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

Abstract

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...

The deubiquitinase ubiquitin–specific protease 20 is a positive modulator of myocardial β1-adrenergic receptor expression and signaling

“…Ub-VME is an activesite-directed probe that has been widely used to assess enzyme activity of DUBs and can distinguish between active and inactive forms of DUBs (26 -28). As expected, Ub-VME did not bind a catalytically inactive USP20 in which the active-site cysteine and histidine residues have been mutated (USP20-CH) (23,29).…”

The deubiquitinating enzyme USP20 stabilizes ULK1 and promotes autophagy initiation

“…Ever since USP20 was identified as a substrate of the cullin‐RING ligase family member CRL VHL , several signaling molecules, including hypoxia‐inducible factor 1α, Rad17, Claspin, TRAF6, and ERK3, have been reported as substrates of USP20 regarding hypoxia, DNA damage responses, and TLR4‐ and mitogen‐signaling pathways . In this study, we demonstrate that the deubiquitinating enzyme USP20 stabilizes ULK1 through deubiquitinating ULK1 at the basal level and is thus a critical factor in inducing autophagy initiation.…”

G Protein–Coupled Receptor Signaling Through β-Arrestin–Dependent Mechanisms

“…31 In addition to conformations provoked in β-arrestin by specific phosphorylation patterns on the GPCR interface, additional modifications on a pre-activated β-arrestin by differential ubiquitination or by other post-translational modifications can expand the conformational repertoire of each active form of β-arrestin. 11,22,53,54 This perhaps allows β-arrestin to possess unlimited conformational plasticity enabling its multi-faceted functions in GPCR signaling.…”

“…140 β-arrestin2 effects on NF-κB signaling are closely associated with its ubiquitination status. 54 Ubiquitinated β-arrestin2 promotes NF-κB signaling in SMCs, whereas deubiquitinated β-arrestin2 attenuates NF-κB signaling by scaffolding the deubiquitinase USP20, that in turn deubiquitinates TRAF6, which blocks the NF-κB signaling cascade. 54 Although these effects of reversible β-arrestin ubiquitination are demonstrated for the Toll-like receptor pathway, these mechanisms may potentially regulate NFκB signaling downstream of GPCRs.…”