Phosphorylation of the α-amino-3-hydroxy-5-methylisoxazole-4-propionic Acid receptor GluR1 subunit by calcium/calmodulin-dependent kinase II

Modulation of α-amino-3-hydroxy-5-methylisoxazole-4-propionic Acid (AMPA) receptors in the brain by protein phosphorylation may play a crucial role in the regulation of synaptic plasticity. Previous studies have demonstrated that calmodulin (CAM) kinase II can phosphorylate and modulate AMPA receptors. However, the sites of CaM kinase phosphorylation have not been unequivocally identified. In the current study, we have generated two phosphorylation site-specific antibodies to analyze the phosphorylation of the glutamate receptor GluR1 subunit. These antibodies recognize GluR1 only when it is phosphorylated on serine residues 831 or 845. We have used these antibodies to demonstrate that serine 831 is specifically phosphorylated by CaM kinase II in transfected cells expressing GluR1 as well as in hippocampal slice preparations. Two-dimensional phosphopeptide mapping experiments indicate that Set-831 is the major site of CaM kinase II phosphorylation on GluR1. In addition, treatment of hippocampal slice preparations with phorbol esters and forskolin increase the phosphorylation of serine 831 and 845, respectively, indicating that protein kinase C and protein kinase A phosphorylate these residues in hippocampal slices. These results identify the site of CaM kinase phosphorylation of the GluR1 subunit and demonstrate that GluR1 is multiply phosphorylated by protein kinase A, protein kinase C, and CaM kinase II in situ.