Date of Award
Doctor of Philosophy
Zoology and Genetics
Janice E. Buss
Signal transduction organizes cell growth, differentiation, and apoptosis. One component of several signaling pathways leading to these outcomes is the Ras protein. Ras is a small GTPase, binding GTP in an active conformation and GDP in an inactive conformation. Upon nucleotide binding, the conformation of Ras changes so that its effector region can bind to proteins directly downstream in several pathways. Several GTP-bound mutants have been discovered, and an activated Ras mutant has been found in at least 50% of human tumors;Ras must reside at the membrane in order to interact with effector proteins. Binding at the plasma membrane is possible because of the addition of an isoprenoid at a C terminal cysteine and, in the case of one type of Ras, H-Ras, the addition of two palmitates at two nearby cysteines. Isoprenylation is essentially permanent, but palmitoylation is labile, with palmitate half-lives being faster than the H-Ras protein half-life. We hypothesize that palmitoylation on H-Ras is regulatable and related to protein activity;These studies have determined that exposure to S-nitrosocysteine and other oxidants increases the palmitate turnover rate on H-Ras by approximately 2-4 fold. The effect of S-nitrosocysteine, a nitrosothiol, is characterized, including its time and dose dependency. Results indicate that S-nitrosocysteine results in increased palmitate turnover on H-Ras while simultaneously causing decreased H-Ras GTP-binding and downstream signaling;The activation state of H-Ras may also contribute to the increased palmitate turnover rate. The palmitate half-lives of wild-type H-Ras (WT), a dominant negative N17 H-Ras, an activated v-H-Ras, and an activated 61L H-Ras were measured. Comparisons of palmitate half-lives indicated that activated H-Ras mutant proteins had palmitate turnover rates 2-4 times faster than WT and N17 H-Ras proteins. By using the measured palmitate half-lives and percentage GTP for each mutant, the palmitate half-life was calculated for a single H-Ras molecule in a GDP (>3h) or GTP-bound (<39') conformation;Results suggest that palmitoylation of H-Ras responds to exogenous S-nitrosocysteine treatment and possibly the activation state of the protein. This may mean that palmitoylation on H-Ras is a regulatable event and related to H-Ras activity. It may be possible to regulate the activity of H-Ras by modifying its palmitate turn-over rates.
Digital Repository @ Iowa State University, http://lib.dr.iastate.edu/
Tara Lynn Baker
Baker, Tara Lynn, "Reversible palmitoylation of H-Ras is affected by oxidants and activation state " (1998). Retrospective Theses and Dissertations. 11592.