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

ArticleinChannels (Austin, Tex.) 4(3):150-4 · May 2010with12 Reads
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.
    • "Endocytosis plays a critical role in the maturation and morphogenesis of many multicellular tubes. In the embryonic Drosophila trachea, Shibire (dynamin), Clathrin, and Rab-5 are required for liquid clearance of dorsal trunk tubes [16], and, likewise, endocytosis plays a role in liquid clearance of lung airway epithelia [17]. Tube length is also regulated by endocytosis: mutations in Drosophila clathrin heavy chain and wurst result in dorsal trunk elongation [18]. "
    [Show abstract] [Hide abstract] ABSTRACT: Most tubes have seams (intercellular or autocellular junctions that seal membranes together into a tube), but "seamless" tubes also exist [1-3]. In Drosophila, stellate-shaped tracheal terminal cells make seamless tubes, with single branches running through each of dozens of cellular extensions. We find that mutations in braided impair terminal cell branching and cause formation of seamless tube cysts. We show that braided encodes Syntaxin7 and that cysts also form in cells deficient for other genes required either for membrane scission (shibire) or for early endosome formation (Rab5, Vps45, and Rabenosyn-5). These data define a requirement for early endocytosis in shaping seamless tube lumens. Importantly, apical proteins Crumbs and phospho-Moesin accumulate to aberrantly high levels in braided terminal cells. Overexpression of either Crumbs or phosphomimetic Moesin induced lumenal cysts and decreased terminal branching. Conversely, the braided seamless tube cyst phenotype was suppressed by mutations in crumbs or Moesin. Indeed, mutations in Moesin dominantly suppressed seamless tube cyst formation and restored terminal branching. We propose that early endocytosis maintains normal steady-state levels of Crumbs, which recruits apical phosphorylated (active) Moe, which in turn regulates seamless tube shape through modulation of cortical actin filaments.
    Article · Jul 2014
    • "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 Bomberger et al. , 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. "
    [Show abstract] [Hide abstract] 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.
    Full-text · Article · Dec 2012
    • "Finally, this recycling is dependent upon a small Nterminal 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 pathway434445 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. "
    [Show abstract] [Hide abstract] 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.
    Full-text · Article · Aug 2012
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