Cadherin cell adhesion molecules link the cytoskeletons of adjacent cells together in solid tissues. In adherens junctions, the catenins link cadherins to the actin-based cytoskeleton, and in desmosomes an analogous protein assembly links cadherins to intermediate filaments. Assembly of adherens junction 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 assembly are responsible for a number of severe skin blistering diseases. The adherens junction protein beta-catenin also serves as a transcriptional coactivator in the Wnt signaling pathway that controls cell fate determination during embryogenesis. Mutations of Wnt signaling components are responsible for many cancers. This proposal aims to develop a biophysical understanding of cell junction architecture and assembly, and also the interactions of beta-catenin in Wnt signaling, using x-ray crystallography to determine structures and physical biochemical methods to determine binding stoichiometries, affinities, and kinetics. 1. The following structures will be determined: a) phosphorylated E-cadherin/beta-catenin complex; b) a ternary complex of E-cadherin, beta-catenin, and alpha-catenin; c) the actin-binding domain of a-catenin; and d) full-length alpha-catenin. The affinity and kinetics of the beta-catenin/phosphorylated E-cadherin interaction will be measured. 2. The structures of domains from desmoplakin, which links the cadherin/plakoglobin complex to intermediate filaments, will be determined to understand how this protein interacts with intermediate filaments and with the cadherin/plakoglobin complex. Complexes of cadherins with plakoglobin will also be studied. 3. The binding properties of different sets of beta-catenin binding repeats of the tumor suppressor APC, both unmodified and phosphorylated, will be determined, and complexes with beta-catenin and Axin will be crystallized. The structure of a ternary complex of beta-catenin, the transcription factor LEF-1, and a cognate DNA sequence will be determined.
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