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Phosphorylation regulates connexin43/ZO-1 binding and release, an important step in gap junction turnover
To investigate whether connexin phosphorylation regulates the known role of zonula occludens-1 protein (ZO-1) in gap junction (GJ) function, we generated and analyzed a series of phosphomimetic and phosphorylation-dead mutants by mutating known conserved regulatory serine (S) residues 255, 279/282,...
Autores principales: | , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
The American Society for Cell Biology
2017
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5706988/ https://www.ncbi.nlm.nih.gov/pubmed/29021339 http://dx.doi.org/10.1091/mbc.E16-07-0496 |
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author | Thévenin, Anastasia F. Margraf, Rachel A. Fisher, Charles G. Kells-Andrews, Rachael M. Falk, Matthias M. |
author_facet | Thévenin, Anastasia F. Margraf, Rachel A. Fisher, Charles G. Kells-Andrews, Rachael M. Falk, Matthias M. |
author_sort | Thévenin, Anastasia F. |
collection | PubMed |
description | To investigate whether connexin phosphorylation regulates the known role of zonula occludens-1 protein (ZO-1) in gap junction (GJ) function, we generated and analyzed a series of phosphomimetic and phosphorylation-dead mutants by mutating known conserved regulatory serine (S) residues 255, 279/282, 365, 368, and 373 located in the C-terminal domain of connexin43 (Cx43) into glutamic acid (E) or alanine (A) residues. All connexin mutants were translated into stable, full-length proteins and assembled into GJs when expressed in HeLa or Madin–Darby canine kidney epithelial cells. However, mutants with S residues exchanged at positions 365, 368, and 373 exhibited a significantly altered ZO-1 interaction profile, while mutants with S residues exchanged at 255 and 279/282 did not. Unlike wild-type Cx43, in which ZO-1 binding is restricted to the periphery of GJ plaques, S365A, S365E, S368A, S368E, and S373A mutants bound ZO-1 throughout the GJ plaques, while the S373E mutant did not bind ZO-1 at all. Inability to disengage from ZO-1 correlated with increased GJ plaque size and increased connexin protein half-life, while maintaining GJ channels in an open, functional state. Quantitative clathrin-binding analyses revealed no significant alterations in clathrin-binding efficiency, suggesting that the inability to disengage from ZO-1 prevented maturation of functional into nonfunctional/endocytic channels, rather than ZO-1 interfering with GJ endocytosis directly. Collectively, our results indicate that ZO-1 binding regulates channel accrual, while disengagement from ZO-1 is critical for GJ channel closure and transitioning GJ channels for endocytosis. Intriguingly, these transitional ZO-1 binding/release and channel-aging steps are mediated by a series of hierarchical phosphorylation/dephosphorylation events at S373, S365, and S368, well-known Cx43 Akt, protein kinase A, and protein kinase C phosphorylation sites located in the vicinity of the ZO-1 binding site. |
format | Online Article Text |
id | pubmed-5706988 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2017 |
publisher | The American Society for Cell Biology |
record_format | MEDLINE/PubMed |
spelling | pubmed-57069882018-02-16 Phosphorylation regulates connexin43/ZO-1 binding and release, an important step in gap junction turnover Thévenin, Anastasia F. Margraf, Rachel A. Fisher, Charles G. Kells-Andrews, Rachael M. Falk, Matthias M. Mol Biol Cell Articles To investigate whether connexin phosphorylation regulates the known role of zonula occludens-1 protein (ZO-1) in gap junction (GJ) function, we generated and analyzed a series of phosphomimetic and phosphorylation-dead mutants by mutating known conserved regulatory serine (S) residues 255, 279/282, 365, 368, and 373 located in the C-terminal domain of connexin43 (Cx43) into glutamic acid (E) or alanine (A) residues. All connexin mutants were translated into stable, full-length proteins and assembled into GJs when expressed in HeLa or Madin–Darby canine kidney epithelial cells. However, mutants with S residues exchanged at positions 365, 368, and 373 exhibited a significantly altered ZO-1 interaction profile, while mutants with S residues exchanged at 255 and 279/282 did not. Unlike wild-type Cx43, in which ZO-1 binding is restricted to the periphery of GJ plaques, S365A, S365E, S368A, S368E, and S373A mutants bound ZO-1 throughout the GJ plaques, while the S373E mutant did not bind ZO-1 at all. Inability to disengage from ZO-1 correlated with increased GJ plaque size and increased connexin protein half-life, while maintaining GJ channels in an open, functional state. Quantitative clathrin-binding analyses revealed no significant alterations in clathrin-binding efficiency, suggesting that the inability to disengage from ZO-1 prevented maturation of functional into nonfunctional/endocytic channels, rather than ZO-1 interfering with GJ endocytosis directly. Collectively, our results indicate that ZO-1 binding regulates channel accrual, while disengagement from ZO-1 is critical for GJ channel closure and transitioning GJ channels for endocytosis. Intriguingly, these transitional ZO-1 binding/release and channel-aging steps are mediated by a series of hierarchical phosphorylation/dephosphorylation events at S373, S365, and S368, well-known Cx43 Akt, protein kinase A, and protein kinase C phosphorylation sites located in the vicinity of the ZO-1 binding site. The American Society for Cell Biology 2017-12-01 /pmc/articles/PMC5706988/ /pubmed/29021339 http://dx.doi.org/10.1091/mbc.E16-07-0496 Text en © 2017 Thévenin et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0). “ASCB®,” “The American Society for Cell Biology®,” and “Molecular Biology of the Cell®” are registered trademarks of The American Society for Cell Biology. |
spellingShingle | Articles Thévenin, Anastasia F. Margraf, Rachel A. Fisher, Charles G. Kells-Andrews, Rachael M. Falk, Matthias M. Phosphorylation regulates connexin43/ZO-1 binding and release, an important step in gap junction turnover |
title | Phosphorylation regulates connexin43/ZO-1 binding and release, an important step in gap junction turnover |
title_full | Phosphorylation regulates connexin43/ZO-1 binding and release, an important step in gap junction turnover |
title_fullStr | Phosphorylation regulates connexin43/ZO-1 binding and release, an important step in gap junction turnover |
title_full_unstemmed | Phosphorylation regulates connexin43/ZO-1 binding and release, an important step in gap junction turnover |
title_short | Phosphorylation regulates connexin43/ZO-1 binding and release, an important step in gap junction turnover |
title_sort | phosphorylation regulates connexin43/zo-1 binding and release, an important step in gap junction turnover |
topic | Articles |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5706988/ https://www.ncbi.nlm.nih.gov/pubmed/29021339 http://dx.doi.org/10.1091/mbc.E16-07-0496 |
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