The long-term goal of this research is to obtain a better understanding of the control of cell proliferation at sites beyond the cell membrane. There appears now to be a limited number of cancer-causing genes or """"""""oncogenes."""""""" However, our knowledge relating the expression of these gene products with neoplastic transformation is deficient in molecular detail. Calcium and calmodulin play key roles as second messengers in cell growth, division, and differentiation. Neoplastic cells are autonomous to the control properties of calcium and have elevated calmodulin levels. Our group has developed a cadre of expertise to now dissect the calcium-calmodulin pathways leading to transformation. We propose to elevate calmodulin levels by microinjecting pure calmodulin, its mRNA, and its structural gene. We can then test the hypothesis that elevated cellular calmodulin results in loss in growth regulation. Oncomodulin, a low molecular weight tumor-specific protein, will be purified and investigated for its growth-promoting properties. In vivo and in vitro phosphorylation studies will be performed using cell lines temperature-sensitive for transformation in order to determine the role of protein kinase C. This calcium-phospholipid kinase represents the major receptor for active phorbol diesters. An additional aspect of this work is the identification and isolation of the calmodulin acceptor proteins (CAPs) associated with neoplasia. Monoclonal antibodies will be produced and quantitative measurements of expression will be performed during neoplastic transformation. These studies will involve sarcoma-virus-infected temperature-sensitive rat kidney cells. Subcellular localization will be determined using immunofluorescence microscopy. Finally, we propose to functionally characterize the growth-related CAP, calplasin. We have shown calplasin to be expressed only during cell growth in the nucleolus. Using an in vitro system, the effects of calcium, calmodulin, oncomodulin, calplasin, and anticalplasin on ribosomal RNA synthesis and processing will be examined. Collectively, the experiments should provide direct evidence which will couple the calcium signal with growth regulation via specific cellular proteins. (N)
