A Cullin-5−TRAF6 Interaction Promotes TRAF6 Polyubiquitination and Lipopolysaccharide Signaling

Cutting Edge: A Cullin-5−TRAF6 Interaction Promotes TRAF6 Polyubiquitination and Lipopolysaccharide Signaling

This information is current as Ziyan Zhu, Lili Wang, Rui Hao, Bo Zhao, Lei Sun and of September 23, 2021. Richard D. Ye J Immunol published online 27 May 2016 http://www.jimmunol.org/content/early/2016/05/27/jimmun ol.1600447 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2016 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published May 27, 2016, doi:10.4049/jimmunol.1600447 Th eJournal of Cutting Edge Immunology

Cutting Edge: A Cullin-5–TRAF6 Interaction Promotes TRAF6 Polyubiquitination and Lipopolysaccharide Signaling Ziyan Zhu,* Lili Wang,* Rui Hao,* Bo Zhao,* Lei Sun,* and Richard D. Ye*,† TNFR-associated factor (TRAF)6 integrates signals from oligomers and Pellino1 bind to TRAF6 and promote its ubiq- multiple cell surface receptors for the activation of NF- uitination in activated T lymphocytes and IL-1–stimulated kB. However, the mechanism underlying LPS-induced HEK293 cells, respectively (6, 7). In addition, IL-17 signaling TRAF6 signaling remains unclear. We report that cullin-5 through TRAF6 requires a U-box ubiquitin ligase Act1 that (Cul-5), a cullin family scaffold protein, binds to TRAF6 catalyzes TRAF6 polyubiquitination (8). Despite these ﬁndings, 63 it remains unclear how an LPS-bound TLR4-MyD88 complex

and promotes TRAF6 polyubiquitination at Lys in re- Downloaded from sponse to LPS stimulation. A direct interaction between activates TRAF6. Given the importance of the TLR-MyD88 the C-terminal domain of Cul-5 and the TRAF-C domain signaling pathways in innate immunity, a better understanding of TRAF6 facilitates polyubiquitination of TRAF6. Hemi- of the upstream activation mechanism for TRAF6 signaling is zygous Cul-5 knockout is associated with improved sur- of significant interest. vival of mice following LPS challenge and significant The cullin (Cul)–RING ligases constitute the largest sub- class of E3 ubiquitin ligases (9). In humans, there are at least delays in the phosphorylation of p65/RelA, ERK, JNK, http://www.jimmunol.org/ seven Culs, including Cul-1–3, Cul-4a, Cul-4b, Cul-5, and and p38 MAPKs in LPS-stimulated macrophages, along k Cul-7, as well as the closely related p53-associated parkin-like with a marked decrease in NF- B activation. These find- cytoplasmic protein. Each Cul protein can associate with ings identify Cul-5 as a signaling component that con- several substrate receptors, allowing regulation of many cel- nects an LPS-activated TLR4-MyD88 complex to TRAF6 k lular activities by targeting a large number of protein sub- for efficient activation of NF- B. The Journal of strates. The Culs typically serve as scaffold proteins for the Immunology, 2016, 197: 000–000. assembly of multisubunit ubiquitin E3s. They interact with other proteins, including RING-box proteins (Rbx)1 and oll-like receptors and TNF-a receptors share several Rbx2, the adaptor proteins, and substrate-binding proteins. by guest on September 23, 2021 signaling components, yet each maintains their distinct Together, these proteins form a Cul-RING domain ubiquitin T signaling profiles. TNFR-associated factor (TRAF)6 is ligase (CRL) that recruits ubiquitin E2s and catalyzes the one of the shared components that integrates signals from ubiquitination of Cul substrates (9, 10). In immune cells, different classes of receptors for the activation of NF-kB Cul-4a is reported to affect the cell cycle and the differenti- (1, 2). Extensive research has been conducted to understand ation of granulocytes (11), whereas Cul-4b maintains the how TRAF6 activates downstream signaling pathways, leading mRNA level of TNF-a (12) and negatively regulates LPS to its identification as a RING domain E3 ubiquitin ligase that signaling in macrophages (13). The biological functions of recruits the ubiquitin-conjugating enzymes Ubc13/Uev1A for other Culs in the immune system remain largely unknown. the synthesis of Lys63 (K63)-linked polyubiquitin chains (3). Gene-targeting approaches have been used in the generation Over the past decade, there has been a wealth of literature on of mice that lack a particular Cul: Cul-5. In the current study, TRAF6-dependent activation of NF-kB (reviewed in Refs. 4, we used the available Cul-5 hemizygous knockout mice and 5). In contrast, relatively little is known about the distinctive short hairpin RNA (shRNA)-mediated gene silencing to pathways connecting a variety of cell surface receptors to TRAF6 evaluate the potential functions of Cul-5 in innate immune polyubiquitination. Published reports showed that MALT1 cells. Our initial observation that hemizygous Cul-5–knockout

*School of Pharmacy, Shanghai Jiao Tong University, Shanghai 200240, China; and Building N22, Room 1049, Macau 999078, China (R.D.Y.) or School of Pharmacy, †Institute of Chinese Medical Sciences, University of Macau, Macau 999078, China Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai 200240, China (L.S.). E-mail addresses: [email protected] (R.D.Y.) or [email protected] (L.S.) ORCID: 0000-0002-5146-2702 (Z.Z.). The online version of this article contains supplemental material. Received for publication March 15, 2016. Accepted for publication May 4, 2016. Abbreviations used in this article: BMDM, bone marrow–derived macrophage; CRL, This work was supported by the National Natural Science Foundation of China (Grants Cul-RING domain ubiquitin ligase; CTD, carboxy-terminal domain; Cul, cullin; IP, 81202316 and 31270941), the National Basic Research Program of China (973 Program, immunoprecipitation; NTD, amino-terminal domain; Rbx, RING box protein; RF, Grant 2012CB518001), and the Specialized Research Fund for the Doctoral Program of RING ﬁnger domain; shRNA, short hairpin RNA; TRAF, TNFR-associated factor; Higher Education of China (Grants 20120073110069 and 20120073120092). R.D.Y. WT, wild-type; ZnF, zinc ﬁnger repeats. received research grant support from the University of Macau (CPG2015-00018-ICMS and SRG2015-00047-ICMS-QRCM). B.Z. received grant support from Shanghai Jiao Ó Tong University (Project 985 Startup Grant WF114117001/004). Copyright 2016 by The American Association of Immunologists, Inc. 0022-1767/16/$30.00 Address correspondence and reprint requests to Dr. Richard D. Ye or Dr. Lei Sun, Institute of Chinese Medical Sciences, University of Macau, Avenida da Universidade,

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1600447 2 CUTTING EDGE: CULLIN-5 REGULATES TRAF6 POLYUBIQUITINATION mice were better protected against systemic LPS challenge led phosphorylated p38 (4631S), ERK (4370S), JNK (4668S), and IkBa (9247S) us to investigate the role of Cul-5 in LPS signaling. We found were from Cell Signaling Technology (Danvers, MA). HEK293T cells (CRL- 11268; American Type Culture Collection, Manassas, VA) were cultured in that LPS potentiates an interaction between Cul-5 and TRAF6, DMEM supplemented with 10% FBS and 1% streptomycin-penicillin. THP-1 resulting in an increase in TRAF6 polyubiquitination, MAPK cells (TIB-202; American Type Culture Collection) were cultured in RPMI phosphorylation, and NF-kB activation. These findings suggest 1640 supplemented with 10% FBS, 1% streptomycin-penicillin, and 10 mM that Cul-5 is an upstream regulator of TRAF6 signaling in 2-ME before use. LPS-stimulated cells. Plasmid construction The cDNAs for Cul-5 and TRAF6 were obtained from human THP-1 cells Materials and Methods using standard PCR techniques and were subsequently inserted into the Mice pcDNA3.1 expression vectors (Invitrogen, Carlsbad, CA). Truncated mutants D D Cul-5 was targeted by gene trapping (www.komp.org). Cryopreserved germplasm of Cul-5 ( CTD and NTD with the C-terminal domain and N-terminal containing trapped Cul-5 alleles (clone 15150A-D10) was recovered by The domain removed) and TRAF6 RF, ZnF, and TRAF-C (containing only the KOMP Repository (Davis, CA). Heterozygous F1 progenies were intercrossed to RING finger domain, zinc finger repeats, and C-terminal domain of TRAF, 2 generate age- and sex-matched Cul-5+/ and Cul-5+/+ littermates. Experiments respectively) were prepared using PCR and standard ligation procedures with involvingmicewereperformedinaccordancewiththeStatuteontheAdminis- the indicated Myc or FLAG tags. Mutants of ubiquitin (K48R and K63R) tration of Laboratory Animals by the Ministry of Science and Technology of were prepared with the KOD-Plus-Mutagenesis Kit (SMK-101; TOYOBO, China. The procedures used were approved by the Institutional Animal Care and Shanghai, China) and subcloned into the pcDNA3.1 vector with the indi- Use Committee at Shanghai Jiao Tong University. cated tags.

Reagents LPS-induced sepsis and acute lung injury in mice Downloaded from The anti–Cul-5 (sc-13014) and anti-TRAF6 (sc-7221) Abs were obtained LPS from Escherichia coli (serotype 0111:B4; Sigma) was diluted in sterile PBS from Santa Cruz Biotechnology (Dallas, TX). The Abs to FLAG tag (8146), for i.p. injection into mice (10 mg/kg body weight) in the sepsis model. Myc tag (2272), ubiquitin (3933), phosphorylated p65/RelA (Ser536; 3031S), Twelve mice were used in each group. http://www.jimmunol.org/ by guest on September 23, 2021

2 FIGURE 1. Effects of Cul-5 hemizygosity on LPS-induced sepsis and cell signaling. (A) Survival of age- and weight-matched male Cul-5+/+ and Cul-5+/ mice (n =12 2 each) after LPS challenge (10 mg/kg, i.p.). (B) Serum levels of IL-1b,IL-6,andTNF-a in Cul-5+/+ and Cul-5+/ mice (n = 5 each) 24 h after i.p. injection of LPS (10 mg/kg) or PBS. NF-kB luciferase activity (Relative Luc) measured in THP-1 cells expressing the Cul-5–specific shRNA (C) or in a THP-1 cell line overexpressing Cul-5 (D) after treatment with LPS (100 ng/ml) or PBS for 12 h. Luciferase assay in HEK293T cells transfected with TRAF6 (E)orp65/RelA(F)alongwithanNF-kB luciferase reporter and increasing concentrations of a Cul-5 expression plasmid (0.2, 0.4, and 1.0 mg DNA/sample). Data in (B)–(F)aremean6 SEM from three independent experiments. *Significant difference versus samples lacking the Cul-5 expression plasmid (lane 2). *p , 0.05, **p , 0.01. (G) Immunoblotting analysis of 2 phosphorylatedp65/RelA,p38,JNK,andERKcomparedwithtotalp65/RelA,p38,JNK,ERK,Cul-5,andb-actin in cell lysates from Cul-5+/+ and Cul-5+/ BMDMs stimulated with LPS (100 ng/ml) for the indicated periods of time. The Abs used are described in Materials and Methods.IkBa degradation was also determined with an 2 anti-IkBa Ab.ThelevelofCul-5inCul-5+/+ and Cul-5+/ BMDMs was determined at the same time points and remained consistent. A representative set of im- munoblots from three independent experiments is shown. See Supplemental Fig. 1B for quantification of the blots. The Journal of Immunology 3

Quantification of cytokines Results and Discussion Bone marrow–derived macrophages (BMDMs) were prepared from wild-type Cul-5 haploinsufficiency improves survival and reduces 2 (WT) and Cul-5+/ mice, as described previously (14). Culture medium of proinflammatory cytokine production in response to LPS challenge BMDMs treated with LPS was collect at the time points indicated in the figure legends. Whole blood was collected from mice, and sera were prepared A literature search of the Cul family proteins found publi- individually. The levels of selected cytokines were measured by ELISA cations suggesting that Cul-4a and Cul-4b contribute to im- (eBioscience, San Diego, CA). mune cell functions (11–13). To investigate the potential functions of other Culs, such as Cul-5, in immune cells, we generated In vitro ubiquitination assay Cul-5–deficient mice using cryopreserved germplasm contain- For the in vitro ubiquitination assay, 500 nM GST–Cul-5, 100 nM His- ing trapped Cul-5 alleles. Of the 148 heterozygous intercrosses TRAF6, 50 nM His-Uba1, 0.3 mM Ubc13-Uev1A, 10 mM ubiquitin, and screened, no homozygotes were obtained. Timed pregnancies 2 2 2 mM ATP were incubated in a reaction buffer (50 mM Tris-HCl [pH 7.5], of heterozygotes yielded no Cul-5 / embryos. Hemizygous 5 mM MgCl2, 2 mM DTT) in Eppendorf tubes at 37˚C for 60 min. The +/2 reaction mixtures with loading buffer were boiled, separated by gel electro- (Cul-5 ) mice, which were viable with no visible abnormal- phoresis, and detected by immunoblotting with an anti-HA Ab (H6908; ities in growth, were used in this study. Sigma). To determine whether Cul-5 regulates innate immunity and 2 inflammation, WT and Cul-5+/ mice were given LPS (10 mg/kg Lentiviral production and transduction body weight, i.p.), and the survival rate was monitored. Although +/2 Cul-5–specific human shRNA (shCul-5, 10 mg; 59-GCTGCAGACTGAA- 100% of the WT mice died within 60 h, 50% of the Cul-5 TTAGTAG-39; Sigma) was transfected into HEK293T cells with pSPAX2 mice survived, with no subsequent deaths (Fig. 1A). Consistent Downloaded from m m (7.5 g) and pMD2G-VSVG (2.5 g). The supernatant containing lentivirus with the above findings, the serum levels of IL-1b,IL-6,and was collected and concentrated. The prepared lentivirus was added to THP-1 a +/2 cells and centrifuged for 90 min at 30˚C, and the medium was changed 4.5 h TNF- were significantly lower in Cul-5 mice than in their later. Stable THP-1/shCul-5 cells were selected with puromycin. The effi- WT littermates (Fig. 1B). ciency of gene silencing was assessed by RT-PCR. Cul-5 regulates NF-kB and MAPK signaling pathways

Transfection and reporter assay NF-kB activation plays an important role in the production http://www.jimmunol.org/ THP-1 cells were transduced with a plasmid encoding human Cul-5– of proinﬂammatory cytokines upon activation of TLRs. We pcDNA3.1–Myc using the Amaxa Nucleofector apparatus (Lonza), according examined Cul-5 for its potential involvement in NF-kBac- to the manufacturer’s instructions. Stable transfectants were selected with tivation using monocytic THP-1 cells expressing a Cul-5– m k G418 (50 g/ml). For the NF- B luciferase reporter assay using THP-1, speciﬁc shRNA (THP-1–Cul-5sh, Fig. 1C) or a Cul-5 cDNA stable THP-1 cells expressing Cul-5 were infected with lentivirus contain- k ing an NF-kB luciferase reporter (Genomeditech, Shanghai, China) for 6 h (THP-1–Cul-5, Fig. 1D). A lentivirus-based NF- B lucifer- and were stimulated with LPS (100 ng/ml) for 72 h postinfection. For the ase reporter was transfected into these stable cell lines prior to luciferase reporter assay using HEK293T cells, the cells were transiently LPS stimulation and measurement of NF-kB luciferase activity. transfectedwithpolyethyleneimine(Polysciences, Badener, Germany), according

k by guest on September 23, 2021 to the manufacturer’s instructions, using NF-kB luciferase reporter and the pRL- The LPS-stimulated NF- B activation was signiﬁcantly atten- TK plasmid (a Renilla luciferase control plasmid; Promega, Fitchburg, WI). The uated in Cul-5–knockdown cells (Fig. 1C), whereas Cul-5 Cul-5 expression plasmid was used at various concentrations, as indicated in the overexpression led to enhanced NF-kB activation upon LPS k legendforFig.1.NF-B promoter activities were analyzed using a Dual- stimulation (Fig. 1D). Luciferase Reporter Assay Kit (Promega). The readings were normalized against k the cotransfected Renilla luciferase activities. TLRs activate NF- B through a complex cascade involving various kinases and adaptor proteins. To identify the molecular k Statistical analysis targets of Cul-5 in TLR4-mediated NF- B activation, different concentrations of a Cul-5 cDNA expression vector were cotrans- Statistical signiﬁcance among different samples was assessed with the paired fected into HEK293T cells together with expression plasmids Student t test or one-way ANOVA with repeated measures if treatment groups k were more than two. Analysis and graphing were performed using Prism soft- coding for TRAF6 or p65/RelA. The NF- B luciferase activity ware (ver. 5.0; GraphPad, San Diego, CA). in cells overexpressing TRAF6 was further enhanced in a Cul-5

FIGURE 2. Molecular interaction between Cul-5 and TRAF6. (A) Co-IP of Myc–Cul-5 with FLAG-TRAF6 in the lysate from HEK293T cells. (B) Detection of FLAG-TRAF6 and Myc–Cul-5 in transiently transfected HeLa cells, using primary anti-FLAG or anti-Myc Abs and FITC- or PE-labeled secondary Abs, respectively. The images with FLAG-TRAF6 (green) and Myc–Cul-5 (red) were superimposed to show colocalization of the two proteins (yellow). Cell nuclei were stained with DAPI (blue). (C) Equal amounts of His-TRAF6 protein were incubated with equal amounts of purified GST–Cul-5 protein or GST alone. After GST pull-down, His-TRAF6 was detected from bound proteins of GST–Cul-5 with an anti-TRAF6 Ab. (D) IP and immunoblot of BMDMs stimulated with LPS (100 ng/ml) for the indicated times. The blots and images shown in this figure are representative of at least three independent experiments, with similar results. 4 CUTTING EDGE: CULLIN-5 REGULATES TRAF6 POLYUBIQUITINATION cDNA dose-dependent manner (Fig. 1E), whereas no significant alteration was seen in cells overexpressing p65/RelA (Fig. 1F). These findings suggest that Cul-5 works together with TRAF6 for NF-kBactivation. 2 BMDMs from Cul-5+/ mice and their WT littermates were prepared for further investigation of the role of Cul-5 in TLR4- mediated signaling through TRAF6, including NF-kB activa- 2 tion and MAPK phosphorylation (Fig. 1G). Cul-5+/ BMDMs responded to LPS stimulation with attenuated production of IL-6 and TNF-a (Supplemental Fig. 1A) compared with 2 their WT counterparts. Following LPS stimulation, Cul-5+/ BMDMs showed less pronounced phosphorylation of p65/ RelA, whereas the degradation of IkBa was not significantly 2 affected in Cul-5+/ BMDMs. The LPS-induced phosphorylation of ERK, JNK, and p38 MAPK was markedly delayed and, in 2 some cases (e.g., p38) it was attenuated in Cul-5+/ BMDMs compared with their WT controls (see Supplemental Fig. 1B for quantification of the blots). Altogether, these findings indicate Downloaded from that Cul-5 potentiates LPS-induced activation of NF-kBand MAPKs, possibly through TRAF6.

Cul-5 interacts with TRAF6 Because overexpression of Cul-5 dose dependently enhanced NF-kB activation through TRAF6 (Fig. 1E), we investigated http://www.jimmunol.org/ whether Cul-5 could interact with TRAF6. FLAG-tagged TRAF6 and Myc-tagged Cul-5 were transiently transfected in- to HEK293T cells for 48 h, and cell extracts were prepared for immunoprecipitation (IP) with an anti-FLAG Ab. A molecular species corresponding to the size of Cul-5 was detected by Western blotting in the immunoprecipitate of FLAG-TRAF6, suggesting Cul-5’s association with TRAF6 (Fig. 2A). In images obtained using a fluorescent confocal microscope, TRAF6 (green by guest on September 23, 2021 fluorescence) and Cul-5 (red fluorescence) were present in the same cytoplasmic compartment, and the superimposed image (yellow) indicated colocalization of the two proteins (Fig. 2B). A direct interaction between Cul-5 and TRAF6 was con- firmed in an in vitro binding assay, because rGST–Cul-5, but not GST, was able to pull down His-tagged TRAF6 (Fig. 2C). In BMDMs, LPS induced the association of Cul-5 with TRAF6 based on a co-IP experiment. Cul-5 was not found in the immunoprecipitate of TRAF6 at time 0, but it appeared at 5 min after LPS stimulation and peaked at 15 min (Fig. 2D). The time course of Cul-5–TRAF6 association was in line with that of LPS-stimulated phosphorylation of major MAPKs, which also appeared at 5 min and peaked at or shortly after 15 min (Fig. 1G). Cul-5 has a long, stalk-like N-terminal domain (NTD) FIGURE 3. Domains involved in the interaction between TRAF6 and Cul-5. consisting of three Cul repeats and a globular carboxy-terminal (A) Lysates from HEK293T cells transiently transfected with FLAG-TRAF6, domain (CTD) that harbors a signature Cul homology domain together with Myc-tagged Cul-5 WT, Cul-5 mutant DCTD, or Cul-5 mutant D (9). To map the region of Cul-5 that interacts with TRAF6, NTD, were subjected to IP with anti-FLAG Ab, followed by Western blot analysis with anti-FLAG Ab and anti-Myc Ab. (B) Interaction of TRAF-C with different portions of Cul-5 were selected for Myc tagging (Fig. Cul-5. Lysates from HEK293T cells transiently transfected with Myc–Cul-5, 3A, upper panel) and expression in HEK293T cells, together together with FLAG-tagged TRAF6 WT or the TRAF6 mutants TRAF6-RF, with FLAG-TRAF6. The full-length Cul-5 strongly bound to TRAF6-ZnF, or TRAF6–TRAF-C were subjected to IP with anti-FLAG Ab, TRAF6. The DNTD construct lacking the NTD also bound followed by Western blotting with anti-FLAG Ab and anti-Myc Ab, as indicated. to TRAF6, but the DCTD construct failed to interact with The blots and images are representative of at least three independent experiments, TRAF6 (Fig. 3A, lower panel). These results show an inter- with similar results. For the input controls in (A)and(B), one-tenth of the action between Cul-5 and TRAF6 through a structural de- samples was loaded for gel electrophoresis and Western blotting. terminant in the CTD of Cul-5. The next experiment focused on the domain(s) of TRAF6 TRAF-C domain (1, 2, 15). Although the N-terminal structure that interact with Cul-5. TRAF6 contains an N-terminal RING (RING finger and zinc finger repeats) contains effector domains finger domain, five zinc finger repeats, a TRAF-N domain, and a for interaction with a ubiquitin E2 (16, 17), the TRAF-C The Journal of Immunology 5 domain is used for TRAF6 dimerization (18). Three deletion the K48- or K63-linked ubiquitin chain was added to TRAF6 in mutants were generated and expressed in transfected the presence of Cul-5. As shown in Supplemental Fig. 2, Cul-5 HEK293T cells along with a Myc-tagged Cul-5 (Fig. 3B, upper potentiation of TRAF6 polyubiquitination was seen in cells panel). Based on immunoblotting results, Cul-5 was coprecipitated expressing the K63 mutant of ubiquitin (with all Lys but K63 with the full-length TRAF6 and TRAF-C but not with TRAF6- mutated to Arg) but not the K48 mutant (with all Lys but RF or TRAF6-ZnF (Fig. 3B, lower panel). These results suggest K48 mutated to Arg) (3). that Cul-5 interacts with TRAF6 through its TRAF-C domain. The above results demonstrate that Cul-5 facilitates LPS- induced signaling through a direct interaction with TRAF6. Cul-5 promotes polyubiquitination of TRAF6 Cul-5 uses its CTD to interact with TRAF6, whereas the TRAF-C TRAF6 is a RING domain ubiquitin ligase that uses the E2 domain in TRAF6 is involved in the binding of Cul-5. TRAF-C is enzyme Ubc13/Uev1A for its autoubiquitination (3). We well conserved among all TRAF proteins (5) and plays a role in performed ubiquitination assays to determine whether Cul-5 TRAF6 homodimerization, which leads to TRAF6 oligomeri- could alter TRAF6 polyubiquitination. Results from in vitro zation (18, 20). Our results support a working model in which ubiquitination assays showed that GST–Cul-5, but not GST, a Cul-5–TRAF6 interaction is induced by LPS stimulation, enhanced TRAF6 polyubiquitination (Fig. 4A). In HEK293T cells which, in turn, promotes rapid signaling through K63-linked that contain endogenous Cul-5 (19), the expression of HA- TRAF6 polyubiquitination, MAPK phosphorylation, and ubiquitin led to polyubiquitination of a FLAG-tagged TRAF6 NF-kB activation.

(Fig. 4B, lane 2), whereas silencing Cul-5 expression by the Cul-5 and other members of the Cul family are best known Downloaded from Cul-5–specific shRNA reduced FLAG-TRAF6 polyubiquitination for their roles in the formation of CRLs, the largest subfamily (Fig. 4B, lane 3). An in vivo ubiquitination assay was also of E3 ligases (9, 21). In CRL5, Cul-5 uses its CTD for as- 2 performed in BMDMs from Cul-5+/ and WT mice (Fig. 4C). sociation with one of the RING box proteins (Rbx1 or Rbx2). 2 Macrophages from Cul-5+/ mice displayed reduced poly- The NTD of Cul-5 contains binding sites for the adaptor ubiquitination upon LPS stimulation compared with WT proteins elongin B and elongin C, which interact with a controls. These results support the notion that Cul-5 facilitates substrate receptor (9, 21). Activation of CRL5 as an ubiquitin http://www.jimmunol.org/ TRAF6 polyubiquitination. ligase involves the recruitment, by the RING finger protein To determine whether Cul-5 binding to TRAF6 is necessary Rbx1 or Rbx2, of a ubiquitin-conjugating enzyme (E2) that for its ubiquitination-potentiating effect, HEK293T cells were catalyzes the transfer of ubiquitin from E2 to a substrate transfected to express full-length Cul-5 or its DCTD or DNTD bound to the substrate receptor (21). How TRAF6 fits into this mutants (Fig. 4D). Cul-5–DCTD, which failed to interact with paradigm remains unclear. Our experimental data suggest that TRAF6, was unable to potentiate TRAF6 polyphosphorylation Cul-5 acts as a scaffold that interacts with TRAF6 and promotes (lane 4). In comparison, full-length Cul-5 and Cul-5–DNTD TRAF6 polyubiquitination. In unstimulated macrophages, Cul-5

enhanced TRAF6 polyubiquitination. We also examined whether and TRAF6 appear to be separate; however, 5 min after LPS by guest on September 23, 2021

FIGURE 4. Promotion of TRAF6 polyubiquitination by Cul-5. (A) TRAF6 ubiquitination in an in vitro assay in the presence or absence of purified GST–Cul-5 or GST alone. Polyubiquitination of TRAF6 was detected with an anti-HA Ab (for HA-ubiquitin). (B) Polyubiquitination of TRAF6 in transfected HEK293T cells with or 2 without a Cul-5–specific shRNA that reduced the level of endogenous Cul-5. (C) Polyubiquitination of TRAF6 in Cul-5+/+ and Cul-5+/ BMDMsstimulatedwithLPS (100 ng/ml) for 4 h. TRAF6 was immunoprecipitated from the cell lysate, and ubiquitin (Ub) was immunoblotted after gel electrophoresis. The protein levels of Cul-5 and TRAF6 in the samples (input) were also determined. (D) Polyubiquitination of TRAF6 in transfected HEK293T cells expressing FLAG-TRAF6, HA-ubiquitin, and Myc-tagged Cul-5, Cul-5–DCTD, or Cul-5–DNTD. The expression of Cul-5 and its mutants was determined by immunoblotting of cell lysate (input). In (B)and(D), 0.5 mg of the TRAF6 plasmid DNA, 1.5 mgoftheHA-UbplasmidDNA,and2.5mg of the Cul-5 and mutants plasmid DNA (or shCul-5) were used for transfection of HEK293T cells. All data are representative of at least three independent experiments that produced similar results. 6 CUTTING EDGE: CULLIN-5 REGULATES TRAF6 POLYUBIQUITINATION stimulation, Cul-5 is identified in the immunoprecipitate of tumor necrosis factor receptor-associated factor protein that mediates signaling from an amino-terminal domain of the CD40 cytoplasmic region. J. Biol. Chem. 271: TRAF6. These results, when compared with the observed in- 28745–28748. teraction of Cul-5 and TRAF6 in the overexpressing 3. Deng, L., C. Wang, E. Spencer, L. Yang, A. Braun, J. You, C. Slaughter, C. Pickart, and Z. J. Chen. 2000. Activation of the IkappaB kinase complex by TRAF6 requires HEK293T cells and in the in vitro binding assays, suggest the a dimeric ubiquitin-conjugating enzyme complex and a unique polyubiquitin chain. presence of regulatory mechanisms that prevent the associa- Cell 103: 351–361. tion of the two proteins in unstimulated macrophages. It is of 4. Walsh, M. C., G. K. Kim, P. L. Maurizio, E. E. Molnar, and Y. Choi. 2008. D TRAF6 autoubiquitination-independent activation of the NFkappaB and MAPK interest to note that a Cul-5– CTD mutant fails to interact pathways in response to IL-1 and RANKL. PLoS One 3: e4064. with TRAF6 or promote its polyubiquitination, whereas a 5. Xie, P. 2013. TRAF molecules in cell signaling and in human diseases. J. Mol. D Signal. 8: 7. Cul-5– NTD mutant that lacks the elongin B– and elongin 6. Sun, L., L. Deng, C. K. Ea, Z. P. Xia, and Z. J. Chen. 2004. The TRAF6 ubiquitin C–interacting domain is still capable of binding TRAF6 and ligase and TAK1 kinase mediate IKK activation by BCL10 and MALT1 in T lymphocytes. Mol. Cell 14: 289–301. promoting its polyubiquitination. Because elongin B and 7. Jiang, Z., H. J. Johnson, H. Nie, J. Qin, T. A. Bird, and X. Li. 2003. Pellino 1 elongin C are required for the interaction of a substrate receptor is required for interleukin-1 (IL-1)-mediated signaling through its interaction inaCRL,ourobservationssuggestthatTRAF6doesnotfitinto with the IL-1 receptor-associated kinase 4 (IRAK4)-IRAK-tumor necrosis factor receptor-associated factor 6 (TRAF6) complex. J. Biol. Chem. 278: the established model for CRL as a substrate that binds to a 10952–10956. substrate receptor. Moreover, TRAF6 itself is an E3 ligase and is 8. Liu, C., W. Qian, Y. Qian, N. V. Giltiay, Y. Lu, S. Swaidani, S. Misra, L. Deng, Z. J. Chen, and X. Li. 2009. Act1, a U-box E3 ubiquitin ligase for IL-17 signaling. capable of recruiting its E2s for autoubiquitination. Therefore, Sci. Signal. 2: ra63. despite the fact that Cul-5 is known to exist in a CRL (CRL5) 9. Petroski, M. D., and R. J. Deshaies. 2005. Function and regulation of cullin-RING ubiquitin ligases. Nat. Rev. Mol. Cell Biol. 6: 9–20. that is an E3 ligase (9, 21), there is no evidence that TRAF6 is a 10. Sarikas, A., T. Hartmann, and Z. Q. Pan. 2011. The cullin protein family. Genome Downloaded from substrate of the ubiquitin-conjugating enzyme recruited to Biol. 12: 220. CRL5. However, it is important to note that the TRAF6- 11. Li, B., F. C. Yang, D. W. Clapp, and K. T. Chun. 2003. Enforced expression of CUL-4A interferes with granulocytic differentiation and exit from the cell cycle. recruited E2 Uev1A may not be sufficient for efficient auto- Blood 101: 1769–1776. ubiquitination (22) and another E3 ligase, such as Act1 (8) or 12. Pfeiffer, J. R., and S. A. Brooks. 2012. Cullin 4B is recruited to tristetraprolin- containing messenger ribonucleoproteins and regulates TNF-a mRNA polysome Pellino3 (23), may potentiate TRAF6 polyubiquitination. loading. J. Immunol. 188: 1828–1839.

Moreover, there is also the possibility that Cul-5 and TRAF6 13. Hung, M. H., Y. R. Jian, C. C. Tsao, S. W. Lin, and Y. H. Chuang. 2014. En- http://www.jimmunol.org/ hanced LPS-induced peritonitis in mice deficiency of cullin 4B in macrophages. forms a functional CRL5 complex that uses the RING domain Genes Immun. 15: 404–412. of TRAF6 for the recruitment of ubiquitin-conjugating en- 14. Sun, L., H. Zhou, Z. Zhu, Q. Yan, L. Wang, Q. Liang, and R. D. Ye. 2015. Ex vivo zymes. Further investigation into these possibilities requires the and in vitro effect of serum amyloid a in the induction of macrophage M2 markers and efferocytosis of apoptotic neutrophils. J. Immunol. 194: 4891–4900. determination of the structural alteration resulting from the 15. Ye, H., J. R. Arron, B. Lamothe, M. Cirilli, T. Kobayashi, N. K. Shevde, D. Segal, CTD–TRAF-C interaction. Moreover, an initial binding of O. K. Dzivenu, M. Vologodskaia, M. Yim, et al. 2002. Distinct molecular mechanism for initiating TRAF6 signalling. Nature 418: 443–447. CTD with TRAF-C might lead to another mode of functional 16. Lamothe, B., A. D. Campos, W. K. Webster, A. Gopinathan, L. Hur, and interaction that contributes to TRAF6 polyubiquitination. B. G. Darnay. 2008. The RING domain and first zinc finger of TRAF6 coordinate signaling by interleukin-1, lipopolysaccharide, and RANKL. J. Biol. Chem. 283:

In conclusion, the current study identifies a novel function 24871–24880. by guest on September 23, 2021 of Cul-5 in its binding to TRAF6 and promotion of TRAF6 17. Yin, Q., S. C. Lin, B. Lamothe, M. Lu, Y. C. Lo, G. Hura, L. Zheng, R. L. Rich, polyubiquitination. Our findings provide a missing piece in the A. D. Campos, D. G. Myszka, et al. 2009. E2 interaction and dimerization in the crystal structure of TRAF6. Nat. Struct. Mol. Biol. 16: 658–666. signaling pathway that connects an LPS-activated TLR4 to the 18. Baud, V., Z. G. Liu, B. Bennett, N. Suzuki, Y. Xia, and M. Karin. 1999. Signaling signaling adaptor TRAF6. It is possible that this regulatory by proinflammatory cytokines: oligomerization of TRAF2 and TRAF6 is sufficient for JNK and IKK activation and target gene induction via an amino-terminal ef- mechanism is also used by other TLRs that use MyD88 for fector domain. Genes Dev. 13: 1297–1308. TRAF6-dependent NF-kB activation. A better understanding 19. Kohroki, J., T. Nishiyama, T. Nakamura, and Y. Masuho. 2005. ASB proteins interact with Cullin5 and Rbx2 to form E3 ubiquitin ligase complexes. FEBS Lett. of Cul-5 and its functions in innate immune cells may help to 579: 6796–6802. develop new therapies for the control of inflammation. 20. Walsh, M. C., J. Lee, and Y. Choi. 2015. Tumor necrosis factor receptor- associated factor 6 (TRAF6) regulation of development, function, and homeostasis of the immune system. Immunol. Rev. 266: 72–92. Disclosures 21. Lamsoul, I., S. Uttenweiler-Joseph, C. Moog-Lutz, and P. G. Lutz. 2016. Cullin 5-RING E3 ubiquitin ligases, new therapeutic targets? Biochimie 122: 339–347. The authors have no financial conflicts of interest. 22. Petroski, M. D., X. Zhou, G. Dong, S. Daniel-Issakani, D. G. Payan, and J. Huang. 2007. Substrate modification with lysine 63-linked ubiquitin chains through the UBC13-UEV1A ubiquitin-conjugating enzyme. J. Biol. Chem. 282: References 29936–29945. 1. Cao, Z., J. Xiong, M. Takeuchi, T. Kurama, and D. V. Goeddel. 1996. TRAF6 is a 23. Siednienko, J., R. Jackson, M. Mellett, N. Delagic, S. Yang, B. Wang, L. S. Tang, signal transducer for interleukin-1. Nature 383: 443–446. J. J. Callanan, B. P. Mahon, and P. N. Moynagh. 2012. Pellino3 targets the IRF7 2. Ishida, T., Si. Mizushima, S. Azuma, N. Kobayashi, T. Tojo, K. Suzuki, S. Aizawa, pathway and facilitates autoregulation of TLR3- and viral-induced expression of T. Watanabe, G. Mosialos, E. Kieff, et al. 1996. Identification of TRAF6, a novel type I interferons. Nat. Immunol. 13: 1055–1062. Zhu et al. Supplemental Figure 1

Supplemental Figure 1. Effects of Cul-5 hemizygosity on LPS-induced signaling. (A) IL-6 and TNF-Į secreted from Cul-5+/+ and Cul-5+/- BMDMs were measured by ELISA after stimulation with LPS (100 ng/ml) for 12 h. (B) Densitometry analysis of data in Figure 1G and repeating experiments (n=3). The results are presented as means ± SEM. *, p<0.05, **, p<0.01 and ***, p<0.001. Zhu et al. Supplemental Figure 2

FLAG-TRAF6 +++++++ HA-Ub - + +-- -- HA-Ub-K63 - --+ + -- HA-Ub-K48 - ---- + + Myc-Cul-5 - - ++-+-

IB:

HA IP:FLAG

(kD) FLAG -58

Myc -85

Input FLAG -58

Supplemental Figure 2. Cul-5 promotes K63-linked ubiquitination of TRAF6. Expression of Cul-5 in transfected HEK293T cells together with the K63 mutant of ubiquitin (all Lys but K63 were mutated to Ala) but not the K48 mutant (all Lys but K48 were mutated to Ala) potentiated TRAF6 polyubiquitination. For input controls, 1/10 of the samples were analyzed by gel electrophoresis and immunoblotting. Data shown are representative of at least 3 independent experiments, each producing similar results.