Wnt Pathway Regulation in Embryos and Cells

描述(由申请人提供)：Wnt信号通路在从调节早期胚胎发育到控制干细胞生长的各种生物学过程中都是至关重要的。对这一途径的错误调控会导致多种癌症，包括结肠癌，这是美国癌症相关死亡的第二大原因。调节这一途径的中心是“破坏复合体”，这是一种蛋白质集合，包括激酶GSK3，它控制转录激活因子β-连环蛋白的水平，β-连环蛋白是Wnt信号的主要效应者。虽然在许多细胞类型中，细胞间Wnt信号通过一种未知的机制调节破坏复合体的活性，但在非洲爪哇发育的早期，该复合体是通过转位含有GSK3结合蛋白GBP的颗粒在细胞内调节的。 这项提案的一个主要目标将建立在我们上一个资助期的结果的基础上，即GBP与Kinesin轻链(KLC)相互作用，我们建议的KLC在易位所需的电机蛋白和Wnt途径的其他调节因子(如Disheveled)之间提供了一座关键的桥梁。我们将使用多种方法来干扰Kinesin功能，以测试Kinesin在粒子运动和轴形成中所起的作用。此外，为了了解这些颗粒是如何在非洲爪哇卵母细胞中组装的，我们将检验酪蛋白激酶1epsilon(CK1epsilon)在颗粒形成中具有重要作用的假设，该蛋白可能由Frizzled7受体激活。最后，使用斑马鱼作为模型系统，我们将测试我们的命题，即这些背靠背的颗粒用于其他低等脊椎动物。 第二个主要研究领域将继续我们对Wnt细胞内途径的结构研究，重点是CK1 epsilon，这是一种在Wnt信号转导中必不可少的激酶。我们将研究CK1epsilon是如何抑制自身活性的，因为最近的研究表明Wnt信号调节CK1epsilon的自身抑制。我们将解决CK1 epsilon活性的主要靶点之一--CK1 epsilon结合散乱的结构。最后，利用结构-功能的方法，我们将确定蓬乱是否是CK1 epsilon活性在调节Wnt途径中的重要靶点。由于Wnt通路在脊椎动物的发育和肿瘤发生中起着关键作用，这些研究将对增进对出生缺陷和癌症的分子基础的认识具有重要意义。

DESCRIPTION (provided by applicant): The Wnt signaling pathway is of critical importance in a wide variety of biological processes from the regulation of early embryonic development to the control of stem cell growth. Misregulation of this pathway leads to a variety of cancers, including colon cancer, the second major cause of cancer-related death in the United States. Central to the regulation of this pathway is the "Destruction Complex", an assembly of proteins including the kinase GSK3, which controls the levels of the transcriptional activator beta-catenin, a principal effector of Wnt signaling. Whereas in many cell types intercellular Wnt signals regulate the activity of the Destruction Complex through an unknown mechanism, in early Xenopus development, the complex is regulated intracellularly by translocating particles containing a GSK3 binding protein, GBP. One major goal of this proposal will build on our result from the last funding period that GBP interacts with kinesin light chain (KLC), which we have proposed provides a critical bridge between a motor protein required for translocation and other regulators of the Wnt pathway such as Dishevelled. Using a variety of approaches to disrupt kinesin function, we will test the proposed role of kinesin in particle movement and axis formation. Furthermore, in order to understand how the particles are assembled in the Xenopus oocyte, we will test the hypothesis that casein kinase 1epsilon (CK1epsilon), potentially activated by the Frizzled 7 receptor, has an essential role in particle formation. Finally, using zebrafish as a model system, we will test our proposition that these dorsalizing particles are used in other lower vertebrates. The second major area of research will continue our structural studies of the Wnt intracellular pathway, focusing on CK1epsilon, a kinase that is essential for transducing Wnt signals. We will examine how CK1epsilon inhibits its own activity, since recent studies have shown that Wnt signaling regulates the autoinhibition of CK1epsilon. We will solve the structure of CK1epsilon bound to Dishevelled, one of the principle targets of CK1epsilon activity. Finally, using a structure-function approach, we will determine if Dishevelled is the essential target of CK1epsilon activity in regulating the Wnt pathway. Since the Wnt pathway plays key roles in vertebrate development and oncogenesis, these studies will be important in advancing knowledge of the molecular basis of birth defects and cancer.