Role of ubiquitination in the Wnt pathway

泛素化在 Wnt 通路中的作用

• 批准号: 8417128
• 负责人: ETHAN LEE
• 金额: $ 29.61万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-06-01 至 2017-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8417128
• 关键词: Affinity; Alanine; Apoptosis Inhibitor; Binding; Biochemistry; Cell Culture Techniques; Cell Maintenance; Cell Nucleus; Cells; Colorectal Cancer; Complex; Cultured Cells; Data; Deubiquitinating Enzyme; Development; Disease; Dissociation; Down-Regulation; Drosophila genus; Embryo; Embryonic Development; Excision; Gene Targeting; Genetic Epistasis; Genetic Transcription; Homologous Gene; In Vitro; Injection of therapeutic agent; Ligands; Lithium; Lysine; Mammalian Cell; Maps; Mass Spectrum Analysis; Mediating; Mediator of activation protein; Modeling; Mutation; N-terminal; Nuclear; Pathway interactions; Phosphorylation; Phosphorylation Site; Play; Proteins; RNA Interference; Reaction; Recombinants; Recruitment Activity; Regulation; Reporting; Role; Signal Pathway; Signal Transduction; Signaling Molecule; Site; Testing; Transcriptional Activation; Ubiquitin; Ubiquitination; Vertebrates; Xenopus; adult stem cell; base; human BIRC4 protein; human disease; mutant; programs; promoter; public health relevance; research study; response; stem; transcription factor; ubiquitin ligase; ubiquitin-protein ligase

DESCRIPTION (provided by applicant): The Wnt pathway plays critical roles in development, stem cell maintenance, and human disease. Over 90% of non-hereditary cases of colorectal cancer have been estimated to have mutations in the Wnt pathway that cause its inappropriate activation. In the prevailing model of Wnt signaling, stabilization of the transcription coactivato, b-catenin, upon Wnt pathway activation results in accumulation of nuclear b-catenin that displaces the co-repressor, Groucho(Gro)/TLE, from the TCF/Lef transcription factor. Binding of b-catenin to TCF/Lef ultimately leads to increased transcription of Wnt target genes. Recently, we performed a screen for ubiquitin E3 ligases and deubiquitinating enzymes that identified X-linked inhibitor of apoptosis (XIAP) and USP47, respectively, as essential mediators of Wnt signaling. We showed that XIAP binds TCF/Lef upon Wnt pathway activation and ubiquitinates Gro/TLE and that ubiquitinated Groucho/TLE has decreased affinity for TCF/Lef. Based on these studies, we propose a new model for transcriptional activation of the Wnt pathway: upon Wnt signaling, XIAP is recruited to TCF/Lef to promote ubiquitin-mediated removal of Gro/TLE from TCF/Lef, allowing for b-catein-TCF/Lef binding. The mechanism by which XIAP is recruited to TCF/Lef is unknown. We found that XIAP coimmunoprecipitates with TCF/Lef in the presence of lithium (inhibits GSK3) and that XIAP is phosphorylated by GSK3. We propose to test our hypothesis that, in the absence of Wnt signaling, GSK3 phosphorylates and inhibits the binding of XIAP to TCF/Lef. We will map the GSK3 phosphorylation sites on XIAP by mass spectrometry and explore the role of GSK3 in regulating binding of XIAP (and XIAP phosphomutants) to TCF/Lef in vitro and in cultured cells. Finally, we propose to perform mass spectrometry to identify proteins that regulate XIAP-TCF/Lef complex formation and Wnt pathway activation. Our proposed model suggests that Gro/TLE is the critical target for XIAP and that the ubiquitin ligase activity of XIAP is necessary for its Wnt pathway function. We will test our model by determining whether inhibition of Wnt signaling via downregulation of XIAP can be suppressed by downregulation of Gro/TLE and whether XIAP ubiquitination mutants have the capacity to mediate Wnt pathway activation. To elucidate the mechanism by which ubiquitination of Gro/TLE (by XIAP) regulates Gro/TLE activity, we also propose to identify the sites on Groucho/TLE ubiquitinated by XIAP and generate mutants that cannot be ubiquitinated by XIAP. Recently, we identified a deubiquitinating (DUB) enzyme, USP47, which is required for Wnt signaling in cultured cells and Xenopus embryos. We found that USP47 coimmunoprecipitates with XIAP. Because E3 ubiquitin ligases are often associated with DUBs that regulate their stability, we will test whether USP47 modulates Wnt signaling by binding to XIAP and regulating its stability and/or activity.

描述（由申请人提供）：Wnt 通路在发育、干细胞维持和人类疾病中发挥着关键作用。据估计，超过 90% 的非遗传性结直肠癌病例中 Wnt 通路存在突变，导致其不适当激活。在 Wnt 信号转导的流行模型中，Wnt 通路激活后转录辅激活因子 b-连环蛋白的稳定会导致核 b-连环蛋白的积累，从而取代 TCF/Lef 转录因子中的共阻遏物 Groucho(Gro)/TLE。 b-连环蛋白与 TCF/Lef 的结合最终导致 Wnt 靶基因的转录增加。最近，我们对泛素 E3 连接酶和去泛素化酶进行了筛选，分别鉴定出 X 连锁凋亡抑制剂 (XIAP) 和 USP47 是 Wnt 信号传导的重要介质。我们发现，XIAP 在 Wnt 通路激活后结合 TCF/Lef，并泛素化 Gro/TLE，并且泛素化的 Groucho/TLE 与 TCF/Lef 的亲和力降低。基于这些研究，我们提出了一种 Wnt 通路转录激活的新模型：在 Wnt 信号传导后，XIAP 被招募到 TCF/Lef 上，以促进泛素介导的 Gro/TLE 从 TCF/Lef 上的去除，从而允许 b-catein-TCF/Lef 结合。 XIAP 被招募到 TCF/Lef 的机制尚不清楚。我们发现 XIAP 在锂存在下与 TCF/Lef 发生共免疫沉淀（抑制 GSK3），并且 XIAP 被 GSK3 磷酸化。我们建议检验我们的假设，即在没有 Wnt 信号传导的情况下，GSK3 磷酸化并抑制 XIAP 与 TCF/Lef 的结合。我们将通过质谱法绘制 XIAP 上的 GSK3 磷酸化位点，并探索 GSK3 在体外和培养细胞中调节 XIAP（和 XIAP 磷酸突变体）与 TCF/Lef 结合的作用。最后，我们建议进行质谱分析来鉴定调节 XIAP-TCF/Lef 复合物形成和 Wnt 通路激活的蛋白质。我们提出的模型表明 Gro/TLE 是 XIAP 的关键靶标，并且 XIAP 的泛素连接酶活性对其 Wnt 通路功能是必需的。我们将通过确定 Gro/TLE 的下调是否可以抑制 XIAP 下调对 Wnt 信号传导的抑制以及 XIAP 泛素化突变体是否具有介导 Wnt 通路激活的能力来测试我们的模型。为了阐明 Gro/TLE（通过 XIAP）泛素化调节 Gro/TLE 活性的机制，我们还建议鉴定 Groucho/TLE 上被 XIAP 泛素化的位点，并生成不能被 XIAP 泛素化的突变体。最近，我们鉴定了一种去泛素化 (DUB) 酶 USP47，它是培养细胞和非洲爪蟾胚胎中 Wnt 信号传导所必需的。我们发现 USP47 与 XIAP 发生免疫共沉淀。由于 E3 泛素连接酶通常与调节其稳定性的 DUB 相关，因此我们将测试 USP47 是否通过与 XIAP 结合并调节其稳定性和/或活性来调节 Wnt 信号传导。