S-Nitrosylation of Mixed Lineage Kinase 3 Contributes to Its Activation after Cerebral Ischemia

Shu-Qun Hu,Jun-Song Ye,Yan-Yan Zong,Chang-Cheng Sun,Dong-Hai Liu,Yong-Ping Wu,Tao Song,Guang-Yi Zhang

doi:10.1074/jbc.m111.227124

Abstract

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.

Highlights

Pounds exert both protective and cytotoxic effects, depending on the cellular context and the nature of the nitric oxide (NO) group
We initially examined whether mixed lineage kinase 3 (MLK3), which is overexpressed in HEK293 cells, is S-nitrosylated by reaction with GSNO at Cys688 and whether this contributes to its dimerization and activation
MLK3 S-Nitrosylation Is Mediated by Endogenous NO during Cerebral Ischemia-Reperfusion—We have reported previously that MLK3 is activated during the assembly of the GluR61⁄7PSD951⁄7MLK3 signaling module following brain ischemia and reperfusion and that its activation may associate with free radicals in the signaling events that take place following brain ischemia

Summary

Introduction

Pounds exert both protective and cytotoxic effects, depending on the cellular context and the nature of the NO group. Based upon the evidence mentioned above, we hypothesize that following brain ischemia/reperfusion, Ca2ϩ influx via NMDAR leads to the formation of calcium-calmodulin signaling compounds, which activate nNOS and increase the release of endogenous NO This augmentation of endogenous NO may S-nitrosylate MLK3 by adding NO to the thiol moiety of reactive cysteine residues and activating this protein MLK3, which in turn facilitates the activation of the JNK signaling pathway and exacerbates ischemic neuron death. When treated with 7-NI and exogenous NO donors (SNP and GSNO), the activation of nNOS was inhibited This leads to a decrease in the levels of S-nitrosylated MLK3 during the early stages of reperfusion, further down-regulates the downstream signaling pathway of JNK, and induces neuroprotection in the hippocampal CA1 region

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Results

Conclusion