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.
CK2 activity is essential for embryonic survival in mice and we hypothesize that it does so through regulation of the Wnt/2-catenin signaling pathway, a key pathway involved in embryonic development, adult tissue homeostasis and in diseases such as congenital diseases and cancer. The results of our experiments will help understand the mechanism of Wnt/2-catenin pathway activation in vivo and will provide important mechanistic information that could be used to prevent and design therapies against diseases that are mediated by CK2 and Wnt/2-catenin signaling such as congenital diseases, cancer and obesity.
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