Activity-Dependent Subcellular Cotrafficking of the Small GTPase Rem2 and Ca2+/CaM-Dependent Protein Kinase IIα

<div><h3>Background</h3><p>Rem2 is a small monomeric GTP-binding protein of the RGK family, whose known functions are modulation of calcium channel currents and alterations of cytoskeletal architecture. Rem2 is the only RGK protein found predominantly in the brain, where it has been linked to synaptic development. We wished to determine the effect of neuronal activity on the subcellular distribution of Rem2 and its interacting partners.</p> <h3>Results</h3><p>We show that Rem2 undergoes activity-and N-Methyl-D-Aspartate Receptor (NMDAR)-dependent translocation in rat hippocampal neurons. This redistribution of Rem2, from a diffuse pattern to one that is highly punctate, is dependent on Ca²⁺ influx, on binding to calmodulin (CaM), and also involves an auto-inhibitory domain within the Rem2 distal C-terminus region. We found that Rem2 can bind to Ca²⁺/CaM-dependent protein kinase IIα (CaMKII) a in Ca²⁺/CaM-dependent manner. Furthermore, our data reveal a spatial and temporal correlation between NMDAR-dependent clustering of Rem2 and CaMKII in neurons, indicating co-assembly and co-trafficking in neurons. Finally, we show that inhibiting CaMKII aggregation in neurons and HEK cells reduces Rem2 clustering, and that Rem2 affects the baseline distribution of CaMKII in HEK cells.</p> <h3>Conclusions</h3><p>Our data suggest a novel function for Rem2 in co-trafficking with CaMKII, and thus potentially expose a role in neuronal plasticity.</p> </div

Possible mechanism underlying Rem2 activity-dependent redistribution.

<p>Top: Representation of the C-terminus of Rem2, with the putative CaM-binding determinant leucine residue highlighted in pink. (A) In the absence of stimulation, Rem2 residues 320–330 (regulatory region) may allosterically inhibit Rem2 association with a putative cytoskeletal regulatory element, and consequently no Rem2 puncta are observed. (B) Upon stimulation, Ca²⁺-CaM may bind the region around residue L317, allowing redistribution to occur by association with the cytoskeleton. (C) The Rem2 mutant 1–320 lacks the regulatory region. This would result in constitutive association with CaM and constitutive puncta. (D) The Rem2 mutant 1–310 lacks the CaM-binding region, and would not redistribute to puncta unless CaMKII is overexpressed. (E) Under basal conditions, Rem2 (blue circles) and CaMKII (red circles) are diffusely distributed in neurons. (F) Upon neuronal stimulation, Ca²⁺ influx through the NMDAR (black brackets) leads to aggregation of Rem2 and CaMKII, possibly at cytoskeletal elements (green lines).</p

Rem2 redistribution is dependent on calcium influx via the activation of NMDA receptors.

<p>(<b>A</b>) Neuronal cultures were stimulated for 60 seconds with either 100 µM glutamate/10 µM glycine (glu/gly and No Ca conditions) or 100 µM NMDA/10 µM glycine. In the No Ca condition, neurons were incubated in EBS without 3 mM CaCl₂, in the presence of 30 nM TTX to reduce spontaneous activity, then stimulated as above. Ratio after/before of Rem2 stimulated with glutamate/glycine, 5.88±0.47, N = 30; with NMDA/glycine, 3.41±0.49, N = 29, one-way ANOVA followed by Bonferroni post-hoc test p<0.005 compared to glutamate/glycine; no calcium + TTX, 1.58±0.28, N = 26, p<0.001 compared to glutamate/glycine. Numbers within the bars denote N, and error bars represent SEM. (<b>B</b>) Redistribution of Rem2 is blocked by MK-801. Neuronal cultures were treated with the NMDAR open-pore blocker MK-801 (62.5 µM) for 20 minutes, and then stimulated for 60 seconds with glutamate/glycine. Controls experienced the same stimulation protocol in the absence of MK-801. This treatment abolished redistribution (ratio after/before of Rem2 alone, 3.17±0.31, N = 50; Rem2+ MK-801, 1.16±0.10, N = 41; t-test p<0.001). (<b>C</b>) Rem2 redistribution is reversed by removing calcium. (Upper) Serial images of individual cells were taken before and after stimulation for 60 seconds with 100 µM glutamate/10 µM glycine. 5 minutes after stimulation, calcium was removed by replacing the bath solution with chelation solution lacking calcium and containing 1 mM EGTA. Images were taken about 5 minutes and 15 minutes after incubating the neurons in chelation solution. (Lower) Quantitation of Rem2 redistribution reversal. Pixel value variance is significantly different after stimulation (882±177 after vs. 164±21 before, one-way repeated measures ANOVA followed by Dunnett’s post-hoc test: N = 27 for each, p<0.001), but goes back to baseline level following calcium chelation (5 min after chelation, 430±130, N = 27; 12 min after chelation, 237±17, N = 9; difference from before stimulation is NS).</p

Rem2 and CaMKII redistribution is co-dependent.

<p>(<b>A</b>) CaMKIIN inhibits redistribution of Rem2 and CaMKII. Rat hippocampal neurons coexpressing <b>(</b>left) GFP-Rem2 and (right) mRuby-CaMKII without (left panels) and with (right panels) HA-CaMKIIN. Cells were imaged before stimulation (top panels), then stimulated with 100 µM glutamate/10 µM glycine and imaged again (middle panels). Neurons were fixed and probed for HA to detect CaMKIIN (bottom panel). The scale bar represents 20 µm. (<b>B</b>) CaMKIIN reduces redistribution of coexpressed CaMKII and Rem2. Clustering factor determined as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041185#pone.0041185-Hudmon1" target="_blank">[2]</a>. Mean ± SEM clustering factor for CaMKII without CaMKIIN: before stimulation, 0.004±0.002; after, 0.021±0.004; with CaMKIIN: before, 0.002±0.0002; after, 0.009±0.002. Rem2 clustering without CaMKIIN: before stimulation, 0.001±0.0002; after, 0.009±0.002; with CaMKIIN: before, 0.001±0.0002; after, 0.003±0.0006. Data is from 4 separate experiments with a total of N = 27 neurons/condition. Error bars are ± SEM; asterisks represent p<0.05 (Kruskal-Wallis test followed by Tukey’s post hoc test). (<b>C</b>) Rem2 and CaMKII redistributions overlap temporally. A timecourse of aggregation of Rem2 and CaMKII shows little clustering before stimulation with glutamate/glycine, and Rem2 and CaMKII clustering occurs at similar rates. N = 27 neurons/condition. (<b>D</b>) CaMKIIN does not interact with Rem2, and expression of CaMKIIN does not interfere with Rem2-CaMKII interaction. HEK cells were transfected with the indicated plasmids. Cell lysates were subjected to co-precipitation assays using a mix of proteinA/G beads and anti-HA,-myc or-GFP. Eluates were separated on SDS-PAGE and probed with anti-HA (top) and anti-myc (bottom). (<b>E</b>) CaMKII aggregates in HEK cells following a pH drop/high Ca protocol <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041185#pone.0041185-Hudmon1" target="_blank">[2]</a> (mean clustering factor before simulation 0.030±0.002; after 0.166±0.013). Aggregation is inhibited by CaMKIIN (before stimulation, 0.024±0.002; after, 0.068±0.010) but unaffected by coexpression of Rem2 (before, 0.031±0.003; after, 0.152±0.009). CaMKIIN inhibition is also insensitive to Rem2 coexpression (before, 0.031±0.003; after, 0.071±0.006). N = 20 cells and p<0.05 for all before-after pairs. (<b>F</b>) Rem2 does not aggregate using the same protocol (without CaMKIIN: before stim, 0.037±0.004; after, 0.032±0.004; with CaMKIIN: before, 0.018±0.002; after, 0.022±0.003), unless CaMKII is also present (without CaMKIIN: before stim, 0.012±0.002; after, 0.073±0.008). CaMKII-induced Rem2 aggregation is also inhibited by CaMKIIN (before, 0.008±0.001; after, 0.021±0.004). Error bars are ± SEM; asterisks represent p<0.05 (Kruskal-Wallis test followed by Tukey’s post hoc test).</p

Calmodulin binding is necessary for Rem2 redistribution.

<p>(<b>A</b>) Predicted calmodulin binding sites in Rem2. An online predictor of calmodulin binding sites (<a href="http://calcium.uhnres.utoronto.ca/" target="_blank">http://calcium.uhnres.utoronto.ca/</a>) suggests that Rem2 has two predominant sites, a higher affinity one close to the C-terminus and a lower affinity one further upstream. The colored numbers under the residues indicate how strongly a stretch of residues is predicted to bind CaM: grey 0 indicates no binding predicted, while red 9 indicates a likely CaM binding site. (<b>B</b>) Rem2 interacts with CaM in the presence of calcium. Untransfected HEK cells (ctrl) and HEK cells expressing full-length HA-tagged Rem2 (Rem2) were lysed, the lysates (<b>input</b>) cleared and passed over calmodulin-conjugated agarose beads in the presence of 3 mM calcium (<b>+calcium</b>) or 2 mM EGTA/2 mM EDTA (<b>–calcium</b>). Beta-actin was used as a loading control (<b>actin</b>). The eluate was subjected to Western blot analysis using HA antibody (top three panels) or actin antibody (bottom panel). (<b>C</b>) The point mutation L317G does not affect the association of full-length Rem2 with CaM. HEK cell lysates containing wild-type HA-Rem2 or the point mutation L317G were used for a pulldown assay with CaM-sepharose beads. Precleared lysates (<b>input</b>) and the eluates pulled down in buffer containing 2 mM EDTA/2 mM EGTA in place of calcium (<b>−calcium</b>) or 3 mM CaCl₂ (<b>+calcium</b>) were separated by SDS-PAGE and transferred to nitrocellulose membranes, then probed with anti-HA antibody. Control cells are untransfected. Note less signal in the presence of EDTA/EGTA. (<b>D</b>) The same mutation abolishes interaction between CaM and the Rem2 C-terminal extension. Upper panel shows cleared lysates (input) from HEK cells expressing Rem2 (284–341) and Rem2 (284–341) L317G. Lower panel (+ calcium) shows Rem2 protein pulled down with CaM-sepharose in the presence of 2 mM calcium. (<b>E</b>) Rem2-calmodulin interaction is necessary for Rem2 redistribution. Neurons expressing either wild-type or Rem2 (L317G) protein were imaged before and after stimulation with 100 µM glutamate/10 µM glycine. Ratio after/before stimulation of WT Rem2, 1.59±0.07, N = 20; Rem2 (L317G), 1.19±0.08, N = 29; t-test p = 0.001. (<b>F</b>) Pixel intensity variances are shown before (dark bars) and after (light bars) stimulation. The variance of full length Rem2 changes upon stimulation (before, 858±53; after, 1429 96, N = 29; paired t-test p<0.001). The truncated mutant Rem2 (1–320) shows high variance before and after stimulation (before, 2035±165; after, 1871±161, N = 39), indicating constitutive puncta, while the Rem2 L317G and the 1–320 truncation carrying the L317G mutation show low variances (Rem2 L317G before, 784±87; after, 918±117, N = 29; Rem2 (1–320) L317G before, 392±101; after, 590±283, N = 10), indicating no constitutive puncta and little redistribution on stimulation. One-way ANOVA + Bonferroni post-hoc test comparing variances before stimulation shows significant differences between full length Rem2 and Rem2 (1–320) (p<0.0001), and no difference between full length and Rem2 L317G or Rem2 (1–320) L317G (p = 1 and 0.42, respectively). Rem2 (1–320) L317G data come from 10 cells from a single transfection. (<b>G</b>) Coexpression of a CaM-binding IQ motif reduces Rem2 redistribution. Neurons cotransfected with YFP-Rem2 and CFP-IQ (TVGKFYATFLIQEYFRKFKKRKEQ) were imaged before and after stimulation with 25 µM glutamate/2.5 µM glycine. Ratio after/before stimulation of Rem2 alone, 3.12±0.22, N = 66; Rem2+ IQ motif, 2.07±0.23, N = 54; t-test p = 0.0013.</p

Rem2 interacts and redistributes with CaMKII.

<p>(<b>A</b>) Calcium-calmodulin is essential for Rem2-CaMKII association. HEK cells were transfected with the indicated GFP-CaMKII, CaM, the calcium-insensitive mutant CaM1234 or HA-Rem2 constructs. Cell lysates were coprecipitated with anti-GFP antibody coupled to proteinA/G beads. Coprecipitated proteins were eluted from beads in sample buffer and separated using 10%/16% tricine SDS-PAGE followed by Western blotting with anti-HA antibody. Precleared lysates (<b>left</b>) and coprecipitated proteins (<b>right</b>) are shown. CaMKII binds only to full length Rem2 (lane 4) and not to a C-terminal fragment of Rem2 (lane 5). CaMKII also shows no binding when Rem2 and CaMKII are coexpressed with CaM1234 (lane 6). (<b>B</b>) Fluorescent signals of GFP-Rem2 and mCherry-CaMKII redistribute together. Rem2 and CaMKII cotransfected neurons were fixed following no stimulation or 60 seconds of 100 µM glutamate/10 µM glycine stimulation and 5 minutes of recovery. Left subpanel of each panel shows a cell coexpressing CaMKII and Rem2. Right subpanel shows a higher resolution of the subcellular distribution of GFP-Rem2 (top), mCherry-CaMKII (center) and an overlay (bottom). Scale bars are 5 µm. (<b>C</b>) Rem2 and CaMKII are colocalized before (dark bars) and after (light bars) stimulation. Intensity correlation of GFP and mCherry signal is given for neurons coexpressing mCherry-CaMKII and GFP-Rem2 wild-type or C-terminal truncations. The higher the intensity correlation quotient (ICQ), the greater the covariance of the two colors across the cell, i.e. higher colocalization within the cell. Correlation of GFP-WT Rem2 and unconjugated mCherry is given as control. ICQ of GFP-WT-Rem2+ mCherry-CaMKII before stimulation, 0.312±0.015; GFP-WT-Rem2+ mCherry alone, 0.137±0.018; two-way ANOVA, p<0.001. Only the constitutively punctate 1–320 Rem2 mutant shows a different distribution from CaMKII before stimulation, although it correlates with CaMKII after. Before stimulation, 0.117±0.026; after stimulation, 0.258±0.030; t-test, p = 0.004. (<b>D</b>) Rem2 alters basal CaMKII distribution. HEK cells expressing GFP-CaMKII alone (left panels) or GFP-CaMKII coexpressed with mCherry-Rem2 (right panels) were fixed and imaged using confocal microscopy. Note the diffuse distribution of GFP-CaMKII when Rem2 is absent. (<b>E</b>) Rem2 alters CaMKII distribution. Mean pixel intensity variance in fixed HEK cells of GFP-CaMKII (alone, 51106±9158, N = 69; with Rem2, 94591±17452, N = 56), mCherry-Rem2 (alone, 90085±16053, N = 60; with CaMKII 74948±10689, N = 60) or GFP fluorescence (alone, 17232±2775, N = 60; with Rem2, 19442±4145, N = 57).</p

The Rem2 C-terminus directs redistribution.

<p>(<b>A</b>) Schematic of the Rem2 protein. The last 30 residues of the C-terminal extension contain a previously identified PIP lipid binding domain as well as a calmodulin binding site. Small triangles in the N-and C-termini represent 14-3-3 binding sites. We created truncated Rem2 proteins ending at residues 310 and 320 to determine if these interaction sites are relevant for Rem2 redistribution. (<b>B</b>) Effect of Rem2 truncations on redistribution. (<b>Left panel</b>) Full-length Rem2 (top) redistributed into puncta on glutamate/glycine stimulation. 1–310 Rem2 (center) showed no redistribution upon stimulation, while 1–320 Rem2 (bottom) formed puncta constitutively. Scale bars indicate 5 µm. (<b>Right panel</b>) WT Rem2 shows a significant difference in pixel intensity variance after stimulation (normalized mean (± SEM) pixel value variance before stimulation, 1.00±0,08; after, 1.74±0.17; paired t-test p<0.001). Additionally, 1–320 Rem2 distribution is different from WT before but not after stimulation (one-way ANOVA followed by Bonferroni’s test; before, p<0.001; after, p = 0.19). Pixel intensity variances were normalized to the variance of full-length Rem2 before stimulation.</p