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Phosphorylation-dependent 14-3-3 protein interactions regulate CFTR biogenesis
Cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP/protein kinase A (PKA)–regulated chloride channel whose phosphorylation controls anion secretion across epithelial cell apical membranes. We examined the hypothesis that cAMP/PKA stimulation regulates CFTR biogenesis posttranslatio...
Autores principales: | , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
The American Society for Cell Biology
2012
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3302758/ https://www.ncbi.nlm.nih.gov/pubmed/22278744 http://dx.doi.org/10.1091/mbc.E11-08-0662 |
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author | Liang, Xiubin Da Paula, Ana Carina Bozóky, Zoltán Zhang, Hui Bertrand, Carol A. Peters, Kathryn W. Forman-Kay, Julie D. Frizzell, Raymond A. |
author_facet | Liang, Xiubin Da Paula, Ana Carina Bozóky, Zoltán Zhang, Hui Bertrand, Carol A. Peters, Kathryn W. Forman-Kay, Julie D. Frizzell, Raymond A. |
author_sort | Liang, Xiubin |
collection | PubMed |
description | Cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP/protein kinase A (PKA)–regulated chloride channel whose phosphorylation controls anion secretion across epithelial cell apical membranes. We examined the hypothesis that cAMP/PKA stimulation regulates CFTR biogenesis posttranslationally, based on predicted 14-3-3 binding motifs within CFTR and forskolin-induced CFTR expression. The 14-3-3β, γ, and ε isoforms were expressed in airway cells and interacted with CFTR in coimmunoprecipitation assays. Forskolin stimulation (15 min) increased 14-3-3β and ε binding to immature and mature CFTR (bands B and C), and 14-3-3 overexpression increased CFTR bands B and C and cell surface band C. In pulse-chase experiments, 14-3-3β increased the synthesis of immature CFTR, reduced its degradation rate, and increased conversion of immature to mature CFTR. Conversely, 14-3-3β knockdown decreased CFTR B and C bands (70 and 55%) and elicited parallel reductions in cell surface CFTR and forskolin-stimulated anion efflux. In vitro, 14-3-3β interacted with the CFTR regulatory region, and by nuclear magnetic resonance analysis, this interaction occurred at known PKA phosphorylated sites. In coimmunoprecipitation assays, forskolin stimulated the CFTR/14-3-3β interaction while reducing CFTR's interaction with coat protein complex 1 (COP1). Thus 14-3-3 binding to phosphorylated CFTR augments its biogenesis by reducing retrograde retrieval of CFTR to the endoplasmic reticulum. This mechanism permits cAMP/PKA stimulation to make more CFTR available for anion secretion. |
format | Online Article Text |
id | pubmed-3302758 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2012 |
publisher | The American Society for Cell Biology |
record_format | MEDLINE/PubMed |
spelling | pubmed-33027582012-05-30 Phosphorylation-dependent 14-3-3 protein interactions regulate CFTR biogenesis Liang, Xiubin Da Paula, Ana Carina Bozóky, Zoltán Zhang, Hui Bertrand, Carol A. Peters, Kathryn W. Forman-Kay, Julie D. Frizzell, Raymond A. Mol Biol Cell Articles Cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP/protein kinase A (PKA)–regulated chloride channel whose phosphorylation controls anion secretion across epithelial cell apical membranes. We examined the hypothesis that cAMP/PKA stimulation regulates CFTR biogenesis posttranslationally, based on predicted 14-3-3 binding motifs within CFTR and forskolin-induced CFTR expression. The 14-3-3β, γ, and ε isoforms were expressed in airway cells and interacted with CFTR in coimmunoprecipitation assays. Forskolin stimulation (15 min) increased 14-3-3β and ε binding to immature and mature CFTR (bands B and C), and 14-3-3 overexpression increased CFTR bands B and C and cell surface band C. In pulse-chase experiments, 14-3-3β increased the synthesis of immature CFTR, reduced its degradation rate, and increased conversion of immature to mature CFTR. Conversely, 14-3-3β knockdown decreased CFTR B and C bands (70 and 55%) and elicited parallel reductions in cell surface CFTR and forskolin-stimulated anion efflux. In vitro, 14-3-3β interacted with the CFTR regulatory region, and by nuclear magnetic resonance analysis, this interaction occurred at known PKA phosphorylated sites. In coimmunoprecipitation assays, forskolin stimulated the CFTR/14-3-3β interaction while reducing CFTR's interaction with coat protein complex 1 (COP1). Thus 14-3-3 binding to phosphorylated CFTR augments its biogenesis by reducing retrograde retrieval of CFTR to the endoplasmic reticulum. This mechanism permits cAMP/PKA stimulation to make more CFTR available for anion secretion. The American Society for Cell Biology 2012-03-15 /pmc/articles/PMC3302758/ /pubmed/22278744 http://dx.doi.org/10.1091/mbc.E11-08-0662 Text en © 2012 Liang 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 of Cell Biology. |
spellingShingle | Articles Liang, Xiubin Da Paula, Ana Carina Bozóky, Zoltán Zhang, Hui Bertrand, Carol A. Peters, Kathryn W. Forman-Kay, Julie D. Frizzell, Raymond A. Phosphorylation-dependent 14-3-3 protein interactions regulate CFTR biogenesis |
title | Phosphorylation-dependent 14-3-3 protein interactions regulate CFTR biogenesis |
title_full | Phosphorylation-dependent 14-3-3 protein interactions regulate CFTR biogenesis |
title_fullStr | Phosphorylation-dependent 14-3-3 protein interactions regulate CFTR biogenesis |
title_full_unstemmed | Phosphorylation-dependent 14-3-3 protein interactions regulate CFTR biogenesis |
title_short | Phosphorylation-dependent 14-3-3 protein interactions regulate CFTR biogenesis |
title_sort | phosphorylation-dependent 14-3-3 protein interactions regulate cftr biogenesis |
topic | Articles |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3302758/ https://www.ncbi.nlm.nih.gov/pubmed/22278744 http://dx.doi.org/10.1091/mbc.E11-08-0662 |
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