Uncontrolled cell proliferation is the basis of malignant transformation of breast epithelium. Ca2+/calmodulin participates in normal cell replication and motility, but the intermediate proteins through which it transduces these effects have not been identified. The levels of calmodulin and its binding proteins (e.g., IQGAP1) are altered in malignancy, suggesting that modification of these interactions could be an effective target for chemotherapy. The hypothesis to be evaluated in this proposal is that defective coupling between calmodulin and selected calmodulin-binding proteins contributes to neoplastic transformation of breast tissue. Our objective is to use a novel, state-of-the-art strategy to identify calmodulin-binding proteins that are directly involved in the regulation of cell transformation and to dissect out their function with biochemical and molecular biology techniques. To determine whether IQGAP1 - the first target identified by this approach - and calmodulin are components of aberrant cell growth, wild-type and mutant IQGAP1 will be overexpressed in breast carcinoma cells; the effects on growth and transformation will be examined in the transfected cells. Because in vitro evidence reveals that Ca2+/calmodulin regulates Cdc42 via IQGAP1, modification of Cdc42 activity by Ca2+/calmodulin through IQGAP1 in intact cells will be analyzed with fusion proteins, cell fractionation, and immunocytochemistry. To establish whether IQGAP1 and calmodulin modulate E-cadherin and beta-catenin function, adhesion assays ion manipulation, transfection and immunocytochemistry will be performed. To identify other calmodulin-binding proteins that may contribute to neoplasia, affinity chromatography or immunoprecipitation with a highly-specific anti-calmodulin antibody will be performed; proteins that are expressed in malignant breast cells at levels different from those in untransformed breast cells will be unambiguously identified by the novel technique of nanoelectrospray tandem mass spectrometry. These studies should provide insight into the molecular mechanisms that underlie the malignant transformation of breast tissue. This knowledge could potential lead to the development of novel markers and specifically targeted pharmacologic agents for breast carcinoma.
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