Many intracellular signaling pathways rely on reactive free radicals for transmitting signals to the nucleus. The concept that reactive oxygen or reactive nitrogen species are required for many ligand-stimulated signaling processes has engendered new paradigms and raised many new questions. The applicant's studies have focused on identifying signaling pathways initiated by reactive free radicals. He has discovered that the reactive nitrogen species, nitric oxide (NO), induces biochemical events characteristic of an activated phenotype in resting human Iymphocytes. NO induced TNF-alpha secretion, NF-kB transcription factor translocation and protein tyrosine kinase activity, all indicators of cellular activation. These activation events were cGMP-independent. Thus, NO may have immune-stimulatory properties which are unrelated to its direct cytotoxic nature and independent of its ability to generate cGMP. The applicant also reported that NO and other free radicals utilize a p21ras (Ras)-dependent signaling pathway to generate nuclear signals, and generated new preliminary data which indicate that NO directly interacts with Ras to induce guanine nucleotide exchange and activation. Accordingly, he proposes to study in detail the NO-Ras interaction. First, the molecular target of NO on Ras will be identified. This will require the use of electrospray ionization-mass spectrometry (ESI-MS) and site-directed mutagenesis. Next, the effector interactions associated with NO-activated Ras will be identified. This will require in vitro binding assays, immunoprecipitation, Western blotting and, possibly, the yeast two-hybrid system. Conformational consequences of Ras modification by NO will also be identified using ESI-MS analysis of hydrogen-exchange experiments and X-ray crystallography. Finally, the generality of these findings will be determined by determining if redox-active environmental toxicants similarly activate Ras and initiate T cell signaling. The proposed experiments are designed to 1) provide a molecular understanding of a redox switch on Ras, and 2) extend these findings to provide a general mechanism by which free radicals, produced endogenously or exogenously, exert their proinflammatory properties.
