Previous studies in our laboratory have shown that mixed lineage kinase 3 (MLK3) can be activated following global ischemia. In addition, other laboratories have reported that the activation of MLK3 may be linked to the accumulation of free radicals. However, the mechanism of MLK3 activation remains incompletely understood. We report here that MLK3, overexpressed in HEK293 cells, is S-nitrosylated (forming SNO-MLK3) via a reaction with S-nitrosoglutathione, an exogenous nitric oxide (NO) donor, at one critical cysteine residue (Cys-688). We further show that the S-nitrosylation of MLK3 contributes to its dimerization and activation. We also investigated whether the activation of MLK3 is associated with S-nitrosylation following rat brain ischemia/reperfusion. Our results show that the administration of 7-nitroindazole, an inhibitor of neuronal NO synthase (nNOS), or nNOS antisense oligodeoxynucleotides diminished the S-nitrosylation of MLK3 and inhibited its activation induced by cerebral ischemia/reperfusion. In contrast, 2-amino-5,6-dihydro-6-methyl-4H-1,3-thiazine (an inhibitor of inducible NO synthase) or nNOS missense oligodeoxynucleotides did not affect the S-nitrosylation of MLK3. In addition, treatment with sodium nitroprusside (an exogenous NO donor) and S-nitrosoglutathione or MK801, an antagonist of the N-methyl-D-aspartate receptor, also diminished the S-nitrosylation and activation of MLK3 induced by cerebral ischemia/reperfusion. The activation of MLK3 facilitated its downstream protein kinase kinase 4/7 (MKK4/7)-JNK signaling module and both nuclear and non-nuclear apoptosis pathways. These data suggest that the activation of MLK3 during the early stages of ischemia/reperfusion is modulated by S-nitrosylation and provides a potential new approach for stroke therapy whereby the post-translational modification machinery is targeted.As a free radical, NO is an endogenous cell signaling molecule involved in the regulation of many physiological and pathophysiological processes (1). NO and NO-related compounds exert both protective and cytotoxic effects, depending on the cellular context and the nature of the NO group. The multifaceted actions of the NO group can be classified into two categories, cGMP-dependent and cGMP-independent. The cGMP-dependent actions play critical roles in a variety of physiological processes, including NO-mediated vasodilation. In contrast, cGMP-independent, nitrosative protein modifications are postulated to be involved in both physiological and pathological responses (2).Endogenous NO is synthesized from L-arginine by NO synthase (NOS) and is associated with S-nitrosylation. S-Nitrosylation, the modification of the covalent attachment to the side chain of cysteine by an NO group, is considered as an important post-translational modification that has profound effects on protein function (3).

No abstract