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.
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