c-Cbl facilitates endocytosis and lysosomal degradation of cystic fibrosis transmembrane conductance regulator in human airway epithelial cells.
Cystic 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.
Department of Physiology, Dartmouth Medical School, Hanover, New Hampshire 03755.
Siying
S
Ye
Department of Nephrology, Children's Hospital of Pittsburgh, Pittsburgh, Pennsylvania 15224.
Kristine
K
Cihil
Department of Cell Biology and Physiology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15201.
Donna Beer
DB
Stolz
Department of Cell Biology and Physiology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15201; Division of Pulmonary, Allergy and Critical Care Medicine, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 152224.
Joseph M
JM
Pilewski
Department of Physiology, Dartmouth Medical School, Hanover, New Hampshire 03755.
Bruce A
BA
Stanton
Department of Nephrology, Children's Hospital of Pittsburgh, Pittsburgh, Pennsylvania 15224; Department of Cell Biology and Physiology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15201. Electronic address: asurban@pitt.edu.
Agnieszka
A
Swiatecka-Urban
D002460:Cell Line; D002462:Cell Membrane / Q000235:genetics / Q000378:metabolism; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000235:genetics / Q000378:metabolism*; D004705:Endocytosis / Q000502:physiology*; D005786:Gene Expression Regulation / Q000502:physiology; D006801:Humans; D008247:Lysosomes / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D050721:Proto-Oncogene Proteins c-cbl / Q000235:genetics / Q000378:metabolism*; D020545:Respiratory Mucosa / Q000378:metabolism*; D054875:Ubiquitination / Q000502:physiology*
C505032:CFTR protein, human; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D050721:Proto-Oncogene Proteins c-cbl; C067419:CBL protein, human