Cargando…

ΔF508 CFTR Surface Stability Is Regulated by DAB2 and CHIP-Mediated Ubiquitination in Post-Endocytic Compartments

The ΔF508 mutant form of the cystic fibrosis transmembrane conductance regulator (ΔF508 CFTR) that is normally degraded by the ER-associated degradative pathway can be rescued to the cell surface through low-temperature (27°C) culture or small molecular corrector treatment. However, it is unstable o...

Descripción completa

Detalles Bibliográficos
Autores principales: Fu, Lianwu, Rab, Andras, Tang, Li ping, Bebok, Zsuzsa, Rowe, Steven M., Bartoszewski, Rafal, Collawn, James F.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2015
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4399842/
https://www.ncbi.nlm.nih.gov/pubmed/25879443
http://dx.doi.org/10.1371/journal.pone.0123131
_version_ 1782366969308119040
author Fu, Lianwu
Rab, Andras
Tang, Li ping
Bebok, Zsuzsa
Rowe, Steven M.
Bartoszewski, Rafal
Collawn, James F.
author_facet Fu, Lianwu
Rab, Andras
Tang, Li ping
Bebok, Zsuzsa
Rowe, Steven M.
Bartoszewski, Rafal
Collawn, James F.
author_sort Fu, Lianwu
collection PubMed
description The ΔF508 mutant form of the cystic fibrosis transmembrane conductance regulator (ΔF508 CFTR) that is normally degraded by the ER-associated degradative pathway can be rescued to the cell surface through low-temperature (27°C) culture or small molecular corrector treatment. However, it is unstable on the cell surface, and rapidly internalized and targeted to the lysosomal compartment for degradation. To understand the mechanism of this rapid turnover, we examined the role of two adaptor complexes (AP-2 and Dab2) and three E3 ubiquitin ligases (c-Cbl, CHIP, and Nedd4-2) on low-temperature rescued ΔF508 CFTR endocytosis and degradation in human airway epithelial cells. Our results demonstrate that siRNA depletion of either AP-2 or Dab2 inhibits ΔF508 CFTR endocytosis by 69% and 83%, respectively. AP-2 or Dab2 depletion also increases the rescued protein half-life of ΔF508 CFTR by ~18% and ~91%, respectively. In contrast, the depletion of each of the E3 ligases had no effect on ΔF508 CFTR endocytosis, whereas CHIP depletion significantly increased the surface half-life of ΔF508 CFTR. To determine where and when the ubiquitination occurs during ΔF508 CFTR turnover, we monitored the ubiquitination of rescued ΔF508 CFTR during the time course of CFTR endocytosis. Our results indicate that ubiquitination of the surface pool of ΔF508 CFTR begins to increase 15 min after internalization, suggesting that CFTR is ubiquitinated in a post-endocytic compartment. This post-endocytic ubiquination of ΔF508 CFTR could be blocked by either inhibiting endocytosis, by siRNA knockdown of CHIP, or by treating cells with the CFTR corrector, VX-809. Our results indicate that the post-endocytic ubiquitination of CFTR by CHIP is a critical step in the peripheral quality control of cell surface ΔF508 CFTR.
format Online
Article
Text
id pubmed-4399842
institution National Center for Biotechnology Information
language English
publishDate 2015
publisher Public Library of Science
record_format MEDLINE/PubMed
spelling pubmed-43998422015-04-21 ΔF508 CFTR Surface Stability Is Regulated by DAB2 and CHIP-Mediated Ubiquitination in Post-Endocytic Compartments Fu, Lianwu Rab, Andras Tang, Li ping Bebok, Zsuzsa Rowe, Steven M. Bartoszewski, Rafal Collawn, James F. PLoS One Research Article The ΔF508 mutant form of the cystic fibrosis transmembrane conductance regulator (ΔF508 CFTR) that is normally degraded by the ER-associated degradative pathway can be rescued to the cell surface through low-temperature (27°C) culture or small molecular corrector treatment. However, it is unstable on the cell surface, and rapidly internalized and targeted to the lysosomal compartment for degradation. To understand the mechanism of this rapid turnover, we examined the role of two adaptor complexes (AP-2 and Dab2) and three E3 ubiquitin ligases (c-Cbl, CHIP, and Nedd4-2) on low-temperature rescued ΔF508 CFTR endocytosis and degradation in human airway epithelial cells. Our results demonstrate that siRNA depletion of either AP-2 or Dab2 inhibits ΔF508 CFTR endocytosis by 69% and 83%, respectively. AP-2 or Dab2 depletion also increases the rescued protein half-life of ΔF508 CFTR by ~18% and ~91%, respectively. In contrast, the depletion of each of the E3 ligases had no effect on ΔF508 CFTR endocytosis, whereas CHIP depletion significantly increased the surface half-life of ΔF508 CFTR. To determine where and when the ubiquitination occurs during ΔF508 CFTR turnover, we monitored the ubiquitination of rescued ΔF508 CFTR during the time course of CFTR endocytosis. Our results indicate that ubiquitination of the surface pool of ΔF508 CFTR begins to increase 15 min after internalization, suggesting that CFTR is ubiquitinated in a post-endocytic compartment. This post-endocytic ubiquination of ΔF508 CFTR could be blocked by either inhibiting endocytosis, by siRNA knockdown of CHIP, or by treating cells with the CFTR corrector, VX-809. Our results indicate that the post-endocytic ubiquitination of CFTR by CHIP is a critical step in the peripheral quality control of cell surface ΔF508 CFTR. Public Library of Science 2015-04-16 /pmc/articles/PMC4399842/ /pubmed/25879443 http://dx.doi.org/10.1371/journal.pone.0123131 Text en © 2015 Fu et al http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
spellingShingle Research Article
Fu, Lianwu
Rab, Andras
Tang, Li ping
Bebok, Zsuzsa
Rowe, Steven M.
Bartoszewski, Rafal
Collawn, James F.
ΔF508 CFTR Surface Stability Is Regulated by DAB2 and CHIP-Mediated Ubiquitination in Post-Endocytic Compartments
title ΔF508 CFTR Surface Stability Is Regulated by DAB2 and CHIP-Mediated Ubiquitination in Post-Endocytic Compartments
title_full ΔF508 CFTR Surface Stability Is Regulated by DAB2 and CHIP-Mediated Ubiquitination in Post-Endocytic Compartments
title_fullStr ΔF508 CFTR Surface Stability Is Regulated by DAB2 and CHIP-Mediated Ubiquitination in Post-Endocytic Compartments
title_full_unstemmed ΔF508 CFTR Surface Stability Is Regulated by DAB2 and CHIP-Mediated Ubiquitination in Post-Endocytic Compartments
title_short ΔF508 CFTR Surface Stability Is Regulated by DAB2 and CHIP-Mediated Ubiquitination in Post-Endocytic Compartments
title_sort δf508 cftr surface stability is regulated by dab2 and chip-mediated ubiquitination in post-endocytic compartments
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4399842/
https://www.ncbi.nlm.nih.gov/pubmed/25879443
http://dx.doi.org/10.1371/journal.pone.0123131
work_keys_str_mv AT fulianwu df508cftrsurfacestabilityisregulatedbydab2andchipmediatedubiquitinationinpostendocyticcompartments
AT rabandras df508cftrsurfacestabilityisregulatedbydab2andchipmediatedubiquitinationinpostendocyticcompartments
AT tangliping df508cftrsurfacestabilityisregulatedbydab2andchipmediatedubiquitinationinpostendocyticcompartments
AT bebokzsuzsa df508cftrsurfacestabilityisregulatedbydab2andchipmediatedubiquitinationinpostendocyticcompartments
AT rowestevenm df508cftrsurfacestabilityisregulatedbydab2andchipmediatedubiquitinationinpostendocyticcompartments
AT bartoszewskirafal df508cftrsurfacestabilityisregulatedbydab2andchipmediatedubiquitinationinpostendocyticcompartments
AT collawnjamesf df508cftrsurfacestabilityisregulatedbydab2andchipmediatedubiquitinationinpostendocyticcompartments