The annexins are a group of homologous, calcium-dependent, membrane- binding proteins that may underlie a diverse array of processes occurring at membrane surfaces. Three members of this protein family, annexin I (lipocortin), annexin II (calpactin), and annexin VI (p68), have been shown to undergo phosphorylation when cells are stimulated to divide by oncogenes or mitogens, suggesting these proteins may play roles in cytoplasmic and membrane changes associated with malignant transformation. In addition, annexins I and II have been shown to be phosphorylated in the chromaffin cell when it is stimulated to carry out exocytosis, suggesting the annexins may also control membrane fusion in differentiated secretory cells. The goal of this project is to understand the role of annexin phosphorylation in control of cellular functions. The sites of phosphorylation on the annexins will be mapped. The effect of phosphorylation on the structure of annexin I will be determined by X-ray diffraction analysis. The effects of phosphorylation on the membrane-binding, membrane-aggregating, and related in vitro activities of the annexins will be determined. The phosphorylation sites will be modified by site-directed mutagenesis to generate recombinant protein models of the phosphorylated annexins. The ability of the native or modified annexins to influence mammalian cells will be examined by introducing these proteins into intact or permeabilized secretory cells and fibroblasts. These studies will provide insights into the cellular functions controlled by the annexins and the role of oncogenes and mitogens in regulating these functions. This knowledge may lead to novel methods of controlling cellular changes associated with malignant transformation.
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