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 malignant tissue, 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 proliferation and to dissect out their function with biochemical and molecular biology techniques. To determine whether IQGAP1 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. To ascertain the clinical relevance of IQGAP1 as a potential tumor suppressor, patient breast carcinoma tissue will be analysed by immunohistochemistry and mass spectrometry. 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 epithelial cells will be unambiguously identified by the novel technique of nanoelectrospray tandem mass spectrometry. Sufficient sequence information will be available to clone the gene of any proteins not in the database. To determine the molecular mechanisms by which selected calmodulin-binding proteins regulate growth, cell lines that do not express or have low levels of the protein will be identified and transfected; the functional effects on growth and proliferation will be studied. Finally, the interactions between calmodulin and the target proteins will be evaluated with GST-fusion proteins and by surface plasmon resonance. These studies should provide insight into the molecular mechanisms that underlie the malignant transformation of breast tissue. This knowledge could potentially lead to the development of novel markers and specifically targeted pharmacologic agents for breast carcinoma.