title: Nucleotides and phosphorylation bi-directionally modulate Ca2+/calmodulin-dependent protein kinase II (CaMKII) binding to the N-methyl-D-aspartate (NMDA) receptor subunit GluN2B.
abstract: The Ca(2+)/calmodulin (CaM)-dependent protein kinase II (CaMKII) and the NMDA-type glutamate receptor are key regulators of synaptic plasticity underlying learning and memory. Direct binding of CaMKII to the NMDA receptor subunit GluN2B (formerly known as NR2B) (i) is induced by Ca(2+)/CaM but outlasts this initial Ca(2+)-stimulus, (ii) mediates CaMKII translocation to synapses, and (iii) regulates synaptic strength. CaMKII binds to GluN2B around S1303, the major CaMKII phosphorylation site on GluN2B. We show here that a phospho-mimetic S1303D mutation inhibited CaM-induced CaMKII binding to GluN2B in vitro, presenting a conundrum how binding can occur within cells, where high ATP concentration should promote S1303 phosphorylation. Surprisingly, addition of ATP actually enhanced the binding. Mutational analysis revealed that this positive net effect was caused by four modulatory effects of ATP, two positive (direct nucleotide binding and CaMKII T286 autophosphorylation) and two negative (GluN2B S1303 phosphorylation and CaMKII T305/6 autophosphorylation). Imaging showed positive regulation by nucleotide binding also within transfected HEK cells and neurons. In fact, nucleotide binding was a requirement for efficient CaMKII interaction with GluN2B in cells, while T286 autophosphorylation was not. Kinetic considerations support a model in which positive regulation by nucleotide binding and T286 autophosphorylation occurs faster than negative modulation by GluN2B S1303 and CaMKII T305/6 phosphorylation, allowing efficient CaMKII binding to GluN2B despite the inhibitory effects of the two slower reactions.
authors.affiliation: Department of Pharmacology, School of Medicine, University of Colorado Denver, Aurora, Colorado 80045, USA.
authors.forename: Heather
authors.initials: H
authors.lastname: O'Leary
authors.forename: Wallace H
authors.initials: WH
authors.lastname: Liu
authors.forename: Jacki M
authors.initials: JM
authors.lastname: Rorabaugh
authors.forename: Steven J
authors.initials: SJ
authors.lastname: Coultrap
authors.forename: K Ulrich
authors.initials: KU
authors.lastname: Bayer
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D001665:Binding Sites; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2 / Q000378:metabolism*; D002460:Cell Line; D006801:Humans; D007700:Kinetics; D009711:Nucleotides / Q000494:pharmacology*; D010766:Phosphorylation; D011485:Protein Binding; D021122:Protein Subunits; D016194:Receptors, N-Methyl-D-Aspartate / Q000378:metabolism*
chemical_list: D009711:Nucleotides; D021122:Protein Subunits; D016194:Receptors, N-Methyl-D-Aspartate; D000255:Adenosine Triphosphate; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2