Protein kinase C activation induces tyrosine phosphorylation of the NR2A and NR2B subunits of the NMDA receptor

The N-methyl-D-aspartate receptor (NMDAR) is an ionotropic glutamate recept or, which plays crucial roles in synaptic plasticity and development. We ha ve recently shown that potentiation of NMDA receptor function by protein ki nase C (PKC) appears to be mediated via activation of non-receptor tyrosine kinases. The aim of this study was to test whether this effect could be me diated by direct tyrosine phosphorylation of the NR2A or NR2B subunits of t he receptor. Following treatment of rat hippocampal CA1 mini-slices with,50 0 nM phorbol 12-myristate 13-acetate (PMA) for 15 min, samples were homogen ized, immunoprecipitated with anti-NR2A or NR2B antibodies and the resultin g pellets subjected to Western blotting with antiphosphotyrosine antibody. An increase in tyrosine phosphorylation of both NR2A (76 +/- 11% above cont rol) and NR2B (41 +/- 11%) was observed. This increase was blocked by pretr eatment with the selective PKC inhibitor chelerythrine, with the tyrosine k inase inhibitor Lavendustin A or with the Src family tyrosine kinase inhibi tor PP2. PMA treatment also produced an increase in the phosphorylation of serine 890 on the NR1 subunit, a known PKC site, at 5 min with phosphorylat ion returning to near basal levels by 10 min while tyrosine phosphorylation of NR2A and NR2B was sustained for up to 15 min. These results suggest tha t the modulation of NMDA receptor function seen with PKC activation may be the result of tyrosine phosphorylation of NR2A and/or NR2B.