Control of embryonic development by CK2: CK2 as a core Wnt/beta-catenin component

描述（由申请人提供）：我们的长期目标是了解激活Wnt/b-连环蛋白通路的分子事件序列。Wnt/β-连环蛋白信号传导对于胚胎发育、成年组织稳态是必不可少的，并且与人类疾病和病症如性逆转、四肢畸形、骨质疏松症、癌症、肥胖症和冠状动脉疾病有关。在过去的二十年中的研究已经导致了许多Wnt/b-连环蛋白组分的发现和信号级联的概述。然而，关键的信息是缺乏关于在体内的Wnt/β-连环蛋白信号传导的重要组成部分，以及它们如何调节Wnt/β-连环蛋白级联反应。这些信息对于理解先天性畸形和异常Wnt/b-连环蛋白信号传导引起的成人疾病的原因至关重要。这项建议将有助于填补这一空白，通过研究高度保守的丝氨酸-苏氨酸激酶CK 2在Wnt/β-连环蛋白途径激活的作用。 关注CK 2是重要的，因为它最近被鉴定为细胞系和非洲爪蟾胚胎中的新Wnt/b-连环蛋白组分;因为CK 2已被描述为在体外作用于两个关键Wnt/b-连环蛋白组分，b-连环蛋白本身和Dishevelled（Dvl）的水平;因为CK 2调节基本的细胞功能如细胞增殖、存活、迁移、命运特化和转化，也受Wnt/β-连环蛋白信号传导调节;并且因为其在小鼠中的遗传缺失导致胚胎死亡，组织中的缺陷显示具有活性Wnt/β-连环蛋白信号传导。基于这些事实，我们假设小鼠中的CK 2缺乏导致体内Wnt/β-连环蛋白途径活化减少，并且CK 2在β-连环蛋白和Dvl活化中起重要作用。 在目的1中，将使用缺乏CK 2激酶基因CK 2a和CK 2a ′的小鼠获得CK 2在体内Wnt/β-连环蛋白途径活化中的作用的遗传证据。三种互补的方法将用于评估Wnt/b-连环蛋白通路的激活：分析核b-连环蛋白水平，Wnt特异性靶基因表达和Wnt报告基因激活使用一种新的Wnt/b-连环蛋白报告基因，LEF-eGFP。在目标2中，将使用细胞系中的生物化学和免疫学分析来确定CK 2用于调节核b-连环蛋白定位的机制的阐明。在目的3中，将评估CK 2等位基因缺失对Dvl水平、磷酸化状态和亚细胞定位的影响。将鉴定Dvl上的CK 2磷酸化位点，并在非洲爪蟾胚胎中确定其在Dvl调节中的功能。 这项提议实现了NIGMS的使命，因为它将拓宽对胚胎发育过程中信号通路的理解。此外，这一提议将产生Wnt/β-连环蛋白通路激活的分子调控机制数据。这些机制数据将提供可用于确定这些分子参与先天性疾病，癌症，肥胖，心脏病和炎症的见解，并可能导致预防和治疗此类疾病的新方法。

DESCRIPTION (provided by applicant): Our long-term goal is to understand the sequence of molecular events that activate the Wnt/b-catenin pathway. Wnt/b-catenin signaling is essential for embryonic development, for adult tissue homeostasis and is linked to human diseases and disorders such as sex reversal, tetramelia, osteoporosis, cancer, obesity and coronary disease. Research in the past two decades has resulted in the discovery of many Wnt/b-catenin components and in the outline of the signaling cascade. However, crucial information is lacking with respect to components that are important in vivo for Wnt/b-catenin signaling and how they regulate the Wnt/b-catenin cascade. This information is critical to understand the cause of congenital malformations and the adult diseases derived from aberrant Wnt/b-catenin signaling. This proposal will contribute to filling this void by studying the role of the highly conserved serine-threonine kinase CK2 in Wnt/b-catenin pathway activation. It is important to focus on CK2 because it was recently identified as a novel Wnt/b-catenin component in cell lines and in Xenopus embryos; because CK2 has been described in vitro to act at the level of two crucial Wnt/b-catenin components, b-catenin itself and Dishevelled (Dvl); because CK2 regulates essential cellular functions such as cell proliferation, survival, migration, fate specification and transformation, also regulated by Wnt/bcatenin signaling; and because its genetic depletion in mice leads to embryonic death with defects in tissues shown to have active Wnt/b-catenin signaling. Based on these facts, we hypothesize that CK2 deficiency in mice leads to diminished Wnt/b-catenin pathway activation in vivo, and that CK2 plays an important role in b-catenin and Dvl activation. In Aim 1, genetic evidence for a role of CK2 in Wnt/b-catenin pathway activation in vivo will be obtained using mice deficient for the CK2 kinase genes, CK2a and CK2a'. Three complementary approaches will be used to evaluate Wnt/b-catenin pathway activation: analysis of nuclear b-catenin levels, Wnt-specific target genes expression and Wnt reporter activation using a novel Wnt/b-catenin reporter, LEF-eGFP. In Aim 2, elucidation of the mechanism utilized by CK2 to regulate nuclear b-catenin localization will be determined using biochemical and immunological analyses in cell lines. In aim 3, the effect of CK2 allelic depletion on Dvl levels, phosphorylation status and subcellular localization will be evaluated. The CK2 phosphorylation site on Dvl will be identified, and its function in Dvl regulation ascertained in Xenopus laevis embryos. This proposal fulfills the NIGMS mission, as it will broaden the understanding of signaling pathways during embryonic development. In addition, this proposal will generate mechanistic data on the molecular regulation of Wnt/b-catenin pathway activation. These mechanistic data will provide insights that can be used to ascertain the involvement of these molecules in congenital diseases, cancer, obesity, heart disease and inflammation, and may lead to novel ways to prevent and treat such diseases.