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

Effect of Multimeric Structure of CaMKII in the GluN2B-Mediated Modulation of Kinetic Parameters of ATP

<div><p>Interaction of GluN2B subunit of N-methyl-D-aspartate receptor with calcium/calmodulin dependent protein kinase II (CaMKII) is critical for the induction of long term potentiation at hippocampal CA3-CA1 synapses. We have previously reported that CaMKII binding to GluN2B increases its affinity but abolishes the cooperativity for ATP. In the present study, we demonstrate that the reduction in S_0.5 for ATP of an individual CaMKII subunit seems to be directly induced by the binding of GluN2B to the same subunit, while any GluN2B induced effects on the cooperativity and maximal velocity would additionally require the CaMKII holoenzyme structure. We measured the apparent kinetic parameters for ATP using an association domain truncated monomeric CaMKII and a heteromultimeric CaMKII (having subunits that are either GluN2B binding defective or ATP binding defective), in the presence of GluN2A or GluN2B substrates. The S_0.5 value for ATP of monomeric CaMKII is reduced ∼ 3 fold by the presence of GluN2B suggesting that the induced change in affinity for ATP is independent of the holoenzyme structure. The heteromultimeric mutant of CaMKII, did not exhibit cooperativity of ATP binding probably because of the interspersing of ATP binding defective subunits in the holoenzyme. In contrast to the wild type holoenzyme, presence of GluN2B increased the V_max of monomeric CaMKII which resulted in an approximately 4.0 fold increase in the apparent catalytic constant (V_max/S_0.5) as compared to GluN2A. The kinetic parameter values of the heteromultimeric CaMKII for ATP, on the other hand, did not show any significant difference between the phosphorylation of GluN2B and GluN2A suggesting that modulation requires binding of GluN2B to the same subunit. Overall, our present study provides insights into the role of multimeric structure of CaMKII in GluN2B-mediated regulation.</p> </div

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

GST-pull down assay shows that α-I205K-CaMKII and β-K43R-CaMKII can form heteromultimers.

<p>The mutants, α-I205K-CaMKII and β-K43R-CaMKII, when expressed from a single vector, form heteromultimers and are pulled down by GST-GluN2B. <b>A: Western blot of (α-I205K)-(β-K43R)-CaMKII expressed in insect cell line.</b> The heteromeric CaMKII was prepared by coexpressing GluN2B binding defective α-subunits (I205K) with β-subunit defective in nucleotide binding (K43R) using pFastBac^TMDual vector. Both the CaMKII mutants were His-tagged at their N-terminii. Lane 2, 3 and 5: Molecular weight marker; Lane 1: Lysate (50 µg) expressing heteromer probed with anti-α-CaMKII antibody; Lane 4: Lysate (50 µg) expressing heteromer probed with anti-β-CaMKII antibody; Lane 6: Lysate (50 µg) expressing heteromer probed with anti-(poly) His antibody. The arrows indicate the positions of α and β subunits in each lane. <b>B: Heteromeric CaMKII binds specifically to GST-GluN2B.</b> Western blot following GST pull down assay of the heteromeric CaMKII mutant is shown. GST-Pull down assays were done with crude cell lysates of fusion proteins and heteromeric CaMKII. Upper panel was probed with anti-(poly) His antibody and lower panel was probed with anti-GST antibody. Lane 1: Pull down with GST-GluN2A; Lane 2: Pull down with GST-GluN2B. Data represents 4 experiments. <b>C: GluN2B binding of heteromeric CaMKII is calcium dependent.</b> Western blot following GST pull down assay of the heteromeric CaMKII mutant with GST- GluN2B is shown. GST-Pull down assays were done with crude cell lysates of fusion proteins and heteromeric CaMKII. Upper panel was probed with anti-(poly) His Antibody and lower panel was probed with anti- GST antibody. Lane 1: Pull down in presence of Ca²⁺; Lane 2: Pull down in the absence of Ca²⁺. Data represents 3 experiments.</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

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

Kinetic parameters of WT, Monomeric and Heteromultimeric CaMKII for ATP.

<p>The ATP kinetic parameters of WT-CaMKII, Δ317-α-CaMKII and heteromeric (α-I205K)-(β-K43R) CaMKII are given. The quantity of CaMKII used for the assays was: WT-CaMKII-0.01–0.017 µg; Monomeric Δ317-α-CaMKII- (0.15 µg); Heteromeric (α-I205K)-(β-K43R) CaMKII-1.9 µg. For heteromeric CaMKII assay, saturating concentrations of purified preparations of GST-GluN2A (6.5 µM) and GST-GluN2B (9.4 µM) were used. For the rest of the assays saturating concentrations of fusion proteins in bacterial lysates were used. p values were calculated in comparison with corresponding GluN2A data in each set of experiments. p<0.05 was considered significant which are indicated with asterisk (*). Representative data used for estimation of kinetic parameters is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045064#pone.0045064.s001" target="_blank">Fig. S1</a>.</p

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