TY - JOUR
T1 - Phosphorylation of Connexin36 near the C-terminus switches binding affinities for PDZ-domain and 14–3–3 proteins in vitro
AU - Tetenborg, Stephan
AU - Wang, Helen Y.
AU - Nemitz, Lena
AU - Depping, Anne
AU - Espejo, Alexsandra B.
AU - Aseervatham, Jaya
AU - Bedford, Mark T.
AU - Janssen-Bienhold, Ulrike
AU - O’Brien, John
AU - Dedek, Karin
N1 - Funding Information:
We thank Josef Meyer, Bettina Kewitz and Irina Fomins for excellent technical assistance and Dr. Beate Grün-berg for constant organizational support. We are also grateful to Prof. Dr. Karl-Wilhelm Koch for help with initial experiments and Dr. Xinran Zhu for providing the MUPP1 expression vector. This work was funded by the Deutsche Forschungsgemeinschaft (DE1154/5-1 to KD, JA854/3-1 to UJB, RTG 1885/1+2 to KD and UJB, RTG 1885/2 startup funding to ST), the US National Institutes of Health (EY012857 to JO’B) and the Cancer Prevention and Research Institute of Texas (PAAC CPRIT Grant RP180804 to MTB).
Publisher Copyright:
© 2020, The Author(s).
PY - 2020/12/1
Y1 - 2020/12/1
N2 - 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.
AB - 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.
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U2 - 10.1038/s41598-020-75375-0
DO - 10.1038/s41598-020-75375-0
M3 - Article
C2 - 33110101
AN - SCOPUS:85094117967
SN - 2045-2322
VL - 10
JO - Scientific reports
JF - Scientific reports
IS - 1
M1 - 18378
ER -