Neuronal L-Type Calcium Channel Signaling to the Nucleus Requires a Novel CaMKIIα-Shank3 Interaction

Abstract

The activation of neuronal plasma membrane Ca²⁺ channels stimulates many intracellular responses. Scaffolding proteins can preferentially couple specific Ca²⁺ channels to distinct downstream outputs, such as increased gene expression, but the molecular mechanisms that underlie the exquisite specificity of these signaling pathways are incompletely understood. Here, we show that complexes containing CaMKII and Shank3, a postsynaptic scaffolding protein known to interact with L-type calcium channels (LTCCs), can be specifically coimmunoprecipitated from mouse forebrain extracts. Activated purified CaMKIIα also directly binds Shank3 between residues 829 and 1130. Mutation of Shank3 residues ⁹⁴⁹Arg-Arg-Lys⁹⁵¹ to three alanines disrupts CaMKII binding in vitro and CaMKII association with Shank3 in heterologous cells. Our shRNA/rescue studies revealed that Shank3 binding to both CaMKII and LTCCs is important for increased phosphorylation of the nuclear CREB transcription factor and expression of c-Fos induced by depolarization of cultured hippocampal neurons. Thus, this novel CaMKII-Shank3 interaction is essential for the initiation of a specific long-range signal from LTCCs in the plasma membrane to the nucleus that is required for activity-dependent changes in neuronal gene expression during learning and memory.SIGNIFICANCE STATEMENT Precise neuronal expression of genes is essential for normal brain function. Proteins involved in signaling pathways that underlie activity-dependent gene expression, such as CaMKII, Shank3, and L-type calcium channels, are often mutated in multiple neuropsychiatric disorders. Shank3 and CaMKII were previously shown to bind L-type calcium channels, and we show here that Shank3 also binds to CaMKII. Our data show that each of these interactions is required for depolarization-induced phosphorylation of the CREB nuclear transcription factor, which stimulates the expression of c-Fos, a neuronal immediate early gene with key roles in synaptic plasticity, brain development, and behavior.

Keywords: CaMKII; calcium; ion channel; scaffolding protein; transcriptional regulation.

Figure 1.

Reciprocal coimmunoprecipitation of Shank3 and CaMKIIα from mouse forebrain extracts. A, Whole forebrain lysates (Input), cytosolic (S1), membrane-associated (S2), and synaptic (P2) subcellular fractions from WT, CaMKIIα-KO, and CaMKIIα^T286A mice were immunoblotted for localization of Shank3 (Cell Signaling Technology antibody), PSD-95, and CaMKIIα. The Shank3 antibody detected two bands (open and solid arrowheads) that are primarily localized in P2 fractions, which were also enriched for CaMKIIα. B, The upper and lower Shank3 bands in whole forebrain lysates from WT and CaMKIIα-KO mice (left) or WT and CaMKIIα^T286A mice (right) were quantified. Signals were corrected for protein loading based on Ponceau-stained membranes and normalized to the levels of the upper Shank3 band in WT samples. C, Synaptic (P2) fractions from WT or CaMKIIα-KO mouse forebrains were immunoprecipitated using control IgG or CaMKIIα-specific antibodies, and immunoblotted using Shank3 (Cell Signaling Technology antibody) and pan-CaMKII antibodies. Arrows indicate CaMKIIα and CaMKIIβ bands. CaMKIIα and coimmunoprecipitated Shank3 were not detected in samples isolated from CaMKIIα-KO mice. D, Synaptic (P2) fractions from WT or CaMKIIα^T286A mouse forebrains were immunoprecipitated using control IgG or Shank3 (Bethyl) antibodies, and immunoblotted using Shank3 (Cell Signaling Technology) and CaMKIIα antibodies. The levels of coprecipitated CaMKIIα from CaMKIIα^T286A mice were significantly reduced (93 ± 4% reduction in lane 6 compared with lane 5, n = 3. p < 0.001, one-sample Student's t test with equal variance compared with theoretical value of 100). All immunoblots are representative of ≥3 biological replicates.

Figure 2.

T286-phosphorylated CaMKIIα specifically binds to Shank3 (829–1130). A, Domain structure of full-length Shank3 and the six GST-Shank3 fusion proteins used in these studies that span the entire Shank3 protein. Gray boxes represent canonical Shank3 domains. Residue numbers are listed in parentheses. ANK, Ankyrin repeats domain; SH3, Src homology 3 domain; PDZ, PSD95/Dlg1/zo-1 domain; PRR, proline-rich region containing binding sites for Homer and Cortactin; SAM, sterile α motif involved in multimerization of Shank3. B, Glutathione agarose cosedimentation assay shows that preactivated (Thr286-autophosphorylated) CaMKIIα specifically binds to GST-Shank3 #4 (829–1130) and positive control GST-GluN2B (1260–1309). *Full-length GST fusion proteins on the GST immunoblot. C, Glutathione agarose cosedimentation assay shows that, in the absence of Ca²⁺/CaM binding or Thr286 autophosphorylation, CaMKIIα (Basal) does not bind to GST-Shank3 #4; in vitro binding of CaMKIIα is partially supported by Ca²⁺/CaM binding (Ca/CaM), and maximally enhanced by pT286 autophosphorylation. Bar graph represents levels of each form of CaMKII bound to GST-Shank3 #4 (or the GST negative control) (mean ± SEM) relative to levels of pT286-CaMKIIα binding to GST-GluN2B. Immunoblots are representative of three or four biological replicates.

Figure 3.

Characterization of the CaMKII binding motif in Shank3. A, Top, Diagram of 3 truncations used to map the CaMKII interaction site within GST-Shank3 #4 (829–1130). Bottom, Sequence alignment of human Shank3 residues 941–959 with the corresponding Shank3 residues in other species and the CaMKII binding domain in the N-terminal domain of the Rattus norvegicus Ca_V1.3 α1 subunit (Wang et al., 2017) and the C-terminal tail of the Rattus norvegicus mGlu5 (Marks et al., 2018). Black represents conserved residues. Gray represents dissimilar residues. Red box represents the conserved tribasic residue motif. B, Glutathione agarose cosedimentation assay comparing binding of activated CaMKIIα to GST-Shank3 #4 (829–1130) and 3 nonoverlapping fragments (4a, 4b, and 4c). *Full-length GST fusion proteins. GST-Shank3 #4 (829–1130) and #4b (931–1014) bind similar amounts of pT286-autophosphorylated CaMKIIα, but there is no detectable binding to the other Shank3 fragments. C, Mutation of amino acids ⁹⁴⁹RRK⁹⁵¹ to AAA in GST-Shank3 #4 (829–1130) blocks CaMKIIα binding in glutathione agarose cosedimentation assay (98 ± 4% reduced compared with WT, n = 3, p < 0.001, one-sample Student's t test with equal variance compared with theoretical value of 100). All immunoblots are representative of three biological replicates.

Figure 4.

A Shank3 ⁹⁴⁹RRK⁹⁵¹ to AAA mutation disrupts association with CaMKIIα but does not with the Ca_V1.3 CTD. A, Soluble fractions of HEK293T cells expressing CaMKIIα with GFP or GFP-Shank3 (WT or ⁹⁴⁹RRK⁹⁵¹ to AAA mutant) were immunoprecipitated using a GFP antibody. Coprecipitation of CaMKIIα with GFP-Shank3-AAA is significantly reduced by 95 ± 10% compared with GFP-Shank3-WT. ***p < 0.001 (one-sample Student's t test with equal variance compared with a theoretical value of 100). B, Soluble fractions of HEK293T cells expressing GFP or GFP-Shank3 (WT or AAA) with the HA-tagged CTD of the Ca_V1.3 α1 subunit (HA-Ca_V1.3-CTD) were immunoprecipitated as in A. The AAA mutation has no significant effect on the coprecipitation of HA-Ca_V1.3-CTD (p = 0.84). All immunoblots are representative of four biological replicates. Error bars indicate the mean ± SEM.

Figure 5.

Shank3 ⁹⁴⁹RRK⁹⁵¹ to AAA mutation disrupts colocalization of activated CaMKIIα. A, Immunoblots of undifferentiated, differentiated, and transfected/differentiated STHdh^+/+ cells, with a WT mouse forebrain lysate as positive control. Shank3 and CaMKIIα are not expressed in nontransfected STHdh^+/+ cells, and transfected mApple-tagged CaMKIIα (mAp-CaMKIIα) is T286-phosphorylated. B, Representative images of differentiated STHdh^+/+ cells expressing GFP-Shank3-WT with mAp-CaMKIIα-WT (left), mAp-CaMKIIα-T286D (middle), or mAp-CaMKIIα-T286A (right). Inset, Regions (dashed line box) of the processes containing punctate GFP signals (arrowheads) that overlap with mApple signal from CaMKIIα-WT and -T286D, but not -T286A. C, Intensity correlation analysis quantifying the colocalization of GFP and mAp signals in transfected and differentiated STHdh^+/+ cells in B. Each data point represents an ICQ value from a single cell, with 7–12 cells analyzed from each of three independent cultures/transfections. ICQ values for GFP-Shank3-WT and either mAp-CaMKIIα-WT (mean ± SEM: 0.29 ± 0.02) or mAp-CaMKIIα-T286D (0.36 ± 0.02) were significantly more colocalized compared with mAp-CaMKIIα-T286A (0.17 ± 0.02) (one-way ANOVA, F_(2,71) = 21.35, p < 0.0001). Tukey's post hoc test: ***p < 0.001, ****p < 0.0001. D, Representative images of differentiated STHdh^+/+ expressing GFP-Shank3-WT or GFP-Shank3-AAA with soluble mAp or mAp-CaMKIIα-WT. Inset, Expanded regions of the processes, as in B. E, Intensity correlation analysis quantifying the colocalization of GFP and mAp signals in transfected and differentiated STHdh^+/+ cells in C. GFP-Shank3-WT and mAp-CaMKIIα-WT (0.31 ± 0.02) are significantly more colocalized than GFP-Shank3-WT and mAp (0.07 ± 0.01) or GFP-Shank3-AAA and mAp-CaMKIIα (0.09 ± 0.02) (one-way ANOVA, F_(2,70) = 39.55, p < 0.0001). Tukey's post hoc test: ****p < 0.0001. Scale bars: B, D, 2.5 μm.

Figure 6.

Effects of Shank3 overexpression on LTCC signaling to the nucleus. A, Shank3 was immunoprecipitated from soluble fractions of HEK293T cells expressing HA-Ca_V1.3-CTD with mAp-Shank3^R-WT or mAp-Shank3^R-ΔPDZ. Immunoblots (representative of three biological replicates) demonstrate that HA-Ca_V1.3-CTD coimmunoprecipitates with mAp-Shank3^R-WT, but not with mAp-Shank3^R-ΔPDZ. B, Schematic of experimental protocols. Primary hippocampal neurons were transfected (see below) and then incubated to stimulate LTCC signaling to the nucleus (see Materials and Methods). Neurons were either fixed and stained using DAPI and pSer133-CREB antibodies after a 90 s depolarization (top: for C), or incubated for an additional 3 h in conditioned media before fixation and staining with DAPI and c-Fos antibodies (bottom: for D). C, Overexpression of mAp-Shank3-WT, but not mAp-Shank3-AAA or mAp-Shank3-ΔPDZ, increases the levels of pCREB staining relative to nontransfected neurons under basal and depolarized conditions (two-way ANOVA with 2 factors [Mutant, Stimulation]: Mutant, F_(3,179) = 17.86, p < 0.0001; Stimulation, F_(1,179) = 1108, p < 0.0001; Interaction, F_(3,179) = 4.442, p < 0.01). Dunnett's multiple-comparisons test: **p < 0.01, ****p < 0.0001. D, The expression of c-Fos is not affected by overexpression of mAp-Shank3-WT, mAp-Shank3-AAA, or mAp-Shank3-ΔPDZ (two-way ANOVA with 2 factors [Mutation, Stimulation]: Mutation, F_(3,166) = 2.152; Stimulation, F_(1,166) = 830.1, p < 0.0001; Interaction, F_(3,166) = 0.3284, Dunnett's multiple-comparisons test). Error bars indicate mean ± SEM. Superimposed data points indicate values from single cells accumulated from three to five independent neuronal cultures/transfections. Images below the bar graphs are of representative nuclei for each condition. Scale bars, 5 μm.

Figure 7.

Shank3 knockdown disrupts pCREB signaling and c-Fos expression. A, Validation of Shank3 shRNA and mApple-Shank3 shRNA-resistant (mAp-Shank3^R) expression vectors. Expression of shRNA, mAp-Shank3, and mAp-Shank3^R in HEK293T cells. Lysates of cells expressing (as indicated above) a control shRNA or Shank3 shRNA, along with mAp-Shank3 constructs with the WT shRNA target sequences (mAp-Shank3-WT and mAp-Shank3-AAA) or contain “silent” mutations that confer shRNA resistance (mAp-Shank3^R constructs) were immunoblotted for Shank3 (NeuroMab antibody). B, DIV13 primary hippocampal neurons transfected at DIV10 with Shank3 shRNA (GFP⁺) and stained for Shank3 (magenta; CST antibody) and CaMKIIα (white; a marker of excitatory neurons). Neurons expressing the Shank3 shRNA contain substantially reduced levels of Shank3 (reduced by 91 ± 2% relative to nearby nontransfected excitatory neurons). ****p < 0.0001 (one-sample unpaired Student's t test with equal variance compared with a theoretical value of 100). Error bars indicate mean ± SEM. Each data point represents a single cell accumulated from three independent neuronal cultures/transfections. Scale bars, 20 μm. C, Shank3 knockdown has little effect on global Ca²⁺ influx. Left, fura-2-loaded hippocampal neurons transfected with control shRNA (nRNA) or Shank3 shRNA were equilibrated with Tyrode's solution containing 5 mm KCl for 3–5 min and switched (black arrow) to Tyrode's solution containing 40 mm KCl for 90 s. The graph plots mean ± SEM ΔF/F0 values for the last 30 s of the equilibration period and during depolarization from four independent experiments (12–90 cells per replicate). The data were analyzed using a two-way repeated-measures ANOVA: Factor 1 (time), F_{(1.342,8.049)} = 32.63, p = 0.0003; Factor 2 (control/shRNA), F_(1,6) = 1.703, p = 0.2398; Interaction, F_(35,210) = 0.8281, p = 0.7423. Right, Comparison of average areas under the curve from each independent experiment revealed no statistically significant difference (n = 4, p = 0.13; paired Student's t test with equal variance). D, The robust increase in pCREB levels following a brief depolarization (as in Fig. 6A) is significantly reduced in cells expressing the Shank3 shRNA (red bar), but not control shRNA (gray bar) (5K vs 40K: unpaired Student's t test with equal variance, ****p < 0.0001; 40K stimulations: one-way ANOVA, F_(2,98) = 38.17, p < 0.0001; Tukey's post hoc test, ****p < 0.0001). E, Similarly, the robust increase in c-Fos expression 3 h after the brief depolarization is significantly reduced in cells expressing Shank3 shRNA (red bar), but not control shRNA (gray bar) (5K vs 40K: unpaired Student's t test with equal variance, ****p < 0.0001; 40K stimulations: one-way ANOVA, F_(2,90) = 9.990, p < 0.001, Tukey's post hoc test, ***p < 0.001). Error bars indicate mean ± SEM. Each data point represents a single cell accumulated from three independent neuronal cultures/transfections. Images below the bar graphs are of representative nuclei for each condition. Scale bars, 5 μm.

Figure 8.

Rescue of pCREB signaling and c-Fos expression after Shank3 shRNA knockdown. The Shank3 shRNA construct was transfected alone or with shRNA-resistant mAp-Shank3^R-WT (green bar), mAp-Shank3^R-AAA (blue bar), or mAp-Shank3^R-ΔPDZ (purple bar). Neurons were depolarized for 90 s, and the levels of pCREB (A) and c-Fos (B) were determined (see Materials and Methods) in transfected (colored bars) and nearby nontransfected (black bars) neurons. A, The expression of mAp-Shank3^R-WT, but not mAp-Shank3^R-AAA or mAp-Shank3^R-ΔPDZ, rescued signaling to pCREB relative to shRNA alone. In addition, neurons expressing Shank3-WT had significantly higher pCREB signal relative to nearby, nontransfected neurons (two-way ANOVA with 2 factors [Mutant, Transfection]: Mutant, F_(3,156) = 10.14, p < 0.0001; Transfection, F_(1,156) = 22.70, p < 0.0001; Interaction, F_(3,156) = 12.29, p < 0.0001, comparison between mutants: Dunnett's multiple-comparisons test, ****p < 0.0001; comparison between transfected/nontransfected cells: Sidak's multiple-comparisons test, *p < 0.05). B, The expression of mAp-Shank3^R-WT, but not mAp-Shank3^R-AAA or mAp-Shank3^R-ΔPDZ, rescued signaling to increase c-Fos expression relative to shRNA alone (two-way ANOVA with 2 factors ([Mutant, Transfection]: Mutant, F_(3,150) = 13.80, p < 0.0001; Transfection, F_(1,150) = 26.52, p < 0.0001; Interaction, F_(3,150) = 8.149, p < 0.0001, comparison between mutants: Dunnett's multiple-comparisons test, ****p < 0.0001; comparison between transfected/nontransfected cells: Sidak's multiple-comparisons test). Error bars indicate mean ± SEM. Each data point represents a single cell, accumulated from four or five independent neuronal cultures/transfections. Images below the bar graphs are of representative nuclei for each condition. Scale bars, 5 μm.