Assembly of cells into tissues depends upon formation of specialized intercellular junctions. In adherens junctions, the catenins link transmembrane cadherin cell adhesion molecules to the actin-based cytoskeleton, and in desmosomes an analogous protein assembly links cadherins to intermediate filaments. Adherens junction assembly is a critical step in the development of cell and tissue morphology, and loss of adherens junction-based cell adhesion is a hallmark of metastasizing cells. Linkage to the intermediate filament system is essential for the mechanical strength and integrity of tissues such as the skin and the heart, and defects in desmosome components are responsible for a number of severe skin blistering diseases. The adherens junction protein beta-catenin is also a transcriptional coactivator in the Wnt signaling pathway that controls cell fate determination during embryogenesis and in the normal renewal of tissues in the adult. In many cancers, stabilization of beta-catenin due to mutations in Wnt pathway components results in inappropriate activation of Wnt target genes. This proposal aims to develop a molecular understanding of cell junction assemblies and beta-catenin in the Wnt pathway by using purified components to reconstitute key interactions. Biochemical and biophysical methods will be used to determine the structures, affinities, and kinetics of these interactions, including the effects of phosphorylation on these properties. 1. The role of alpha-catenin in regulating adherens junction assembly will be investigated by studying how the binding of certain ligands to a-catenin alters its affinity for others. The conformational changes associated with these responses will be investigated by biochemical and structural methods. 2. The reported interactions among desmosomal cadherins, plakoglobin, plakophillins, and desmoplakin will- be investigated using the purified proteins. The structure of the desmoplakin N-terminal domain will be determined, alone and in complex with partners identified in the biochemical experiments. The interactions between the desmoplakin Cterminal domain and intermediate filaments will be investigated using structure-based mutagenesis and cocrystallization with small intermediate filament fragments. 3. The mechanism by which the tumor suppressors axin and APC promote efficient phosphorylation and degradation of beta-catenin will be studied, in particular how phosphorylation modulates the interactions among these proteins. 4. The higher-order transcriptional assemblies of beta-catenin will be examined, as well as the molecular mechanism by which beta-catenin changes Tcf/Lef-family transcription factors from co-repressors to co-activators.
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