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

Toll-like receptor 4 (TLR4) promotes vascular inflammatory disorders such as neointimal hyperplasia and atherosclerosis. TLR4 triggers NFκB 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 β-arrestin2. Although β-arrestin2 effects on TRAF6 suggest an anti-inflammatory role, physiologic β-arrestin2 promotes inflammation in atherosclerosis and neointimal hyperplasia. We hypothesized that anti- and proinflammatory dimensions of β-arrestin2 activity could be dictated by β-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 β-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 NFκB activation and neointimal hyperplasia. We found that β-arrestin2 ubiquitination was promoted by TLR4 and reversed by USP20. The association of USP20 with β-arrestin2 was augmented when β-arrestin2 ubiquitination was prevented and reduced when β-arrestin2 ubiquitination was rendered constitutive. Constitutive β-arrestin2 ubiquitination also augmented NFκB activation. We infer that pro- and anti-inflammatory activities of β-arrestin2 are determined by β-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 β-arrestin2.

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 could help explain the ability of ␤arr2 to inhibit proinflammatory signaling that culminates in the activation of NFB (6 -8). Canonical activation of NFB involves agonist-mediated TLR4 or interleukin-1 receptor dimerization, which engenders MyD88-dependent activation of the E3 ubiquitin ligase TRAF6 -a process that involves TRAF6 oligomerization, autoubiquitination, and subsequent synthesis of Lys-63-linked polyubiquitin chains that are either covalently or noncovalently attached to other proteins (9). Such Lys-63-linked polyubiquitin chains activate TAK1 and co-localize TAK1 with IB kinase (IKK) through noncovalent interactions (9). Consequently, TAK1 phosphorylates and thereby activates IKK␤. IKK␤-mediated phosphorylation of IB␣ triggers Lys-48-linked polyubiquitination and proteasomal degradation of IB␣, with subsequent deinhibition of NFB p65/p50 heterodimers (9,10). In this schema, ␤arr2 can inhibit NFB signaling at two levels: by binding to and thereby impeding oligomerization and autoubiquitination of TRAF6 (6) and by binding to and preventing the degradation of IB␣ (7,8). Whether the association of ␤arr2 with TRAF6 and/or IB␣ facilitates deubiquitination of these proteins remains enigmatic.
Although ␤arr2 appears to inhibit NFB activation and consequent inflammation in certain systems (6 -8, 11-13), it also appears to augment inflammatory signaling in distinct systems (14 -16). Physiologic ␤arr2 expression in SMCs of endothelium-denuded arteries promotes neointimal hyperplasia, a pathology involving inflammation-induced proliferation and migration of SMCs from the tunica media into the subendothelial tunica intima (14,17). In Ldlr Ϫ/Ϫ mice, ␤arr2 promotes atherosclerosis (14), a chronic vasculitis that fundamentally involves canonical NFB signaling (18 -20). Similar proinflammatory roles of ␤arr2 have been reported in allergic asthma and in lysophosphatidic acid-induced NFB activation (16,21). Thus, current data paint a paradoxical picture for ␤arr2 with respect to NFB signaling and inflammation.
Apparent paradoxes in ␤arr2-regulated inflammatory signaling may be reconciled by extrapolating from studies demonstrating reciprocal functions of ubiquitinated ␤arr2 and nonubiquitinated ␤arr2 in 7TMR signaling (22,23). Does reversible ubiquitination of ␤arr2 explain the proinflammatory versus anti-inflammatory dimensions of ␤arr2 activity? Deubiquitination of ␤arr2 itself is mediated by USP33 (23), and the USP33 homolog USP20 has been shown to deubiquitinate TRAF6 in heterologous systems (24). By scaffolding TRAF6 and USP20, could ␤arr2 block canonical NFB activation? Conversely, by sequestering USP20, could ␤arr2 inhibit TRAF6 deubiquitination and thereby promote canonical NFB activation? And could these reciprocal roles of ␤arr2 be regulated by reversible ubiquitination of ␤arr2? This study uses a variety of in vivo and in vitro approaches to address these questions and to determine whether USP20 and ␤arr2 function in concert to regulate TRAF6 ubiquitination and canonical NFB activation.

Experimental Procedures
Generation of Transgenic Mice-All animal experiments were performed in accordance with protocols approved by the Duke University Institutional Animal Care and Use Committee. Transgenic mice were generated to overexpress mouse USP20 or its catalytically inactive mutant, dominant negative USP20 (DN-USP20), which possesses two mutations (C154S and H645Q) in the catalytic domain of the protein. The QuikChange TM site-directed mutagenesis kit (Stratagene) was used to insert the mutations on the basis of protocols described previously (25). N-terminal HA-tagged USP20 or DN-USP20 coding sequences were inserted into a cloning vector, pBluescript II, so that they were flanked upstream by a 481-base pair portion of the SM22␣ promoter (Ϫ440 to ϩ41 relative to transcription start) for smooth muscle cell-specific expression and downstream by a bovine growth hormone poly(A) signal (26 -28). The plasmid constructs were linearized, purified, and microinjected into the pronuclei of B6SJLF1/J zygotes and subsequently implanted into surrogate mice by the Duke Transgenic Core Facility. Positive animals were identified by PCR amplification using a 5Ј primer in the SM22␣ promoter region and a 3Ј primer in the USP20 transgene.
Purified Proteins-C-terminal MYC/DDK-tagged recombinant human TRAF6 (TP319528) and C-terminal MYC/DDKtagged human USP20 (TP308051) were purchased from Ori-Gene Technologies, Inc. Both proteins were supplied at Ͼ80% purity. (DDK is an alternative appellation for the FLAG epitope DYKDDDDK and is referred to as FLAG in this article.) Purified rat ␤-arrestin2 was provided by Dr. Robert J. Lefkowitz (Duke University) (30). Using protocols we have reported previously (23,25), we purified HA-USP20 from COS-7 cells transfected with a pcDNA3-HA construct that contained the human USP20 cDNA insert (23,25).
Cell Lines-Smooth muscle cells (SMCs) were isolated by enzymatic digestion of aortas stripped of adventitia and endothelial cells, according to published protocols (14,31,32). They were split 1:4 for each passage and not used after passage 7 (freshly isolated cells ϭ passage 1). ␤-Arrestin1/2 double knockout mouse embryo fibroblasts (MEFs) were obtained from Dr. Robert J. Lefkowitz (Duke University) and were characterized previously (29,33,34). SMCs and double knockout MEFs were kept in Dulbecco's modified Eagle's medium with 10% fetal bovine serum and 1% penicillin/streptomycin. HEK-293 cells were obtained from the American Type Culture Collection. These cells were grown in minimal essential medium supplemented with 10% fetal bovine serum and 1% penicillin/ streptomycin. For generation of stable cell lines expressing TRAF6, HEK-293 cells were transfected with a plasmid encoding the mouse TRAF6 with a FLAG epitope at its N terminus. Transfectant clones were selected by cultivation in growth medium supplemented with G418: initially at 1 mg/ml and in later passages at 400 g/ml, as described previously (35).
Immunoprecipitation and Immunoblotting-Plasmid transfections were performed in HEK-293 cells and MEFs at 50% confluency with Lipofectamine 2000 TM 48 h before experiments. For signaling experiments, SMCs and MEFs were starved overnight in serum-free medium. HEK-293 cells were starved for 4 h prior to stimulation with LPS or vehicle. Cells were washed with ice-cold phosphate-buffered saline (pH 7.4) and solubilized in an ice-cold lysis buffer (50 mM HEPES (pH 7.5), 2 mM EDTA, 250 mM NaCl, 10% (v/v) glycerol, and 0.5% (v/v) IGEPAL CA-630) that was supplemented with phosphatase and protease inhibitors (1 mM sodium orthovanadate, 1 mM sodium fluoride, 1 mM phenylmethylsulfonyl fluoride, 5 g/ml leupeptin, 5 g/ml aprotinin, 1 g/ml pepstatin A, and 100 M benzaminidine; all from Sigma-Aldrich). The lysis buffer used in the ubiquitination assays for immunoprecipitating TRAF6 and ␤arr2 was supplemented with 10 mM N-ethylmaleimide and 20 M MG132 to inhibit cellular DUB and 26S proteasome activities, respectively. The cell lysates were centrifuged at 13,000 rpm for 20 min at 4°C to remove cell debris, and protein concentrations were determined on the resulting supernatant whole cell extracts by Bradford protein assay. Cell lysate proteins (ϳ800 g) were immunoprecipitated using either anti-FLAG M2 resin or A1CT antibody with protein G Plus/protein A-agarose beads. Samples were incubated overnight (4°C) with end-over-end rotation for immunoprecipitation. Immune complexes were washed three times with lysis buffer, and bound proteins were eluted in 2ϫ SDS-PAGE sample buffer. Samples were resolved on 4 -20% gradient or 10% Tris-glycine gels along with 20 g of corresponding lysates (which corresponded to ϳ2.5% of that used for the IP) and then transferred onto nitrocellulose membranes. Membranes were blocked and probed in 5% (w/v) dried skim milk powder dissolved in TTBS (2% (v/v) Tween 20, 10 mM Tris-Cl, (pH 8.0), and 150 mM NaCl), and washes were performed in TTBS. Enhanced chemiluminescence (SuperSignal West Pico reagent, Pierce) was used for protein detection. Blot imaging was performed with a charge-coupled device camera system (Bio-Rad Chemidoc-XRS), and band densities were quantified with Image-Lab software (Bio-Rad).
In Vitro Binding of USP20 -For the analysis of USP20/␤arr2 binary interaction, 125 ng of purified FLAG-USP20 was incubated for 30 min with increasing doses of ␤arr2 in a total volume of 50 l of KOAc buffer (34) containing 100 mM K ϩ acetate, 50 mM HEPES, 0.5 mM MgSO 4 , 0.2 mM DTT, 0.2% bovine serum albumin, and protease inhibitors (pH 7.4). The protein complex was subsequently diluted to 500 l with lysis buffer supplemented with 10 mM N-ethylmaleimide (as in immunoprecipitation assays) and rotated with M2 anti-FLAG affinity agarose beads for 2 h at 4°C. Beads were pelleted, washed three times in lysis buffer, eluted in SDS sample buffer, run on 4 -12% gradient Tris-glycine gels, and immunoblotted. Reactions containing either only USP20 or only ␤arr2 served as negative controls. To determine USP20-TRAF6-␤arr2 complex formation, 80 ng of purified FLAG-TRAF6 was mixed with 125 ng of HAtagged USP20 and incubated for 30 min in KOAc buffer with varied doses of purified ␤arr2. FLAG pull-down and subsequent steps were similar to those used for the binary complex above.
RNA Interference-Non-targeting control siRNA and siRNA targeting ␤arr2, USP20, or USP33 were purchased from Dharmacon GE Healthcare Life Sciences and described previously (25,36,37). For rescue experiments, siRNA specifically targeting human ␤arr2 or human USP20 were co-transfected along with siRNA-resistant, YFP-tagged rat ␤arr2 (2 g in a 100-mm dish) or HA-tagged mouse USP20 (2 g in a 100-mm dish). GeneSilencer was used for ␤arr2 silencing, and Lipofectamine 2000 TM was used for USP20/USP33 silencing, following the protocol of the manufacturer. Early-passage cells that were 40 -50% confluent were transfected with 20 g of siRNA with or without plasmid DNA and incubated for 4 h (HEK-293) or 12-14 h (SMCs) at 37°C in serum-free medium and then for 48 h in serum-containing medium prior to assays. Cells with Ͼ85% reduction in target protein expression were used for experimental analyses.
In SMC experiments, each well of a 6-well dish was independently transfected with siRNA because trypsinizing siRNA-transfected SMCs engendered excessive cell toxicity. Consequently, each well of SMCs constituted a single experimental replicate. For this reason, interassay variability was greater in these assays than in assays of transgenic SMC lines. To compensate for this variability, SMC RNAi experiments included a greater number of experimental replicates (Fig. 9). Carotid Endothelial Denudation-Carotid endothelial denudation was performed on mice anesthetized with pentobarbital (50 mg/kg) using a 0.36-mm-diameter coronary guide wire (Cordis), as we described previously (14,31). Four weeks after endothelial denudation, injured common carotids were harvested from anesthetized mice after 20 min of perfusion-fixation (80 mm Hg) with 10% formalin in PBS. Subsequently, carotids were fixed in formalin for 20 h and then embedded in paraffin.
Histology-For carotid artery morphometry, paraffin-embedded specimens were sliced at 5 m and stained with a modified Masson's trichrome and Verhoeff's elastic tissue stain as we described previously (14,31). Computerized planimetry with ImageJ TM was performed as described previously (14,31) by observers blinded to sample identity. Immunofluorescence staining was performed on aortas embedded in OCT compound or paraffin-embedded carotids after samples were sliced at 5 m (14, 31). Tissue sections were probed with rabbit IgGs specified above, followed by anti-rabbit IgG conjugated to Alexa-488, Alexa-548, or Alexa-594, as described previously (14,31), or with Cy3-conjugated 1A4 anti-SMC ␣-actin (Sigma-Aldrich), as described previously (14,31). Nuclei were counterstained with Hoechst 33342 (10 g/ml) during the secondary antibody incubation. Nonspecific fluorescence (determined on serial sections probed with equivalent concentrations of nonimmune rabbit IgG) was subtracted from the total signal to obtain antigen-specific fluorescence. Imaging and analyses were performed by observers blinded to specimen identity. Specimens from all three groups (non-Tg, USP20-Tg, and DN-USP20-Tg) were stained and imaged batchwise to minimize variation in staining and imaging among groups.
Statistical Analyses-All experiments were reproduced at least three independent times. Data averaged from three or more independent experiments are presented as means Ϯ S.E. Statistical significance was determined by analysis of variance followed by post hoc test for multiple comparisons (GraphPad Prism 6 from GraphPad, Inc.), and p Ͻ 0.05 was considered significant.

Results
␤arr2, USP20, and TRAF6 Associate with Each Other-In the course of regulating 7TMR trafficking and endocytosis, ␤arr2 associates with and is deubiquitinated by ubiquitin-specific protease 33 (USP33) (23). To determine whether ␤arr2 could be regulated by other USP family members, we asked whether ␤arr2 associates with USP20, which shares 59% identity with USP33 (25). Co-immunoprecipitation of ␤arr2-FLAG in HEK-293 cells showed that endogenous USP20 interacted with ␤arr2 (Fig. 1A). ␤arr2 also associates with TRAF6 and thereby inhibits FIGURE 2. The relative abundance of ␤arr2 determines whether ␤arr2 forms binary or ternary complexes with TRAF6 and USP20 in purified preparations. A, the indicated concentration of purified ␤arr2 was incubated in 50 l (final volume) with or without 20 nM purified FLAG-USP20, as described under "Experimental Procedures." FLAG pull-downs were immunoblotted (IB) sequentially for ␤arr2 and USP20. Nonspecific ␤arr2 pull-down was determined from lanes lacking FLAG-USP20. Shown is an experiment representative of four performed. B, the indicated concentrations of purified ␤arr2 were incubated in 50 l (final volume) with or without purified HA-USP20 (20 nM) and/or FLAG-TRAF6 (20 nM), as described under "Experimental Procedures." FLAG pull-downs were successively immunoblotted for HA, ␤arr2, and TRAF6. Nonspecific HA-USP20 pull-down was determined from lanes lacking FLAG-TRAF6. Shown are results of a single experiment representative of four performed. C, for the USP20 pull-downs in A, specific (total minus nonspecific) ␤arr2 band intensities were quantified and normalized to USP20 band intensities. These ratios were normalized to those obtained with 4 nM ␤arr2 to obtain percent of maximum (% max), plotted (filled squares) as mean Ϯ S.E. from four independent experiments. Compared with 0.4 nM: *, p Ͻ 0.05 (analysis of variance). For the TRAF6 pull-downs in B, specific (total minus nonspecific) USP20 band intensities were normalized to cognate TRAF6 band intensities. These ratios were normalized to those obtained with 2 nM ␤arr2 to obtain the percentage of maximum (% max), plotted (empty circles) as means Ϯ S.E. from four independent experiments. Compared with control: *, p Ͻ 0.01. D, Coomassie-stained gels show 1 g of purified proteins separated on 4 -20% gradient Tris-Glycine polyacrylamide gels. Arrows indicate the mobility of purified proteins. The asterisks indicate light chain IgG bands that co-elute during the purification of HA-tagged proteins. not only TRAF6 oligomerization and autoubiquitination but also NFB signaling (6). As reported before (6), ␤arr2-FLAG co-immunoprecipitated with endogenous TRAF6 in our experiments (Fig. 1B). Furthermore, FLAG-TRAF6 co-immunoprecipitated with endogenous USP20 (Fig. 1C). These protein-protein interaction studies suggest that USP20, ␤arr2 and TRAF6 bind each other and might function together in NFB signaling.
To determine whether ␤arr2 binds to USP20 directly, we tested the interaction of purified ␤arr2 with purified USP20 (Fig. 2, A-D). As shown in Fig. 2A, the association of ␤arr2 with USP20 increased roughly linearly with [␤arr2] and then saturated over the range of ␤arr2 concentrations used. We then used this purified protein approach to determine whether ␤arr2 is required for the interaction of USP20 and TRAF6. In the absence of ␤arr2, minimal amounts of USP20 were detected in TRAF6 pull-downs. At low concentrations, ␤arr2 augmented the association of USP20 with TRAF6 by ϳ5-fold (Fig. 2B). However, at higher concentrations, ␤arr2 failed to augment the association of USP20 with TRAF6 at all (Fig. 2B). Thus, under conditions wherein the concentration of ␤arr2 is limiting, ␤arr2 can function as a scaffold that conjoins USP20 and TRAF6 in a ternary complex (with ␤arr2). However, at high concentrations, ␤arr2 forms only binary complexes with USP20 or perhaps with TRAF6 (Fig. 2, A-C).
␤arr2 Functions as a Scaffold for USP20-mediated Deubiquitination of TRAF6 -␤arr2 serves as a multifunctional scaffold and adaptor in 7TMR signaling. For example, ␤arr2 recruits to the receptor different components of the ERK pathway or elements of the endocytosis machinery (4,38,39). We hypothesized that the scaffolding abilities of ␤arr2 were critical for TLR4-dependent NFB signaling and asked whether ␤arr2 affects the interaction of USP20 and TRAF6. We first silenced ␤arr2 in HEK-293 cells and assayed USP20/TRAF6 association. In control cells, immunoprecipitation of TRAF6 pulled down both ␤arr2 and USP20. This observation suggested the possibility that these proteins form a ternary complex in intact cells (Fig. 3A). Remarkably, silencing ␤arr2 reduced the amount of endogenous USP20 that co-immunoprecipitated with TRAF6 (by 30 Ϯ 2 [unstimulated] to 60 Ϯ 3% [ϩLPS]) even though cellular levels of TRAF6 and USP20 did not change with ␤arr2 knockdown (Fig. 3, A and B). Our siRNA knockdown was specific for ␤arr2 because the levels of its homolog ␤arr1 were unchanged. Thus, ␤arr2 appears to promote the binding of USP20 with TRAF6. On the other hand, USP20 knockdown did not significantly alter the amount of endogenous TRAF6 coimmunoprecipitating with ␤arr2 (Fig. 3, C and D).
If ␤arr2 affects the association of USP20 with TRAF6, then one should expect ␤arr2 to affect the ubiquitination of TRAF6. Indeed, TRAF6 ubiquitination increased after siRNA-mediated knockdown of either ␤arr2 or USP20 (Fig. 4A). In these assays, there was no additive augmentation of TRAF6 ubiquitination when ␤arr2 and USP20 were knocked down simultaneously (Fig. 4, A and B). Thus, ␤arr2 and USP20 appear to use a shared mechanism to prevent TRAF6 ubiquitination (Fig. 4B). In these experiments, silencing USP20 or ␤arr2 had no effect on the expression level of USP33 (lysate blots in Fig. 4A). Hence, USP20 appears to have a distinct role from its homolog USP33 in deubiquitinating TRAF6.
To ascertain the specificity of our knockdown experiments, we performed rescue experiments for both ␤arr2 (Fig. 4, C and D) and USP20 (Fig. 4, E and F). In these assays, ␤arr2 knockdown induced an increase in TRAF6 ubiquitination that was reversed when a plasmid encoding rat ␤arr2 cDNA was cotransfected with the siRNA (Fig. 4, C and D). Similarly, cotransfection of a mouse HA-USP20 construct reversed the increase of TRAF6 ubiquitination observed with USP20 knockdown (Fig. 4, E and F). Together our results strongly suggest that USP20 is a deubiquitinase for TRAF6 and that ␤arr2 func- These ratios were normalized to that obtained with non-stimulated (NS) cells transfected with control siRNA, and plotted as the mean Ϯ S.E. from six experiments. Compared with control: *, p Ͻ 0.05. C, HEK-293 cells stably expressing FLAG-TRAF6 were transfected with siRNA targeting no protein or human ␤arr2. Simultaneously, these cells were transfected with pcDNA3 containing only YFP or ␤arr2-YFP (rat ␤arr2 cDNA). D, the ubiquitin smear in each IP was normalized to the cognate TRAF6 (FLAG) band in each lane. These ratios were normalized to those obtained in stimulated cells cotransfected with control siRNA and the YFP vector to obtain fold over control, plotted as mean Ϯ S.E. from five independent experiments. Compared with control: *, p Ͻ 0.05. E, HEK-293 cells stably expressing FLAG-TRAF6 were transfected with siRNA targeting no protein or human USP20. Simultaneously, these cells were transfected with pcDNA3 containing only HA tag or HA-USP20 (mouse USP20 cDNA). After LPS stimulation (37°C, 10 min), cells were solubilized for IP. TRAF6 (FLAG) immunoprecipitates and whole cell lysates were immunoblotted for the indicated proteins. Shown are results of a single experiment representative of three performed. F, the ubiquitin smear in each IP was normalized to the cognate TRAF6 (FLAG) band in each lane. These ratios were normalized to those obtained in stimulated cells co-transfected with control siRNA and the HA vector to obtain fold over control, plotted as mean Ϯ S.E. from three independent experiments. Compared with control: *, p Ͻ 0.05. tions as a critical adaptor for TRAF6 deubiquitination by USP20.
SMC-specific Expression of USP20 and DN-USP20 in Transgenic Mice-To determine the vascular effects of USP20, we generated transgenic mice overexpressing either USP20 or a catalytically inactive, dominant-negative mutant USP20 (DN-USP20-Tg) (Fig. 5A) (25) under the control of the SMC-specific SM22␣ promoter (28,40,41). Immunoblots of aortic extracts demonstrated that the total level of USP20 in the USP20-Tg was 2 Ϯ 1-fold more than endogenous USP20 levels in non-Tg and that DN-USP20-Tg expression was about 3 Ϯ 1-fold greater than endogenous USP20 levels (Fig. 5B). By immunostaining for the HA tag of the transgenes in aortic crosssections, we found that actin and DNA staining were comparable in littermate control and Tg aortas, but only Tg aortas stained for HA-USP20. Furthermore, ϳ90% of HA-USP20 (WT or DN) co-localized with SMC-actin (colocalization plugin, ImageJ software), indicating SMC-specific expression (Fig. 5C).
Antagonizing USP20 Activity Augments Neointimal Hyperplasia and Vascular NFB Activation-In model cell lines, USP20 can inhibit TRAF6-dependent NFB activation (24), which regulates a multitude of inflammatory signaling pathways (20,42). To determine whether SMC USP20 activity suppresses vascular inflammation, we subjected the transgenic animals and their non-Tg littermates to carotid endothelial denudation. This wire-mediated procedure triggers adhesion to the subendothelial extracellular matrix by neutrophils and platelets, which secrete cytokines and growth factors that provoke proliferation of medial SMCs that migrate across the internal elastic lamina and into the subendothelial, "neointimal" space to create "neointimal hyperplasia," an inflammatory lesion that compromises the efficacy of arterial stenting (14,20,31,42,43). Although the carotid arteries of each Tg mouse were morphologically equivalent before intervention, neointimal and medial areas 4 weeks after endothelial denudation were 2.6and 1.4-fold greater, respectively, in SMC-DN-USP20-Tg than in either Non-Tg or SMC-USP20-Tg mice (Fig. 6). Correspondingly, the luminal area was 2-fold less in SMC-DN-USP20-Tg mice (Fig. 6). Thus, antagonizing USP20 activity in SMCs augments neointimal hyperplasia, which is triggered by vascular inflammation (14,20,42,43).
USP20 Inhibits TLR4-induced NFB Activation in SMCs-In the pathogenesis of arterial injury, one of the important triggers for neointimal hyperplasia and SMC NFB activity is TLR4 signaling in SMCs (48). Therefore, we studied the effects of USP20 on NFB activation in SMCs in vitro by assaying NFB p65 Ser-536 phosphorylation (as in our in vivo studies above) as well as the rate of IB␣ degradation (31) triggered upon TLR4 stimulation with LPS. In SMCs from SMC-DN-USP20-Tg mice, LPS induced NFB p65 phosphorylation on Ser-536 to an extent that was 3-and 10-fold greater, respectively, than that in non-Tg and SMC-USP20-Tg SMCs (Fig. 8, A and B). Congruently, the LPS-induced rate of IB␣ degradation followed this rank order: SMC-DN-USP20-Tg Ͼ Non-Tg Ͼ SMC-USP20-Tg. Indeed, after 30 min of LPS stimulation, the levels of IB␣ in non-Tg and SMC-DN-USP20-Tg SMCs were 5-fold lower than those in SMC-USP20-Tg SMCs (Fig. 8, C and D). These shortterm signaling data were corroborated by long-term NFB activity data, assessed as the expression level of the NFBdependent VCAM-1. In response to 24 h of LPS stimulation, SMCs from non-Tg and SMC-DN-USP20-Tg mice evinced VCAM-1 protein levels that were 2.7-fold higher than those in SMCs from SMC-USP20-Tg mice (Fig. 8, E and F). Thus, USP20 activity in SMCs inhibits TLR4-induced NFB activation.
To evaluate whether USP20 activity affects LPS-induced signaling upstream of TRAF6, we quantitated in our three SMC lines the protein levels of three cell-surface proteins required for LPS-induced signaling: TLR4, CD14, and MD-2 (49). As shown in Fig. 8G, all three SMC lines expressed equivalent levels of TLR4, CD14, and MD-2. Consequently, differences among SMCs with regard to LPS-evoked signaling were not attributable to differences in LPS-binding proteins; rather, these differences accorded with their relative levels of USP20 activity in the SMCs.
NFB Signaling Is Reduced in ␤arr2 Ϫ/Ϫ SMCs-To determine whether the effects of USP20 on NFB signaling are regulated by ␤arr2, we performed USP20 RNAi in SMCs from congenic WT and ␤arr2 Ϫ/Ϫ mice. In WT and ␤arr2 Ϫ/Ϫ SMCs, silencing USP20 reduced levels of IB␣ in unstimulated SMCs as well as in LPS-stimulated SMCs (Fig. 9). Thus, USP20 appears to regulate NFB activity in SMCs in the absence or presence of ␤arr2. However, with or without USP20 silencing, ␤arr2 Ϫ/Ϫ SMCs demonstrated less IB␣ degradation than WT SMCs at each time point (Fig. 9). Nevertheless, ␤arr2 Ϫ/Ϫ and WT SMCs expressed equivalent levels of TLR4 (Fig. 9). These findings suggest that ␤arr2 contributes to NFB activation and, thereby, to proinflammatory phenotypic changes in SMCs.
LPS-induced ␤arr2 Ubiquitination Is Reversed by USP20 -␤arr2 seems to play paradoxically reciprocal roles in TLR4-dependent NFB signaling in SMCs. The first apparent role is anti-inflammatory: ␤arr2 scaffolds USP20 and TRAF6 and thereby facilitates TRAF6 deubiquitination and, consequently, diminishes NFB activation. The second ␤arr2 role is proinflammatory: ␤arr2 appears to promote IB␣ degradation. To elucidate how ␤arr2 could be either antior proinflammatory, we investigated whether the reciprocal effects of ␤arr2 in TLR4-dependent NFB signaling could be influenced by ubiquitination of ␤arr2 itself. We pursued this strategy because ubiquitination regulates the function of ␤arr2 in GPCR trafficking and endocytosis (4,5). To examine ␤arr2 ubiquitination in NFB signaling, we immunoprecipitated endogenous ␤arr isoforms from non-Tg and SMC-DN-USP20-Tg SMCs challenged with LPS. Both basal and LPS-induced ␤arr ubiquitination were greater in SMC-DN-USP20-Tg than in non-Tg SMCs (Fig. 10,  A and B). Thus, ␤arr isoforms are ubiquitinated downstream of TLR4 activation, and ␤arr appears to be deubiquitinated by USP20. With prolonged stimulation, ␤arr ubiquitination was undetectable in both non-Tg and SMC-DN-USP20-Tg SMCs, suggesting that deubiquitinases distinct from USP20 may also deubiquitinate ␤arr isoforms. . Antagonizing USP20 activity augments neointimal hyperplasia. A, congenic C57BL/6 mice with SM22␣-driven expression of USP20, DN-USP20, or no transgene (non-Tg) were subjected to wire-mediated carotid endothelial denudation. Carotids were harvested 4 weeks later after perfusion-fixation, and sections were stained with a modified connective tissue stain. Scale bars ϭ 50 m. B, the areas of the indicated arterial layers were measured by observers blinded to specimen identity. Areas are plotted as means Ϯ S.E. of Ն6 carotids per group. Compared with non-Tg: *, p Ͻ 0.01.
To corroborate findings obtained with DN-USP20 in SMCs, we tested ␤arr2 ubiquitination in HEK-293 cells. First, just as we found in SMCs, DN-USP20 in HEK-293 cells increased the ubiquitination of ␤arr2 (Fig. 10, C and D). We then silenced USP20 with siRNA transfection and observed similar effects: ␤arr2 ubiquitination increased when USP20 expression was reduced (Fig. 10, E and F). To determine whether USP33 can deubiquitinate ␤arr2 and whether USP20 and USP33 jointly effect ␤arr2 deubiquitination, we silenced USP33 alone or in combination with USP20 (Fig 10, E and F). Interestingly, ␤arr2 ubiquitination levels increased with silencing of either USP20 or USP33 even though USP20 RNAi did not decrease USP33 levels and USP33 RNAi did not decrease USP20 levels. Although these findings suggested the possibility that USP20 and USP33 deubiquitinate distinct sites in ␤arr2, silencing USP20 and USP33 simultaneously failed to augment ␤arr2 ubiquitination above levels observed with individual USP silencing (Fig 10, E and F). (This latter observation may be attributable, in part, to incomplete efficacy of the double knockdown). Together, these data strongly suggest that, upon TLR4 stimulation, USP20 deubiquitinates ␤arr2 as well as TRAF6.
Non-ubiquitinated ␤arr2 Is an Efficient Scaffold for USP20 -Because ␤arr2 ubiquitination promoted TLR4-induced activation of NFB, we asked whether ␤arr2 ubiquitination affects the ternary complex of USP20, ␤arr2, and TRAF6. To this end, we first immunoprecipitated FLAG-tagged ␤arr2-WT, ␤arr2-Ub, and ␤arr2-0K from HEK-293 cells and immunoblotted for endogenous TRAF6. This approach showed that the association of TRAF6 with ␤arr2 was greatest when ␤arr2 was not ubiquitinated (Fig. 11, C and D). The same approach showed that the association of ␤arr2 with endogenous USP20 is FIGURE 7. Antagonizing USP20 activity promotes neointimal NFB activation in vivo. A, serial sections from the injured carotid arteries used for Fig.  6 were fluorescently stained for DNA (blue) and immunostained with rabbit IgG specific for phospho-p65(Ser-536) (p-p65), total p65, VCAM-1, or no particular protein (Control). In addition, serial sections were stained for SMC ␣-actin. Scale bars ϭ 50 m. B, the ratios of protein immunofluorescence to DNA fluorescence intensity within the neointima were normalized to those obtained from WT samples from the same immunostaining batch to yield percent of non-Tg. Plotted are the means Ϯ S.E. from four or more carotid arteries of each genotype. Compared with non-Tg: *, p Ͻ 0.05.
2-fold greater when ␤arr2 is not ubiquitinated (Fig. 11, E and  F). In contrast, the association of ␤arr2 with endogenous USP20 was equivalent whether the ␤arr2 construct was constitutively ubiquitinated (␤arr2-Ub) or the WT (presumably because a substantial fraction of the WT ␤arr2 was ubiquiti-nated, as seen in Fig. 10, C and E). These results indicate that the non-ubiquitinated form of ␤arr2 is a better scaffold for simultaneously engaging TRAF6 and USP20 than the ubiquitinated form of ␤arr2. Thus, perhaps by scaffolding TRAF6 and USP20, deubiquitinated (or non-ubiquitinated) ␤arr2 FIGURE 8. Modulation of USP20 activity alters LPS-induced NFB signaling in SMCs. SMCs from non-Tg, SMC-USP20-Tg, and SMC-DN-USP20-Tg mice were serum-starved overnight and then exposed at 37°C to serum-free medium lacking (Ϫ, control) or containing 1 g/ml LPS for 10 min (A and B), 10 and 30 min (C and D), and 24 h (E and F). Cells were solubilized in 2ϫ sample buffer and immunoblotted for the indicated proteins. A, SMC lysates were serially immunoblotted (IB) for phospho-p65(Ser-536), total p65, and ␤-actin. B, band intensities for phospho-p65 were normalized to those of cognate ␤-actin. These ratios were normalized to those obtained in unstimulated non-Tg SMCs to obtain fold over control basal, plotted as mean Ϯ S.E. from six independent experiments. Compared with control (*) or with USP20-Tg (#): p Ͻ 0.0001. C, SMC lysates were serially blotted for IB␣ and ␤-actin. D, band intensities for IB␣ were normalized to those of corresponding ␤-actin bands. These ratios were normalized to those obtained from unstimulated SMCs of the cognate genotype to obtain fold over basal, plotted as mean Ϯ S.E. from seven independent experiments. Compared with non-Tg and USP20-Tg: #, p Ͻ 0.05. Compared with Non-Tg and DN-USP20-Tg: *, p Ͻ 0.05. E, SMCs stimulated with LPS for 24 h were immunoblotted for VCAM-1. F, band intensities for VCAM-1 were normalized to those of corresponding ␤-actin bands. These ratios were normalized to those obtained from unstimulated non-Tg SMCs to obtain fold over basal, plotted as mean Ϯ S.E. from six independent experiments. Compared with non-Tg (*) or DN-USP20-Tg (#): p Ͻ 0.0001. G, equivalent protein samples of the indicated SMC lysates were immunoblotted for TLR4, MD-2, CD14, and ␤-actin.

Discussion
Our data demonstrate that USP20 inhibits inflammatory signaling in SMCs, and that USP20 may do so, in part, by deubiquitinating TRAF6 in a manner that requires scaffolding by non-ubiquitinated ␤arr2. Non-ubiquitinated ␤arr2 thereby serves to diminish inflammatory signaling. However, ubiquitinated ␤arr2 augments inflammatory signaling in a manner that can be triggered by TLR4 signaling. TLR4 signaling promotes ␤arr2 ubiquitination, reduces USP20/␤arr2 association, and thereby potentiates TRAF6 ubiquitination and downstream NFB signaling (Fig. 12). Consequently, our study helps to elucidate apparently paradoxical findings showing that ␤arr2 can be antiinflammatory in some systems (6 -8, 11-13) and proinflammatory in models of vascular disease (14 -16, 21).
In earlier studies, ␤arr2 failed to deubiquitinate TRAF6 in cell-free assays (6). Consequently, the inhibitory effect of ␤arr2 on TLR4-dependent NFB signaling was previously attributed to ␤arr2-mediated inhibition of TRAF6 oligomerization and subsequent TRAF6 autoubiquitination (6). In this study, however, we report that ␤arr2 facilitates TRAF6 deubiquitination by serving as a scaffold for the deubiquitinase USP20. Thus, the ternary complex of TRAF6-␤arr2-USP20 conforms to a common theme: that deubiquitinases associate with scaffolding proteins to facilitate association with their substrate and, consequently, to enhance their substrate affinity and specificity (51). Although the absence of USP20 did not affect the association of ␤arr2 with TRAF6, the absence of ␤arr2 abrogated the association of USP20 with TRAF6 in cells and reduced by 5-fold the association of purified USP20 with purified TRAF6. Indeed, the ␤arr2 dependence of TRAF6/USP20 association may, along with possible ␤arr2-mediated inhibition of TRAF6 oligomerization, account for the increase in TRAF6 ubiquitination observed in ␤arr2-deficient cells (6).
␤arr2 appears to regulate NFB activation through cell-and signaling context-specific mechanisms. In response to LPS, bone marrow-derived macrophages from ␤arr2 Ϫ/Ϫ mice show more IKK activity than WT macrophages (6), but they show equivalent LPS-induced secretion of the NFB-dependent gene products TNF and IL-6 (11). Although ␤arr2 appears to reduce secretion of TNF and IL-6 from fibroblast-like synoviocytes (13), it has no effect on the secretion of the NFB-dependent gene products (52)(53)(54) hyaluronan and plasminogen activator inhibitor-1 from lung fibroblasts (15). However, in SMCs, ␤arr2 augments TLR4-dependent IB degradation and inflammation-associated SMC proliferation ( Fig. 9 and Ref. 14). By performing ␤arr2 reconstitution experiments in ␤arr1/2-double knockout MEFs, distinct groups have shown both (a) that ␤arr2 decreases LPS-induced TRAF6 ubiquitination and IB␣ phosphorylation (6), and (b) that ␤arr2 increases lysophosphatidic acid-induced activation of nuclear NFB (21). In the intact mouse, ␤arr2 exerts similarly diverse effects on a variety of endpoints regulated substantially by canonical NFB activation (20,55,56). ␤arr2 attenuates the effects of LPS-induced or septic shock, which transpires over hours (6,11). However, ␤arr2 has no effect on LPS-induced asthma (16), and ␤arr2 augments allergic asthma (16), arterial neointimal hyperplasia, and atherosclerosis (14), which develop over many weeks. To reconcile these diverse (and in some cases divergent) findings in vitro and in vivo, one could in some cases invoke the diversity of signaling mechanisms in play. However, the current work with dynamic ␤arr2 ubiquitination enables us to invoke more specific and novel mechanisms, too. We speculate that (a) ␤arr2 augments FIGURE 9. NFB signaling is reduced in ␤arr2 ؊/؊ SMCs. A, SMCs of the indicated genotype were transfected with siRNA targeting no mRNA (Control) or USP20 mRNA. Cells were stimulated with 100 ng/ml LPS at 37°C for the indicated times. Lysates were resolved by SDS-PAGE on BisTris gels (10% polyacrylamide) and immunoblotted (IB) for the indicated proteins. Arrowheads indicate the electrophoretic mobility for USP20 (second panel) and ␤arr2 (fourth panel). B, band intensities for IB␣ were normalized to corresponding ␤-actin band intensities. These ratios were normalized to those obtained from unstimulated WT SMCs transfected with control siRNA to obtain percent of control, plotted as mean Ϯ S.E. from a total of nine independent siRNA transfections in three independent pairs of WT and ␤arr2 Ϫ/Ϫ SMC lines. From two-way analysis of variance with Tukey's multiple comparison test, we found p Ͻ 0.05 for the following comparisons, designated by SMC genotype/(siRNA transfected): *, compared with WT/(control); ‡, compared with ␤arr2 Ϫ/Ϫ /(control); and †, compared with ␤arr2 Ϫ/Ϫ /(USP20).
NFB activity under conditions where deubiquitination of ␤arr2 is relatively slow, or impaired, so that ␤arr2 cannot serve to tether USP20 to TRAF6 and (b) ␤arr2 attenuates NFB activity under conditions where ␤arr2-mediated scaffolding of USP20 is important for negatively regulating NFB activation. Such conditions may be found in systems wherein the ratios of ␤arr2:USP20 and ␤arr2:TRAF6 are sufficiently low to favor the ternary complex of ␤arr2-USP20-TRAF6 rather than the binary complexes of ␤arr2-USP20 and ␤arr2-TRAF6, as demonstrated by our studies with purified proteins (Fig. 2). Our transgenic mice with SMC-specific expression of USP20 or DN-USP20 provide the first in vivo evidence that USP20 serves an antiinflammatory role. This finding is remarkable because USP20 is only one of ϳ85 DUBs in the mammalian proteome (57,58), and very few of these have been implicated in the regulation of NFB signaling. For example, the USP-family DUB known as CYLD can bind to p62/TRAF6 complexes, inhibit TRAF6 ubiquitination, and regulate RANK signaling in osteoclast precursor cells (59). Furthermore, CYLD also inhibits TNF receptor-triggered NFB signaling by deubiquitinating TRAF2 (60, 61). A somewhat contrary example is provided by the ovarian tumor protease DUB subfamily member A20, FIGURE 10. USP20 reverses TLR4-induced ubiquitination of ␤arr2. A, SMCs from congenic non-transgenic or SMC-DN-USP20-Tg mice were exposed to serum-free medium lacking (Ϫ) or containing 1 g/ml LPS at 37°C for the indicated times and then solubilized. SMC lysates were immunoprecipitated with anti-␤arr1/2 IgG, and IPs were immunoblotted (IB) serially for Ub with FK1 IgG and then for ␤arr1/2. Tris-glycine 4 -20% gradient gels were used to facilitate resolution of ubiquitinated proteins. However, these gels do not optimally resolve the two ␤arr isoforms. (Nonetheless, the A1CT IgG used for IP pulls down ␤arr2 as well as or even better than it does ␤arr1 (69)). B, ubiquitin smears (M r Ն64) in each lane were normalized to their corresponding ␤arr band intensities. These ratios were normalized to those obtained from unstimulated non-Tg SMCs to obtain "ubiquitinated ␤arr," plotted as mean Ϯ S.E. from three independent experiments. Compared with unstimulated Non-Tg: *, p Ͻ 0.05. C, HEK-293 cells were transfected with FLAGtagged ␤arr2 and either empty vector plasmid (control) or untagged DN-USP20 plasmid, exposed to serum-free medium lacking or containing LPS (1 g/ml) for 10 min at 37°C, and then solubilized. ␤arr2 was immunoprecipitated with anti-FLAG IgG, and immunoprecipitates were immunoblotted serially for total ubiquitin and then ␤arr2. Lysates were immunoblotted for USP20. D, for each ␤arr2 IP, the density of the entire lane of bands staining for ubiquitin was normalized to the cognate ␤arr2 band. Data were plotted as mean Ϯ S.E. from five experiments. Compared with unstimulated control cells: *, p Ͻ 0.05. E, HEK-293 cells were transfected with FLAG-tagged ␤arr2 and siRNA targeting no known protein (control, CTL), USP20, USP33, or USP20 ϩ USP33. Cells were challenged Ϯ LPS and processed as in C. F, ␤arr2 ubiquitination was calculated as in D and plotted as mean Ϯ S.E. from five experiments. Compared with unstimulated control cells: *, p Ͻ 0.05, ** , p Ͻ 0.01. which can also deubiquitinate TRAF6, regulate NFB signaling (62)(63)(64), and reduce NFB-dependent gene product expression and atherosclerosis in Apoe Ϫ/Ϫ mice (65). Knockin studies with a deubiquitinase-defective A20 demonstrate that domains distinct from the DUB domain appear to achieve A20-mediated NFB regulation (66). Whether ␤arr2 functions as an adaptor for additional DUBs that may regulate the NFB pathway remains to be determined.
This study reveals the importance of dynamic ubiquitination as a major modulator of the reciprocal roles of ␤arr2 in NFB signaling. Although ubiquitinated ␤arr2 scaffolds proteins in 7TMR pathways (22), non-ubiquitinated ␤arr2 appears to scaffold USP20 and its substrate TRAF6 in canonical NFB pathways. Our data also suggest the possibility that activation and inactivation of USP20 may regulate the signaling properties of ␤arr2 in the canonical NFB pathways. In the context of 7TMR FIGURE 11. Reciprocal roles of ␤arr2 in NFB signaling are defined by the scaffolding efficiency of ␤arr2 for USP20. A, ␤arr1 Ϫ/Ϫ /␤arr2 Ϫ/Ϫ MEFs were transfected with plasmids encoding YFP (control) or the indicated YFP-tagged construct: WT-␤arr2; a ␤arr2-ubiquitin chimera that is resistant to deubiquitination (␤arr2-Ub); or ␤arr2 in which all Lys residues are mutated to Arg (␤arr2-0K). MEFs were stimulated Ϯ LPS (1 g/ml) for 10 min (37°C), and extracts were immunoblotted (IB) serially for phospho-p65(Ser-536), ␤arr2, and ␤-actin. B, band intensities for p-p65(Ser-536) were normalized to corresponding ␤-actin band intensities. These ratios were normalized to those obtained from unstimulated, control-transfected ␤arr1 Ϫ/Ϫ /␤arr2 Ϫ/Ϫ MEFs to obtain fold over control, plotted as mean Ϯ S.E. from five independent experiments. Compared with the cognate basal signal (*) or compared with all LPS-stimulated groups (#): p Ͻ 0.05. C, HEK-293 cells were transiently transfected with plasmids encoding no protein (Ϫ) or the indicated FLAG-tagged construct: ␤arr2 WT, ␤arr2-Ub (Ub), or ␤arr2-0K (0K). ␤arr2 immunoprecipitates and cognate lysates were immunoblotted serially for (endogenous) TRAF6 and ␤arr2. D, band intensities for coimmunoprecipitated TRAF6 were normalized to corresponding ␤arr2 band intensities. These ratios were normalized to those obtained in WT ␤arr2 IPs to obtain fold over control, plotted as mean Ϯ S.E. from three independent experiments performed in triplicate. *, p Ͻ 0.05 compared with ␤arr2 WT or with ␤arr2-Ub. E, HEK-293 cells were transfected and immunoprecipitated as in C, but ␤arr2 immunoprecipitates and cognate whole cell lysates were immunoblotted serially for (endogenous) USP20 and ␤arr2. F, band intensities for co-immunoprecipitated USP20 were normalized to corresponding ␤arr2 band intensities. These ratios were normalized to those obtained in WT ␤arr2 IPs to obtain fold over control, plotted as mean Ϯ S.E. from three independent experiments performed in triplicate. *, p Ͻ 0.05 compared with ␤arr2 WT; #, p Ͻ 0.01 compared with ␤arr2-Ub.
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.