Regulation of Epidermal Growth Factor Receptor Ubiquitination and Trafficking by the USP8·STAM Complex

Berlin, Ilana; Schwartz, Heather L.; Nash, Piers

doi:10.1074/jbc.m109.016287

Cited by 93 publications

(113 citation statements)

References 53 publications

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“…FLAG-ubiquitin, Myc-Cbl (BC032851), and MycNrdp1⌬C (BC032637) constructs were generated by PCR and cloned into pcDNA3.1 at BamHI/EcoRI. USP8 and STAM2 constructs used in the bimolecular fluorescence complementation (BiFC) assay (35) have also been previously described (18).…”

Section: Methodsmentioning

confidence: 99%

“…We have recently shown that USP8 associates with STAM to mediate direct deubiquitination of activated EGFR, thereby stabilizing the receptor against down-regulation by the lysosome (18). The work presented herein investigates a different role of USP8 in endocytosis through the examination of its effects on trafficking and turnover of the chemokine receptor, CXCR4.…”

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confidence: 99%

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The Deubiquitinating Enzyme USP8 Promotes Trafficking and Degradation of the Chemokine Receptor 4 at the Sorting Endosome

Berlin¹,

Higginbotham

Dise

et al. 2010

Journal of Biological Chemistry

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Reversible ubiquitination orchestrated by the opposition of ubiquitin ligases and deubiquitinating enzymes mediates endocytic trafficking of cell surface receptors for lysosomal degradation. Ubiquitin-specific protease 8 (USP8) has previously been implicated in endocytosis of several receptors by virtue of their deubiquitination. The present study explores an indirect role for USP8 in cargo trafficking through its regulation of the chemokine receptor 4 (CXCR4). Contrary to the effects of USP8 loss on enhanced green fluorescent protein, we find that USP8 depletion stabilizes CXCR4 on the cell surface and attenuates receptor degradation without affecting its ubiquitination status. In the presence of ligand, diminished CXCR4 turnover is accompanied by receptor accumulation on enlarged early endosomes and leads to enhancement of phospho-ERK signaling. Perturbation in CXCR4 trafficking, resulting from USP8 inactivation, occurs at the ESCRT-0 checkpoint, and catalytic mutation of USP8 specifically targeted to the ESCRT-0 complex impairs the spatial and temporal organization of the sorting endosome. USP8 functionally opposes the ubiquitin ligase AIP4 with respect to ESCRT-0 ubiquitination, thereby promoting trafficking of CXCR4. Collectively, our findings demonstrate a functional cooperation between USP8, AIP4, and the ESCRT-0 machinery at the early sorting phase of CXCR4 and underscore the versatility of USP8 in shaping trafficking events at the earlyto-late endosome transition.Endocytosis and trafficking of cell surface receptors is essential for organized signal transduction and maintenance of homeostasis. Malfunctions along the molecular pathways governing endocytosis can lead to a wide range of human pathologies including metabolic disorders, autoimmune diseases, and cancer (1, 2). Ubiquitination, a reversible post-translational modification of proteins (3), mediates spatial and temporal aspects of endocytosis by dictating macromolecular complex assembly and cargo fate (4). Although polyubiquitination linked through Lys 48 of ubiquitin targets substrates for proteasomal degradation (5), mono-and Lys 63 -linked ubiquitination signal cargo trafficking (6), and modulate function of endocytic sorting machinery (7). The reversible nature of ubiquitination is imparted by deubiquitinating enzymes (DUBs) 4 and enables dynamic regulation of these ubiquitin-dependent processes in the cell (8).Trafficking of endocytosed cargo for degradation by the lysosome occurs in a series of discrete selection stages that utilize the endosomal sorting complexes required for transport (ESCRTs) -0, -I, -II, and -III. The ESCRT-0 complex, consisting of the adaptor proteins hepatocyte growth factor-regulated substrate (Hrs) and signal transducing adaptor molecule (STAM), resides at the sorting endosome and functions in the first step of cargo selection. ESCRT-0 partitions the endocytosed material arriving on the early endosomes between recycling and the multivesicular body (9 -11) and interacts with downstream ESCRT machinery (12, 13) respon...

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Section: Methodsmentioning

confidence: 99%

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confidence: 99%

The Deubiquitinating Enzyme USP8 Promotes Trafficking and Degradation of the Chemokine Receptor 4 at the Sorting Endosome

Berlin¹,

Higginbotham

Dise

et al. 2010

Journal of Biological Chemistry

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“…The best candidate mammalian Doa4 ortholog is Usp8/UBPY, which can also bind ESCRT-III; however, its role in rescuing Ub from lysosomal degradation has not been specifically determined (Wright et al 2011). Indeed, USP8/ UBPY as well as a similarly positioned DUb, AMSH, can bind both ESCRT-0 and ESCRT-III and a host of experiments looking at different cargoes suggest they can play a role in deubiquitinating cargo before MVB sorting to promote cargo recycling (McCullough et al 2004;Mizuno et al 2005;Bowers et al 2006;Berlin et al 2010;Zhou et al 2013), cargo deubiquitination after sorting as a way to release them from the ESCRT apparatus and promote sorting into MVBs (Alwan and van Leeuwen 2007;Balut et al 2011), or as a way of deubiquitinating ESCRT components to rescue them from degradation or inactive conformations (Row et al 2007;Sierra et al 2010). One of the interesting aspects of cargo deubiquitination in the final stages of sorting is that it predicts that cargo is sorted into a domain capable of trapping Ub cargo such that it cannot escape upon deubiquitination.…”

Section: Ubiquitin-dependent Sorting In Endocytosismentioning

confidence: 99%

Ubiquitin-Dependent Sorting in Endocytosis

Piper

Đikić

Lukacs

2014

Cold Spring Harbor Perspectives in Biology

190

176

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When ubiquitin (Ub) is attached to membrane proteins on the plasma membrane, it directs them through a series of sorting steps that culminate in their delivery to the lumen of the lysosome where they undergo complete proteolysis. Ubiquitin is recognized by a series of complexes that operate at a number of vesicle transport steps. Ubiquitin serves as a sorting signal for internalization at the plasma membrane and is the major signal for incorporation into intraluminal vesicles of multivesicular late endosomes. The sorting machineries that catalyze these steps can bind Ub via a variety of Ub-binding domains. At the same time, many of these complexes are themselves ubiquitinated, thus providing a plethora of potential mechanisms to regulate their activity. Here we provide an overview of how membrane proteins are selected for ubiquitination and deubiquitination within the endocytic pathway and how that ubiquitin signal is interpreted by endocytic sorting machineries. HOW PROTEINS ARE SELECTED FOR UBIQUITINATIONU biquitin (Ub) is covalently attached to substrate proteins by the concerted action of E2-conjugating enzymes and E3 ligases (Varshavsky 2012). Most of the specificity and regulation of ubiquitination is at the level of the E3 ligases, which vastly outnumber the E2-conjugating enzymes. Ubiquitination results from the transfer of Ub, held either by the E2 or in some cases by the E3 as a high-energy thioester bond, onto a substrate protein, typically on the terminal amide of a substrate acceptor lysine side chain. Owing to the intense interest in the ubiquitination system, many of the simple rules and distinctions concerning different types of ligases, their specificity for forming particular polyUb chains, and even the kinds of residues in substrate proteins that can be ubiquitin modified, have been blurred with the discovery of mixed poly-Ub chains, new enzymatic mechanisms for Ub transfer, and ubiquitination of nonlysine residues (Kulathu and Komander 2012;Wenzel and Klevit 2012;McDowell and Philpott 2013). Nonetheless, in basic terms, Ub ligases function as platforms that coordinate substrate recognition with Ub transfer as well as configuring poly-Ub chain topology by the E2-conjugating enzyme. Substrates can be bound directly by the E3 ligase or by adaptor proteins that in turn bind to ligases, and selecEditors:

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“…In addition, it has been reported that Usp8 can interact with the ESCRT-0 component STAM via its SH3 domain and that its depletion results in accelerated degradation of EGF receptor in the lysosome, strongly suggesting that Usp8-dependent deubiquitylation of EGF receptor prevents EGF receptor from being recognized by the ESCRT machinery, a recognition depending on ESCRT-0 component Hrs (Berlin et al, 2010, Rao et al, 2011. Furthermore, the ability of Usp8 to bind both ESCRT-I and ESCRT-III suggests a dual function for Usp8 in receptor sorting.…”

Section: Usp8mentioning

confidence: 99%

“…However, other studies have shown that USP8 can also act , earlier at the level of ESCRT-I, by preventing entry into multivesicular bodies promoting recycling. The interaction of USP8 to both ESCRT-I and ESCRT-III indicates a complex role for this DUB in sorting of endosomal cargo (Berlin et al, 2010).…”

Section: Introductionmentioning

confidence: 99%