The deubiquitinating enzyme USP10 regulates the endocytic recycling of CFTR in airway epithelial cells

Abstract

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a cyclic AMP-regulated chloride channel that plays an important role in regulating the volume of the lung airway surface liquid, and thereby mucociliary clearance and elimination of pathogens from the lung. In epithelial cells, cell surface CFTR abundance is determined in part by regulating both CFTR endocytosis from the apical plasma membrane and recycling back to the plasma membrane. We recently reported, using an activity-based chemical screen to identify active deubiquitinating enzymes (DUBs) in human airway epithelial cells, that Ubiquitin Specific Protease-10 (USP10) is located and active in the early endosomal compartment and regulates the deubiquitination of CFTR and thereby promotes its endocytic recycling. siRNA-mediated knockdown of USP10 increased the multi-ubiquitination and lysosomal degradation of CFTR and decreased the endocytic recycling and the half-life of CFTR in the apical membrane, as well as CFTR-mediated chloride secretion. Overexpression of wild-type USP10 reduced CFTR multi-ubiquitination and degradation, while overexpression of a dominant-negative USP10 promoted increased multi-ubiquitination and lysosomal degradation of CFTR. In the current study, we show localization and activity of USP10 in the early endosomal compartment of primary bronchial epithelial cells, as well as an interaction between CFTR and USP10 in this compartment. These studies demonstrate a novel function for USP10 in facilitating the deubiquitination of CFTR in early endosomes, thereby enhancing the endocytic recycling and cell surface expression of CFTR.

Figure 1

USP10 is expressed and active in early endosomes of primary human bronchial epithelial (HBE) cells. (A) Early endosomes were isolated from HBE cells using a sucrose gradient. The isolation and purification of early endosomes was confirmed by western blot analysis. The isolated fractions contained the early endosomal antigen (EEA)-1 and Rab5a, which are located in early endosomes, but did not contain LAMP-1, a lysosomal protein, or actin, a cytoplasmic protein. The early endosome fraction was positive for USP10. Lysates (LYSATE), which contains cytoplasm, lysosomes and early endosomes, were positive for USP10, EEA-1, Rab5, LAMP-1 and actin. Rab11a and Rab7 protein were not detected in the early endosomal fraction (Data not shown). (B) Representative confocal images of HBE cells infected with a baculovirus Rab5a-eGFP construct, which is expressed in early endosomes (green). USP10 was immunolocalized using an anti-USP10 antibody and an Alexa-568 secondary antibody (red). Infection with the baculovirus expressing the empty vector had no effect on cellular morphology, and fluorescence in the green channel in cells infected with this baculovirus was similar to background (data not shown). Scale bar equals 10 μm. (C) Early endosomes were isolated and incubated with the HA-UbVME probe to identify active DUBs. The HA-UbVME-DUB complex was immunoprecipitated with an anti-HA antibody, and the immunoprecipitated complex was analyzed by SDS-PAGE followed by western blot analysis using an anti-USP10 antibody, an anti-USP8 antibody or an anti-HA antibody (lane labeled HA-IP). Lane labeled LYSATE represent lysates of early endosomes blotted with the anti-USP10, the anti-USP8 or the anti-HA antibody. Although USP10 and USP8 were expressed in early endosomes, only USP10 was active as determined by the chemical probe technique. The non-immune IgG did not immunoprecipitate USP10, USP8 or HA-labeled complexes, and thus served as a negative control. Experiments were performed three times. Representative blots are shown.

Figure 2

USP10 immunoprecipitates CFTR. HBE cells were lysed, USP10 was immunoprecipitated using an anti-USP10 antibody, and western blot analysis was performed for USP10 and CFTR. LYSATE, cell lysates (2.5% of lysate run on gel). USP10 IP indicates proteins that were immunoprecipitated using the USP10 antibody. The non-immune IgG did not immunoprecipitate USP10 or CFTR, and thus served as a negative control. Experiments were performed three times. Representative blots are shown.

Figure 3

Ubiquitin-mediated regulation of CFTR intracellular trafficking. Based on our studies and those of other laboratories, we propose that CFTR is ubiquitinated at the plasma membrane and endocytosed. Multi-ubiquitinated CFTR is detected in the early endosome, where USP10 deubiquitinates CFTR and thereby promotes its endocytic recycling. A reduction of USP10 activity decreases the endocytic recycling of CFTR thereby increasing the targeting of CFTR for lysosomal degradation.