The neuronal connexin36 interacts with and is phosphorylated by CaMKII in a way similar to CaMKII interaction with glutamate receptors

Abstract

Electrical synapses can undergo activity-dependent plasticity. The calcium/calmodulin-dependent kinase II (CaMKII) appears to play a critical role in this phenomenon, but the underlying mechanisms of how CaMKII affects the neuronal gap junction protein connexin36 (Cx36) are unknown. Here we demonstrate effective binding of (35)S-labeled CaMKII to 2 juxtamembrane cytoplasmic domains of Cx36 and in vitro phosphorylation of this protein by the kinase. Both domains reveal striking similarities with segments of the regulatory subunit of CaMKII, which include the pseudosubstrate and pseudotarget sites of the kinase. Similar to the NR2B subunit of the NMDA receptor both Cx36 binding sites exhibit phosphorylation-dependent interaction and autonomous activation of CaMKII. CaMKII and Cx36 were shown to be significantly colocalized in the inferior olive, a brainstem nucleus highly enriched in electrical synapses, indicating physical proximity of these proteins. In analogy to the current notion of NR2B interaction with CaMKII, we propose a model that provides a mechanistic framework for CaMKII and Cx36 interaction at electrical synapses.

Fig. 1.

CaMKII binds effectively to cytoplasmic loop (GST-Cx36CL) and carboxyl-terminal (GST-Cx36CT) fusion proteins immobilized on GST agarose beads. (A) Schematic representation of the putative CaMKII binding (CB) and phosphorylation domains (C-Phos) of Cx36 as indicated by in silico screens. (B) Representative autoradiographs and quantitative densitometry of binding of in vitro translated [³⁵S]-CaMKII to the GST-Cx36CL fusion protein. Binding is effective with translated CaMKII protein plus Ca²⁺ (second lane with CaM in brackets) because in vitro translated CaMKII contains sufficient amounts of calmodulin from the erythrocyte lysate as assessed by Western blotting (data not shown). Addition of Ca²⁺/CaMKII does not increase binding significantly. (See the third lane in B labeled with an asterisk. This band was used for quantification of signal intensity set to 100%; see also in C.) Autophosphorylation was achieved by incubation of CaMKII with Ca²⁺/CaM at 30 °C in the presence of ATP. In the control, ATP was added after incubation. Autophosphorylated CaMKII binds more effectively than the unphosphorylated CaMKII form. (C) Binding of [³⁵S]-CaMKII to the carboxyl-terminal Cx36 fusion protein (GST-Cx36CT). Autophosphorylation leads to a 2.5-fold increase of CaMKII binding. (D) Deletion of the putative CaMKII binding domains (Del) of Cx36 leads to a clearly reduced affinity of CaMKII. Quantification of signal intensity was in relation to the respective wild-type constructs (100%, indicated with *). Incubation with GST alone served for control. Note that the autoradiograph shows duplicate lanes from the same experiment.

Fig. 2.

Activated CaMKII phosphorylates Cx36 and exhibits autonomous kinase activity by interacting with binding fragments of Cx36. (A) GST fusion protein of the cytoplasmic loop site (GST-Cx36CL) was in vitro phosphorylated by CaMKII in the presence of Ca²⁺/CaM. Ca²⁺/CaM is sufficient to phosphorylate the cytoplasmic loop fusion protein. Autophosphorylation of CaMKII reveals no significant effect (set to 100% for quantification and labeled with an asterisk). Peptide inhibition using a sequence corresponding to the Cx36 binding site (CLB) inhibits phosphorylation almost completely, whereas peptide CLP corresponding to the phosphorylation domain of Cx36 shows a lower effect. (B) Autophosphorylation of CaMKII is essential for phosphorylation of the Cx36CT fusion protein (Cx36CT-GST). Phosphorylation is reduced by competition with both cognate peptides (CTP and CTB). (C) Point mutations, including (S110A) and (T111A) for the cytoplasmic loop and (S293A) and (S315A) of the carboxyl-terminal fusion proteins, were studied. Densitometric quantification revealed a significant reduction in phosphorylation at the cytoplasmic loop residues after alanine substitution. Note that the carboxyl-terminal GST-Cx36CT revealed a lower band in the autoradiogram, which is considered to represent a degradation product. The specific band is indicated by an arrow. In the carboxyl terminus, mutation of S293 had no effect on the phosphorylation efficacy. S315A mutation severely reduced phosphorylation. (D) Immobilized biotinylated peptides of cytoplasmic binding sites (CLB and CTB) and phosphorylation sites (CLP and CTP) were incubated with CaMKII without Ca²⁺ and calmodulin. Trapped CaMKII was then tested for activity to phosphorylate the CaMKII substrate autocamtide in the presence of [γ-³²P]ATP. Both peptides derived from the Cx36 binding sites exhibited a profound effect on autonomous activity of CaMKII, whereas the peptides covering the phosphorylation sites did not.

Fig. 3.

Sequence similarities between the Cx36 binding domains and the autoinhibitory region of CaMKII. (A) The cytoplasmic loop (CL) binding site of Cx36 reveals similarities to core residues of the pseudotarget site of CaMKII surrounding T(286). (B) The carboxyl-terminal binding site of Cx36 reveals a conserved motif, which is similar to the pseudosubstrate binding site of CaMKII. Notably, both the CT binding site of Cx36 and the pseudosubstrate binding site of CaMKII contain consensus motifs for calmodulin binding, indicated with underlying bars. (C) The CL binding site of Cx36 is also highly similar to the CaMKII binding sequence of the NR2B subunit of the NMDA receptor. Neutral amino acid exchanges are indicated by dots.

Fig. 4.

Cx36 and CaMKII are found to colocalize in immunostained inferior olive. (A) Merged image of anti-Cx36 (green) and CaMKII (red) immunolabeling depicts frequent overlapping localization of both proteins. Single neurons are labeled with white arrows. (B and C) Individual image pairs of CaMKII and Cx36 immunoreactivity. (D) Low magnification of a cresylviolet-stained cryostate section consecutive to the immunolabeled section shown in A–C. Black arrows show the area depicted in A–C. IOA, subnucleus A; IOB, subnucleus B; IOC, subnucleus C; py, pyramidal tract. (Scale bars: 40 μm in C for A–C and 500 μm in D.) Quantification of colocalization and pull-down experiments of CaMKII and Cx36 are indicated in Fig. S3.

Fig. 5.

Model for the interaction of CaMKII with Cx36. The model follows the “gate” concept developed for interaction of CaMKII with the NMDA receptor (see also text). (A) Upon local Ca²⁺ elevation Ca²⁺/calmodulin, either from cytosolic sources or Cx36 bound, binds to the calmodulin binding site of CaMKII that overlaps with its pseudosubstrate site. (B) Binding of Ca²⁺/CaM to CaMKII opens the gate and exposes the target site (T-site) and the substrate site (S-site) of the kinase. Exposure of the T-site allows binding of the cytoplasmic loop binding site (CLB) of Cx36, a process that does not require autophosphorylation of the kinase. (C) Autophosphorylation of CaMKII at T(286) leads to enhanced binding of the carboxyl-terminal binding site (CTB) of Cx36 to the S-site of the activated kinase. Increasing autophosphorylation of CaMKII may also facilitate shifting of Ca/CaM from a Cx36-bound position to kinase binding because of an increase in CaM affinity (trapping phenomenon) of CaMKII.