Nitric oxide (NO) has been shown to negatively regulate c-Jun N-terminal kinase (JNK) through S-nitrosylation. Here, we show that disruption of an interaction between JNK and its substrate c-Jun is an important mechanism underlying the NO-mediated inhibition of JNK signaling. Endogenous NO, which was generated by interferon-gamma treatment, suppressed anisomycin-stimulated JNK activity in microglial BV-2 cells. The interferon-gamma-induced suppression of JNK1 activation in BV-2 cells was prevented completely by treatment with N-G-nitro-L-arginine, an inhibitor of NO synthase. A NO donor S-nitro-N-acetyl-DL-penicillamine (SNAP) inhibited JNK activity in vitro, and this inhibition was reversed by a thiol-reducing agent, dithiothreitol. Nitric oxide disrupts a physical interaction between JNK and its substrate c-Jun both in vitro and in intact cells without affecting an interaction between SEK1 and JNK. Collectively, our results suggest that the inhibition of the interaction between JNK and c-Jun may be an integral part of the mechanism underlying the negative regulation of the JNK signaling pathway by NO. (c) 2006 Elsevier Inc. All rights reserved.