P21(RAS) AS A COMMON SIGNALING TARGET OF REACTIVE FREE-RADICALS AND CELLULAR REDOX STRESS

Reactive free radicals have been implicated in mediating signal transduction by a variety of stimuli. We have investigated the role of p21(ras) in mediating free radical signaling. Our studies revealed that signaling by oxidative agents which modulate cellular redox status, such as H2O2, hemin, Hg2+, and nitric oxide was prevented in cells in which p21(ras) activity was blocked either through expression of a dominant negative mutant or by treating with a farnesyltransferase inhibitor, as assessed by NF-kappa B binding activity. Furthermore, the NP-kappa B response to these oxidative stress stimuli was found to be enhanced when cells from the human T cell line, Jurkat, were pretreated with L-buthionine-(S,R)-sulfoximine, an inhibitor of glutathione synthesis. We directly assayed p21(ras) and mitogen-activated protein kinase activities in jurkat cells and found both of these signaling molecules to be activated in cells treated with the redox modulating agents. Blocking glutathione synthesis made cells 10- to 100-fold more sensitive to these agents. Finally, using recombinant p21(ras) in vitro, we found that redox modulators directly promoted guanine nucleotide exchange on p21(ras). This study suggests that direct activation of p21(ras) may be a central mechanism by which a variety of redox stress stimuli transmit their signal to the nucleus.