Enzymatic activity of CaMKII is not required for its interaction with the glutamate receptor subunit GluN2B

Abstract

Binding of the Ca²⁺/calmodulin (CaM)-dependent protein kinase II (CaMKII) to the NMDA-type glutamate receptor subunit GluN2B is an important control mechanism for the regulation of synaptic strength. CaMKII binding to GluN2B and CaMKII translocation to synapses are induced by an initial Ca²⁺/CaM stimulus, which also activates the kinase. Indeed, several mechanistically different CaMKII inhibitors [tatCN21 and KN-93 (N-[2-[[[3-(4-chlorophenyl)-2-propenyl]methylamino]methyl]phenyl]-N-(2-hydroxyethyl)-4-methoxybenzenesulphonamide)] and inactivating mutations (K42M, A302R, and T305/T306D) impair this interaction, suggesting that it requires CaMKII enzymatic activity. However, this study shows that two general kinase inhibitors, H7 [1-(5-isoquinolinylsulfonyl)-2-methylpiperazine] and staurosporine (Sta), which inhibit CaMKII activity by yet another mechanism, did not interfere with GluN2B binding in vitro or within cells. In contrast to a previous report, we found that Sta, like H7, inhibited CaMKII in an ATP-competitive manner. Nucleotide binding significantly enhances CaMKII/GluN2B binding in vitro, but the nucleotide competition by H7 or Sta did not prevent this effect and instead even mimicked it. H7 (700 µM) and Sta (2 µM) efficiently blocked enzymatic activity of CaMKII, both in vitro and within cells. However, neither H7 nor Sta prevented Ca²⁺-induced translocation of CaMKII to GluN2B in heterologous cells or to synapses in hippocampal neurons. Thus, activity of CaMKII (or of any other kinase inhibited by H7 or Sta) is not required for stimulation-induced GluN2B-binding or synaptic translocation of CaMKII, despite previous indication to the contrary. This shows that results with inhibitors and inhibiting mutants can be caused by structural effects independent from catalytic activity, and that detailed understanding of the mechanisms is required for their interpretation.

Fig. 1.

H7 and Sta inhibit CaMKII enzymatic activity in vitro. To test kinase activity, recombinant, purified CaMKIIα (2.5 nM) was used to phosphorylate syntide-2 peptide (75 μM) in the presence of control (Ctl), 700 μM H7, or 2 μM Sta. Results were measured in phosphorylation reactions per minute. Both H7 and Sta significantly reduced the activity from control. One-way ANOVA; ***P < 0.001

Fig. 2.

Sta is competitive with ATP. To determine the inhibitory mechanism of Sta, concentration-response curves of kinase activity were measured over a range of ATP concentrations. (A) Increasing concentrations of Sta increase the apparent K_m for ATP without an effect on V_max, suggesting a competitive model by Michaelis-Menten kinetics. (B) Using the Lineweaver-Burk model, the lines all intersect at the same y-intercept with varying x-intercepts, mathematically suggesting a competitive model. (C) Testing the data under tight-binding equations shows a mixed model of inhibition that is predominantly competitive with ATP.

Fig. 3.

H7 and Sta mimic the nucleotide enhancement of CaMKII/GluN2B binding in vitro. CaMKII-binding to GluN2B was assessed by immobilizing GST-tagged GluN2B C-tail to anti–GST-coated plates and adding CaMKII in the presence of Ca²⁺ and calmodulin. Bound protein was measured by Western blot. (A) Representative Western blot of bound fractions from an in vitro binding assay run in biologic replicates. The membranes were probed for CaMKII (above) and GST-GluN2B (below). Binding reactions contained varied combinations of inhibitors (Inh)—700 μM H7 or 2 μM Sta—and nucleotide (Nuc)—100 μM ADP or ATP. These conditions are indicated above the lanes. (B) Quantification of the Western blot results normalized to no-nucleotide control showing enhancement of binding under all conditions relative to no-nucleotide control (one-way ANOVA; **P < 0.01; ***P < 0.001). No differences existed within H7 or Sta treatments, though binding in the presence of ADP was greater than ATP (one-way ANOVA; ^‡P < 0.05). (n = 6–17 per group)

Fig. 4.

H7 and Sta inhibit CaMKII enzymatic activity within cells. To assess inhibition of CaMKII enzymatic activity within cells, HEK293 cells were cotransfected with CaMKII and GluA1. These cells were incubated with inhibitor and then stimulated with ionomycin (Iono). After these treatments, the cells were harvested with phosphatase inhibitors, and the resultant extracts were analyzed by Western blotting. (A) H7 reduced S831-phosphorylation in a dose-dependent manner, seen comparing stimulated cells in the absence of inhibitor to stimulated cells incubated with 700 μM H7 (one way ANOVA; *P < 0.05) in the graph of Western blot quantification. Below the graph is a representative Western blot of phospho-S831 GluA1 (top) and total GluA1 (bottom). H7 treatment was from 7 to 700 μM; H7 IC₅₀ = 7 μM (n = 4 per group). (B) Sta also reduced S831-phosphorylation in a dose-dependent manner, seen comparing stimulated cells in the absence of inhibitor to stimulated cells incubated with 200 nM H7 (one-way ANOVA; *P < 0.05; **P < 0.01) in the graph of Western blot quantification. Below the graph is a Western blot of phospho-S831 GluA1 (top) and total GluA1 (bottom), run in biologic replicates. Sta treatment was from 2 to 200 nM; Sta IC₅₀ = 20 nM (n = 3 per group).

Fig. 5.

H7 and Sta permit translocation of GFP-CaMKII in heterologous expression systems. (A) Representative time-course of stimulation-induced GFP-CaMKII translocation to GluN2B in HEK293 cells. Ionomycin-stimulation induces distinctive puncta and increased signal intensity in the perinuclear region. (B) Quantitation of the translocation data shows an increase in ionomycin-induced translocation of CaMKII. There were no differences in time courses between control (Ctl) and H7; Sta induced greater translocation at minutes 1 and 2 (two-way repeated measures ANOVA; **P < 0.01). (n = 7–14 per group). (C) Representative images of HEK293 cells fixed without (top) and with (below) ionomycin stimulation. The latter condition displays increased colocalization of GFP-CaMKII (left) and immunostained GluN2B (center), as seen in the merged image (right; GFP-CaMKII shown in green, GluN2B shown in red). (D) Quantitation of colocalization between GFP-CaMKII signal and GluN2B staining shows that stimulation increased colocalization in the control condition (indicating translocation), which was further increased when cells were pretreated with 700 μM H7 or 2 μM Sta (one-way ANOVA; *P < 0.05; ***P < 0.001), (n = 12–100 per group; note that ionomycin stimulation decreased cellular adherence, decreasing the number of cells able to be visualized). (E) Comparison of colocalization after stimulation of CaMKII with either wild-type (WT) or S1303A mutant GluN2B. S1303A displayed increased colocalization with CaMKII relative to WT in the control condition but not after pretreatment with H7 or Sta, which likely prevent S1303 phosphorylation (one-way ANOVA; ***P < 0.001), (n = 12–47 per group). Scale bar = 10 μM.

Fig. 6.

H7 and Sta permit synaptic translocation of CaMKII in primary hippocampal neurons. (A) Representative time-course of stimulation-induced synaptic translocation of GFP-CaMKII. The GFP-CaMKII signal moves from a diffuse pattern throughout the dendrite to concentrated puncta at dendritic spines. (B) The spine-to-shaft ratio of GFP-CaMKII signal at 0 and 240 seconds after glut/gly stimulation shows an increase in signal from 0 to 240 seconds in all conditions and no differences between control (Ctl), H7, and Sta (n = 10 and 11). Statistics relative to the nonstimulated condition within each treatment (two-way ANOVA; *P < 0.05; **P < 0.01; ***P < 0.001). (C) Representative images of neurons fixed with (top) or without (bottom) glut/gly stimulation and stained for CaMKII (left) and the synaptic marker Shank (center); merge (right) shows overlay of CaMKII (green) and Shank (red). (D) Synaptic enrichment of CaMKII increases after stimulation of increasing duration in both control and H7-pretreated neurons. Synaptic CaMKII was greater for H7 than control at 120 seconds; no other differences existed between treatments (one-way ANOVA; *P < 0.05), (n = 23–62 per condition). Scale bar = 10 μM.