Understanding the function of the Ras oncogene at the molecular level is a high priority of cancer research because mutational activation of Ras is involved in large number of neoplasias. Ras proteins are low molecular weight GTP binding proteins that function as on/off switches to regulate signal transduction pathways. Ras is active when in the GTP bound state. Hydrolysis of GTP to GDP inactivates Ras as a signaling molecule. In addition to the regulation of Ras by guanine nucleotides, Ras function requires a series of posttranslational modifications. Work accomplished during the first funding period of this grant elucidated the posttranslational events required to generate functional Ras protein. The steps include; (1) farnesylation of a cysteine that occurs in a conserved C-terminal sequence motif referred to as the CaaX box (C is Cys, a is any aliphatic residue, and X is the C-terminal residue), (2) proteolytic removal of the -aaX sequence, (3) carboxyl methylation of the newly exposed cysteinyl carboxyl, and (4) palmitoylation of a second cysteine residue often found close to the CaaX box. One function of these modifications is to redirect Ras from the cytoplasm to the plasma membrane where it interacts with cellular effectors to modulate signal transduction pathways. The goal of this project is to determine how posttranslational events regulate the function of Ras. The work described in this proposal will focus on the Ras gene homologs found in Saccharomyces cerevisiae. The yeast system has been chosen so that a combination of biochemical and genetic techniques can be employed. Ras mutants will be constructed and their activity measured by in vivo and in vitro techniques. In work leading up to this proposal we have found that addition of the palmitoyl moiety to Ras plays an important role in directing Ras to its site of action in the plasma membrane. In the current study, we propose methods to isolate the palmitoyl transferase gene and to study the biochemical activity of the palmitoyl transferase enzyme. Although the mechanism by which CaaX box processing controls Ras function is still unclear, inhibitors of the Ras processing pathway are being developed as potential cancer chemotherapeutic agents. The experiments we propose will improve our understanding of Ras function and will contribute to efforts to design drugs that interfere with the activity of activated Ras alleles.
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