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

描述(申请人提供)：我们的长期目标是了解激活Wnt/b-catenin途径的分子事件序列。Wnt/b-catenin信号对胚胎发育和成人组织动态平衡是必不可少的，并与人类疾病和疾病有关，如性反转、四肢萎缩症、骨质疏松症、癌症、肥胖症和冠心病。在过去的二十年中，研究发现了许多Wnt/b-catenin组分，并勾勒出了信号级联的轮廓。然而，关于体内对Wnt/b-catenin信号转导具有重要作用的成分以及它们如何调节Wnt/b-catenin级联反应，缺乏关键信息。这些信息对于了解先天畸形和由Wnt/b-catenin信号异常引起的成人疾病的原因至关重要。这一建议将有助于通过研究高度保守的丝氨酸-苏氨酸激酶CK2在Wnt/b-catenin途径激活中的作用来填补这一空白。有必要关注CK2，因为它最近在细胞系和非洲爪哇胚胎中被发现是一个新的Wnt/b-catenin组分；因为CK2在体外被描述在两个关键的Wnt/b-catenin组分的水平上发挥作用，b-catenin本身和DVL；因为CK2调控细胞的基本功能，如细胞增殖、存活、迁移、命运指定和转换，也受Wnt/b-catenin信号的调控；还因为它在小鼠中的基因缺失导致胚胎死亡，组织中显示具有活跃的Wnt/b-catenin信号的缺陷。基于这些事实，我们假设CK2缺乏导致体内Wnt/b-catenin途径激活减弱，并且CK2在b-catenin和DVL的激活中起重要作用。在目标1中，利用缺乏CK2激酶基因CK2a和CK2a‘的小鼠，将获得CK2在体内Wnt/b-连环蛋白途径激活中的作用的遗传学证据。三种互补的方法将被用来评估Wnt/b-catenin途径的激活：分析核b-catenin水平，Wnt特异的靶基因表达，以及使用新的Wnt/b-catenin报告基因Lef-EGFP激活Wnt报告。在目标2中，将通过细胞系的生化和免疫学分析来确定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。将确定DVL上的CK2磷酸化位点，并确定其在非洲爪哇胚胎DVL调节中的功能。这项提议完成了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，这一建议还将产生关于Wnt/b-catenin途径激活的分子调控的机制数据。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、肥胖症、心脏病和炎症的洞察力，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.