Activity of Junb in T Lymphocytes Sumoylation Regulates The

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SUMOylation Regulates the Transcriptional Activity of JunB in T Lymphocytes Johan Garaude, Rosa Farrás, Guillaume Bossis, Seyma Charni, Marc Piechaczyk, Robert A. Hipskind and Martin This information is current as Villalba of September 29, 2021. J Immunol 2008; 180:5983-5990; ; doi: 10.4049/jimmunol.180.9.5983 http://www.jimmunol.org/content/180/9/5983 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 © 2008 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

SUMOylation Regulates the Transcriptional Activity of JunB in T Lymphocytes1

Johan Garaude,2,3 Rosa Farra´s,4 Guillaume Bossis, Seyma Charni, Marc Piechaczyk, Robert A. Hipskind, and Martin Villalba2

The AP-1 family member JunB is a critical regulator of T cell function. JunB is a transcriptional activator of various cytokine genes, such as IL-2, IL-4, and IL-10; however, the post-translational modiﬁcations that regulate JunB activity in T cells are poorly characterized. We show here that JunB is conjugated with small ubiquitin-like modiﬁer (SUMO) on lysine 237 in resting and activated primary T cells and T cell lines. Sumoylated JunB associated with the chromatin-containing insoluble fraction of cells, whereas nonsumoylated JunB was also in the soluble fraction. Blocking JunB sumoylation by mutation or use of a dominant- negative form of the SUMO-E2 Ubc-9 diminished its ability to transactivate IL-2 and IL-4 reporter genes. In contrast, nonsumoylable JunB mutants showed unimpaired activity with reporter genes controlled by either synthetic 12-O-tetradecanoylphorbol- Downloaded from 13-acetate response elements or NF-AT/AP-1 and CD28RE sites derived from the IL-2 promoter. Ectopic expression of JunB in activated human primary CD4؉ T cells induced activation of the endogenous IL-2 promoter, whereas the nonsumoylable JunB mutant did not. Thus, our work demonstrates that sumoylation of JunB regulates its ability to induce cytokine gene transcription and likely plays a critical role in T cell activation. The Journal of Immunology, 2008, 180: 5983–5990.

ctivator protein-1 collectively describes a class of struc- giving rise to transactivating or transrepressing complexes with http://www.jimmunol.org/ turally and functionally related proteins that are charac- different biochemical properties. A terized by a basic DNA-binding domain and a leucine- Jun proteins are implicated in numerous cellular processes, such zipper dimerization motif. It principally comprises members of the as transcriptional control, proliferation, differentiation, and tumor- Jun (c-Jun, JunB, and JunD) and Fos (c-Fos, FosB, Fra-1, and igenesis (3–6). In particular, JunB has a unique, nonredundant Fra-2) protein families. These proteins bind as dimers to specific function in vivo, because its inactivation in mice causes vascular sequences in the promoter and enhancer regions of target genes; defects in extra-embryonic tissues that lead to embryonic lethality the canonical site mediates activation by 12-O-tetradecanoylphor- (7). In adult mice, JunB plays an important regulatory role in the 5 bol-13-acetate (TPA) and is termed a TPA response element hematopoietic system and tumorigenesis. The embryonic lethality by guest on September 29, 2021 (TRE). Additionally, some members of the activating transcription of JunBϪ/Ϫ mice is reversed by expression of a junB transgene factor and Jun dimerization protein families can form AP-1 com- under the control of the Ubiquitin promoter (Ubi-JunB). However, plexes, primarily with Jun proteins, and bind to TRE-like se- these animals develop a myeloid hyperproliferation that progresses quences (1, 2). Unlike the Jun proteins, Fos family members can- to a disease resembling chronic myeloid leukemia; remarkably, not form homodimers, but heterodimerize with Jun partners, this corresponds to silencing of the Ubi-JunB transgene (8, 9). In agreement with this, junB expression is diminished in some human chronic myeloid leukemia (10) and B cell leukemias (11, 12). Sim- Institut de Geńe´tique Molećulaire de Montpellier, Centre National de la Recherche ilarly, JunB expression blocks leukemia induced by knockdown of Scientifique, Unite´Mixte de Recherche 5535, Montpellier, France PU.1 in mouse hematopoietic stem cells (13). Although this sug- Received for publication August 27, 2007. Accepted for publication March 4, 2008. gests that JunB is a tumor suppressor, it appears to be oncogenic The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance in T cells, because some hyperproliferative T cell lymphomas with 18 U.S.C. Section 1734 solely to indicate this fact. show JunB overexpression (14–18). Accordingly, JunB is a criti- 1 This work was supported by the l’Institut National du Cancer/Cance´ropoˆle Grand cal regulator of the expression of cytokines important for T lym- Sud-Ouest (to M.V.), “La Ligue Nationale Contre le Cancer. Comite´de la Loze`re” (to phocyte proliferation and differentiation, namely IL-4 (5, 19, 20) M.V.), “Association pour la Recherche sur le Cancer” (to R.A.H.), and fellowships from “La Ligue Nationale Contre le Cancer. Comite Nationale” (to J.G.) and “La and IL-2 (21–23). Moreover, JunB expression and binding capac- Ligue Nationale Contre le Cancer. Region Languedoc-Rousillon” (to S.C.). M.P. is an ity are decreased in anergic T cells, which do not produce IL-2 “Equipe labelliseé” by the “La Ligue Nationale Contre le Cancer”. R.F. was a Human Frontier Science Program postdoctoral fellow. (24–27). 2 Address correspondence and reprint requests to Dr. Johan Garaude or Dr. Martin The activity of AP-1 is critically modulated by post-translational Villalba, Institut de Geńe´tique Molećulaire de Montpellier, 1919 route de Mende, modifications, in particular, phosphorylation mediated by MAPK 34293 Montpellier cedex 5, France. E-mail addresses: [email protected] cascades. The JNK pathway is an important regulator of c-Jun and [email protected] function; however, its role in JunB phosphorylation remains un- 3 Current address: Mount Sinai Medical School, New York, NY 10029. clear, even though JunB contains a docking site for JNKs (5, 28, 4 Current address: Centro de Investigacioń Prıńcipe Felipe, Fundacioń Valenciana de Investigaciones Biome´dicas, Valencia, Spain. 29). Instead, JNKs regulate the E3 ubiquitin ligase Itch that targets JunB for ubiquitylation in T cells (30). Thus, mice lacking Itch 5 Abbreviations used in this paper: TPA, 12-O-tetradecanoylphorbol-13-acetate; SUMO, small ubiquitin-like modifier; TRE, TPA response element; HA, hemagglu- show an accumulation of JunB in helper T cells, which leads to an tinin; wt, wild type; DN, dominant negative; NEM, N-ethylmaleimide; Q-PCR, quan- increase in Th2 differentiation (31). titative-PCR; IMAC, immobilized metal affinity chromatography. Sumoylation is a reversible modification that conjugates an Copyright © 2008 by The American Association of Immunologists, Inc. 0022-1767/08/$2.00 ubiquitin-like peptide, SUMO (for small ubiquitin-like modifier), www.jimmunol.org 5984 SUMOYLATION OF JunB IN T CELLS

onto speciﬁc lysine residues of substrate proteins (32–34). Three the DNA mix and electroporated at 260 mV, 960 mF in 400 ␮l of RPMI SUMO isoforms are expressed in mammalian cells. SUMO-1 1640. Murine primary T cells were isolated from spleens using a murine T shows 47% homology at the protein level with SUMO-2 and -3, cell negative isolation kit (Dynal Biotech) according to the manufacturer’s protocol. Human PBMCs were isolated as previously described (45). Hu- whereas SUMO-2 and -3 are 95% homologous (33). Sumoylation man CD4ϩ T cells were isolated from fresh whole blood using RosetteSep of target proteins is a multistep process that is mechanistically human CD4ϩ T cell enrichment (Stem Cell Technologies) according to the analogous to ubiquitination. An E1-activating enzyme, namely supplier’s protocol. Five to ten million CD4ϩ T cells were transfected Aos1/Uba2, and an E2-conjugating enzyme, Ubc9, are sufﬁcient using a Human T cell Nucleofector kit (Amaxa) following the manufacturer’s protocol. When required, CD4ϩ T cells were activated 6 h post- for SUMO conjugation in vitro. In vivo, this sometimes requires transfection as described below. E3 factors, such as PIAS proteins, PC2 and RanBP2 (32). Sumoy- lation affects target protein function in a variety of ways and has T cell activation now been implicated in many cellular processes, notably regula- Plates were coated with 2 ␮g/ml each anti-human or -mouse CD3 and tion of gene expression (33, 35). Numerous proteins involved in CD28 Abs in PBS for4hat4°C. T cells were incubated on these plates for transcriptional control are sumoylated, including transcription fac- the indicated times. Plates were placed on ice, and cells were lysed with tors, coactivators, corepressors, and histones, to name a few. In SDS-PAGE sample buffer (2% SDS, 0.1 M DTT, 50 mM Tris [pH 6.8], and 10% glycerol). PMA (100 ng/ml) and ionomycin (1 ␮g/ml) were added most cases, sumoylation represses transcription; the mechanisms to the cell suspension for the indicated times. Cells were washed once with include recruiting repressor complexes directly on promoters (35) PBS and processed. or by sequestering proteins in nuclear subcompartments. Although Reporter gene assays sumoylation represses the activity of the AP-1 components c-Fos

and c-Jun (36), it enhances that of other transcription factors, such In all experiments, cells were transfected with a ␤-galactosidase reporter Downloaded from ϫ 6 as NF-AT1 and Oct4 (37, 38). plasmid as control of transfection (40). Transfected cells (1 10 ) were harvested after 2 days and washed twice with PBS. Cells were lysed in 100 In the present study, we show that endogenous and ectopically ␮l luciferase lysis buffer (Promega) and luciferase assays (40 ␮l) per- expressed JunB is sumoylated in both resting and activated T lym- formed according to the manufacturer’s instructions (Promega) using a phocytes. Of the three SUMO consensus sites, the site containing Berthold luminometer. For ␤-galactosidase assays, 40 ␮l of lysates were lysine 237 is the primary site of conjugation of either SUMO-1 or added to 200 ␮lof␤-galactosidase assay buffer (50 mM phosphate buffer ␮ [pH 7.4], ortho-nitro-phenyl-galactopyranoside 200 g, 1 mM MgCl2, and -2 in T cells. Remarkably, blocking JunB sumoylation, either by 50 mM 2-ME) and the absorbance measured at 400 nm. The results were http://www.jimmunol.org/ mutation or by coexpression with inactive Ubc9, strongly dimin- expressed as luciferase units normalized to the corresponding ␤-galactosi- ishes JunB-mediated transactivation of reporter genes controlled dase activity. The expression level of the transfected proteins was routinely by the IL-2 and IL-4 promoters. In contrast, JunB sumoylation controlled by immunobloting analysis. plays no role in its activity on synthetic TRE or AP-1 binding sites Small-scale fractionation derived from the IL-2 promoter. These data suggest that sumoylation of JunB plays an important role in the transcriptional acti- Small-scale biochemical fractionation was performed as described previously (46). Brieﬂy, 20 ϫ 106 cells were collected, washed with precold vation of certain cytokine genes in T cells. PBS, and resuspended in 0.5 ml of buffer A (10 mM HEPES [pH 7.9], 10

mM KCl, 1.5 mM MgCl2, 0.34 M sucrose, 10% glycerol, 1 mM DTT, Materials and Methods “complete” protease inhibitor mixture, and 20 mM NEM). After addition by guest on September 29, 2021 Plasmids of Triton X-100 (0.1% final concentration), cells were incubated on ice for 8 min, and nuclei (fraction P1) were collected by centrifugation (5 min, Human hemagglutinin (HA)-tagged JunB wild type (wt), the single mu- 1300g, 4°C). The supernatant (fraction S1) was clarified by high-speed tants (K237R, K267R, and K301R) and the JunB triple mutant (K237R/ centrifugation (5 min, 20000g, 4°C), and the supernatant (fraction S2) was K267R/K301R, termed JunB-3R) were cloned in the pcDNA3 vector (In- collected. The P1 nuclei were washed once in buffer A and lysed on ice for vitrogen). Point mutations were made using the QuickChange site-directed 30 min in buffer B (3 mM EDTA, 0.2 mM EGTA, 1 mM DTT, protease mutagenesis kit (Stratagene) according to the manufacturer’s protocol. The inhibitor mixture, 20 mM NEM, and 20 mM iodoacetamide). The insoluble expression vectors pcDNA3-His6-SUMO-2 and pCDNA3-His6-SUMO-1 chromatin (fraction P3) and soluble (fraction S3) fractions were separated (39), the TRE-luciferase reporter (40), and IL-2 Luc reporter containing the by centrifugation (5 min, 1.700g, 4°C). The P3 fraction was washed once 300bp upstream of the initiation site (41) have been described. The IL-4 with buffer B and resuspended in SDS-PAGE sample buffer. Luc reporter plasmid was provided by Dr. Glimcher (42). wt Ubc9 in pCDNA3 was previously described (36). Dominant negative Ubc9 (Ubc9 Purification of SUMO conjugates DN) was obtained by mutating cysteine 93 to serine and leucine 97 to Forty million cells were lysed in 2 ml of Solution A (6 M guanidium-HCl, serine. 0.1MNa2HPO4/NaH2PO4, 0.1 M Tris-HCl [pH 8.0], 20 mM NEM, 20 Abs and reagents mM iodoacetamide, and 10 mM imidazole). Samples were sonicated (30 s) and centrifuged for 15 min at 14,000 rpm at 4°C. Supernatants were trans- The anti-human CD3, anti-mouse CD3, and anti-mouse CD28 mAbs were ferred to a new tube containing 30 ␮l of prewashed Ni2ϩ-NTA magnetic purified as previously described (43, 44). The anti-human CD28 mAb was beads (Promega) and incubated for4hatroom temperature on a rocking from BD Biosciences. The JunB rabbit polyclonal Ab was from Santa Cruz platform. Twenty five ␮l of the supernatants were used as inputs. Beads Biotechnology, and the mouse monoclonal JunB Ab was a kind gift from were sequentially washed with 2 ml of the following solutions: A, B (8 M

the laboratory of Dr. M. Yaniv. The rat monoclonal anti-HA (3F10) was urea, 0.1 M Na2HPO4/NaH2PO4, 0.1 M Tris-HCl [pH 8.0], 20 mM NEM, from Roche. Donkey anti-rabbit or sheep anti-mouse IgG Abs were from 20 mM iodoacetamide, and 10 mM imidazole), and C (8 M urea, 0.1 M

Amersham Biosciences, and the goat anti-hamster IgG was from Pierce. Na2HPO4/NaH2PO4, 0.1 M Tris-HCl [pH 6.3], 20 mM NEM, 20 mM io- N-ethylmaleimide (NEM), iodoacetamide, imidazole, PMA, DTT, and the doacetamide, 10 mM imidazole, and 0.2% Triton X-100). After two addi- ionomycin were from Sigma-Aldrich. The “Complete” protease inhibitor tional washes in buffer C without Triton X-100, proteins were eluted by mixture was from Roche. adding 50 ␮l of SDS-PAGE sample buffer and analyzed by SDS-PAGE. Cell culture and transfection RNA isolation and quantitative-PCR (Q-PCR) All cells were grown in RPMI 1640 medium (Sigma-Aldrich) supple- Total RNA was isolated from 3 ϫ 106 human CD4ϩ T cells using a mented with 10% FBS, 2 mM glutamine, 1 mM sodium pyruvate, 10 mM GenElute Mammalian total RNA Miniprep kit (Sigma-Aldrich) using the HEPES [pH 7.4], 1ϫ MEM nonessential amino acid (Invitrogen), 50 ␮M supplier’s instructions. One to 2 ␮g of RNA were treated with DNase I 2-ME, and 100 U/ml each of penicillin G and streptomycin. Jurkat cells in (Promega), and 1 ␮g RNA was used to synthesize cDNA using pdN9 logarithmic growth phase were transfected with the indicated amounts of primers and M-Mulv reverse transcriptase (New England Biolabs) using plasmid by electroporation (40). In each experiment, cells were transfected the supplier’s protocol. Q-PCR for IL-2 and S26 was performed using Taq with the same total amount of DNA by adding the required quantities of polymerase and SYBR green reagent (Invitrogen) according to the manu- empty vector. Cells were incubated for 10 min at room temperature with facturer’s instructions. The Q-PCR was performed in a 96-well plate using The Journal of Immunology 5985

the Stratagene MX3000P, and analysis was performed with MXPro soft- ware. All samples were analyzed in duplicates, and the results were ex-

pressed as fold induction compare with the empty pCDNA3 vector. Primers used in this study were hIL-2, sense: TCA CCA GGA TGC TCA CAT TTA AGT; antisense: GA GGT TTG AGT TCT TCT TCT AGA CAC TGA; and for hRPS26, sense: GAA CGC ATT TCC ACC CTA GA; antisense: GCA CGA CCA TTG TTC CTT CT. ELISA Human CD4ϩ T cells were seeded at 2.106 cells/ml in 24-well plates (Pharmingen) precoated with 2 ␮g/ml anti-CD3 and anti-CD28 Abs and left at 37°C for 12 h. IL-2 secretion was measured using Human IL-2 quantikine ELISA kit (R&D systems) following the manufacturer’s instructions. All samples were analyzed in duplicates, and the results were expressed as fold induction compare with cells transfected with empty

pcDNA3 vector. Statistical analysis The statistical analysis of the differences between means of paired samples was performed using the paired t test. The results are given as the conﬁ- dence interval (p). All the experiments described in the ﬁgures were per-

formed at least three times with similar results. Downloaded from Results JunB is sumoylated in primary and leukemic T lymphocytes JunB migrates in SDS-PAGE as a doublet with an apparent molecular mass of 40 kDa in the murine leukemic T cell line EL4, as

well as in mouse and human primary T cells (Fig. 1). This com- FIGURE 1. JunB is sumoylated in T cells. A, JunB expression in stim- http://www.jimmunol.org/ plements the list of resting T cells of different origins that express ulated EL-4 cells. One million EL-4 cells were treated with 100 ng/ml this protein constitutively (5, 25, 27). In EL4 cells, activation with PMA and 1 ␮g/ml ionomycin for the indicated times and then lysed. Equiv- PMA/ionomycin treatment increases JunB expression, as previ- alent amounts of protein were separated by SDS-PAGE, transferred to ␤ ously described in other cells (47, 48). Moreover, we observed a membranes and incubated with Abs against JunB and -Actin. B, JunB expression in stimulated human PBMCs and murine T lymphocytes. Hu- species of JunB migrating at 60 kDa that is more easily visualized man PBMCs or murine T lymphocytes were purified and treated with 100 after PMA/ionomycin treatment (Fig. 1A). This band was also ob- ng/ml PMA and 1 ␮g/ml ionomycin or plated on anti-(CD3ϩCD28)-coated served when we used a mouse monoclonal JunB Ab against the dishes for the indicated periods. Cells were processed as described in A. JunB N-terminal. The 20 kDa increase in size indicated a post- The arrow indicates sumoylated JunB. C, Endogenous JunB is sumoylated translational modification, such as sumoylation, which has already in Jurkat cells. Forty million Jurkat cells were transfected with 2 ␮gof by guest on September 29, 2021 ␮ been described for ectopically expressed JunB in Hela cells (49). His6-SUMO-2 and 0.5 g of Ubc-9. Two dats later, cells were treated with Importantly, the 60 kDa JunB band was also observed in mouse 100 ng/ml PMA and 1 ␮g/ml ionomycin for 4 h and lysed. Sumoylated and human primary T cells activated either with PMA/ionomycin proteins were purified by immobilized metal affinity chromatography or anti-CD3/anti-CD28. Maximal activation with the latter was (IMAC) and JunB visualized by immunoblotting (top panel). Immunoblot observed 2 h after stimulation; at 1 h, we found a small increase in analysis of the inputs, representing 10% of the total protein, is shown in the bottom panel. nonmodified and modified proteins (Fig. 1B). To confirm that this band comigrates with sumoylated JunB, we

cotransfected Jurkat T cells with expression vectors for His6- SUMO-2 and the SUMO E2 enzyme Ubc9. After activation with nous JunB was conjugated to SUMO-2 in both resting and acti- PMA/Ionomycin, cells were lysed under denaturing conditions to vated T cells (Fig. 2A). Similar results were obtained using an

prevent desumoylation, and SUMO-2 conjugates were purified by expression vector for His6-SUMO-1 (Fig. 3B). Although JunB was metal affinity chromatography. A fraction of endogenous JunB was predominantly monosumoylated, SUMO-2 also generated a ladder detected in the latter by immunoblot analysis, where it migrated at reflecting conjugation of multiple peptides (Fig. 2A, upper panel). 60 kDa (Fig. 1C). This strongly suggests that the 60 kDa band This could arise from addition of SUMO-2 chains to a single site observed in Fig. 1, A and B, represents monosumoylated JunB. in JunB, monosumoylation on multiple sites, or a combination of Most studies have needed to use protein enrichment or overex- the two (see below). pression of the protein or SUMO isoforms to detect the sumoylated forms. These include the transcription factors NF-AT, Oct-4, and Lysine 237 is the primary sumoylation site on JunB in vivo Elk-1 (38, 50, 51). In contrast, we readily detected the upper JunB Sumoylation generally occurs on the lysine residue within a ␺KXE band in total cell extracts without enrichment of sumoylated pro- consensus motif, where ␺ is an aliphatic amino acid and X any teins or ectopic expression of JunB or SUMO isoforms. Hence, the amino acid (33). Three motifs are present in JunB, surrounding proportion of endogenous JunB appearing to be sumoylated is lysines 237, 267, and 301 (Fig. 2B). To determine which sites are higher than many other proteins. used in vivo, these lysines were mutated to arginines individually To facilitate further molecular analysis, we checked whether (JunB-K237R, -K267R, and -K301R, Fig. 2B) or in combination ectopically expressed JunB is also sumoylated in Jurkat cells. (JunB-K237R/K267R/K301R, termed JunB-3R). The expression Transfections were performed as described above with the addition vectors were transfected into Jurkat cells, which were then acti- of an expression vector for HA-tagged JunB wt. Affinity-purified vated by PMA/ionomycin and lysed. Overexpressed wt or mutant His-tagged proteins were separated by SDS-PAGE, and ectopic JunB in the whole cell extracts was detected in Western blots using JunB was visualized in Western blots by immunodetection of the an anti-HA Ab. Mutation of lysine 237, but not that of lysines 267 HA epitope. Like the endogenous protein, a proportion of exoge- or 301, strongly diminished JunB modification by endogenous 5986 SUMOYLATION OF JunB IN T CELLS Downloaded from

FIGURE 3. Lysine 237 is the SUMO acceptor in Jurkat cells. A, Ten million Jurkat cells were transiently transfected with 5 ␮g of expression vectors encoding the indicated HA-Tagged JunB proteins. Cells were activated for 4 h with 100 ng/ml PMA and 1 ␮g/ml ionomycin, then processed as described in the legend to Fig. 2 to visualize HA-tagged JunB. http://www.jimmunol.org/ FIGURE 2. Mutation of the three consensus sumoylation sites of JunB The panel contains a long exposure to reveal the band of sumoylated JunB, prevents sumoylation. A, Forty million Jurkat cells were transiently trans- indicated by the arrow. B, Forty million Jurkat cells were transiently trans- ␮ ␮ ␮ fected with 2 gofaHis6-SUMO-2 expression vector, together with 5 g fected with 2 g of an expression vector for His6-SUMO-1, together with of vectors encoding either wt HA-Tagged JunB (JunB wt) or the mutant 5 ␮g of vector encoding HA-Tagged JunB wt or -K237R. Two days later, HA-JunB-3R, where lysines 237, 267, and 301 are changed to arginine. cells were treated with 100 ng/ml PMA and 1 ␮g/ml ionomycin for4hand Half of the cells were stimulated with 100 ng/ml PMA and 1 ␮g/ml iono- lysed. Sumoylated proteins were purified by IMAC, separated on SDS- mycin for 4 h. After lysis, sumoylated proteins were purified by IMAC, PAGE, and JunB visualized by immunoblotting. The asterisk indicates separated by SDS-PAGE, and visualized by immunoblotting with an anti- nonspecific binding of JunB.

HA Ab (top panel). Immunoblot analysis of the inputs, representing 10% by guest on September 29, 2021 of the total protein (WCE), is shown in the bottom panel. The arrow indicates sumoylated JunB. B, Diagram depicting the major structural fea- showed endogenous JunB to be primarily nuclear in primary T tures of JunB. The consensus site for Ubc9 binding and sumoylation is cells and Jurkat cells, without revealing any preferential sub- shown, where ␺ is an aliphatic amino acid, X is any amino acid, and K the nuclear association (data not shown). This conﬁrms fractionation lysine conjugated to SUMO. Also indicated are the sequences harboring experiments in Jurkat cells, where JunB was exclusively in the the three major consensus sites in JunB, surrounding lysines 237, 267, and nucleus (40). 301 (bold letters). DBD: DNA-binding domain; NLS: Nuclear Localization Signal; and bZip: basic domain-leucine zipper. Like JunB, the AP-1 family members c-Jun and c-Fos are conjugated to SUMO (36, 49), and sumoylated c-Fos preferentially localizes in an insoluble nuclear fraction in HeLa cells (36). To SUMO (Fig. 3A). Nevertheless, these two lysines were used, albeit investigate whether this was also the case for sumoylated JunB, we weakly, because the residual level of the 60 kDa band seen with performed nuclear fractionation experiments (Fig. 4). Because we JunB-K237R completely disappeared in JunB-3R (Fig. 3A). To wished to localize sumoylated vs nonsumoylated endogenous conﬁrm this, we overexpressed either JunB wt, K237R, or 3R in

Jurkat cells together with His6-SUMO-1 or His6-SUMO-2 and pu- riﬁed SUMO conjugates from cell lysates by nickel afﬁnity chromatography under denaturing conditions (Figs. 2A and 3B). In- deed, mutation of lysine 237 strongly impaired JunB conjugation with SUMO-1 (Fig. 3B) or SUMO-2 (data not shown). A low level of sumoylation was still found with JunB-3R (Fig. 2A), indicating the presence of another weak, nonconsensus site in JunB. Taken together, these data show that lysine 237 in JunB is the preferred conjugation site for SUMO.

Sumoylated JunB is enriched in a nonsoluble nuclear fraction FIGURE 4. Subcellular distribution of sumoylated JunB in human PBMCs. Human PBMCs were either untreated or stimulated for 1 h with SUMO conjugation has been linked to nuclear localization of nu- PMA (100 ng/ml) and ionomycin (1 ␮g/ml), followed by a small-scale cell merous transcription factors (33), such as the T cell regulatory fractionation. The S2 fraction contains soluble proteins, P3 is the Triton- protein NF-AT1 (38). Moreover, certain sumoylated transcrip- insoluble nuclear fraction, and W is the wash of P3. Cell equivalent tional regulators are found associated with a nuclear subdomain amounts of each fraction was separated by SDS-PAGE and proteins visu- that is implicated in leukemia, namely promyelocytic leukemia alized by immunoblotting with the indicated Abs. For the input, 2% of total nuclear bodies (Ref. 33). Indirect immunoﬂuorescence microscopy protein was analyzed the same way. The arrow indicates sumoylated JunB. The Journal of Immunology 5987 Downloaded from http://www.jimmunol.org/

FIGURE 6. Sumoylation does not affect JunB transcriptional activity on minimal AP-1-driven reporter genes. Twenty million Jurkat cells were transfected with 5 ␮g of expression vectors for HA-tagged JunB wt, JunB- K237R, and JunB-3R as indicated, together with 2 ␮g of a reporter gene controlled by the 4ϫTRE (A), ARRE-2 (NF-AT/AP-1) (B), or CD28RE (C). The latter two are derived from the IL-2 promoter. Two days after by guest on September 29, 2021 FIGURE 5. Sumoylation controls JunB transcriptional activity on the transfection, luciferase activities were measured. The data are presented as minimal IL-2 and IL-4 promoters. Twenty million Jurkat cells were trans- the mean Ϯ SD of at least three independent experiments. fected with expression vectors for HA-Tagged JunB wt, JunB-K237R, JunB-3R, and DN Ubc9 as indicated, along with 2 ␮g of a luciferase reporter gene linked to either the IL-2 gene promoter (A and B) or the IL-4 gene promoter (C). Two days after transfection, luciferase activities were (Fig. 3). Therefore, sumoylation of JunB was constitutive and did not measured. Ectopic expression was conﬁrmed by Western blotting (right require T cell activation; still, as the total amount of JunB increased in p Ͻ 0.01 activated T cells via the de novo expression of the protein (Fig. 1), the ,ء :panels). The data were evaluated using the student’s t test compared with cells transfected with JunB wt alone. The data are pre- amount of sumoylated JunB increased comparably. sented as the mean Ϯ SD of at least three independent experiments. Sumoylation activates JunB on IL-2 and IL-4 promoters Sumoylation regulates transactivation by numerous transcription JunB in activated vs nonactivated cells, we performed a 1-h stim- factors and, in many cases, has a repressive effect (35). To evaluate ulation of PBMCs with anti-CD3/CD28 Abs (Fig. 1). Although the effect of SUMO conjugation on trans-activation by JunB, we unconjugated JunB was equally distributed in the soluble and in- performed cotransfection experiments with reporter genes con- soluble nuclear fractions (S2 and P3, respectively, left panels), trolled by the IL-2 and IL-4 promoters, two well-known JunB tar- sumoylated JunB was detectable uniquely in the insoluble fraction. gets in T cells (5, 22, 52). In Jurkat cells, wt JunB activated the The purity of the two fractions was conﬁrmed by the selective IL-2 promoter by 70-fold (Fig. 5A). Interestingly, induction was presence of the chromatin- and matrix-associated protein Topo- half as strong with JunB-K237R, and mutation of all three con- isomerase I in P3 and the nucleoplasmic protein PHAX in S2. This sensus lysines almost abolished activation of the IL-2 promoter. indicates that SUMO-conjugated JunB associates with the insolu- All three proteins were expressed at similar levels (Fig. 5A). Our ble fraction of the nucleus, possibly a subdomain tightly associated results show that K237 was the primary site for sumoylation; when with chromatin. In addition, we detected similar levels of the 60 this site was mutated to alanine, we detected a minor level of kDa band representing endogenous JunB conjugated to SUMO sumoylated JunB (Fig. 3). This indicates that other lysines, espe- (Fig. 4, right panel) in resting and stimulated cells. These data cially K267 and K301 in the other sumoylation consensus sites, show that T cell activation did not increase the percentage of were also conjugated with SUMO. Therefore, we used the triple sumoylated JunB or change JunB intracellular localization. The mutant that was barely sumoylated to investigate the role of sumoylated form of JunB was easily observed after activation be- sumoylation on the transcriptional activity of JunB. This mutant cause activated cells expressed more JunB (Fig. 1). Consistent activated the TRE, NF-AT/AP-1, and CD28 reporters to the same with this, ectopically expressed JunB was sumoylated to a similar extent as wt JunB, indicating that the mutation did not affect basal extent in resting Jurkat cells as endogenous JunB in activated cells function (Fig. 6). In agreement with Li et al. (5), wt JunB activated 5988 SUMOYLATION OF JunB IN T CELLS

respectively, in combination with AP-1, in particular JunB (22, 25, 26). Surprisingly, JunB-K237R and JunB-3R transactivated reporter genes driven by these composite sites at the same level as wt JunB (Fig. 6B and C). Taken together, these results suggest that sumoylation controls JunB transcriptional activity on certain cytokine promoters without intrinsically affecting JunB transactivation.

Sumoylation is essential for endogenous IL-2 promoter activation by JunB To unambiguously investigate the role of JunB sumoylation in primary T cells, we transfected human CD4ϩ T cells with JunB and JunB-3R. Resting CD4ϩ T cells did not express measurable levels of IL-2, and ectopically expressed JunB did not induce its expression (data not shown). Therefore, in the next set of experiments, we used activated CD4ϩ T cells. Like in Jurkat T cells, ectopically expressed JunB wt, but not JunB-3R, was sumoylated ϩ in CD4 T cells (Fig. 7A). wt JunB, but not JunB-3R, signiﬁcantly Downloaded from increased expression of endogenous IL-2 mRNA in CD4ϩ T cells (Fig. 7B). We conﬁrmed this differential effect of JunB wt and -3R by measuring IL-2 levels in supernatants of transfected cells (Fig. 7C). Thus, in primary CD4ϩ T cells, sumoylation of JunB cooperates with other endogenous signals to fully activate the IL-2

promoter. http://www.jimmunol.org/

Discussion We report here that JunB is covalently modiﬁed by either SUMO-1 or SUMO-2 in both human and mouse T cells. The major acceptor site is lysine 237, although weak levels of sumoylation are detectable on other sites. Like the majority of sumoylated proteins (32, FIGURE 7. JunB sumoylation is required for JunB-induced expression 33, 35), a minor proportion of JunB, both endogenous and exog- ϩ

␮ by guest on September 29, 2021 of endogenous IL-2. Ten million CD4 T cells were transfected with 5 g enous, was conjugated with SUMO under steady-state conditions. of expression vectors for HA-Tagged JunB wt or JunB-3R. Ectopic ex- However, we could detect sumoylated JunB in whole cell extracts, pression was confirmed by Western blotting (A). Cells were activated with which was enhanced by enrichment of SUMO-conjugated pro- anti-CD3 plus anti-CD28 Abs as described in Materials and Methods. Eight hours later, IL-2 mRNA expression was measured by Q-PCR using teins. Activation of T cells also allowed us to more easily visualize S26 as the reference gene (B). IL-2 secretion was evaluated by ELISA after sumoylated JunB. This reflected the increase in JunB expression, 12-h stimulation (C). The data were evaluated using the student’s t test: because the ratio between sumoylated and nonsumoylated JunB Ͻ ءء , p 0.005 compared with cells transfected with pCDNA3. The data are remained the same as found in resting cells. The latter observation presented as the mean Ϯ SD of two independent experiments. suggests that this modification of JunB is constitutive and does not require T cell activation. However, this does not exclude that other signals or stresses could regulate JunB sumoylation, as observed the IL-4 reporter gene 4-fold (Fig. 5C); this was reduced to control for c-Fos and c-Jun in Hela cells (36, 39). levels with JunB-3R (Fig. 5C). These data suggested that sumoy- Sumoylation is often linked to a specific intranuclear distribu- lation of JunB potentiates its ability to transactivate the IL-4 and tion of the target proteins. Many sumoylated nuclear proteins lo- IL-2 promoters. calize in nuclear subdomains, such as promyelocytic leukemia nu- Ubc9 is the only SUMO-E2 identified in human cells. As ex- clear bodies, which may serve to sequester them and thereby pected, cotransfection of Ubc-9DN led to inhibition of sumoyla- suppress their activity. Endogenous JunB is nuclear in primary T tion of ectopically expressed JunB (Fig. 5B). Consistent with the cells and Jurkat cells but showed no concentration in a subdomain. results using nonsumoylable mutants, cotransfection of Ubc-9DN Nevertheless, the modified protein does show a striking localiza- led to a 2-fold reduction in JunB-driven activation of the IL-2 tion, as it was found exclusively in the insoluble nuclear fraction reporter gene (Fig. 5B). Although this strongly suggests that of human primary T cells, i.e., associated with a detergent-resistant Ubc9-DN inhibits JunB activity in this context, we cannot exclude nuclear matrix and/or chromatin structure. In contrast, nonmodi- that part of the Ubc9-DN effect was mediated by an effect on fied JunB was equally distributed between soluble and nonsoluble another protein. nuclear chromatin fractions. Thus, JunB resembles c-Fos and sev- Surprisingly, wt JunB and JunB-3R showed indistinguishable eral other nuclear factors in the preferential binding of the sumoy- activity on the reference AP-1 reporter gene, namely the TRE related protein to this insoluble nuclear structure (36, 53). Further porter plasmid that is driven by four canonical binding sites up- work will determine whether sumoylation is a cause or a conse- stream of the TATA box (Fig. 6A). Thus, sumoylation regulates quence of this localization. JunB activity on specific cytokine promoters. The proximal region In certain transcriptional regulators, such as Elk-1, the lysines in of the IL-2 promoter contains several regulatory elements targeted the sumoylation consensus motif were initially identified as being by different transcription factors (23). The composite elements essential for repressing their activity (54). Similarly, this motif is ARRE-2 and CD28RE are recognized by NF-AT and NF-␬B, found in synergy control domains present in c-Myb, C/EBP, SP3, The Journal of Immunology 5989 and nuclear receptors (55). Synergy controls down-regulate syn- Disclosures ergistic activation of reporter genes driven by compound transcrip- The authors have no financial conflict of interest. tional regulatory elements but do not affect the activity of single response elements. Subsequently, this negative regulation was References shown to be dependent upon sumoylation, and, in fact, SUMO 1. Wagner, E. F. 2001. AP-1: introductory remarks. 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Chronic myeloid leukemia with increased granulocyte pro- cytokine promoters but not isolated elements? It could be that genitors in mice lacking junB expression in the myeloid lineage. Cell 104: 21–32. Downloaded from 9. Passegue, E., E. F. Wagner, and I. L. Weissman. 2004. JunB deficiency leads to sumoylated JunB is targeting a different regulatory element than a myeloproliferative disorder arising from hematopoietic stem cells. Cell 119: AP-1, NF-AT, or NF-␬B, or that sumoylated JunB interacts with 431–443. different proteins than nonsumoylated JunB. This will be difficult 10. Yang, M. Y., T. C. Liu, J. G. Chang, P. M. Lin, and S. F. Lin. 2003. JunB gene expression is inactivated by methylation in chronic myeloid leukemia. Blood 101: to establish, because JunB might interact with several AP-1 com- 3205–3211. ponents, e.g., c-Jun and c-Fos. Moreover, the resulting AP-1 dimer 11. Szremska, A. P., L. Kenner, E. Weisz, R. G. Ott, E. Passegue, M. Artwohl, M. Freissmuth, R. Stoxreiter, H. 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