CaMKII-dependent phosphorylation regulates SAP97/NR2A interaction.
Synapse-associated protein 97 (SAP97), a member of membrane-associated guanylate kinase protein family, has been implicated in the processes of targeting ionotropic glutamate receptors at postsynaptic sites. Here we show that SAP97 is enriched at the postsynaptic density where it co-localizes with both ionotropic glutamate receptors and downstream signaling proteins such as Ca2+/calmodulin-dependent protein kinase II (CaMKII). SAP97 and alphaCaMKII display a high co-localization pattern in hippocampal neurons as well as in transfected COS-7 cells. Metabolic labeling of hippocampal cultures reveals that N-methyl-D-aspartic acid (NMDA) receptor activation induces CaMKII-dependent phosphorylation of SAP97; co-incubation with the CaMKII-specific inhibitor KN-93 reduces SAP97 phosphorylation to basal levels. Our results show that SAP97 directly interacts with the NR2A subunit of NMDA receptor both in an in vitro "pull-out" assay and in co-immunoprecipitation experiments from homogenates and synaptosomes purified from hippocampal rat tissue. Interestingly, in the postsynaptic density fraction, SAP97 fails to co-precipitate with NR2A. We show here that SAP97 is directly associated with NR2A through its PDZ1 domain, and CaMKII-dependent phosphorylation of SAP97-Ser-232 disrupts NR2A interaction both in an in vitro pull-out assay and in transfected COS-7 cells. Moreover, expression of SAP97(S232D) mutant has effects similar to those observed upon constitutively activating CaMKII. Our findings suggest that SAP97/NR2A interaction is regulated by CaMKII-dependent phosphorylation and provide a novel mechanism for the regulation of synaptic targeting of NMDA receptor subunits.
Gardoni
Fabrizio
F
Center of Excellence on Neurodegenerative Diseases and Department of Pharmacological Sciences, University of Milano, via Balzaretti 9, 20133 Milano, Italy. Fabrizio.Gardoni@unimi.it
Mauceri
Daniela
D
Fiorentini
Chiara
C
Bellone
Camilla
C
Missale
Cristina
C
Cattabeni
Flaminio
F
Di Luca
Monica
M
D048868:Adaptor Proteins, Signal Transducing; D000595:Amino Acid Sequence; D000818:Animals; D001596:Benzylamines / Q000494:pharmacology; D019556:COS Cells; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000032:analysis / Q000037:antagonists & inhibitors / Q000378:metabolism*; D002478:Cells, Cultured; D003712:Dendrites / Q000737:chemistry; D004622:Embryo, Mammalian; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D006624:Hippocampus; D007163:Immunosorbent Techniques; D008565:Membrane Proteins; D016297:Mutagenesis, Site-Directed; D016202:N-Methylaspartate / Q000494:pharmacology; D009419:Nerve Tissue Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D009474:Neurons / Q000648:ultrastructure; D010761:Phosphorus Radioisotopes; D010766:Phosphorylation; D051381:Rats; D016194:Receptors, N-Methyl-D-Aspartate / Q000032:analysis / Q000378:metabolism*; D011993:Recombinant Fusion Proteins; D013329:Structure-Activity Relationship; D013449:Sulfonamides / Q000494:pharmacology; D013574:Synaptosomes / Q000737:chemistry; D014162:Transfection; D000096926:Benzenesulfonamides
D048868:Adaptor Proteins, Signal Transducing; D001596:Benzylamines; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D004791:Enzyme Inhibitors; D008565:Membrane Proteins; D016202:N-Methylaspartate; D009419:Nerve Tissue Proteins; D010761:Phosphorus Radioisotopes; D016194:Receptors, N-Methyl-D-Aspartate; D011993:Recombinant Fusion Proteins; D013449:Sulfonamides; C501119:Dlg1 protein, rat; C120997:NR2A NMDA receptor; C072105:KN 93; D000096926:Benzenesulfonamides