Calcium-calmodulin-dependent protein kinase II phosphorylation modulates PSD-95 binding to NMDA receptors.
At the postsynaptic membrane of excitatory synapses, NMDA-type receptors are bound to scaffolding and signalling proteins that regulate the strength of synaptic transmission. The cytosolic tails of the NR2A and NR2B subunits of NMDA receptor bind to calcium-calmodulin-dependent protein kinase II (CaMKII) and to members of the MAGUK family such as PSD-95. In particular, although NR2A and NR2B subunits are highly homologous, the sites of their interaction with CaMKII as well as the regulation of this binding differ. We identified PSD-95 phosphorylation as a molecular mechanism responsible for the dynamic regulation of the interaction of both PSD-95 and CaMKII with the NR2A subunit. CaMKII-dependent phosphorylation of PSD-95 occurs both in vitro, in GST-PSD-95 fusion proteins phosphorylated by purified active CaMKII, and in vivo, in transfected COS-7 as well as in cultured hippocampal neurons. We identified Ser73 as major phosphorylation site within the PDZ1 domain of PSD-95, as confirmed by point mutagenesis experiments and by using a phospho-specific antibody. PSD-95 Ser73 phosphorylation causes NR2A dissociation from PSD-95, while it does not interfere with NR2B binding to PSD-95. These results identify CaMKII-dependent phosphorylation of the PDZ1 domain of PSD-95 as a mechanism regulating the signalling transduction pathway downstream NMDA receptor.
Gardoni
Fabrizio
F
Center of Excellence on Neurodegenerative Diseases and Department of Pharmacological Sciences, University of Milan, via Balzaretti 9, 20133 Milano, Italy. fabrizio.gardoni@unimi.it
Polli
Federica
F
Cattabeni
Flaminio
F
Di Luca
Monica
M
D000818:Animals; D001596:Benzylamines / Q000494:pharmacology; D015153:Blotting, Western / Q000379:methods; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism*; D002478:Cells, Cultured; D002522:Chlorocebus aethiops; D000076122:Disks Large Homolog 4 Protein; D004347:Drug Interactions; D004622:Embryo, Mammalian; D018690:Excitatory Amino Acid Agonists / Q000494:pharmacology; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006624:Hippocampus / Q000187:drug effects / Q000378:metabolism; D007150:Immunohistochemistry / Q000379:methods; D047468:Immunoprecipitation / Q000379:methods; D066298:In Vitro Techniques; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism*; D016296:Mutagenesis / Q000502:physiology; D016202:N-Methylaspartate / Q000494:pharmacology; D010766:Phosphorylation / Q000187:drug effects; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D051381:Rats; D016194:Receptors, N-Methyl-D-Aspartate / Q000502:physiology*; D012694:Serine / Q000378:metabolism; D013449:Sulfonamides / Q000494:pharmacology; D014162:Transfection / Q000379:methods; D000096926:Benzenesulfonamides
D001596:Benzylamines; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D000076122:Disks Large Homolog 4 Protein; D018690:Excitatory Amino Acid Agonists; D049452:Green Fluorescent Proteins; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; D016202:N-Methylaspartate; D047428:Protein Kinase Inhibitors; D016194:Receptors, N-Methyl-D-Aspartate; D012694:Serine; D013449:Sulfonamides; C501449:Dlg4 protein, rat; C120997:NR2A NMDA receptor; C121001:NR2B NMDA receptor; C072105:KN 93; D000096926:Benzenesulfonamides