Phosphorylation of Connexin36 near the C-terminus switches binding affinities for PDZ-domain and 14–3–3 proteins in vitro

Abstract

Connexin36 (Cx36) is the most abundant connexin in central nervous system neurons. It forms gap junction channels that act as electrical synapses. Similar to chemical synapses, Cx36-containing gap junctions undergo activity-dependent plasticity and complex regulation. Cx36 gap junctions represent multimolecular complexes and contain cytoskeletal, regulatory and scaffolding proteins, which regulate channel conductance, assembly and turnover. The amino acid sequence of mammalian Cx36 harbors a phosphorylation site for the Ca2+/calmodulin-dependent kinase II at serine 315. This regulatory site is homologous to the serine 298 in perch Cx35 and in close vicinity to a PDZ binding domain at the very C-terminal end of the protein. We hypothesized that this phosphorylation site may serve as a molecular switch, influencing the affinity of the PDZ binding domain for its binding partners. Protein microarray and pulldown experiments revealed that this is indeed the case: phosphorylation of serine 298 decreased the binding affinity for MUPP1, a known scaffolding partner of connexin36, and increased the binding affinity for two different 14–3–3 proteins. Although we did not find the same effect in cell culture experiments, our data suggest that phosphorylation of serine 315/298 may serve to recruit different proteins to connexin36/35-containing gap junctions in an activity-dependent manner.

Trafficking of Connexin36 (Cx36) in the early secretory pathway

“…PTPN13 consists of multiple of PDZ domains and functions as a tumor suppressor (32). We have previously shown that perch Cx35, which has an identical PBM to Cx36, bound to the second PDZ domain of PTPN13 in a micro array screen (33). Consistent with this finding, we observed extensive colocalization of transiently expressed Cx36 and endogenous PTPN13 at gap junctions in transfected HEK293T cells (Figure 4F).…”

Shank3mutations impair electrical synapse scaffolding and transmission in mouse brain

“…The sensitivity of Cx36 to local domains of Ca 2+ influx and activated CaMKII underlies activity-dependent strengthening of electrical synapses in IO neurons (Turecek et al, 2014; Welsh and Turecek, 2017). Recent work demonstrated that phosphorylation of the CaMKII consensus site within Cx36 also increases PDZ binding (Tetenborg et al, 2020). The combined evidence implicates a feed-forward process in which CaMKII activation simultaneously increases electrical synapse strength and stabilizes gap junction intercellular channels within the extended PSD via interactions involving Cx36, SHANK3, and NMDAR1.…”

Craniofacial studies in chicken embryos confirm the pathogenicity of Frizzled2 variants associated with Robinow syndrome

“…To study the effects of the FZD2 viruses, we investigated stage 28 (E5.5, Table S4) and 38 (E12.5, Table S3) after performing overexpression of high titre (>2 x 10 8 ) FZD2 viruses at stage 15 (E2.5). The location of FZD2 viruses was only determined retrospectively on histological sections stained with a Group-associated antigens (GAG), an antibody that recognizes specific proteins in the RCAS virus or (Gignac et al, 2023; Gignac et al, 2019; Hosseini-Farahabadi et al, 2017; Geetha-Loganathan et al, 2014; Hosseini-Farahabadi et al, 2013; Chen et al, 2013; Kowalik and Hudspeth, 2011) antibody against the FLAG sequence (Gignac et al, 2023; Tetenborg et al, 2020) (Table S7).…”