Inhibition of SNAP-25 phosphorylation at Ser187 is involved in chronic morphine-induced down-regulation of SNARE complex formation.
Opiate abuse has been shown to cause adaptive changes in presynaptic release and protein phosphorylation-mediated synaptic plasticity, but the underlying mechanisms remain unclear. Neuronal SNARE proteins serve as important regulatory molecules underlying neural plasticity in view of their major role in the process of neurotransmitter release. In the present study, the expression of SNAP-25, a t-SNARE protein essential for vesicle release, was found to be dramatically regulated in hippocampus after chronic morphine treatment, which was visualized with two-dimensional gel electrophoresis. The spots of SNAP-25 in the gel were shifted along the dimension of isoelectric point, indicating a likely change of the post-transcriptional modification. Immunoblotting analysis with specific antibody to Ser187, a protein kinase C (PKC) phosphorylation site of SNAP-25, revealed that the specific phosphorylation was correspondingly decreased, which was correlated with morphine-induced inhibition of PKC activity. Moreover, the level of ternary complex of SNARE proteins in either synaptosomes or PC12 cells was significantly reduced after chronic morphine treatment. This suggests a causal relationship between the inhibition of PKC-dependent SNAP-25 phosphorylation and the down-regulation of SNARE complex formation after chronic morphine treatment. Further analysis of SNARE complex formed by transfection of the wild-type or Ser187 mutants of SNAP-25 showed that only wild-type-formed complex was inhibited by morphine treatment. Thus, these results indicate that chronic morphine treatment inhibits phosphorylation of SNAP-25 at Ser187 and leads to a down-regulation of SNARE complex formation, which presents a potential molecular mechanism for the alteration of exocytotic process and neural plasticity during opiate abuse.
Laboratory of Molecular Cell Biology, Institute of Biochemistry and Cell Biology, Institute of Neuroscience, Graduate School of the Chinese Academy of Sciences, Shanghai, People's Republic of China.
Nan-Jie
NJ
Xu
Yong-Xin
YX
Yu
Jian-Mei
JM
Zhu
Hua
H
Liu
Li
L
Shen
Rong
R
Zeng
Xu
X
Zhang
Gang
G
Pei
D000701:Analgesics, Opioid / Q000494:pharmacology*; D000818:Animals; D015153:Blotting, Western; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D015536:Down-Regulation; D015180:Electrophoresis, Gel, Two-Dimensional; D005786:Gene Expression Regulation; D006624:Hippocampus / Q000378:metabolism; D015151:Immunoblotting; D007525:Isoelectric Focusing; D008297:Male; D008565:Membrane Proteins / Q000737:chemistry* / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D009020:Morphine / Q000494:pharmacology*; D009154:Mutation; D009419:Nerve Tissue Proteins / Q000737:chemistry* / Q000378:metabolism; D009474:Neurons / Q000378:metabolism; D016716:PC12 Cells; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism; D012323:RNA Processing, Post-Transcriptional; D051381:Rats; D012694:Serine / Q000737:chemistry; D050825:Synaptosomal-Associated Protein 25; D013574:Synaptosomes / Q000378:metabolism; D013997:Time Factors; D014162:Transfection
D000701:Analgesics, Opioid; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; C494209:Snap25 protein, mouse; C494210:Snap25 protein, rat; D050825:Synaptosomal-Associated Protein 25; D012694:Serine; D009020:Morphine; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C