The goals of the proposed studies are to understand the function of the highly conserved membrane-associated cellular tyrosine kinase, c-src, at the cell and tissue level, and to elucidate its molecular mechanism of action. The key to our approach is the use of highly differentiated tissue-like structures which can respond morphologically to changes in c-src expression. Madin Darby Canine Kidney (MDCK) epithelial cells form multicellular epithelial structures-that have a fixed, uniformly spherical 3-D structure. If c-src is elevated from 2 to 9-fold over the endogenous level, the MDCK cell cysts become pleomorphic: the epithelial monolayer exhibits multiple folds, but there is no alteration of cell proliferation or cell-cell adhesion. It is hypothesized that c-src may regulate the structural plasticity of multicellular structures, and thus may act as a """"""""morphoregulator"""""""" of the 3-D topography of epithelial tissues. This idea will be tested using immunocytochemistry, and in situ hybridization on epithelial cysts in vitro, and on tissues that show normal and abnormal states of epithelial morphogenesis (e.g. histogenesis, regeneration, neoplasia). If the theory is valid, the regions or epithelial monolayers that are undergoing dynamic changes in 3-D structure should be enriched with c-src. We plan to elucidate the molecular mechanism by which c-src induces structural plasticity in MDCK epithelial cells. A 160-170 kd phosphotyrosine-containing protein has been identified that is more heavily phosphorylated in MDCK cells expressing elevated levels of c-src than in MDCK cells expressing low levels of c-src. Biochemical techniques, monoclonal antibody production and molecular biological techniques will be used to localize, purify and clone the cDNA of this putative substrate of c-src. We will test the hypothesis that this, and/or other c-src induced phosphorylation(s) convert the fodrin-based subcortical membrane skeleton of MDCK cells from a relatively stable, rigid structure to a dynamic structure that is capable of undergoing the structural reorganizations necessary to generate diverse morphologies (i.e. pleomorphism). Immunoblotting, immunofluorescence microscopy, metabolic labelling/immunoprecipitation, and 2-D electrophoresis will be used to achieve these goals. The approach outlined here will provide a rational basis for future investigations of the molecular alterations that underlie states of aberrant epithelial morphogenesis (e.g. neoplasia).
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