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.

Ca2+ influx through NMDA‐type glutamate receptor at excitatory synapses causes activation of post‐synaptic Ca2+/calmodulin‐dependent protein kinase type II (CaMKII) and its translocation to the NR2B subunit of NMDA receptor. The major binding site for CaMKII on NR2B undergoes phosphorylation at Ser1303, in vivo. Even though some regulatory effects of this phosphorylation are known, the mode of dephosphorylation of NR2B‐Ser1303 is still unclear. We show that phosphorylation status at Ser1303 enables NR2B to distinguish between the Ca2+/calmodulin activated form and the autonomously active Thr286‐autophosphorylated form of CaMKII. Green fluorescent protein–α‐CaMKII co‐expressed with NR2B sequence in human embryonic kidney 293 cells was used to study intracellular binding between the two proteins. Binding in vitro was studied by glutathione‐S‐transferase pull‐down assay. Thr286‐autophosphorylated α‐CaMKII or the autophosphorylation mimicking mutant, T286D‐α‐CaMKII, binds NR2B sequence independent of Ca2+/calmodulin unlike native wild‐type α‐CaMKII. We show enhancement of this binding by Ca2+/calmodulin. Phosphorylation or a phosphorylation mimicking mutation on NR2B (NR2B‐S1303D) abolishes the Ca2+/calmodulin‐independent binding whereas it allows the Ca2+/calmodulin‐dependent binding of α‐CaMKII in vitro. Similarly, the autonomously active mutants, T286D‐α‐CaMKII and F293E/N294D‐α‐CaMKII, exhibited Ca2+‐independent binding to non‐phosphorylatable mutant of NR2B under intracellular conditions. We also show for the first time that phosphatases in the brain such as protein phosphatase 1 and protein phosphatase 2A dephosphorylate phospho‐Ser1303 on NR2B.

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Calcium/calmodulin dependent protein kinase II (CaMKII) is implicated to play a key role in learning and memory. NR2B subunit of N-methyl-D-aspartate receptor (NMDAR) is a high affinity binding partner of CaMKII at the postsynaptic membrane. NR2B binds to the T-site of CaMKII and modulates its catalysis. By direct measurement using isothermal titration calorimetry (ITC), we show that NR2B binding causes about 11 fold increase in the affinity of CaMKII for ATPγS, an analogue of ATP. ITC data is also consistent with an ordered binding mechanism for CaMKII with ATP binding the catalytic site first followed by peptide substrate. We also show that dephosphorylation of phospho-Thr286-α-CaMKII is attenuated when NR2B is bound to CaMKII. This favors the persistence of Thr286 autophosphorylated state of CaMKII in a CaMKII/phosphatase conjugate system in vitro. Overall our data indicate that the NR2B- bound state of CaMKII attains unique biochemical properties which could help in the efficient functioning of the proposed molecular switch supporting synaptic memory.

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