S-nitrosylation of mixed lineage kinase 3 contributes to its activation after cerebral ischemia.

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.

• Authors: Shu-Qun Hu, Jun-Song Ye, Yan-Yan Zong, Chang-Cheng Sun, Dong-Hai Liu, Yong-Ping Wu, Tao Song, Guang-Yi Zhang
• Journal: The Journal of biological chemistry (J Biol Chem) Vol. 287 Issue 4 Pg. 2364-77 (Jan 20 2012) ISSN: 1083-351X [Electronic] United States
• PMID: 22123824 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)

Chemical References

• 2-amino-5,6-dihydro-6-methyl-4H-1,3-thiazine
• Enzyme Inhibitors
• Neuroprotective Agents
• Oligodeoxyribonucleotides, Antisense
• Thiazines
• S-Nitrosoglutathione
• Dizocilpine Maleate
• Nitric Oxide Synthase Type I
• Nos1 protein, rat
• MAP Kinase Kinase Kinases
• mitogen-activated protein kinase kinase kinase 11

Topics

• Animals
• Brain Ischemia (enzymology, genetics, pathology)
• Dizocilpine Maleate (pharmacology)
• Enzyme Activation (drug effects, genetics)
• Enzyme Inhibitors (pharmacology)
• HEK293 Cells
• Humans
• MAP Kinase Kinase Kinases (genetics, metabolism)
• Male
• Neuroprotective Agents (pharmacology)
• Nitric Oxide Synthase Type I (antagonists & inhibitors, genetics, metabolism)
• Oligodeoxyribonucleotides, Antisense (pharmacology)
• Protein Multimerization
• Protein Processing, Post-Translational
• Rats
• Rats, Sprague-Dawley
• Reperfusion Injury (enzymology, genetics, pathology)
• S-Nitrosoglutathione (metabolism)
• Thiazines (pharmacology)