ye-ccblfacilitates-2010.pdf (3.68 MB)
c-Cbl facilitates endocytosis and lysosomal degradation of cystic fibrosis transmembrane conductance regulator in human airway epithelial cells
journal contribution
posted on 2010-08-27, 00:00 authored by Siying Ye, K Cihil, D Stolz, J Pilewski, B Stanton, A Swiatecka-UrbanCystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-activated Cl− channel expressed in the apical membrane of fluid-transporting epithelia. The apical membrane density of CFTR channels is determined, in part, by endocytosis and the postendocytic sorting of CFTR for lysosomal degradation or recycling to the plasma membrane. Although previous studies suggested that ubiquitination plays a role in the postendocytic sorting of CFTR, the specific ubiquitin ligases are unknown. c-Cbl is a multifunctional molecule with ubiquitin ligase activity and a protein adaptor function. c-Cbl co-immunoprecipitated with CFTR in primary differentiated human bronchial epithelial cells and in cultured human airway cells. Small interfering RNA-mediated silencing of c-Cbl increased CFTR expression in the plasma membrane by inhibiting CFTR endocytosis and increased CFTR-mediated Cl− currents. Silencing c-Cbl did not change the expression of the ubiquitinated fraction of plasma membrane CFTR. Moreover, the c-Cbl mutant with impaired ubiquitin ligase activity (FLAG-70Z-Cbl) did not affect the plasma membrane expression or the endocytosis of CFTR. In contrast, the c-Cbl mutant with the truncated C-terminal region (FLAG-Cbl-480), responsible for protein adaptor function, had a dominant interfering effect on the endocytosis and plasma membrane expression of CFTR. Moreover, CFTR and c-Cbl co-localized and co-immunoprecipitated in early endosomes, and silencing c-Cbl reduced the amount of ubiquitinated CFTR in early endosomes. In summary, our data demonstrate that in human airway epithelial cells, c-Cbl regulates CFTR by two mechanisms: first by acting as an adaptor protein and facilitating CFTR endocytosis by a ubiquitin-independent mechanism, and second by ubiquitinating CFTR in early endosomes and thereby facilitating the lysosomal degradation of CFTR.
History
Journal
Journal of biological chemistryVolume
285Pagination
27008 - 27018Publisher
American Society for Biochemistry and Molecular BiologyLocation
Bethesda, Md.Publisher DOI
Link to full text
ISSN
0021-9258eISSN
1083-351XLanguage
engNotes
Article first published online 4th June, 2010Publication classification
C1.1 Refereed article in a scholarly journalCopyright notice
2010, The American Society for Biochemistry and Molecular Biology, Inc.Usage metrics
Categories
No categories selectedKeywords
ABC transporterE3 ubiquitin Ligaseendocytosisprotein Sortingubiquitinationlysosomal degradationScience & TechnologyLife Sciences & BiomedicineBiochemistry & Molecular BiologyGROWTH-FACTOR RECEPTORCLATHRIN-COATED PITSEGF RECEPTORMEDIATED ENDOCYTOSISNEGATIVE REGULATIONDOWN-REGULATIONRING FINGERCFTRMEMBRANEUBIQUITIN