然后直接与果蝇中称为Armadillo/ARM的β-catenin结合。之上 WG/WNT刺激途径和产生的途径激活，所有这些因素彼此共定位 和β-catenin，并将其与微型毛细血管（MTS）结合在一起。驱动蛋白2，IFT-AS或双重突变细胞或双突变细胞 KAP3无法在果蝇和MEF中正确激活WG/WNT信号目标。此外，斧头双重 具有IFT-A或驱动蛋白2的突变背景揭示了高水平的细胞质ARM/β-catenin，但无法激活 WG/WNT靶标，由于核β-catenin定位降低。这些数据表明Kinesin-2/IFT-A 复合物通过保护其免受细胞质系绳/抑制剂的影响来促进ARM/β-catenin的核定位。我们 因此，已经鉴定 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 有可能接受药物治疗。但是，仍然存在几个问题：（i）Kinesin-2/ift-a如何 复杂促进核定位；我们的数据表明这是沿着微管运动，但这需要 要完善； （ii）与IFT140竞争与ARM/β-catenin结合并提供的因素是什么 ARM/β-catenin核定位的“抑制”功能。我们将在AIM 1中解决此细胞生物学 使用优雅的离体怀有信号系统的机制，AIM 2量身定制以识别拮抗作用 与IFT140结合竞争的因子。我们的初步数据表明，这些知识可以作为 新药开发的入口点可抑制过度活跃的Wnt/β-catenin信号传导。那是获取的信息 在此应用中，将提高我们对Wnt/β-catenin信号传导的机械理解，并有可能 导致后续研究以开发新药物，从而使疾病相关环境中的治疗受益。