): An early event in the development of colon cancer is loss of heterozygosity for the normal adenomatous polyposis coli (APC) gene. APC is normally expressed in the proliferative zone of the crypt border in the colon. It is felt to have important functions in cell migration, anchorage and cell-cell interactions. One of APC's known binding partners is EB1, product of a gene found by the yeast two hybrid interaction trap using APC as bait. EB1 has no known function, but it is expressed in many tissues and conserved from humans to the highly homologous yeast protein Yer0l6p. In unrelated work, EB1 was independently isolated in the lab of Barbara Bierer by immunoprecipitation with a specific form of tubulin, tyrosinated tubulin, thought to represent the dynamic pool of tubulin. The interaction between EB1 and tyrosinated tubulin was established in T-cells, suggesting a role for EB1 in lymphocytes. This proposal outlines the approach to determining the function of EB1. To test the hypothesis that EB1 coordinates the binding of APC with the cytoskeleton, members of the macromolecular complex involving APC, EB1, and tubulin, will be analyzed for direct interactions. Mutational analysis will be used to dissect the domains of EB1 which interact with each specific partner. Changes in EB1 expression and cellular localization during cell division and differentiation will be investigated. The yeast homolog of EB1, Yer016p, has been deleted in S. cerevisiae by homologous recombination. Haploid yeast lacking Yer016p are viable. Their phenotype is currently under study. The yeast model system will allow rapid analysis of the phenotype of Yer016p deletion, assessment of the capacity of the human EB1 gene to replace the function of the yeast homolog, and mutational analysis of human EB1. To assess the role of EB1 within the intact colon and in mammalian development, a murine model of targeted germline deletion of EB1 will be generated. These studies bear directly on the understanding of mitosis, cell cycle progression, growth control, and tumorigenesis. Determining the function of EB1 will elucidate how an early lesion in colon cancer positions the cell to undergo malignant transformation.
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