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S-nitrosylation of c-Src via NMDAR-nNOS module promotes c-Src activation and NR2A phosphorylation in cerebral ischemia/reperfusion

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Abstract

Previous studies suggested that activated c-Src promote the tyrosine phosphorylation of NMDA receptor subunit NR2A, and thus aggravate the injury induced by transient cerebral ischemia/reperfusion (I/R) in rat hippocampus CA1 region. In this study, we examined the effect of nitric oxide (NO) on the activation of c-Src and the tyrosine phosphorylation of NMDA receptor NR2A subunit. The results show that S-nitrosylation and the phosphorylation of c-Src were induced after cerebral I/R in rats, and administration of nNOS inhibitor 7-NI, nNOS antisense oligonucleotides and exogenous NO donor sodium nitroprusside diminished the increased S-nitrosylation and phosphorylation of c-Src during cerebral I/R. The cysteine residues of c-Src modified by S-nitrosylation are Cys489, Cys498, and Cys500. On the other hand, NMDAR antagonist MK-801 could attenuate the S-nitrosylation and activation of c-Src. Taken together, the S-nitrosylation of c-Src is provoked by NO derived from endogenous nNOS, which is activated by Ca2+ influx from NMDA receptors, and promotes the auto-phosphorylation at tyrosines and further phosphorylates NR2A. The molecular mechanism we outlined here is a novel postsynaptic NMDAR-nNOS/c-Src-mediated signaling amplification, the ‘NMDAR-nNOS → NO → SNO-c-Src → p-c-Src → NMDAR-nNOS’ cycle, which presents the possibility as a potential therapeutic target for stroke treatment.

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Abbreviations

I/R:

Ischemia/reperfusion

AS-ODNs:

Antisense oligodeoxynucleotides

MS-ODNs:

Missenses oligodeoxynucleotides

DTT:

1,4-Dithiothreitol

IB:

Immunoblotting

SDS-PAGE:

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis

PMSF:

Phenylmethylsulfonyl fluoride

SNP:

Sodium nitroprusside

GSNO:

S-nitrosoglutathione

NO:

Nitric oxide

NOS:

NO synthase

NMDA:

N-methyl-d-aspartate

NR:

NMDA receptor

AMPA:

α-Amino-3-hydroxy-5-methyl-4-soxazolepropionic acid

KA:

Kainite

MK-801:

(5R,10S)-(+)-5-Methyl-10,11-dihydro-5H-dibenzo [a,d] cyclohepten-5,10-imine hydrogen maleate

7-NI:

7-Nitroindazole

NEM:

N-Ethylmaleimide

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Acknowledgments

This work was supported by grants from the National Natural Science Foundation of China (Nos. 30870543, 31000360), the Natural Science Funds of Jiangsu Province (Nos. BK2010171, BK2010176), the Graduate Innovation Project of Jiangsu Province (CX08S-004Z) and the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD). Dr. Chong Li was supported by “New Century Excellent Talents in University (NCET)” program of Chinese Ministry of Education and “Six Talent Peaks Program” of Jiangsu Province.

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Authors and Affiliations

  1. Research Center of Biochemistry and Molecular Biology and Jiangsu Key Laboratory of Brain Disease Bioinformation, Xuzhou Medical College, 84 West Huai-hai Road, Xuzhou, 221002, Jiangsu, People’s Republic of China

    Li-Juan Tang, Chong Li, Shu-Qun Hu, Yan-Yan Zong, Chang-Cheng Sun, Fa Zhang & Guang-Yi Zhang

  2. Jiangsu Key Laboratory of Anesthesiology, Xuzhou Medical College, Xuzhou, Jiangsu, People’s Republic of China

    Yong-Ping Wu

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  1. Li-Juan Tang
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Correspondence to Guang-Yi Zhang.

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Li-Juan Tang, Chong Li, and Shu-Qun Hu contributed equally to this work.

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Tang, LJ., Li, C., Hu, SQ. et al. S-nitrosylation of c-Src via NMDAR-nNOS module promotes c-Src activation and NR2A phosphorylation in cerebral ischemia/reperfusion. Mol Cell Biochem 365, 363–377 (2012). https://doi.org/10.1007/s11010-012-1280-4

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