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

Department of Physiology, Dartmouth Medical School, Hanover, NH, USA.
Channels (Austin, Tex.) (Impact Factor: 2.32). 05/2010; 4(3):150-4. DOI: 10.4161/chan.4.3.11223
Source: PubMed


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.

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    • "Additional genetic and biochemical studies will be needed to identify its direct substrate protein. The vertebrate ortholog of CG32479 is USP10, which is known to regulate membrane protein trafficking and deubiquitination of tumor suppressor p53 (Faus et al. 2005; Bomberger et al. 2009, 2010; Yuan et al. 2010; Draker et al. 2011; Liu et al. 2011). As the regulatory functions of two other DUBs identified in our screen on Notch signaling are conserved, it would be interesting to investigate whether USP10 also participates in the regulation of Notch signaling activity in mammals. "
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    ABSTRACT: Notch signaling is highly conserved in all metazoan animals and plays critical roles in cell fate specification, cell proliferation, apoptosis, and stem cell maintenance. Although core components of the Notch signaling cascade have been identified, many gaps in the understanding of the Notch signaling pathway remain to be filled. One form of posttranslational regulation, which is controlled by the ubiquitin-proteasome system, is known to modulate Notch signaling. The ubiquitination pathway is a highly coordinated process in which the ubiquitin moiety is either conjugated to or removed from target proteins by opposing E3 ubiquitin ligases and deubiquitinases (DUBs). Several E3 ubiquitin ligases have been implicated in ubiquitin conjugation to the receptors and the ligands of the Notch signaling cascade. In contrast, little is known about a direct role of DUBs in Notch signaling in vivo. Here, we report an in vivo RNA interference screen in Drosophila melanogaster targeting all 45 DUBs that we annotated in the fly genome. We show that at least four DUBs function specifically in the formation of the fly wing margin and/or the specification of the scutellar sensory organ precursors, two processes that are strictly dependent on the balanced Notch signaling activity. Furthermore, we provide genetic evidence suggesting that these DUBs are necessary to positively modulate Notch signaling activity. Our study reveals a conserved molecular mechanism by which protein deubiquitination process contributes to the complex posttranslational regulation of Notch signaling in vivo.
    G3-Genes Genomes Genetics 12/2012; 2(12):1563-75. DOI:10.1534/g3.112.003780 · 3.20 Impact Factor
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    • "Finally, this recycling is dependent upon a small N-terminal domain whose absence leads to a decreased association with RME-1 (EHD1) and subsequent more rapid degradation [18] (Fig. 8). These results lead to several additional questions, including whether the endocytosis and recycling of KCa2.3 is associated with a cycle of ubiquitylation/deubiquitylation as has been described for other ion channels that enter the recycling pathway [43]–[45] and whether this post-translational modification is manipulated physiologically as a means of altering N. In addition, it will be important to define the components involved in removing KCa2.3 from the recycling pathway and to determine whether this channel is ultimately destined for ubiquitylation and lysosomal degradation in a manner analogous to what we recently described for KCa3.1 [20] or some other degradative fate. "
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    ABSTRACT: Regulation of the number of ion channels at the plasma membrane is a critical component of the physiological response. We recently demonstrated that the Ca2+-activated K+ channel, KCa2.3 is rapidly endocytosed and enters a Rab35- and EPI64C-dependent recycling compartment. Herein, we addressed the early endocytic steps of KCa2.3 using a combination of fluorescence and biotinylation techniques. We demonstrate that KCa2.3 is localized to caveolin-rich domains of the plasma membrane using fluorescence co-localization, transmission electron microscopy and co-immunoprecipitation (co-IP). Further, in cells lacking caveolin-1, we observed an accumulation of KCa2.3 at the plasma membrane as well as a decreased rate of endocytosis, as assessed by biotinylation. We also demonstrate that KCa2.3 and dynamin II are co-localized following endocytosis as well as demonstrating they are associated by co-IP. Further, expression of K44A dynamin II resulted in a 2-fold increase in plasma membrane KCa2.3 as well as a 3-fold inhibition of endocytosis. Finally, we evaluated the role of Rab5 in the endocytosis of KCa2.3. We demonstrate that expression of a dominant active Rab5 (Q79L) results in the accumulation of newly endocytosed KCa2.3 on to the membrane of the Rab5-induced vacuoles. We confirmed this co-localization by co-IP; demonstrating that KCa2.3 and Rab5 are associated. As expected, if Rab5 is required for the endocytosis of KCa2.3, expression of a dominant negative Rab5 (S34N) resulted in an approximate 2-fold accumulation of KCa2.3 at the plasma membrane. This was confirmed by siRNA-mediated knockdown of Rab5. Expression of the dominant negative Rab5 also resulted in a decreased rate of KCa2.3 endocytosis. These results demonstrate that KCa2.3 is localized to a caveolin-rich domain within the plasma membrane and is endocytosed in a dynamin- and Rab5-dependent manner prior to entering the Rab35/EPI64C recycling compartment and returning to the plasma membrane.
    PLoS ONE 08/2012; 7(8). DOI:10.1371/journal.pone.0044150 · 3.23 Impact Factor
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