Tyrosine Phosphorylation of LRP6 and its role in regulation Wnt signaling

后生动物使用Wnt/β-连环蛋白信号传导来控制细胞行为的关键方面，并且失调的Wnt信号传导与人类疾病密切相关。我们进行基于细胞培养的表达筛选，以确定新的修饰剂，影响特定的Wnt途径的组成部分。我们工作的一个重点是了解细胞如何通过低密度脂蛋白受体相关蛋白6（LRP 6）控制Wnt信号的接收。虽然丝氨酸和苏氨酸磷酸化已被充分研究，但LRP 6的酪氨酸磷酸化的潜在作用尚未研究。我们使用泛磷酸化特异性抗体，通过SDS-PAGE /免疫印迹分析筛选激酶cDNA文库，并鉴定了一种新的LRP 6酪氨酸激酶。初步工作表明，它特异性地与LRP 6相互作用，抑制LRP 6/Wnt信号传导，并且本身受Wnt信号传导调节。这些发现表明LRP 6的酪氨酸磷酸化在调节Wnt信号传导中起重要作用。在这里，我们建议详细分析这种激酶的功能，以调节LRP 6，使用分子生物学，细胞生物学以及体内实验。将使用细胞培养进行生化实验，并使用斑马鱼和非洲爪蟾进行生理相关性研究。

Metazoans use Wnt/β-catenin signaling to control critical aspects of cell behavior and deregulated Wnt signaling is strongly implicated in human disease. We perform cell culturebased expression screening to identify new modifiers that effect specific Wnt pathway components. A key focus of our work is to understand how cells control reception of Wnt signaling via low-density lipoprotein receptor-related-protein 6 (LRP6). Although serine and threonine phosphorylation has been well studied, the potential role of tyrosine phosphorylation of LRP6 has not been investigated. Using pan phospho-specific antibodies we screened a kinase cDNA library by SDS-PAGE / Immunoblot analysis and identified a novel LRP6 tyrosine kinase. Preliminary work demonstrates that it specifically interacts with LRP6, inhibits LRP6/Wnt signaling and is itself regulated by Wnt signaling. These finding indicate that tyrosine phosphorylation of LRP6 plays an important role in regulating Wnt signaling. Here we propose to analyze in detail how this kinase functions to regulate LRP6, using molecular biology, cell biology as well as in-vivo experiments. Biochemical experiments will be performed using cell culture and physiological relevance will be addressed using both zebrafish and Xenopus.