CaMKII Binding to GluN2B Is Differentially Affected by Macromolecular Crowding Reagents

<div><p>Binding of the Ca²⁺/calmodulin(CaM)-dependent protein kinase II (CaMKII) to the NMDA-type glutamate receptor (NMDAR) subunit GluN2B controls long-term potentiation (LTP), a form of synaptic plasticity thought to underlie learning and memory. Regulation of this interaction is well-studied biochemically, but not under conditions that mimic the macromolecular crowding found within cells. Notably, previous molecular crowding experiments with lysozyme indicated an effect on the CaMKII holoenzyme conformation. Here, we found that the effect of molecular crowding on Ca²⁺/CaM-induced CaMKII binding to immobilized GluN2B <i>in vitro</i> depended on the specific crowding reagent. While binding was reduced by lysozyme, it was enhanced by BSA. The ATP content in the BSA preparation caused CaMKII autophosphorylation at T286 during the binding reaction; however, enhanced binding was also observed when autophosphorylation was blocked. Importantly, the positive regulation by nucleotide and BSA (as well as other macromolecular crowding reagents) did not alleviate the requirement for CaMKII stimulation to induce GluN2B binding. The differential effect of lysozyme (14 kDa) and BSA (66 kDa) was not due to size difference, as both dextran-10 and dextran-70 enhanced binding. By contrast, crowding with immunoglobulin G (IgG) reduced binding. Notably, lysozyme and IgG but not BSA directly bound to Ca²⁺/CaM in an overlay assay, suggesting a competition of lysozyme and IgG with the Ca²⁺/CaM-stimulus that induces CaMKII/GluN2B binding. However, lysozyme negatively regulated binding even when it was instead induced by CaMKII T286 phosphorylation. Alternative modes of competition would be with CaMKII or GluN2B, and the negative effects of lysozyme and IgG indeed also correlated with specific or non-specific binding to the immobilized GluN2B. Thus, the effect of any specific crowding reagent can differ, depending on its additional direct effects on CaMKII/GluN2B binding. However, the results of this study also indicate that, in principle, macromolecular crowding enhances CaMKII binding to GluN2B.</p></div

Ca²⁺/CaM stimulation is still required for CaMKII to GluN2B binding.

<p><i>A</i>, CaMKIIα (40 nM subunits) was incubated with immuno-immobilized GST-GluN2B-C as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096522#pone-0096522-g001" target="_blank">Figure 1A</a>, but in the presence or absence of Ca²⁺/CaM (1 mM/1 µM), ADP (100 µM), or BSA (100 mg/ml). Bound CaMKII was eluted and detected by Western-analysis. BSA and ADP alone were insufficient to induce CaMKII to GluN2B binding. GST-GluN2B detection is shown as a loading control. Representative images are from the same experiment and Western-blots. <i>B</i>, CaMKIIα (40 nM subunits) was incubated with immuno-immobilized GST-GluN2B-C as in panel A. Different crowding agents, BSA, dextran-10 (DEX10) and dextran-70 (DEX70) (all at 100 mg/ml) in the presence of ADP (100 µM) were not sufficient to induce detectable CaMKII binding to GluN2B without CaMKII stimulation.</p

Induction of binding by T286 phosphorylation, instead of Ca²⁺/CaM.

<p>CaMKIIα was pre-incubated with ATP and Ca²⁺/CaM to induce autophosphorylation at T286; then, Ca²⁺ was chelated with EGTA and further phosphorylation was inhibited by staurosporine. Phospho-T286 CaMKIIα (40 nM subunits) was bound to GST-GluN2B as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096522#pone-0096522-g001" target="_blank">Figure 1A</a>, but in the presence of 1.5 mM EGTA, 2 µM staurosporine, and without addition of Ca²⁺/CaM or nucleotide. Lysozyme (100 mg/ml) decreased CaMKII binding to GluN2B. T286-phosphorylation was verified using a phospho-T286-specific antibody. Phospho-T286 immunoblots are from the same experiment run on a separate gel to avoid reprobing blots at the same molecular weight. Dotted lines represent IDVs from CaMKII/GluN2B binding when instead stimulated with Ca²⁺/CaM and nucleotide (from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096522#pone-0096522-g001" target="_blank">Figure 1A</a>). n = 6 or 7 per group; **: p<0.01, difference from control, one-way ANOVA followed by Tukey's HSD. #: no difference compared to the same binding condition, but with binding induced by Ca²⁺/CaM (compare Fig. 1A). Bar graphs indicate mean ± s.e.m, and GST-GluN2B detection is shown as a loading control.</p

Two differently sized dextrans both enhance CaMKII binding to GluN2B, indicating that the differential effects of BSA and lysozyme were not due to size differences.

<p>Ca²⁺/CaM-stimulated CaMKII binding to GluN2B was tested as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096522#pone-0096522-g001" target="_blank">Figure 1A</a>. Molecular crowding with dextran-10 (DEX10) or dextran-70 (DEX 70), both at 100 mg/ml, both increased CaMKII binding to GluN2B to an equal extent. n = 6–7; *: p<0.05, **: p<0.01 in one-way ANOVA followed by Tukey's HSD; n.s.: no significant difference between the two dextrans. Bar graphs indicate mean ± s.e.m, and GST-GluN2B detection is shown as a loading control.</p

Lysozyme but not BSA binds to the GST immobilization scaffold.

<p>100 mg/ml Lysozyme (6.8 mM) or BSA (1.5 mM) were tested for binding to immuno-immobilized GST-GluN2B-C under the same conditions as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096522#pone-0096522-g001" target="_blank">Figure 1A</a>. Samples were eluted and subjected to SDS-PAGE, after which the gel was fixed and stained for total protein (silver stain). Binding was detected for lysozyme but not BSA. Lysozyme binding was detected also to immobilized GST (without GluN2B fusion) and to empty wells, indicating non-specific binding to the immobilization scaffold.</p

IgG binds to GluN2B and decreases CaMKII binding to GluN2B.

<p><i>A</i>, Ca²⁺/CaM-stimulated CaMKII binding to GluN2B was tested as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096522#pone-0096522-g001" target="_blank">Figure 1A</a>. Molecular crowding with PVP-40 (100 mg/ml) had no effect on CaMKII binding to GluN2B, while rabbit immunoglobulin (IgG) (100 mg/ml) decreased binding. n = 7 (PVP-40) or 4 (IgG); *: p<0.05 in Kruskal Wallis test followed by Dunn-Bonferroni multiple comparison. Bar graphs indicate mean ± s.e.m, and GST-GluN2B detection is shown as a loading control. The immuno-detection examples are cropped from the same exposure of the same blots. <i>B</i>, 100 mg/ml IgG or Lysozyme were tested for binding to immuno-immobilized GST-GluN2B-C under the same conditions as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096522#pone-0096522-g001" target="_blank">Figure 1A</a>. Samples were eluted and subjected to SDS-PAGE, after which the gel was fixed and stained for total protein (silver stain). Binding was detected for both IgG and lysozyme.</p

Lysozyme but not BSA binds Ca²⁺/CaM.

<p><i>A</i>, The chicken lysozyme amino acid sequence with predicted CaM-binding sites marked. Prediction utilized the “Calmodulin Target Database” (<a href="http://calcium.uhnres.utoronto.ca/ctdb/ctdb/home.html" target="_blank">http://calcium.uhnres.utoronto.ca/ctdb/ctdb/home.html</a>). Numbers from 1 to 9 indicate increasing likelihood of predicted CaM binding. <i>B</i>, Lysozyme and BSA (0, 1, 2, 4, and 8 µg) were subjected to SDS-PAGE and transferred to a PVDF membrane. The membrane was first stained for total protein (ponceau, bottom panel), then incubated with biotin-labeled CaM in presence of CaCl₂. Bound CaM was detected by chemi-luminescence (top panel). Ca²⁺/CaM bound Lysozyme, but not BSA.</p