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 Tyr⁵²³ 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 (Tyr⁵²³), Tyr⁵²³ 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 Tyr⁵²³ 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.