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-catenin通路的分子事件序列。Wnt/b-连环蛋白信号传导对胚胎发育和成人组织稳态至关重要，并与人类疾病和失调有关，如性逆转、四足症、骨质疏松症、癌症、肥胖和冠心病。在过去二十年的研究中，发现了许多Wnt/b-连环蛋白成分，并勾勒出信号级联的轮廓。然而，关于体内Wnt/b-catenin信号传导的重要成分以及它们如何调节Wnt/b-catenin级联的关键信息缺乏。这一信息对于了解先天性畸形的原因和由异常Wnt/b-连环蛋白信号引起的成人疾病至关重要。本研究通过研究高度保守的丝氨酸-苏氨酸激酶CK2在Wnt/b-catenin通路激活中的作用，将有助于填补这一空白。关注CK2很重要，因为它最近被鉴定为细胞系和非洲爪蟾胚胎中的一种新的Wnt/b-连环蛋白成分；因为CK2在体外被描述为在两个关键的Wnt/b-catenin成分水平上起作用，b-catenin本身和disheveled (Dvl)；因为CK2调节细胞增殖、存活、迁移、命运规范和转化等基本细胞功能，也受Wnt/bcatenin信号的调控；还因为它在小鼠体内的基因缺失会导致胚胎死亡，并在具有活跃Wnt/b-连环蛋白信号的组织中出现缺陷。基于这些事实，我们假设小鼠CK2缺乏导致体内Wnt/b-catenin通路激活减弱，CK2在b-catenin和Dvl激活中起重要作用。在Aim 1中，将使用缺乏CK2激酶基因CK2a和CK2a'的小鼠，获得CK2在体内Wnt/b-catenin通路激活中作用的遗传证据。三种互补的方法将用于评估Wnt/b-catenin通路的激活：核b-catenin水平分析，Wnt特异性靶基因表达和使用新的Wnt/b-catenin报告基因LEF-eGFP激活Wnt报告基因。在Aim 2中，将通过细胞系的生化和免疫学分析来阐明CK2调节核b-连环蛋白定位的机制。在目标3中，将评估CK2等位基因缺失对Dvl水平、磷酸化状态和亚细胞定位的影响。在非洲爪蟾胚胎中，我们将确定CK2在Dvl上的磷酸化位点，并确定其在Dvl调控中的作用。这一建议完成了NIGMS的任务，因为它将扩大对胚胎发育过程中信号通路的理解。此外，本提案将产生Wnt/b-catenin通路激活的分子调控机制数据。这些机制数据将为确定这些分子在先天性疾病、癌症、肥胖、心脏病和炎症中的作用提供见解，并可能导致预防和治疗这些疾病的新方法。

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.