Rap1 signaling is required for suppression of Ras-generated reactive oxygen species and protection against oxidative stress in T lymphocytes.

Transient production of reactive oxygen species (ROS) plays an important role in optimizing transcriptional and proliferative responses to TCR signaling in T lymphocytes. Conversely, chronic oxidative stress leads to decreased proliferative responses and enhanced transcription of inflammatory gene products, and is thought to underlie the altered pathogenic behavior of T lymphocytes in some human diseases, such as rheumatoid arthritis (RA). Although the signaling mechanisms regulating ROS production in T lymphocytes has not been identified, activation of the small GTPase Ras has been shown to couple agonist stimulation to ROS production in other cell types. We find that Ras signaling via Ral stimulates ROS production in human T lymphocytes, and is required for TCR and phorbol ester-induced ROS production. The related small GTPase Rap1 suppresses agonist, Ras and Ral-dependent ROS production through a PI3K-dependent pathway, identifying a novel mechanism by which Rap1 can distally antagonize Ras signaling pathways. In synovial fluid T lymphocytes from RA patients we observed a high rate of endogenous ROS production, correlating with constitutive Ras activation and inhibition of Rap1 activation. Introduction of dominant-negative Ras into synovial fluid T cells restored redox balance, providing evidence that deregulated Ras and Rap1 signaling underlies oxidative stress and consequent altered T cell function observed in RA.

Effect of redox balance alterations on cellular localization of LAT and downstream T-cell receptor signaling pathways.

The integral membrane protein linker for activation of T cells (LAT) is a central adapter protein in the T-cell receptor (TCR)-mediated signaling pathways. The cellular localization of LAT is extremely sensitive to intracellular redox balance alterations. Reduced intracellular levels of the antioxidant glutathione (GSH), a hallmark of chronic oxidative stress, resulted in the membrane displacement of LAT, abrogated TCR-mediated signaling and consequently hyporesponsiveness of T lymphocytes. The membrane displacement of LAT is accompanied by a considerable difference in the mobility of LAT upon native and nonreducing denaturing polyacrylamide gel electrophoresis analysis, a finding indicative of a conformational change. Targeted mutation of redox-sensitive cysteine residues within LAT created LAT mutants which remain membrane anchored under conditions of chronic oxidative stress. The expression of redox-insensitive LAT mutants allows for restoration of TCR-mediated signal transduction, whereas CD28-mediated signaling pathways remained impaired. These results are indicative that the membrane displacement of LAT as a result of redox balance alterations is a consequence of a conformational change interfering with the insertion of LAT into the plasma membrane. Conclusively, the data suggest a role for LAT as a crucial intermediate in the sensitivity of TCR signaling and hence T lymphocytes toward chronic oxidative stress.