Role of ubiquitination in the Wnt pathway

描述(由申请人提供)：Wnt途径在发育、干细胞维持和人类疾病中发挥关键作用。据估计，超过90%的非遗传性结直肠癌病例存在Wnt途径的突变，导致其不适当的激活。在Wnt信号的流行模型中，转录辅助激活因子b-catenin在Wnt途径激活时的稳定导致核b-catenin的积累，从而取代Tcf/Lef转录因子中的辅助抑制因子Groucho(Gro)/TLE。B-catenin与Tcf/Lef的结合最终导致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，发现X连锁的凋亡抑制因子(XIAP)和USP47分别是Wnt信号的重要介质。我们发现XIAP在Wnt途径激活时结合TCF/LEF并泛素化Gro/TLE，泛素化的Groucho/TLE降低了与TCF/LEF的亲和力。在这些研究的基础上，我们提出了一个新的Wnt途径转录激活模型：在Wnt信号转导下，XIAP被招募到Tcf/Lef，以促进泛素介导的Gro/TLE从Tcf/Lef中移除，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，在锂的存在下，XIAP与TCF/LEF共沉淀(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。我们将通过质谱仪定位XIAP上的GSK3磷酸化位点，并在体外和培养细胞中探讨GSK3在调节XIAP(和XIAP磷酸化突变体)与TCF/Lef结合中的作用。最后，我们建议用质谱学的方法来鉴定调节XIAP-TCF/Lef复合体形成和Wnt途径激活的蛋白质。我们提出的模型表明，Gro/Tle是XIAP的关键靶点，XIAP的泛素连接酶活性是其Wnt途径功能所必需的。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Wnt信号，以及XIAP泛素化突变体是否具有介导Wnt途径激活的能力来测试我们的模型。为了阐明XIAP泛素化Gro/Tle调节Gro/Tle活性的机制，我们还建议确定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，它在培养细胞和非洲爪哇胚胎的Wnt信号中是必需的。我们发现USP47与XIAP共沉淀。由于E3泛素连接酶经常与调节其稳定性的DUB相关，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.