S-Nitrosylation of proline-rich tyrosine kinase 2 involves its activation induced by oxygen-glucose deprivation.
Previous studies have demonstrated that activation of proline-rich tyrosine kinase 2 (PYK2) in cerebral ischemia is involved in the modulation of N-methyl-d-aspartate-type (NMDA) glutamate receptor activity and Ca(2+) dynamics, resulting in ischemic neuron death ultimately. A number of reports indicate that PYK2 is a redox sensitive kinase that must be activated by an estrogen-induced reactive oxygen species (ROS). However, the mechanism of PYK2 activation remains incompletely illustrated. Accumulating attention is focused on nitric oxide (NO, a free radical) which plays a critical role in cellular signal transduction through stimulus-coupled S-nitrosylation of cysteine residues. Here we reported that PYK2 over-expressed in human embryonic kidney (HEK293) cells was S-nitrosylated (forming SNO-PYK2) by reacting with GSNO, an exogenous NO donor, at one critical cysteine residue (Cys534) with a biotin switch assay. Moreover, our results showed that S-nitrosylation and phosphorylation of PYK2 over-expressed in SH-SY5Y cells was significantly increased after oxygen-glucose deprivation (OGD). We further investigated whether the activation (phosphorylation) of PYK2 was associated with S-nitrosylation following SH-SY5Y cells OGD. Our results showed that the cysteine534 residue (site of S-nitrosylation) mutant PYK2 over-expressed in SH-SY5Y cells diminished S-nitrosylation of PYK2 and inhibited its phosphorylation induced by OGD. In addition, overexpression of the mutant PYK2 protein could prevent nuclear accumulation and abrogate neuronal cell death compared to wild type PYK2 in SH-SY5Y cells induced by OGD. These data suggest that the activation of PYK2 following OGD may be modulated by S-nitrosylation, which provides a new avenue for stroke therapy by targeting the post-translational modification machinery.
Department of Emergency, Qilu Hospital, Shandong University, Jinan 250000, China; Institute of Emergency Rescue Medicine, Xuzhou Medical College, Xuzhou 221002, China.
Xian-Liang
XL
Yan
Jiangsu Key Laboratory of Brain Disease Bioinformation, Xuzhou Medical College, Xuzhou 221004, China.
Dong-Hai
DH
Liu
Department of Physiology, College of Basic Medical Science, Anhui Medical University, 81 Meishan Road, Hefei, Anhui 230032, China.
Gong-Liang
GL
Zhang
Institute of Emergency Rescue Medicine, Xuzhou Medical College, Xuzhou 221002, China; Jiangsu Key Laboratory of Brain Disease Bioinformation, Xuzhou Medical College, Xuzhou 221004, China. Electronic address: hushuqun88@126.com.
Shu-Qun
SQ
Hu
Department of Emergency, Qilu Hospital, Shandong University, Jinan 250000, China. Electronic address: chen919085@126.com.
Yu-Guo
YG
Chen
Institute of Emergency Rescue Medicine, Xuzhou Medical College, Xuzhou 221002, China. Electronic address: xutie889@163.com.
Tie
T
Xu
D015687:Cell Hypoxia; D045744:Cell Line, Tumor; D004789:Enzyme Activation; D051418:Focal Adhesion Kinase 2 / Q000378:metabolism*; D005947:Glucose / Q000172:deficiency*; D006801:Humans; D009569:Nitric Oxide / Q000378:metabolism*; D020030:Nitric Oxide Donors / Q000378:metabolism; D052248:Nitric Oxide Synthase Type I / Q000378:metabolism; D010100:Oxygen / Q000378:metabolism*; D026422:S-Nitrosoglutathione / Q000378:metabolism
D020030:Nitric Oxide Donors; D009569:Nitric Oxide; D026422:S-Nitrosoglutathione; D052248:Nitric Oxide Synthase Type I; D051418:Focal Adhesion Kinase 2; D005947:Glucose; D010100:Oxygen
OGD; PYK2; Phosphorylation; S-Nitrosylation