Received for publication August 31, 2005.
Revised November 8, 2005.
Accepted for publication November 8, 2005.

A Glutathione S-Transferase Activated Pro-drug Causes Kinase Activation Concurrent with S-glutathionylation of Proteins

Abstract

Nitric oxide (NO) is an endogenous, diffusible, trans-cellular messenger shown to impact 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. Alternatively, 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 GST activated pro-drug, O²-{2,4-dinitro-5-[4-(N-methylamino)benzoyloxy]phenyl} 1-(N,N-dimethylamino)diazen-1-ium-1,2-diolate or 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 (<5 mins) and is sustained for ~ 7h, implying a half-life for the deglutathionylation process of approximately 3h. 2D SDS-PAGE and immunoblotting with a monoclonal antibody to S-glutathionylated residues indicated that numerous proteins were S-glutathionylated. Subsequent MALDI-TOF analysis identified 10 proteins, including -lactate dehydrogenase, Rho GDP dissociation inhibitor , ATP synthase, elongation factor 2, protein disulfide isomerase, nucleophosmin-1, chaperonin, actin, 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, since MRP1 over-expressing cells were less sensitive to the drug and had reduced levels of S-glutathionylated proteins.

Key words: Nitric oxide, MAP Kinase, Glutathione S-transferases, Phase II enzymes, Regulation - post-transcriptional, Glutathione, Oxidative stress/antioxidants, Oxidative stress, Mechanisms of cell killing/apoptosis, Pharmacokinetics, metabolism and activation

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