Identification of a Phosphorylation Site for Calcium/Calmodulindependent Protein Kinase II in the NR2B Subunit of the N-Methyl-D-aspartate Receptor

The N-methyl-D-aspartate (NMDA) subtype of excitatory glutamate receptors plays critical roles in embryonic and adult synaptic plasticity in the central nervous system. The receptor is a heteromultimer of core subunits, NR1, and one or more regulatory subunits, NR2A-D. Protein phosphorylation can regulate NMDA receptor function (Lieberman, D. N., and Mody, I. (1994) Nature 369, 235-239; Wang, Y. T., and Salter, M. W. (1994) Nature 369, 233-235; Wang, L.-Y., Orser, B. A., Brautigan, D. L., and MacDonald, J. F. (1994) Nature 369, 230-232). Here we identify a major phosphorylation site on subunit NR2B that is phosphorylated by Ca2+/calmodulin-dependent protein kinase II (CaM kinase II), an abundant protein kinase located at postsynaptic sites in glutamatergic synapses. For the initial identification of the site, we constructed a recombinant fusion protein containing 334 amino acids of the C terminus of the NR2B subunit and phosphorylated it with CaM kinase II in vitro. By peptide mapping, automated sequencing, and mass spectrometry, we identified the major site of phosphorylation on the fusion protein as Ser-383, corresponding to Ser-1303 of full-length NR2B. The Km for phosphorylation of this site in the fusion protein was ~50 nM, much lower than that of other known substrates for CaM kinase II, suggesting that the receptor is a high affinity substrate. We show that serine 1303 in the full-length NR2B and/or the cognate site in NR2A is a major site of phosphorylation of the receptor both in the postsynaptic density fraction and in living hippocampal neurons

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

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

CaMKIINα inhibits the dephosphorylation of CaMKII.

<p>A: Autophosphorylated CaMKII was dephosphorylated in presence of either GST as control or GST-CaMKIINα. The same dephosphorylation condition was applied to all samples but, with and without treatment of PP1. B: Difference between the densitometric values of autoradiographic bands obtained with and without PP1 was used for calculating dephosphorylation. The values plotted are the mean ± standard deviation of data of four experiments. In each set, the intensity of the band without PP1 treatment was taken as 100% phosphorylation (p < 0.05 in two-tailed student’s t-test).</p

Calcium dependent binding of CaMKIINα to CaMKII.

<p>A: GST-pulldown assay was carried out to show binding of α-CaMKII to GST-CaMKIINα. Blot was cut into two pieces. Upper part of the blot was probed with anti-α-CaMKII antibody and the lower part was probed with anti-GST antibody. Marker is from the same blot. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0162011#pone.0162011.s001" target="_blank">S1 Fig</a> shows SDS-PAGE pattern of CaMKIINα. B: GluN2B reduced CaMKII binding to CaMKIINα. GST-pulldown assay was performed as described in methods. Lane 1: GST-pulldown of CaMKII with GST-CaMKIINα. Lane 2: CaMKIINα was allowed to bind to CaMKII first, followed by incubation with His-GluN2B. Lane 3: GST-CaMKIINα and His-GluN2B were allowed to bind to CaMKII simultaneously. C: The mean ± standard deviation of densitometric values of the band intensities obtained from three independent experiments as shown in B are plotted. The binding of CaMKII with GST-CaMKIINα taken as the control is considered as 100% binding. The binding of CaMKII in 2 and 3 are presented as the relative binding with respect to control in the bar graphs. The p value for the difference between 1 and 3 is less than 0.05 in two-tailed student’s t-test.</p

Comparison of the effect of GluN2B on the rates of dephosphorylation of WT and E96A.

<p>A and C: The extent of dephosphorylation of WT and E96A mutant of α-CaMKII were determined at 1, 2 and 3 minutes in presence of GST-S1303A-GluN2B or GST-S1291A-GluN2A. Phosphorylation intensity without PP1 at 0 time was taken as 100%. The values plotted were from the densitometric data of autoradiographic images as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0162011#pone.0162011.s005" target="_blank">S5 Fig</a>, from three experiments. B and D: The slope values of the plots of the time course of dephosphorylation in presence of GluN2B and GluN2A sequences are presented as the bar graphs (For B, p<0.05 in a two—tailed student’s t-test).</p

Protection of α-CaMKII from Dephosphorylation by GluN2B Subunit of NMDA Receptor Is Abolished by Mutation of Glu⁹⁶ or His²⁸² of α-CaMKII

<div><p>Interaction of CaMKII and the GluN2B subunit of NMDA receptor is essential for synaptic plasticity events such as LTP. Synaptic targeting of CaMKII and regulation of its biochemical functions result from this interaction. GluN2B binding to the T-site of CaMKII leads to changes in substrate binding and catalytic parameters and inhibition of its own dephosphorylation. We find that CaMKIINα, a natural inhibitor that binds to the T-site of CaMKII, also causes inhibition of dephosphorylation of CaMKII similar to GluN2B. Two residues on α-CaMKII, Glu⁹⁶ and His²⁸², are involved in the inhibition of CaMKII dephosphorylation exerted by binding of GluN2B. E96A-α-CaMKII is known to be defective in GluN2B-induced catalytic modulation. Data presented here show that, in both E96A and H282A mutants of α-CaMKII, GluN2B-induced inhibition of dephosphorylation is impaired.</p></div

Influence of a mutation in the ATP-binding region of Ca<SUP>2+</SUP>/calmodulindependent protein kinase II on its interaction with peptide substrates

CaMKII (Ca<SUP>2+</SUP>/calmodulin-dependent protein kinase II) is expressed in high concentrations in the brain and is found enriched in the postsynaptic densities. The enzyme is activated by the binding of calmodulin to the autoregulatory domain in the presence of high levels of intracellular Ca<SUP>2+</SUP>, which causes removal of auto-inhibition from the N-terminal catalytic domain. Knowledge of the 3D (three-dimensional) structure of this enzyme at atomic resolution is restricted to the association domain, a region at the extreme C-terminus. The catalytic domain of CaMKII shares high sequence similarity with CaMKI. The 3D structure of the catalytic core of CaMKI comprises ATP-and substrate-binding regions in a cleft between two distinct lobes, similar to the structures of all protein kinases solved to date. Mutation of Glu-60, a residue in the ATP-binding region of CaMKII, to glycine exerts different effects on phosphorylation of two peptide substrates, syntide and NR2B (N-methyl-D-aspartate receptor subunit 2B) 17-mer. Although the mutation caused increases in the K<SUB>m</SUB> values for phosphorylation for both the peptide substrates, the effect on the k<SUB>cat</SUB> values for each was different. The kcat value decreased in the case of syntide, whereas it increased in the case of the NR2B peptide as a result of the mutation. This resulted in a significant decrease in the apparent k<SUB>cat</SUB>/K<SUB>m</SUB> value for syntide, but the change was minimal for the NR2B peptide. These results indicate that different catalytic mechanisms are employed by the kinase for the two peptides. Molecular modelling suggests structural changes are likely to occur at the peptide-binding pocket in the active state of the enzyme as a consequence of the Glu-60Gly mutation