Regulation of Ca2+/calmodulin-dependent protein kinase II catalysis by N-methyl-D-aspartate receptor subunit 2B

Abstract

Binding of CaMKII (Ca(2+)/calmodulin-dependent protein kinase II) to the NR2B subunit of the NMDAR (N-methyl-D-aspartate-type glutamate receptor) in the PSD (postsynaptic density) is essential for the induction of long-term potentiation. In this study, we show that binding of NR2B to the T-site (Thr(286)-autophosphorylation site binding pocket) of CaMKII regulates its catalysis as reflected in the kinetic parameters. The apparent S(0.5) (substrate concentration at half maximal velocity) and V(max) values for ATP were lower for phosphorylation of a GST (glutathione transferase)-fusion of NR2B((1271-1311)) (with the phosphorylation site Ser(1303)) when compared with phosphorylation of the analogous sequence motif from NR2A. The co-operative behaviour exhibited by the CaMKII holoenzyme towards ATP for phosphorylation of GST-NR2A was significantly altered by the interaction with GST-NR2B. Disrupting the T-site-mediated binding by mutagenesis of either NR2B or CaMKII abolished the modulation of CaMKII activity by NR2B. The active site residue of alpha-CaMKII, Glu(96), participates in effecting the modulation. The CaMKII-binding motif of the Drosophila voltage-gated potassium channel Eag interacted with the T-site of CaMKII with lower affinity and caused catalytic modulation to a lesser extent. The kinetic parameters of ATP for the Thr(286)-autophosphorylation reaction of CaMKII were also altered by NR2B in a similar manner. Interestingly, the NR2B sequence motif caused increased sensitivity of CaMKII activity to ATP, and saturation by lower concentrations of ATP, which, in effect, resulted in a constant level of activity of CaMKII over a broad range of ATP concentrations. Our findings indicate that CaMKII at the PSD may be regulated by bound NR2B in a manner that supports synaptic memories.

Calcium/Calmodulin Dependent Protein Kinase II Bound to NMDA Receptor 2B Subunit Exhibits Increased ATP Affinity and Attenuated Dephosphorylation

“…We have previously reported that CaMKII shows enhanced activity at low [ATP] in the presence of saturating concentrations of non-phosphorylatable GST-NR2B (S1303A) [8]. When pretreated with subsaturating concentrations of GST-NR2B (S1303A) also, CaMKII showed enhanced activity at lower [ATP] compared with control CaMKII pretreated with non-phosphorylatable GST-NR2A (S1291A) (Fig.…”

“…1 inset). It has previously been shown by GST pull down assay that NR2A sequence does not bind to the T-site of CaMKII [3], [ 8]. The activity of the enzyme achieves saturation at very low [ATP] in presence of NR2B sequence and stays constant for a broad range of [ATP] whereas in the absence of NR2B the activity attained saturation only at much higher [ATP].…”

“…Binding of CaMKII to NR2B, by a non-catalytic site called T-site, enables it to remain autonomously active [7]. In addition, the interaction between CaMKII and NR2B through the T-site has been found to modulate the kinetics of catalysis by the enzyme [8]. It was proposed that CaMKII in combination with protein phosphatase 1 (PP1), a phosphatase enriched in PSD, can form a Ca 2+ -sensitive molecular switch that can respond with specificity to the type of Ca 2+ signals and provide stability to molecular memories [9]–[12].…”

Nucleotides and Phosphorylation Bi-directionally Modulate Ca2+/Calmodulin-dependent Protein Kinase II (CaMKII) Binding to the N-Methyl-d-aspartate (NMDA) Receptor Subunit GluN2B

“…This interaction of CaMKII with GluN2B is indeed important in regulation of synaptic strength (28 -30). Binding to GluN2B keeps CaMKII in an active conformation, which allows phosphorylation of GluN2B even after the initial Ca 2ϩ stimulus has subsided, and even when T286 is no longer phosphorylated (15,17,31,32). In turn, CaMKII activity is thought to regulate NMDA-receptor currents (33)(34)(35).…”

Isoflurane/nitrous oxide anesthesia induces increases in NMDA receptor subunit NR2B protein expression in the aged rat brain

“…Therefore, anesthesia-induced changes in NR2B protein levels in the hippocampus and cortex may initiate dysfunction in NMDAR-ERK mediated learning and memory signaling pathways, corresponding to delayed spatial learning deficits in 18-month-old rats treated with anesthesia. Disruption of the NMDA receptor signaling through changes in NR2B may also interfere with other important learning and memory mechanisms, such as CaMKII activation (Santucci and Raghavachari, 2008; Pradeep et al, 2009) and AMPA receptor trafficking and regulation (Hall et al, 2007) that may offer additional explanations for the observed impairment in spatial learning in anesthesia-treated 18-month-old rats in this study.…”