Increased tyrosine phosphorylation of PSD-95 by Src family kinases after brain ischaemia.
PSD (postsynaptic density)-95, a scaffold protein that tethers NMDA (N-methyl-D-aspartate) receptors to signal molecules, is implicated in pathological events resulting from excitotoxicity. The present study demonstrates that brain ischaemia and reperfusion increase the tyrosine phosphorylation of PSD-95 in the rat hippocampus. PP2, a specific inhibitor of SrcPTKs (Src family protein tyrosine kinases), prevents the ischaemia-induced increases not only in the tyrosine phosphorylation of PSD-95, but also in the interaction between PSD-95 and Src kinases. PSD-95 is phosphorylated either by purified Src/Fyn kinases in vitro or by co-expression of constitutively active Src/Fyn in COS7 cells. The results suggest that SrcPTKs are involved in PSD-95 phosphorylation. The single Tyr(523) mutation to phenylalanine (Y523F) reduces the Src/Fyn-mediated phosphorylation of PSD-95 in COS7 cells and in vitro. As shown with a rabbit polyclonal antibody against phospho-PSD-95 (Tyr(523)), Tyr(523) phosphorylation is responsible for the increased tyrosine phosphorylation of PSD-95 induced by ischaemia in the rat hippocampus. In cultured hippocampal neurons, overexpression of PSD-95 Y523F, but not PSD-95 Y533F, abolishes the facilitating effect of PSD-95 on the glutamate- or NMDA-mediated currents, implying that PSD-95 Tyr(523) phosphorylation contributes to the post-ischaemic over-activation of NMDA receptors. Thus the present study reveals an additional mechanism for the regulation of PSD-95 by tyrosine phosphorylation. This mechanism may be of pathological significance since it is associated with excitotoxicity in the ischaemic brain.
Du
Cai-Ping
CP
Jiangsu Key Laboratory of Brain Disease Bioinformation, Research Center for Biochemistry and Molecular Biology, Xuzhou Medical College, 84 West Huaihai Road, Jiangsu 221002, China.
Gao
Jin
J
Tai
Jian-Min
JM
Liu
Yong
Y
Qi
Jing
J
Wang
Wei
W
Hou
Xiao-Yu
XY
D000818:Animals; D001665:Binding Sites / Q000235:genetics; D002545:Brain Ischemia / Q000503:physiopathology*; D019556:COS Cells; D002478:Cells, Cultured; D002522:Chlorocebus aethiops; D000076122:Disks Large Homolog 4 Protein; D018698:Glutamic Acid / Q000494:pharmacology; D006624:Hippocampus / Q000166:cytology / Q000378:metabolism; D015151:Immunoblotting; D047468:Immunoprecipitation; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics / Q000378:metabolism*; D008297:Male; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D016202:N-Methylaspartate / Q000494:pharmacology; D009474:Neurons / Q000187:drug effects / Q000378:metabolism / Q000502:physiology; D018408:Patch-Clamp Techniques; D010766:Phosphorylation; D051381:Rats; D017207:Rats, Sprague-Dawley; D016194:Receptors, N-Methyl-D-Aspartate / Q000378:metabolism / Q000502:physiology; D014443:Tyrosine / Q000378:metabolism*; D019061:src-Family Kinases / Q000378:metabolism*
D000076122:Disks Large Homolog 4 Protein; C501449:Dlg4 protein, rat; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; D016194:Receptors, N-Methyl-D-Aspartate; D018698:Glutamic Acid; D014443:Tyrosine; D016202:N-Methylaspartate; D019061:src-Family Kinases