The upregulation of NR2A-containing N-methyl-D-aspartate receptor function by tyrosine phosphorylation of postsynaptic density 95 via facilitating Src/proline-rich tyrosine kinase 2 activation.

The activation of postsynaptic N-methyl-D-aspartate (NMDA) receptors is required for long-term potentiation (LTP) of synaptic transmission. Postsynaptic density 95 (PSD-95) serves as a scaffold protein that tethers NMDA receptor subunits, kinases, and signal molecules. Our previous study proves that PSD-95 is a substrate of Src/Fyn and identifies Y523 on PSD-95 as a principal phosphorylation site. In this paper, we try to define an involvement and molecular consequences of PSD-95 phosphorylation by Src in NMDA receptor regulation. We found that either NMDA or chemical LTP induction leads to rapid phosphorylation of PSD-95 by Src in cultured cortical neurons. The phosphorylation of Y523 on PSD-95 potentiates NR2A-containing NMDA receptor current amplitude, implying an important role of Src-mediated PSD-95 phosphorylation in NMDA receptor activation. Comparing to wild-type PSD-95, overexpression of nonphosphorylatable mutant PSD-95Y523F attenuated the NMDA-stimulated NR2A tyrosine phosphorylation that enhances electrophysiological responses of NMDA receptor channels, while did not affect the membrane localization of NR2A subunits. PSD-95Y523D, a phosphomimetic mutant of PSD-95, induced NR2A tyrosine phosphorylation even if there was no NMDA treatment. In addition, the deficiency of Y523 phosphorylation on PSD-95 impaired the facilitatory effect of PSD-95 on the activation of Src and proline-rich tyrosine kinase 2 (Pyk2) and decreased the binding of Pyk2 with PSD-95. These results indicate that PSD-95 phosphorylation by Src facilitates the integration of Pyk2 to PSD-95 signal complex, the activation of Pyk2/Src, as well as the subsequent tyrosine phosphorylation of NR2A, which ultimately results in the upregulation of NMDA receptor function and synaptic transmission.