PHOSPHORYLATION OF AMPA-TYPE GLUTAMATE RECEPTORS BY CALCIUM/CALMODULIN-DEPENDENT PROTEIN-KINASE-II AND PROTEIN-KINASE-C IN CULTURED HIPPOCAMPAL-NEURONS

Phosphorylation of glutamate receptors (GluRs) is emerging as an important regulatory mechanism. In this study P-32 labeling of non-NMDA GluRs was investigated in cultured hippocampal neurons stimulated 2-15 min with agonists that selectively stimulate either Ca2+/calmodulin-dependent protein kinase II (CaM-kinase II), Ca2+/phospholipid-dependent protein kinase C (PKC), or cAMP-dependent protein kinase A (PKA). Treatment of hippocampal neurons with glutamate/ glycine (Glu/Gly), ionomycin, or 12-O-tetradecanoylphorbol 13-acetate (TPA) increased P-32 labeling of immunoprecipitated alpha-amino-3-hydroxy-5-methyl-4-isoxazoleproprionate (AMPA)-type GluRs by 145%, 180%, and 227%, respectively, of control values. This increased phosphorylation of GluRs was predominantly P-32-Ser with little P-32-Thr and no detectable P-32-Tyr. Glu/Gly and ionomycin, but not TPA, also increased P-32 labeling of CaM-kinase II by 175% and 195%, respectively, of control values. Of these three agonists, only TPA stimulated phosphorylation of MARCKS (225% of control), a specific substrate of PKC. Forskolin treatment gave a three- to fourfold increase in the active catalytic subunit of PKA but did not result in the P-32 labeling of AMPA-type GluRs, CaM-kinase II, or MARCKS. Phosphorylation of GluRs in response to Glu/Gly was blocked by a specific NMDA receptor/ion channel antagonist (DL-2-amino-5-phosphonovaleric acid) or by a cell-permeable inhibitor of CaM-kinase II (1-[N,O-bis(1,5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phenylpiperazine, KN-62). These results are consistent with the hypothesis that Ca2+ influx through the NMDA-type ion channel can activate CaM-kinase II, which in turn can phosphorylate and regulate AMPA-type GluR ion channels (McGlade-McCulloh et al., 1993). Such a mechanism could contribute to the postsynaptic component of long-term potentiation and other forms of synaptic plasticity.