Nitric oxide negatively regulates c-Jun N-terminal kinase/stress-activatedprotein kinase by means of S-nitrosylation

NO, produced from L-arginine in a reaction catalyzed by NO synthase, is an endogenous free radical with multiple functions in mammalian cells. Here, w e demonstrate that endogenously produced NO can suppress c-Jun N-terminal k inase (JNK) activation in intact cells. Treatment of BV-2 murine microglial cells with IFN-gamma induced endogenous NO production, concomitantly suppr essing JNK1 activation. Similarly, IFN-gamma induced suppression of JNK1 ac tivation in RAW264.7 murine macrophage cells and rat alveolar macrophages. The IFN-gamma -induced suppression of JNK1 activation in BV-2, RAW264.7, or rat alveolar macrophage cells was completely prevented by N-G-nitro-L-argi nine, a NO synthase inhibitor. Interestingly, the IFN-gamma -induced suppre ssion of JNK1 activation was not affected by 1H-[1.2,4]oxadiazolo[4.3-a]qui noxalin-1-one, an inhibitor of guanylyl cyclase. 8-Bromo-cGMP, a membrane-p ermeant analogue of cGMP, did not change JNK1 activation in intact cells ei ther. In contrast, S-nitro-N-acetyl-DL-penicillamine (SNAP), a NO donor, in hibited JNK1 activity in vitro. Furthermore, a thiol reducing agent, DTT, r eversed not only the in vitro inhibition of JNK1 activity by SNAP but also the in vivo suppression of JNK1 activity by IFN-gamma. Substitution of seri ne for cysteine-116 in JNK1 abolished the inhibitory effect of IFN-gamma or SNAP on JNK1 activity in vivo or in vitro, respectively. Moreover. IFN-gam ma enhanced endogenous S-nitrosylation of JNK1 in RAW264.7 cells. Collectiv ely, our data suggest that endogenous NO mediates the IFN-gamma -induced su ppression of JNK1 activation in macrophage cells by means of a thiol-redox mechanism.