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 impor tant regulatory mechanism. In this study P-32 labeling of non-NMDA Glu Rs was investigated in cultured hippocampal neurons stimulated 2-15 mi n with agonists that selectively stimulate either Ca2+/calmodulin-depe ndent protein kinase II (CaM-kinase II), Ca2+/phospholipid-dependent p rotein kinase C (PKC), or cAMP-dependent protein kinase A (PKA). Treat ment of hippocampal neurons with glutamate/ glycine (Glu/Gly), ionomyc in, or 12-O-tetradecanoylphorbol 13-acetate (TPA) increased P-32 label ing of immunoprecipitated ha-amino-3-hydroxy-5-methyl-4-isoxazolepropr ionate (AMPA)-type GluRs by 145%, 180%, and 227%, respectively, of con trol 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 ago nists, only TPA stimulated phosphorylation of MARCKS (225% of control) , a specific substrate of PKC. Forskolin treatment gave a three- to fo urfold increase in the active catalytic subunit of PKA but did not res ult 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 spec ific NMDA receptor/ion channel antagonist (DL-2-amino-5-phosphonovaler ic acid) or by a cell-permeable inhibitor of CaM-kinase II esulfonyl)- N-methyl-L-tyrosyl]-4-phenylpiperazine, KN-62). These results are cons istent with the hypothesis that Ca2+ influx through the NMDA-type ion channel can activate CaM-kinase II, which in turn can phosphorylate an d regulate AMPA-type GluR ion channels (McGlade-McCulloh et al., 1993) . Such a mechanism could contribute to the postsynaptic component of l ong-term potentiation and other forms of synaptic plasticity.