Role of ubiquitination in the Wnt pathway

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起关键作用。据估计，超过90%的非遗传性结直肠癌病例在Wnt通路中存在导致其不适当激活的突变。在Wnt信号传导的主流模型中，在Wnt通路激活后，转录协同激活因子b-连环蛋白的稳定导致核b-连环蛋白的积累，从而取代TCF/Lef转录因子中的协同抑制因子Groucho(Gro)/TLE。b-连环蛋白与TCF/Lef结合最终导致Wnt靶基因转录增加。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-linked inhibitor of apoptosis （XIAP）和USP47是Wnt信号传导的重要介质。我们发现XIAP在Wnt通路激活时结合TCF/Lef并泛素化Gro/TLE，泛素化的Groucho/TLE降低了TCF/Lef的亲和力。基于这些研究，我们提出了一种新的Wnt通路转录激活模型：在Wnt信号传导后，XIAP被募集到TCF/Lef上，促进泛素介导的Gro/TLE从TCF/Lef上去除，从而允许b- catin -TCF/Lef结合。XIAP被招募到TCF/ left的机制尚不清楚。我们发现在锂的存在下，XIAP与TCF/Lef共免疫沉淀（抑制GSK3），并且XIAP被GSK3磷酸化。我们提出验证我们的假设，即在缺乏Wnt信号的情况下，GSK3磷酸化并抑制XIAP与TCF/ left的结合。我们将通过质谱法绘制XIAP上的GSK3磷酸化位点，并在体外和培养细胞中探索GSK3在调节XIAP（和XIAP磷化物）与TCF/ left结合中的作用。最后，我们建议采用质谱法鉴定调节XIAP-TCF/Lef复合物形成和Wnt通路激活的蛋白。我们提出的模型表明，Gro/TLE是XIAP的关键靶点，而XIAP的泛素连接酶活性是其Wnt通路功能所必需的。我们将通过确定通过下调XIAP对Wnt信号的抑制是否可以通过下调Gro/TLE来抑制，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泛素化（被XIAP）调控Gro/TLE活性的机制，我们还提出鉴定被XIAP泛素化的Groucho/TLE上的位点，并产生不能被XIAP泛素化的突变体。最近，我们发现了一种去泛素化（DUB）酶USP47，它是培养细胞和爪蟾胚胎中Wnt信号转导所必需的。我们发现USP47与XIAP共免疫沉淀。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。

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.