Effect of multimeric structure of CaMKII in the GluN2B-mediated modulation of kinetic parameters of ATP

Abstract

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.

Figure 1. Monomeric CaMKII can bind to GluN2B sequence. A: Sequence alignment of GluN2A and GluN2B.

The sequence motifs of GluN2A and GluN2B used for the study are aligned. The conserved residues are underlined with black lines and identical residues are shown in bold. The phosphorylatable residues are shown in a larger font. B: GST-pull down assay followed by phosphorylation shows that monomeric CaMKII binds to GST-GluN2B. GST-pull down assay was done with crude cell lysates of fusion proteins and monomeric CaMKII. Since western blotting could not detect the pulled down monomeric enzyme, we resorted to utilizing the calcium independent activity of the GluN2B bound CaMKII for detection. Upper panel shows the SDS-PAGE and lower panel shows the autoradiogram of the gel. Molecular weight marker positions in the autoradiogram are indicated with black lines. Phosphorylation has happened only in the reactions that have pulled down CaMKII. Arrow mark shows the position of GST-GluN2B. Lane 1: phosphorylation of GST-GluN2B (1271–1311) by WT-α-CaMKII before pull down; lanes 2 and 3: phosphorylation after pull down performed as negative control using GST-GluN2A (1265–1301) for Δ317-α-CaMKII and WT-α-CaMKII respectively; lane MW: Molecular weight marker. Molecular weights are indicated in kDa. Lanes 4 and 5: phosphorylation after pull down using GST-GluN2B (1271–1311) of Δ317-α-CaMKII and WT-α-CaMKII respectively. Data represents 2 experiments.

Figure 2. GST-pull down assay shows that α-I205K-CaMKII and β-K43R-CaMKII can form heteromultimers.

The mutants, α-I205K-CaMKII and β-K43R-CaMKII, when expressed from a single vector, form heteromultimers and are pulled down by GST-GluN2B. A: Western blot of (α-I205K)-(β-K43R)-CaMKII expressed in insect cell line. 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: Heteromeric CaMKII binds specifically to GST-GluN2B. 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. C: GluN2B binding of heteromeric CaMKII is calcium dependent. 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.