A central focus in studies of the molecular control of proliferation is the cellular ras gene. The critical importance of this gene in the control of cellular proliferation is indicated by the frequency of its mutation in naturally occurring tumors, and the fact that injection of inhibitory antibodies or mutant blocks cellular proliferation. The goal of this proposal is to further investigate the mechanism by which this gene is controlled. The ras protein is believed to be biologically active when bound to GTP, until the native GTPase activity of ras converts the bound nucleotide to GDP. Other cellular factors are apparently involved in the control of this conversion, including a GTPase activating protein (GAP). Past studies from this laboratory indicate that GAP activity is inhibited in the presence of certain lipids which are known to be produced at the time of mitogenic stimulation. In the course of lipid studies a factor was identified which had the ability to inhibit GTPase activity of ras in a lipid- dependent manner. It is the first objective of the work described here to purify this GTPase inhibitory protein, clone the gene, and further characterize its activity. Important progress toward this goal has been made since the initial submission of this proposal. The information gained in these studies might help define the role of this protein in the normal control proliferation, and alterations of this normal control mechanism during the development of tumors. The presence of multiple cellular factors required for the activity of cellular ras was clearly demonstrated in microinjection studies with two separate dominant inhibitory ras mutant proteins. A second objective of this proposal is to determine whether GAP, the GTPase inhibitory protein, or NF-1 protein is the cellular target of either of these inhibitors. The critical importance of this effort relates to the fact that one of these mutants is specifically inhibitory to mutant ras proteins. Definition of its target might provide a means to selectively inhibit the activity of mutant ras in tumors. Finally, recent evidence indicates that the gene mutated in neurofibromatosis type 1 (NF-1 is related to GAP both in nucleotide sequence and in the ability to stimulate the GTPase activity of ras. Furthermore, recent studies from this laboratory indicate that lipids inhibit the NF-1 protein. There were similarities and significant differences in the types of lipids able to inhibit NF-1 compared to GAP. Unfortunately, because the NF-1 protein is extremely large, only the catalytic fragment of NF-1 has been available for study. The full-length protein must now be analyzed. Mutations in this gene are common and have important clinical consequences. Information gained in this proposal will be essential to determine the role of lipids in the control of NF-1, alterations in this type of control which might result from naturally occurring mutations, and if such alterations play a role in clinical manifestations. In summary, the information gained in the studies outlined here are expected to aid in understanding the general molecular mechanisms involved in the control of proliferation, and provide clues as to how these processes might be controlled.
