Biophysical Training: Investigation into the Molecular Mechanism of APC Regulation in β-catenin Proteolysis

描述(申请人提供)：结直肠癌的一种常见的早期突变是Wnt/？-catenin生长因子信号通路的失控。该途径通过稳定或降解双转录因子和黏附蛋白--连环蛋白来控制细胞生长控制基因的活性。特别是，80%的结直肠癌 通过在称为突变簇区(MCR)的区域截断支架蛋白腺瘤性息肉病结肠(APC)来解除对Wnt信号的调控。尽管经过了20多年的研究，但在理解这种截断如何扰乱ç-catenin的调节蛋白水解性破坏方面仍然存在一个关键的空白。了解这种截断为什么会导致ç-catenin蛋白分解停止，可能会使结直肠癌的新疗法成为可能。我对这个项目的总体目标是获得生化和生物物理技术方面的培训，以明确为什么APC截断会导致Wnt信号中？连环蛋白泛素化和蛋白水解性破坏的中断。我的中心假设是，APC MCR区域内的连环蛋白抑制域(CID)在整个细胞周期中与不同的泛素化蛋白相互作用，这种相互作用是由CID内的磷酸化事件介导的。根据DDB1/CUL4复合体和Skp1/Cul1复合体平行作用于Skp1/Cul1复合体的假设，在细胞周期和Wnt失活过程中，DDB1/CUL4复合体与Skp1/Cul1复合体平行作用，以破坏？连环蛋白。我还计划确定APC中CID区域的磷酸化如何影响？连环蛋白的蛋白分解。我假设APC磷酸化位点T1438是AE-catenin结合所必需的，并通过支撑AE-catenin/？-catenin异二聚体来定位？-catenin，以被泛素连接酶？TrCP识别，从而促进？？连环蛋白的蛋白降解。这个项目的完成将为我提供广泛的生物物理技术培训。此外，他们还将阐明APC和ç-catenin蛋白分解机制之间的两个特定的分子相互作用。这些数据将提供为什么结直肠癌通常在同一地区截短APC的洞察，并可能为癌症治疗和诊断提供新的靶点。预期的结果是识别和验证新的泛素蛋白，这些蛋白与ç-catenin蛋白分解相互作用。此外，该项目将定义MCR区域的磷酸化的新作用，并将其与ç-连环蛋白的蛋白分解联系起来。

DESCRIPTION (provided by applicant): A common early mutation in colorectal cancer is the deregulation of the Wnt/ ß-catenin growth factor signaling pathway. This pathway controls the activity of cellular growth control genes through stabilization or degradation of the protein ß-catenin, a dual transcription factor and adhesion protein. In particular, 80% of colorectal cancers deregulate Wnt signaling by truncating the scaffolding protein Adenomatous Polyposis Coli (APC) in a region called the Mutational Cluster Region (MCR). Despite over 20 years of study, there remains a critical gap in understanding how this truncation disrupts the regulated proteolytic destruction of ß-catenin. Understanding why this truncation leads to cessation of ß-catenin proteolysis could enable new treatments for colorectal cancers. My overall objective for this project is to obtain training in biochemical and biophysical techniques to understand specifically why APC truncation leads to interruption of ß-catenin ubiquitination and proteolytic destruction in Wnt signaling. My central hypothesis is that the Catenin Inhibitory Domain (CID) within the MCR region of APC interacts with different ubiquitination proteins throughout the cell cycle, and that this interaction is mediated by phosphorylation events within the CID. I aim to identify the ubiquitin transfer mechanism controlling ß-catenin proteolysis in the cytoplasm and nucleus during the cell cycle and Wnt signaling following the hypothesis that the DDB1/Cul4 complex acts in parallel with the SKP1/Cul1 complex to destroy ß-catenin during homeostasis and Wnt deactivation. I also plan to identify how phosphorylation of the CID region in APC affects ß-catenin proteolysis. I hypothesize the APC phosphorylation site T1438 is required for aE-catenin binding and enhances ß-catenin proteolysis by scaffolding the aE-catenin/ß-catenin heterodimer to position ß-catenin for recognition by the ubiquitin ligase ß-TrCP. Fulfillment of this project will provide me with extensive training in biophysical techniques. Furthermore, they will elucidate two specific molecular interactions between APC and ß-catenin proteolysis machinery. These data will provide insight into why colorectal cancers usually truncate APC in the same region, and may provide new targets for cancer treatments and diagnostics. The expected outcomes are the identification and validation of new ubiquitin proteins that interact with ß-catenin proteolysis. Additionally, this project will define a new role for phosphorylation n the MCR region and relate it to ß-catenin proteolysis.