MOLECULAR AND CELLULAR BIOLOGY OF THE APC PROTEIN

APC 蛋白的分子和细胞生物学

• 批准号: 6344748
• 负责人: RAYMOND L WHITE
• 金额: $ 10.06万
• 依托单位: UNIVERSITY OF UTAH
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-08-16 至 2001-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6344748
• 关键词: adenomatous polyps; biological signal transduction; cadherins; cell adhesion; cell biology; cell nucleus; clinical research; cytoplasm; gene expression; genetic regulation; genetic transcription; human tissue; immunofluorescence technique; molecular biology; nucleic acid hybridization; proteasome; protein structure function; tissue /cell culture

The goal of this project is to understand the cellular roles of the Adenomatous Polyposis Coli (APC) protein, with special attention to its potential functions in the cell nucleus. These studies have a central theme: the interaction between APC and the cell adhesion/signaling molecule beta-catenin. Beta-catenin constitutes the final step in the Wnt signaling pathway, complexing with Tcf/LEF-1 transcription factors, translocating into the nucleus and activating expression of target genes. In addition to its role in Wnt signaling, beta-catenin is a functional component of cell-cell adhesion junction, placing it in a prime position to link intercellular communication with intracellular signal transduction. Because APC can promote proteasome-dependent degradation of Beta-catenin, it is the ultimate regulator of beta-catenin activity. Given its ability to regulate beta-catenin protein, it is likely that control of beta-catenin mediated transcription is a major function of APC. Aim 1 proposes to employ powerful genomic screening technologies in a comprehensive scan of beta-catenin/Tcf-mediated transcription in the presence of absence of functional APC protein. The rationale is two-fold: the events downstream of APC/beta-catenin are key mechanisms in tumorigenesis, and the beta-catenin-induced genes are potential targets for anti-polyp drug development. APC protein and beta-catenin can interact in the cell's nucleus as well in the cytoplasm, raising the possibility that APC targets nuclear beta- catenin for proteasome-dependent degradation. Aim 2 uses a combination of protein half-life assays, cell fractionation procedures, and proteasome- inhibiting agents to explore the possibility. The presence of APC in both the cytoplasm and the nucleus suggests that APC relays Wnt signaling information between these two compartments. This hypothesis, combined with the identification of a nuclear export signal in the APC protein, provides the basis for Aim 3, an exploration of whether APC is engaged as a nuclear shuttle protein. Microinjection of nuclei with purified peptides and antibodies, followed by immunofluorescence analysis will be used to track the movement of APC and beta-catenin between the nuclear and cytoplasmic compartments under a variety of conditions. Together, the experiments proposed here focus on understanding the molecular mechanisms underlying the regulatory functions of APC protein and determining how mutations in APC alter these functions, leading to colon polyps and cancer.

该项目的目的是了解 腺瘤性息肉病（APC）蛋白质，特别注意其 细胞核中的潜在功能。这些研究有一个中心 主题：APC和单​​元格粘附/信号传导之间的相互作用 分子β-catenin。 β-catenin构成WNT的最后一步 信号通路，与TCF/LEF-1转录因子络合， 易位到核并激活靶基因的表达。 除了其在Wnt信号传导中的作用外，β-catenin是功能 细胞细胞粘附连接的组成部分，将其放置在质量位置 将细胞间通信与细胞内信号联系起来 转导。因为APC可以促进蛋白酶体依赖性降解 β-catenin，它是β-catenin活性的最终调节剂。 鉴于其能够调节β-catenin蛋白的能力，很可能 控制β-catenin介导的转录是APC的主要功能。 AIM 1提议在A中采用强大的基因组筛选技术 β-catenin/TCF介导的转录的全面扫描 存在功能性APC蛋白的存在。理由是两个方面： APC/β-catenin下游的事件是关键机制 肿瘤发生，β-catenin诱导的基因是潜在的靶标 用于抗类药物开发。 APC蛋白和β-catenin也可以在细胞的核中相互作用 细胞质增加了APC靶向核β-的可能性 用于蛋白酶体依赖性降解的蛋白酶。 AIM 2使用 蛋白质半衰期测定，细胞分级程序和蛋白酶体 抑制代理人探索可能性。 APC在两者中的存在 细胞质和细胞核表明APC继电器Wnt信号传导 这两个隔间之间的信息。这个假设，结合 APC蛋白中核输出信号的鉴定提供了 AIM 3的基础，探索APC是否作为核 穿梭蛋白。用纯化的肽和 抗体，然后进行免疫荧光分析来跟踪 APC和β-catenin在核和细胞质之间的运动 在各种条件下的隔室。一起实验 这里提出的重点是理解基本机制 APC蛋白的调节功能并确定突变如何 APC改变了这些功能，导致结肠息肉和癌症。