Phosphorylation of the Deubiquitinase USP20 by Protein Kinase A Regulates Post-endocytic Trafficking of β2 Adrenergic Receptors to Autophagosomes during Physiological Stress

Kommaddi, Reddy Peera; Jean-Charles, Pierre-Yves; Shenoy, Sudha K.

doi:10.1074/jbc.m114.630541

Cited by 37 publications

(54 citation statements)

References 60 publications

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“…trafficking, USP20 activity is regulated by cAMP-dependent kinase (PKA)-mediated phosphorylation of USP20 (67). Whether seryl phosphorylation of USP20 by TLR4-activated kinases such as IRAK1 (68) can modulate USP20 activity and thereby regulate ␤arr2 functions remains an interesting possibility that warrants further scrutiny.…”

Section: Discussionmentioning

confidence: 99%

Ubiquitin-specific Protease 20 Regulates the Reciprocal Functions of β-Arrestin2 in Toll-like Receptor 4-promoted Nuclear Factor κB (NFκB) Activation

Jean-Charles

Zhang

et al. 2016

Journal of Biological Chemistry

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Toll-like receptor 4 (TLR4) promotes vascular inflammatory disorders such as neointimal hyperplasia and atherosclerosis. TLR4 triggers NFB signaling through the ubiquitin ligase TRAF6 (tumor necrosis factor receptor-associated factor 6). TRAF6 activity can be impeded by deubiquitinating enzymes like ubiquitin-specific protease 20 (USP20), which can reverse TRAF6 autoubiquitination, and by association with the multifunctional adaptor protein ␤-arrestin2. Although ␤-arrestin2 effects on TRAF6 suggest an anti-inflammatory role, physiologic ␤-arrestin2 promotes inflammation in atherosclerosis and neointimal hyperplasia. We hypothesized that anti-and proinflammatory dimensions of ␤-arrestin2 activity could be dictated by ␤-arrestin2's ubiquitination status, which has been linked with its ability to scaffold and localize activated ERK1/2 to signalosomes. With purified proteins and in intact cells, our protein interaction studies showed that TRAF6/USP20 association and subsequent USP20-mediated TRAF6 deubiquitination were ␤-arrestin2-dependent. Generation of transgenic mice with smooth muscle cell-specific expression of either USP20 or its catalytically inactive mutant revealed anti-inflammatory effects of USP20 in vivo and in vitro. Carotid endothelial denudation showed that antagonizing smooth muscle cell USP20 activity increased NFB activation and neointimal hyperplasia. We found that ␤-arrestin2 ubiquitination was promoted by TLR4 and reversed by USP20. The association of USP20 with ␤-arrestin2 was augmented when ␤-arrestin2 ubiquitination was prevented and reduced when ␤-arrestin2 ubiquitination was rendered constitutive. Constitutive ␤-arrestin2 ubiquitination also augmented NFB activation. We infer that pro-and anti-inflammatory activities of ␤-arrestin2 are determined by ␤-arrestin2 ubiquitination and that changes in USP20 expression and/or activity can therefore regulate inflammatory responses, at least in part, by defining the ubiquitination status of ␤-arrestin2.␤-Arrestin2 (␤arr2) is an ϳ46-kDa multifunctional scaffolding protein that was discovered originally for its ability to desensitize G protein-mediated signaling evoked by seventransmembrane receptors (7TMRs) 4 (1, 2). However, ␤arr2 modulates the signaling and/or endocytosis of not only most 7TMRs but also of several receptor protein tyrosine kinases, cytokine receptors, ion channel receptors, and the LDL receptor (3, 4). Both the endocytic and signaling functions of ␤arr2 are intertwined with its ubiquitination, which in turn is stimulus-driven and regulated by specific E3 ubiquitin ligases or deubiquitinases (DUBs) (4). ␤arr2 not only undergoes dynamic ubiquitination/deubiquitination but also recruits E3 ubiquitin ligases to other substrates. Indeed, ␤arr2 is integral to the ubiquitination of cell surface receptors, channels, and non-receptor proteins (4, 5). However, thus far there is no clear demonstration that ␤arr2 can scaffold a DUB to specific substrates and affect signal transduction by mediating deubiquitination.A role in deubiquitination c...

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Section: Discussionmentioning

confidence: 99%

Ubiquitin-specific Protease 20 Regulates the Reciprocal Functions of β-Arrestin2 in Toll-like Receptor 4-promoted Nuclear Factor κB (NFκB) Activation

Jean-Charles

Zhang

et al. 2016

Journal of Biological Chemistry

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“…The ␤2-adrenergic receptor also appears to sort to an autophagic pathway under stress conditions. Stimulation of the ␤2-adrenergic receptor stably expressed in HEK293 cells with isoproterenol for 4 h in the presence of bafilomycin and chloroquine, which inhibit the vacuolar type H ϩ -ATPase that prevents acidification and inhibit fusion of late endosomes, multivesicular bodies, and autophagosomes, was recently shown to induce ␤2-adrenergic receptor colocalization and association with LC3-II (46). Colocalization and interaction with LC3-II also requires ␤2-adrenergic receptor ubiquitination.…”

Section: Discussionmentioning

confidence: 99%

Recycling and Endosomal Sorting of Protease-activated Receptor-1 Is Distinctly Regulated by Rab11A and Rab11B Proteins

Grimsey¹,

Coronel²,

Cordova³

et al. 2016

Journal of Biological Chemistry

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Protease-activated receptor-1 (PAR1) is a G protein-coupled receptor that undergoes proteolytic irreversible activation by coagulant and anti-coagulant proteases. Given the irreversible activation of PAR1, signaling by the receptor is tightly regulated through desensitization and intracellular trafficking. PAR1 displays both constitutive and agonist-induced internalization. Constitutive internalization of PAR1 is important for generating an internal pool of naïve receptors that replenish the cell surface and facilitate resensitization, whereas agonist-induced internalization of PAR1 is critical for terminating G protein signaling. We showed that PAR1 constitutive internalization is mediated by the adaptor protein complex-2 (AP-2), whereas AP-2 and epsin control agonist-induced PAR1 internalization. However, the mechanisms that regulate PAR1 recycling are not known. In the present study we screened a siRNA library of 140 different membrane trafficking proteins to identify key regulators of PAR1 intracellular trafficking. In addition to known mediators of PAR1 endocytosis, we identified Rab11B as a critical regulator of PAR1 trafficking. We found that siRNA-mediated depletion of Rab11B and not Rab11A blocks PAR1 recycling, which enhanced receptor lysosomal degradation. Although Rab11A is not required for PAR1 recycling, depletion of Rab11A resulted in intracellular accumulation of PAR1 through disruption of basal lysosomal degradation of the receptor. Moreover, enhanced degradation of PAR1 observed in Rab11B-deficient cells is blocked by depletion of Rab11A and the autophagy related-5 protein, suggesting that PAR1 is shuttled to an autophagic degradation pathway in the absence of Rab11B recycling. Together these findings suggest that Rab11A and Rab11B differentially regulate intracellular trafficking of PAR1 through distinct endosomal sorting mechanisms.Protease-activated receptor-1 (PAR1) 5 is a G protein-coupled receptor (GPCR) that elicits cellular responses to coagulant and anti-coagulant proteases (1, 2). Thrombin, the key effector protease of the coagulation cascade, mediates hemostasis, thrombosis, and inflammatory responses to vascular injury predominantly through PAR1 (3). The mechanisms by which proteases activate PAR1 and subsequent signal regulatory mechanisms are best understood for thrombin. Thrombin binds to and cleaves the N terminus of PAR1, revealing a new N-terminal domain that acts as a tethered ligand by binding intramolecularly to the receptor to initiate signaling and irreversibly activates the receptor (4, 5). In addition to rapid desensitization, trafficking of PAR1 is critical for regulating thrombin signaling and appropriate cellular responses. Unlike classic GPCRs, which are internalized and recycled back to the cell surface after agonist stimulation, activated PAR1 is internalized and sorted from endosomes to lysosomes and degraded. Internalization and lysosomal sorting is critical for terminating activated PAR1 signaling to heterotrimeric G proteins (6, 7). In addition to agonist-indu...

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“…Agonist‐stimulated ubiquitination of the β 2 AR is (i) targeted to specific lysines in the third intracellular loop and the carboxyl tail, (ii) a prerequisite for the trafficking of internalized β 2 AR complexes into autophagosomes and lysosomes, (iii) mediated by a HECT‐domain containing E3 ligase NEDD4, and (iv) dependent on β‐arrestin2 which functions as an adaptor for escorting NEDD4 to agonist‐activated β 2 AR (Shenoy et al, ; Xiao and Shenoy, ; Han et al, ; Kommaddi et al, ). NEDD4 family members contain two to four characteristic WW‐domains with two conserved tryptophan residues that are essential for binding proline rich PPxY (PY) motifs or phospho‐serine/threonine residues in substrates (Lu et al, ).…”

Section: β‐Arrestins As Adaptors For E3 Ligasesmentioning

confidence: 99%

Ubiquitin‐Related Roles of β‐Arrestins in Endocytic Trafficking and Signal Transduction

Jean-Charles

Rajiv

Shenoy

2016

Journal Cellular Physiology

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The non-visual arrestins, β-arrestin1, and β-arrestin2 were originally identified as proteins that bind to seven-transmembrane receptors (7TMRs, also called G protein-coupled receptors, GPCRs) and block heterotrimeric G protein activation, thus leading to desensitization of transmembrane signaling. However, as subsequent discoveries have continually demonstrated, their functionality is not constrained to desensitization. They are now recognized for their critical roles in mediating intracellular trafficking of 7TMRs, growth factor receptors, ion transporters, ion channels, nuclear receptors, and non-receptor proteins. Additionally, they function as crucial mediators of ubiquitination of 7TMRs as well as other receptors and non-receptor proteins. Recently, emerging studies suggest that a class of proteins with predicted structural features of β-arrestins regulate substrate ubiquitination in yeast and higher mammals, lending support to the idea that the adaptor role of β-arrestins in protein ubiquitination is evolutionarily conserved. β-arrestins also function as scaffolds for kinases and transduce signals from 7TMRs through pathways that do not require G protein activation. Remarkably, the endocytic and scaffolding functions of β-arrestin are intertwined with its ubiquitination status; the dynamic and site specific ubiquitination on β-arrestin plays a critical role in stabilizing β-arrestin-7TMR association and the formation of signalosomes. This review summarizes the current findings on ubiquitin-dependent regulation of 7TMRs as well as β-arrestins and the potential role of reversible ubiquitination as a "biological switch" in signal transduction. J. Cell. Physiol. 231: 2071-2080, 2016. © 2016 Wiley Periodicals, Inc.

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Phosphorylation of the Deubiquitinase USP20 by Protein Kinase A Regulates Post-endocytic Trafficking of β2 Adrenergic Receptors to Autophagosomes during Physiological Stress

Cited by 37 publications

References 60 publications

Ubiquitin-specific Protease 20 Regulates the Reciprocal Functions of β-Arrestin2 in Toll-like Receptor 4-promoted Nuclear Factor κB (NFκB) Activation

Ubiquitin-specific Protease 20 Regulates the Reciprocal Functions of β-Arrestin2 in Toll-like Receptor 4-promoted Nuclear Factor κB (NFκB) Activation

Recycling and Endosomal Sorting of Protease-activated Receptor-1 Is Distinctly Regulated by Rab11A and Rab11B Proteins

Ubiquitin‐Related Roles of β‐Arrestins in Endocytic Trafficking and Signal Transduction

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