Dissecting the Molecular Mechanisms of Canonical Wnt Signaling

剖析经典 Wnt 信号转导的分子机制

• 批准号: 8009140
• 负责人: Raymond Habas
• 金额: $ 28.88万
• 依托单位: TEMPLE UNIV OF THE COMMONWEALTH
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-07-01 至 2014-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8009140
• 关键词: Anterior; Applications Grants; Area; Behavior; Binding; Biochemical; Biological; Biological Models; Cell Culture Techniques; Cell Nucleus; Cell Proliferation; Cell membrane; Cells; Characteristics; Complex; Cultured Cells; Cytoplasm; Cytoplasmic Protein; Defect; Development; Developmental Process; Embryo; Embryonic Development; Embryonic Pattern Specification; Family; Felis catus; Fibrinogen; Gene Targeting; Glycoproteins; Head; Human; Human Development; In Vitro; Lead; Malignant Neoplasms; Mammalian Cell; Mammary gland; Mediating; Membrane Proteins; Modeling; Molecular; Mutation; Nature; Neoplasm Metastasis; Nuclear; Nuclear Envelope; Nuclear Translocation; Pathology; Pathway interactions; Pattern Formation; Phosphoproteins; Phosphorylation Site; Play; Principal Investigator; Process; Proteins; Regulation; Role; Signal Pathway; Signal Transduction; Signal Transduction Pathway; Signaling Molecule; System; Testing; Transcriptional Regulation; Wnt proteins; Work; Xenopus; Xenopus laevis; Yeasts; base; cancer cell; casein kinase I; cell motility; gain of function; in vitro Assay; in vivo; insight; loss of function; malignant breast neoplasm; mutant; prevent; programs; protein function; public health relevance; receptor; research study; response; trafficking; tumor; tumor growth; tumorigenesis; xenopus development; yeast two hybrid system

DESCRIPTION (provided by applicant): A central characteristic of cancer cells is the ability of the cells to become deregulated in their normal function and subsequently undergo rapid division (tumor growth) and changes in their migratory behavior (tumor metastasis). Several signaling molecules along with components of their signaling pathways have been implicated as causative factors for, one of these being Wnt proteins. There is important evidence for a role of Wnt signaling in cancers as Wnt proteins can transform mammary cells and mutations in components of the Wnt signaling pathway such as 2-Catenin have been shown to play causative roles in cancers in humans. In fact, the first Wnt protein, Wnt-1, was identified as a factor when aberrantly expressed in the mammary gland resulted in breast cancer. We therefore propose that identifying the molecular components and understanding the mechanisms of Wnt signaling remains crucial for our understanding of human cancer formation. Studies have shown that Wnt signaling through the cytoplasmic protein Dishevelled (Dvl) induces the stabilization and nuclear translocation of 2-catenin (2-cat), which then regulates transcriptional induction of Wnt-target genes. However, the regulation of 2-cat nuclear translocation remains poorly defined and also the regulation of 2-cat transcriptional activity is not fully understood. This proposal is directed towards the characterization of a new modulator of Wnt signaling. We have identified this molecule termed DBP, for Dishevelled Binding Molecule, via a yeast-two hybrid screen using Dvl as a bait. In mammalian cells, DBP interacts with key components of the Wnt signaling pathway including Dvl and Casein kinase 1 (CK1), and antagonizes Wnt-stimulated transcriptional induction. Cellular localization studies of DBP demonstrate that this protein is normally present in both the cytoplasmic and nuclear compartments of cells. However, in response to Wnt stimulation, DBP becomes predominantly localized to the nucleus. Furthermore, DBP influences the subcellular localization of 2- cat, a crucial factor required for the transcriptional induction of Wnt-target genes. In vivo analysis of DBP during Xenopus development perturbs embryonic pattern formation and depletion of DBP prevents embryonic head formation. Based on our preliminary studies, we hypothesize that DBP is an essential component required for Wnt signaling. We propose two specific aims in this grant proposal and our studies are directed towards a biochemical, cell biological and in-vivo characterization of DBP to delineate its mode of action along with a recently identified effector for DBP. These studies will utilize mammalian culture cells and the Xenopus embryo as model systems. Towards this end, our studies are directed towards uncovering and understanding the role of these two new components of the Wnt signaling pathway. We propose that analyzing these new components will help to define the molecular nature of Wnt signaling from the plasma membrane to the cell nucleus and ultimately lead to a better understanding of how deregulated Wnt signaling results in cancer formation. PUBLIC HEALTH RELEVANCE: Understanding the development of human cancers remains dependent on identifying key signaling molecules and their signal transduction pathways that contribute to this pathology. One key-signaling molecule that has been demonstrated to play causative roles in human cancer is the Wnt protein. Our studies are focused on understanding two new molecules that we have identified that function in the Wnt signaling pathway, and we propose that these studies can provide important new insights into cancer formation.

描述（由申请人提供）：癌细胞的一个主要特征是细胞能够在其正常功能中变得失调，随后经历快速分裂（肿瘤生长）和其迁移行为的变化（肿瘤转移）。几种信号传导分子沿着其信号传导途径的组分被认为是致病因子，其中之一是Wnt蛋白。有重要证据表明Wnt信号传导在癌症中的作用，因为Wnt蛋白可以转化乳腺细胞，并且Wnt信号传导途径的组分如2-连环蛋白的突变已显示在人类癌症中起致病作用。事实上，第一个Wnt蛋白Wnt-1被鉴定为乳腺中异常表达导致乳腺癌的因素。因此，我们建议，确定分子组成部分和了解Wnt信号传导的机制仍然是我们了解人类癌症形成的关键。 研究表明，通过胞质蛋白Dishevelled（Dvl）的Wnt信号传导诱导2-连环蛋白（2-cat）的稳定和核转位，其然后调节Wnt靶基因的转录诱导。然而，2-cat核转位的调节仍然不清楚，2-cat转录活性的调节也没有完全理解。该提议是针对Wnt信号传导的新调制器的表征。我们已经通过使用Dvl作为诱饵的酵母双杂交筛选鉴定了这种称为DBP的分子，即紊乱结合分子。在哺乳动物细胞中，DBP与Wnt信号通路的关键组分（包括Dvl和酪蛋白激酶1（CK 1））相互作用，并拮抗Wnt刺激的转录诱导。DBP的细胞定位研究表明，这种蛋白质通常存在于细胞的细胞质和细胞核区室。然而，响应于Wnt刺激，DBP变得主要定位于细胞核。此外，DBP影响2- cat的亚细胞定位，2-cat是Wnt靶基因转录诱导所需的关键因子。在非洲爪蟾发育过程中DBP的体内分析扰乱了胚胎模式的形成，DBP的缺失阻止了胚胎头的形成。基于我们的初步研究，我们假设DBP是Wnt信号传导所需的重要组分。我们提出了两个具体的目标，在这个补助金的建议和我们的研究是针对DBP的生化，细胞生物学和体内表征，描绘其作用模式沿着最近确定的DBP效应。这些研究将利用哺乳动物培养细胞和非洲爪蟾胚胎作为模型系统。 为此，我们的研究旨在揭示和理解Wnt信号通路的这两个新组分的作用。我们认为，分析这些新的成分将有助于确定Wnt信号从质膜到细胞核的分子性质，并最终导致更好地理解Wnt信号失调如何导致癌症形成。 公共卫生关系：了解人类癌症的发展仍然依赖于确定关键信号分子及其信号转导通路，有助于这种病理。已被证明在人类癌症中发挥致病作用的一种关键信号分子是Wnt蛋白。我们的研究重点是了解我们已经确定的在Wnt信号通路中起作用的两种新分子，我们认为这些研究可以为癌症形成提供重要的新见解。