TY - JOUR T1 - A Glutathione <em>S</em>-Transferase π-Activated Prodrug Causes Kinase Activation Concurrent with <em>S</em>-Glutathionylation of Proteins JF - Molecular Pharmacology JO - Mol Pharmacol SP - 501 LP - 508 DO - 10.1124/mol.105.018523 VL - 69 IS - 2 AU - Danyelle M. Townsend AU - Victoria J. Findlay AU - Farit Fazilev AU - Molly Ogle AU - Jacob Fraser AU - Joseph E. Saavedra AU - Xinhua Ji AU - Larry K. Keefer AU - Kenneth D. Tew Y1 - 2006/02/01 UR - http://molpharm.aspetjournals.org/content/69/2/501.abstract N2 - Nitric oxide (NO) is an endogenous, diffusible, transcellular messenger shown to affect regulatory and signaling pathways with impact on cell survival. Exposure to NO can impart direct post-translational modifications on target proteins such as nitration and/or nitrosylation. As an alternative, after interaction with oxygen, superoxide, glutathione, or certain metals, NO can lead to S-glutathionylation, a post-translational modification potentially critical to signaling pathways. A novel glutathione S-transferase π (GSTπ)-activated pro-drug, O2-{2,4-dinitro-5-[4-(N-methylamino)benzoyloxy]phenyl}1-(N,N-dimethylamino)diazen-1-ium-1,2-diolate (PABA/NO), liberates NO and elicits toxicity in vitro and in vivo. We now show that PABA/NO induces nitrosative stress, resulting in undetectable nitrosylation, limited nitration, and high levels of S-glutathionylation. After a single pharmacologically relevant dose of PABA/NO, S-glutathionylation occurs rapidly (&lt;5 min) and is sustained for ∼7 h, implying a half-life for the deglutathionylation process of approximately 3 h. Two-dimensional SDS-polyacrylamide gel electrophoresis and immunoblotting with a monoclonal antibody to S-glutathionylated residues indicated that numerous proteins were S-glutathionylated. Subsequent matrix-assisted laser desorption ionization/time of flight analysis identified 10 proteins, including β-lactate dehydrogenase, Rho GDP dissociation inhibitor β, ATP synthase, elongation factor 2, protein disulfide isomerase, nucleophosmin-1, chaperonin, actin, protein tyrosine phosphatase 1B (PTP1B), and glucosidase II. In addition, we showed that sustained S-glutathionylation was temporally concurrent with drug-induced activation of the stress kinases, known to be linked with cell death pathways. This is consistent with the fact that PABA/NO induces S-glutathionylation and inactivation of PTP1B, one phosphatase that can participate in deactivation of kinases. These effects were consistent with the presence of intracellular PABA/NO or metabolites, because cells overexpressing MRP1 were less sensitive to the drug and had reduced levels of S-glutathionylated proteins. ER -