of March 9, 2018.This information is current as in Normal Human T Lymphocytes

180 Site of the IL-2 Promoter−Binding to the Element-Binding Protein and Its Subsequent

Activation of Cyclic AMP-Responsive Participates in the θProtein Kinase C-

TsokosElena E. Solomou, Yuang-Taung Juang and George C.

http://www.jimmunol.org/content/166/9/5665doi: 10.4049/jimmunol.166.9.5665

2001; 166:5665-5674; ;J Immunol 

Referenceshttp://www.jimmunol.org/content/166/9/5665.full#ref-list-1

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Print ISSN: 0022-1767 Online ISSN: 1550-6606. Immunologists All rights reserved.Copyright © 2001 by The American Association of1451 Rockville Pike, Suite 650, Rockville, MD 20852The American Association of Immunologists, Inc.,

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Protein Kinase C-u Participates in the Activation of CyclicAMP-Responsive Element-Binding Protein and Its SubsequentBinding to the 2180 Site of the IL-2 Promoter in NormalHuman T Lymphocytes1

Elena E. Solomou, Yuang-Taung Juang, and George C. Tsokos2

IL-2 gene expression is regulated by the cooperative binding of discrete transcription factors to the IL-2 promoter/enhancer andis predominantly controlled at the transcriptional level. In this study, we show that in normal T cells, the2180 site (2164/2189)of the IL-2 promoter/enhancer is a p-cAMP-responsive element-binding protein (p-CREB) binding site. Following activation of theT cells through various membrane-initiated and membrane-independent pathways, protein kinase C (PKC)-u phosphorylatesCREB, which subsequently binds to the2180 site and associates with the transcriptional coactivator p300. Rottlerin, a specificPKC-u inhibitor, diminished p-CREB protein levels when normal T cells were treated with it. Rottlerin also prevented theformation of p-CREB/p300 complexes and the DNA-CREB protein binding. Cotransfection of fresh normal T cells with luciferasereporter construct driven by two tandem 2180 sites and a PKC-u construct caused a significant increase in the transcription ofthe reporter gene, indicating that this site is functional and regulated by PKC-u. Cotransfection of T cells with a luciferaseconstruct driven by the 2575/157 region of the IL-2 promoter/enhancer and a PKC-u construct caused a similar increase in thereporter gene transcription, which was significantly limited when two bases within the2180 site were mutated. These findingsshow that CREB plays a major role in the transcriptional regulation of IL-2 and that a major pathway for the activation of CREBand its subsequent binding to the IL-2 promoter/enhancer in normal T cells is mediated by PKC-u. The Journal of Immunology,2001, 166: 5665–5674.

I nterleukin-2 is a growth factor for both T and B lymphocytesthat is exclusively produced by T cells upon T cell stimula-tion through both the TCR and the CD28 costimulatory mol-

ecule (1). Although control of IL-2 synthesis occurs at differentlevels, the inducible expression of IL-2 is tightly regulated by mul-tiple transcription factors that bind at distinct sites on the IL-2promoter/enhancer, including AP-1, NF-kB, NF-AT, and Oct (2,3). The2180 site (2164–189 bp) of the IL-2 promoter/enhancerhad been proposed to be a distal AP-1 binding site (4), but re-cently, it was reported that in anergic cells this site binds het-erodimers of cAMP-responsive element-binding protein/cAMP-re-sponsive element modulator (CREB/CREM)3 and not AP-1 (5).

CREB, CREM, inducible cAMP early repressor (ICER), andactivating transcription factor-1 are members of the cAMP-respon-sive NFs and exhibit a high degree of sequence homology. One

common feature is the basic domain/leucine zipper motifs, whichbind an 8-bp regulatory palindromic DNA sequence (cAMP-re-sponsive elements). These NFs are activated following phosphor-ylation by several kinases in response to different signaling routes,including protein kinase A (PKA), protein kinase C (PKC), ribo-somal S6 kinase pp90rsk, mitogen- and stress-activated kinase, mi-togen-activated protein kinase-activated protein kinase-2 (MAP-KAP-K2), and Ca21/calmodulin-dependent kinase IV.Phosphorylation of Ser133 in CREB and Ser117 in CREM acts as amolecular switch, because it dictates the ability of these factors tointeract with the ubiquitously expressed coactivators CREB-bind-ing protein (CBP) and p300 that form a bridge with the basaltranscriptional machinery (6–8).

CREB and CREM consist of the transcriptional activation do-main (Q1, P-box, Q2) and the DNA-binding/dimerization domain(bZip region). Both the CREB and CREM genes encode multipleisoforms. CREB isoforms act as transcription activators, whereasCREM isoforms can act either as activators or repressors. CREMisoforms containing only the P-box or the Q2 domain act as re-pressors. ICER is produced by alternative promoter usage withinthe CREM gene and acts only as a repressor (6–8).

Stimulation of T cells through the TCR leads to immediatephosphorylation and activation of several cytoplasmic protein ty-rosine kinases. Subsequently, phospholipase C is activated and hy-drolyzes inositol phospholipids into inositol polyphosphates anddiacylglycerol (DAG). Inositol polyphosphate leads to elevation ofintracellular Ca21, which acts synergistically with DAG to activatemultiple kinases, including PKC (9, 10). PKC isoforms are serine/threonine-specific protein kinases that can be divided into threesubclasses (11): the conventional PKCs (PKC-a, PKC-b I and II,PKC-g) are activated by DAG, phosphatidylserine, and Ca21; thenovel PKCs (PKC-d, PKC-e, PKC-u, PKC-h, and PKC-m), which

Department of Cellular Injury, Walter Reed Army Institute of Research, SilverSpring, MD 20910; and Department of Medicine, Uniformed Services University ofHealth Sciences, Bethesda, MD 20814

Received for publication December 1, 2000. Accepted for publication February20, 2001.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby markedadvertisementin accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.1 This work was supported by Public Health Service Grant RO1 AI-42269.2 Address correspondence and reprint requests to Dr. George C. Tsokos, Walter ReedArmy Institute of Research, Department of Cellular Injury, Building 503, Room1A32, 503 Robert Grant Road, Silver Spring, MD 20910-7500. E-mail address:gtsokos@usa.net3 Abbreviations used in this paper: CREB, cAMP-responsive element-binding pro-tein; CREM, cAMP-responsive element modulator; CBP, CREB-binding protein;DAG, diacylglycerol; ICER, inducible cAMP early repressor; MAPKAP-K2, mito-gen-activated protein kinase-activated protein kinase-2; MEK, mitogen-activated pro-tein/extracellular signal-regulated kinase kinase; PKA, protein kinase A; PKC, proteinkinase C.

Copyright © 2001 by The American Association of Immunologists 0022-1767/01/$02.00

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are activated by DAG and phosphatidylserine, but not Ca21; andthe atypical PKCs (PKC-z, PKC-i, and PKC-l), which only re-spond to phosphatidylserine.

Expression patterns and differences in their potential substratespecificity suggest that each isoenzyme may be involved in spe-cific regulatory processes (12–14). High protein levels of PKC-uare present in muscle cells, hemopoietic cells, and T but not Blymphocytes (15, 16). Recent studies show that PKC-u (17) is theonly isoform to translocate to the site of contact between T cellsand APCs (11), which occurs only upon exposure to Ag. Further-more, it has been shown that AP-1 (18) and NF-kB (19, 20) areactivated through PKC-u and subsequently bind to the IL-2 pro-moter/enhancer (21).

Mice expressing a dominant-negative form of CREB have de-fective thymocyte proliferation and IL-2 production (22), suggest-ing that CREB is crucial in the transcription of the IL-2 gene. Inthis study, we investigated whether CREB is important in the tran-scription of the IL-2 gene in normal human T cells. Our studiesshow that the2180 site of the IL-2 promoter/enhancer binds p-CREB, and that PKC-u is involved in its phosphorylation.

Materials and MethodsLymphocyte isolation and stimulation conditions

Heparinized peripheral venous blood was obtained from the study subjects.PBMC were separated from RBC on Lymphoprep gradient (NycomedPharma, Oslo, Norway), and T cells were separated subsequently by mag-netic depletion of non-T cells, as recommended by the manufacturer(MACS Pan T cell isolation kit; Miltenyi Biotec, Auburn, CA). Briefly,non-T cells (B cells, monocytes, NK cells, dendritic cells, early erythroidcells, platelets, and basophils) from PBMC were indirectly magneticallylabeled using a cocktail of hapten-conjugated CD11b, CD16, CD19, CD36,and CD56 Abs, and MACS microbeads coupled to an anti-hapten mAb.The magnetically labeled cells were depleted by retaining them on aMACS column in the magnetic field of MidiMACS. The purified T cellswere.95% positive for CD3, as tested using flow cytometry. Where men-tioned, stimulation of T cells was performed using anti-CD3 (OKT3) (10mg/ml) and anti-CD28 (2.5mg/ml) Abs, or 10 ng/ml PMA and 0.5mg/mlionomycin.

Antibodies

Anti-phospho-CREB (rabbit polyclonal IgG), anti-p300 (rabbit polyclonalIgG), and murine anti-human CREB-binding protein (rabbit polyclonalIgG, CBP-NT) Abs were purchased from Upstate Biotechnology (LakePlacid, NY). Anti-phospho-CREM (rabbit polyclonal IgG), anti-CREB(rabbit monoclonal IgG), anti-actin (goat polyclonal IgG), as well as thegoat anti-rabbit and goat anti-mouse HRP-conjugated mAbs were pur-chased from Santa Cruz Biotechnology (Santa Cruz, CA).

Inhibitors

Calphostin C (0.05mM) and rottlerin (30mM) were used for the inhibitionof PKC and PKC-u, respectively (20, 23–25). KT5720 (5mM) was used forthe inhibition of PKA pathway (26); SB 203580 (10mM) and PD 98059(50 mM) for the inhibition of MAPKAP kinase-2 and mitogen-activatedprotein/extracellular signal-regulated kinase kinase (MEK), respectively(27, 28); and W7 (15mM) for the inhibition of calmodulin (29). All in-hibitors were purchased from Calbiochem (La Jolla, CA). Where men-tioned, freshly isolated normal T cells were incubated with the above con-centrations of the inhibitors for 30 min at 37°C (95% O2, 5% CO2),followed by stimulation with PMA alone, PMA and ionomycin, or anti-CD3 and anti-CD28 Abs for 6 h.

Preparation of nuclear extracts

At least five million T cells were used for preparation of extracts for eachexperimental point. T cells following treatment with the appropriate stim-ulus were washed twice in PBS and resuspended in 300ml buffer A (10mM HEPES-KOH (pH 7.9), 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA)with mixture of protease inhibitors (1 mM DTT, 0.5 mM PMSF, 10mg/mlaprotinin, 10 mM NaF, and 1 mM Na3VO4) at 4°C. Cells were incubatedon ice for 15 min, and after adding 25ml of 10% Nonidet P-40 werevigorously vortexed for 10 s and centrifuged to homogenate at 13,000 rpmat 4°C for 30 s. The supernatant cytoplasmic extract was transferred in a

new tube, and the nuclear pellet was resuspended in 30ml buffer B (20 mMHEPES-KOH (pH7.9), 0.4 M NaCl, 1 mM EDTA, 1 mM EGTA) withprotease inhibitors (1 mM DTT, 1 mM PMSF, 10mg/ml aprotinin). Theresuspended nuclear pellet was incubated, rocking for 15 min at 4°C, andthen was spun at 13,000 rpm in a microcentrifuge to remove insolublematerial. The extracts were frozen at280°C until they were assayed. Theprotein content of the extracts was determined using the Bio-Rad (Her-cules, CA) protein assay.

EMSAs

The dsDNA probe of the2180 site on the IL-2 promoter used was 59-catccattcagtcagtctttgggggt-39. The M1 and M2 oligonucleotides representthe 2180 site dsDNA probes in which a base pair was mutated: M1, 59-catccataaagtcagtctttgggggt-39, and M2, 59-catccattcagtcaccctttgggggt-39.GATA dsDNA probe was used as control in the cold-inhibition experi-ments: 59-attcttatctaattcctatcttgattgg-39. The probes were synthesized byLife Technologies (Grant Island, NY). Nuclear extracts (2mg) were incu-bated with 30,000 cpm end-labeled probe, 4ml buffer (Hi-Density 53Tris-borate-EDTA sample buffer; Novex, San Diego, CA), 1ml KCl 1 M,and 1 mg of poly(dG)zpoly(dC) (Sigma, St. Louis, MO) as nonspecificcompetitor for 30 min at room temperature in total volume 20ml. Gelelectrophoresis was then run on 6% DNA retardation gels (Novex) in 0.53Tris-borate-EDTA buffer. The gel was then dried under vacuum on blottingpaper, and the protein-DNA complexes were visualized using phosphorimager (Bio-Rad).

The comparison concerning band density was made for each individualgel, and quantitation of each of the bands was determined using the Quan-tityOne software (Bio-Rad). In all the assays performed, the backgroundwas determined for each individual lane and subtracted from the banddensity.

Supershift analysis

Nuclear and cytoplasmic extracts were preincubated with 3mg of the in-dicated Abs for 15 min at room temperature before the addition of thelabeled probe. Supershift analysis was then completed as described above.

Immunoblotting and immunoprecipitation

Nuclear proteins (10mg/lane) were resolved by 10% Tris-glycine SDS gel(Novex) electrophoresis at 125 V. Resolved proteins from the gel weretransferred on Immobilon polyvinylidene difluoride membrane (Millipore,Bedford, MA; Sigma) in transfer buffer at 14 V overnight at 4°C. Themembrane was blocked for 2 h with 5% BSA in PBS and 0.05% Tween 20and incubated in primary Ab. For the detection of CREB, the blot was firstincubated with anti-p-CREB Ab for 2 h at room temperature. The mem-brane was then washed for 15 min in PBS, by changing the buffer every 5min, and subsequently was incubated with anti-rabbit Ab HRP conjugated.Secondary HRP-conjugated Ab incubation was performed at 1/1000 dilu-tion at room temperature for 1 h. Following the washing of the membranewith PBS and 0.05% Tween 20 for 1 h by changing the buffer four timesand PBS for another 15 min, detection of the bands of interest was per-formed with the ECL system (Amersham Pharmacia Biotech, Bucking-hamshire, U.K.). Membranes were stripped after the first blotting in Im-munoPure IgG elution buffer (Pierce, Rockford, IL) for 1 h shaking at roomtemperature, reblocked, and reblotted with anti-p-CREB Ab, as describedabove, to detect the p-CREB protein levels. To evaluate equal loading ofthe lanes with protein, membranes were restripped, reblocked, and reblot-ted with anti-actin goat polyclonal Ab.

To immunoprecipitate (30) p300, 50mg of nuclear or cytoplasmic pro-tein extract, obtained as described earlier, was incubated with 2mg of p300polyclonal Ab at 4°C shaking for 1 h. A total of 25ml of 50% slurry ofprotein A/G plus agarose (Santa Cruz) was added to capture immune com-plexes and incubated for 1 h at 4°C on a rotator. Agarose-bound immunecomplexes were collected, washed four times with lysis buffer (20 mMHEPES-KOH (pH 7.9), 0.4 M NaCl, 1 mM EDTA, 1 mM EGTA) withprotease inhibitors (1 mM DTT, 1 mM PMSF, 10mg/ml aprotinin), and thepellet was boiled for 3 min with 50ml Laemmli sample buffer to dissociatethe agarose beads from the immune complexes. Agarose was discarded,and the supernatants containing the immune complexes were brought to afinal concentration of 5% with 2-ME. Electrophoretic protein fractionationof equal sample volumes (25ml of sample per lane) on 10% Tris-glycineSDS gels (Novex) was followed by transfer of the membranes on Immo-bilon polyvinylidene difluoride membrane (Millipore; Sigma) and immu-noblotting with rabbit polyclonal anti-p-CREB Ab. Detection was per-formed as described above. Densitometric measurements were performedusing QuantityOne software (Bio-Rad).

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Transfection and luciferase assays

Freshly isolated normal T cells were left overnight in medium containing10% FCS and PHA (1mg/ml) at 37°C. We used PHA because we havefound that it permits maximal transfection efficacy compared with cellstransfected in the absence of PHA. However, our transfection experimentsdescribed in this work were performed without PHA, and the patterns ofluciferase activity were similar (31, 32). Luciferase reporter plasmid(pGL2; Promega, Madison, WI) driven by either two2180 sites on theIL-2 promoter, or the whole IL-2 promoter freed from pIL2 CAT (kind giftof Dr. A. Rao, Harvard University, Cambridge, MA), plasmids expressingconstitutively active PKC-u, a, ande isoforms (kind gift of Dr. G. Baier(University of Innsbruck, Innsbruck, Austria), Dr. A. Altman (La JollaInstitute of Allergy and Immunology, San Diego, CA), and Dr. W. C.Greene (University of California, San Francisco, CA), and plasmid ex-pressing CREMa (kind gift of Dr. P. Sassone-Corsi (Centre National de laRecherche Scientifique, Strasbourg, France)) were used for transfectionexperiments. Normal T cells (53 106) were transiently transfected byelectroporation (Gene pulser II; Bio-Rad) at 250 mV, 950mF in 0.25 ml ofcomplete medium (RPMI, 10% FCS). The total amount of plasmids usedin each sample was 8mg. After 18 h, T cells were stimulated with PMAand ionomycin for 6 h, as we have found in preliminary experiments to bethe optimal time for greater luciferase activity. Cytoplasmic extracts wereprepared using a luciferase assay kit (Promega). Briefly, cells were resus-pended in lysis solution (Tropix, Bedford, MA) with DTT (0.01 M) andincubated at room temperature for 15 min. After a brief centrifugation, 30ml of the supernatant was used with 100ml of luciferase assay reagent.Luminescence was measured immediately for 30 s using a luminometer.Transfection efficiency was established in all samples by cotransfection ofa plasmid encodingb-galactosidase (2mg for each sample). The luciferaseactivity was normalized using theb-galactosidase readings. Jurkat cells(5 3 106 for each sample) were transfected with the same constructs asdescribed above. After transfection, Jurkat cells were rested overnight, fol-lowed by stimulation with PMA and ionomycin for 6 h. Cells were col-lected, and luciferase activity was examined, as described earlier.

Data analysis

Analysis of the OD of the CREB/CREM band was performed using Quan-tityOne software (Bio-Rad) after background subtraction from each band.Data were evaluated for statistical significance by Student’st test.

ResultsThe2180 site of the IL-2 promoter binds p-CREB in normalhuman T cells

The site on the IL-2 promoter/enhancer that lies between NF-kBand CD28 response element binding sites (2164 to2189 bp,known as2180 site) has been proposed to be a distal AP-1 bindingsite because of the homology that shares with the consensus AP-1sequence; nevertheless, this binding was never clearly shown (4).PKC-u has been shown to be involved in the activation of tran-scription factors that bind to the2150 (proximal AP-1 bindingsite) (18, 33) and2200 (NF-kB binding site) binding sites (19,20). To understand the role of the transcription factor that binds tothe 2180 site on the IL-2 promoter/enhancer in normal human Tcells in the transcriptional regulation of the IL-2 gene, and deter-mine whether PKC-u is involved in the activation of this transcrip-tion factor, we studied the DNA-protein interactions using nuclearextracts from freshly isolated normal T cells and an oligonucleo-tide that spans from2164 to2189 bp (2180 site) on the humanIL-2 promoter/enhancer.

The2180 site is located on a minor groove of the DNA (5), andthis may have been the reason that no definite binding had beenpreviously detected by using poly(dIz dC) as nonspecific compet-itor in shift assay experiments (4, 38). We first examined the bind-ing of nuclear extracts from normal T cells stimulated with PMAfor 6 h to the 2180 site oligonucleotide incubated either withpoly(dG)zpoly(dC) or poly(dIz dC) as nonspecific competitor. Thebinding observed when using poly(dG)zpoly(dC) could not be de-tected when using poly(dIz dC) (Fig. 1). Therefore, all subsequentshift assay experiments were performed with poly(dG)zpoly(dC) asnonspecific competitor.

Nuclear extracts from unstimulated normal T cells displayedminimal or no binding to the2180 oligonucleotide (Figs. 2 and 3),whereas nuclear extracts from stimulated T cells bound to it. Spe-cifically, nuclear extracts from T cells stimulated with anti-CD3and anti-CD28 Abs displayed significant binding that reachedmaximal values at 6 h and decreased thereafter (Fig. 2A). Similarresults were obtained when we stimulated the T cells with PMAand ionomycin, to bypass membrane-mediated signaling events(Fig. 2B). This band decreased by 80% in the presence of 10-foldand disappeared in the presence of 100-fold molar excess of un-labeled oligonucleotide, indicating that the binding was specific(Fig. 2C).

Normal T cells were also stimulated with PMA alone to excludeCa21 interference, or forskolin, a PKA activator, or both. Maximalintensity of the specific binding was reached after 3 h when stim-ulated with PMA and forskolin (Fig. 2E) and after 6 h when T cellswere stimulated with PMA (Fig. 2F) or forskolin (not shown)alone. As above, the presence of 100-fold molar excess of unla-beled oligonucleotide or the presence of anti-p-CREB Ab dimin-ished the binding (data not shown).

To determine the composition of the shifted bands, we used Absdirected against p-CREB, p-CREM, AP-1, and Ets transcriptionfactor-1. The binding of nuclear proteins from stimulated T cells to

FIGURE 1. Poly(dI z dC) prevents the2180 site binding to the IL-2promoter. Nuclear extracts from normal T cells stimulated with PMA for6 h were analyzed using an oligonucleotide that spans from the2164 to the2189 bp on the human IL-2 promoter. The presence of poly(dIz dC) (1mg)prevented the observed binding when using poly(dG)zpoly(dC) (1mg) asnonspecific competitor. Nuclear extracts from unstimulated cells were ex-amined using 1mg poly(dG)zpoly(dC). Shift assay was performed as de-scribed inMaterials and Methods.

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the 2180 oligonucleotide diminished by 90% in the presence ofanti-p-CREB Ab and only by 10% in the presence of anti-p-CREMAb (Figs. 2D, 3B, and 3D). The presence of anti-junor anti-fosAbin the shift assay reaction failed to change significantly the inten-sity of the band. Specifically, the mean intensity (OD) of the bandusing nuclear extracts from cells stimulated with PMA or PMAand ionomycin was 169 and 184, respectively. The mean intensityof the band in the presence of anti-jun Ab was 136,p 5 0.03,whereas the mean intensity of the band in the presence of anti-fosAb was 145,p 5 0.185 (Fig. 3,B andD). Similarly, the presenceof anti-Elf Ab (control) did not have any effect on this binding(Fig. 3B). These data indicate that the2180 site of the IL-2 pro-moter/enhancer represents a p-CREB binding site. The specificityof this binding was also examined using excess unlabeled GATA,M1, and M2 oligonucleotides. The presence of excess unlabeled

FIGURE 2. p-CREB binds to the2180 site of the IL-2 promoter in normalhuman T cells. Nuclear extracts from normal T cells were analyzed using anoligonucleotide that spans from2164 to2189 bp on the IL-2 promoter. Nuclearextracts from unstimulated cells displayed minimal or no binding, whereas nuclearextracts from stimulated cells displayed strong binding. Specifically, nuclear ex-tracts from T cells stimulated with a combination of mAbs against TCR and CD28(A) or with PMA and ionomycin (B) displayed significant binding that reachedmaximal values at 6 h and decreased later. Specificity of the binding was examinedusing excess unlabeled oligonucleotide (C). To determine the composition of theshifted bands, we used Abs directed against p-CREB, p-CREM. The contributionof p-CREM in this binding is minimal (D). When T cells were stimulated withPMA, maximal intensity was also reached at 6 h (E) and after 3 h when we usedcombination of PMA and forskolin for stimulation (F). The data shown are rep-resentative of experiments performed using nuclear extracts from T cells from 20normal individuals.

FIGURE 3. p-CREB binds to the2180 site of the IL-2 promoter innormal human T lymphocytes.A, Nuclear extracts from normal T cellsstimulated with PMA and ionomycin were examined in EMSA experi-ments using the2180 site oligonucleotide in the presence of excess un-labeled oligonucleotides. The presence of 103excess unlabeled M1, M2,or GATA oligonucleotides failed to change significantly the binding com-pared with the binding in the absence of any unlabeled oligonucleotide.B,The presence of anti-fosor anti-Ets transcription factor-1 Abs did notchange binding to the2180 site. In contrast, anti-p-CREB Ab completelyinhibited the binding.C, Binding of nuclear extracts from normal T cellsstimulated with PMA to end-labeled2180 or the M2 oligonucleotides. Thedetected binding of nuclear extracts to the labeled M2 oligonucleotide iscomparable with that of nuclear extracts from unstimulated normal T cellsexamined in shift assay experiments using end-labeled2180 oligonucle-otide.D, The presence of anti-p-CREB, but not anti-jun, Ab inhibited theintensity of the band.

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GATA, M1, or M2 oligonucleotide, unlike the excess2180 oli-gonucleotide, failed to change significantly the intensity of thebinding (Fig. 3A, compare to Fig. 2C). Moreover, when we labeledthe M2 probe and used it in shift assay experiments using nuclearextracts from T cells stimulated with PMA, the detected bindingwas comparable with the binding of nuclear extracts from unstimu-lated T lymphocytes to the2180 oligonucleotide (Fig. 3C). Theseresults suggest that the2180 site of the IL-2 promoter representsa p-CREB binding site.

PKC-u is involved in the phosphorylation of CREB and itssubsequent binding to the2180 site of the IL-2 promoter

To examine whether PKC-u participates in CREB activation andleads to IL-2 promoter/enhancer binding, normal T cells were in-cubated with specific PKC and PKC-u inhibitors before stimula-tion with PMA, or PMA and ionomycin, as described inMaterialsand Methods, and nuclear proteins were examined in immunoblot-ting and shift assay experiments. It has been proposed that MEKand MAPKAP-K2 lie downstream of PKC in CREB activation(34). To examine also the role of these kinases in CREB activationin normal T cells, we used nuclear extracts from T cells that weretreated with SB203580 or PD98059, specific MAPKAP-K2 andMEK inhibitors, respectively, prior to stimulation with PMA orPMA and ionomycin. KT5720 was used as a specific PKA inhib-itor and W7 as a calcium/calmodulin inhibitor.

First, we performed immunoblot analysis of nuclear extractsfrom unstimulated and PMA- and ionomycin-stimulated T cellstreated with or without inhibitors prior to stimulation. p-CREBprotein levels were abundant in nuclear extracts from stimulatednormal T cells, but not in nuclear extracts from unstimulated Tcells (Fig. 4). The presence of rottlerin or calphostin C decreasedthe p-CREB protein levels by 60% (Fig. 4A) and 90% (Fig. 4B),respectively. The presence of PD98059, W7, KT5720, orSB203580 did not affect significantly the p-CREB protein levels(Fig. 4B). Total CREB protein levels were not affected in the pres-ence of these inhibitors, indicating that they only inhibited CREBactivation. This experiment suggested that CREB activation in-volves PKC-u, which does not require Ca21 to be activated (9, 10).Subsequently, we determined whether p-CREB protein levels areaffected when normal T cells are stimulated only with PMA in thepresence of the same inhibitors. As expected, total CREB proteinlevels were not affected, but the p-CREB protein levels were di-minished by 95% in the presence of calphostin C and by 90% inthe presence of rottlerin (Fig. 5). The presence of SB203580,PD98059, KT5720, or W7 did not affect the CREB protein levelsand did not decrease significantly the p-CREB protein levels(Fig. 5).

Subsequently, we determined whether p-CREB binding to the2180 site of the IL-2 promoter is affected in the presence of thesame inhibitors. The presence of calphostin prevented p-CREBbinding to the2180 oligonucleotide, whereas rottlerin decreasedp-CREB binding by 80%, compared with the binding of extractsfrom stimulated cells with PMA and ionomycin in the absence ofany inhibitor (Fig. 6A). Similar results were obtained when normalT cells were stimulated with anti-CD3 and anti-CD28 Abs (datanot shown). In nuclear extracts from T cells that were treated withPD98059 or SB203580 before stimulation, p-CREB binding de-creased by 25% (Fig. 6A). p-CREB binding of nuclear extractsfrom normal T cells treated with forskolin in the presence ofKT5720, a specific PKA inhibitor, decreased by 50% (data notshown). Similar results were obtained from cells treated withKT5720 before stimulation with PMA and ionomycin (Fig. 6A). Ithas been proposed that calcium/calmodulin kinase IV can phos-phorylate and therefore activate CREB. When normal T cells were

FIGURE 4. p-CREB protein levels are affected by the presence of PKCand PKC-u inhibitors. Nuclear extracts from unstimulated normal T cells,from stimulated T cells with PMA and ionomycin, and from T cells thatwere treated with inhibitors prior to stimulation with PMA and ionomycinwere examined in immunoblots.A, The presence of rottlerin decreasedp-CREB protein levels by 60%.B, The presence of calphostin C decreasedp-CREB protein levels by 90%, whereas the presence of W7, or KT5720,or SB203580, or PD98059 did not significantly affect the p-CREB proteinlevels (the % represents mean values of six experiments).Left margin,Molecular size marker migration. Ionom, ionomycin.

FIGURE 5. Rottlerin diminishes the p-CREB protein levels in normalhuman T lymphocytes. Nuclear extracts from T cells stimulated with PMAand from T cells that were treated with inhibitors, before stimulation withPMA, were examined in immunoblots. The presence of calphostin C de-creased p-CREB protein levels by 95%, whereas the presence of rottlerindecreased p-CREB protein levels by 90%. The presence of W7, orKT5720, or SB203580, or PD98059 did not affect significantly thep-CREB protein levels.Left margin, Molecular size marker migration.

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treated with W7, a calcium/calmodulin inhibitor, before stimula-tion with PMA and ionomycin, p-CREB binding to the2180 oli-gonucleotide decreased by 25% (Fig. 6A). We treated T cells withthe same inhibitors and then stimulated them with anti-CD3 andanti-CD28 Abs. EMSA and subsequent densitometric analysis re-vealed inhibitory patterns similar to those obtained when T cellswere stimulated with PMA and ionomycin (data not shown). Fi-nally, we examined p-CREB binding to the2180 oligonucleotideusing nuclear extracts from normal T cells that were treated withPMA alone and with the same inhibitors. W7, SB203580, andPD98059 did not affect p-CREB binding, whereas calphostin Cprevented the binding to the2180 oligonucleotide and rottlerin

decreased this binding by 70% (Fig. 6B). The results describedabove were generated with doses that had been found in prelimi-nary experiments to be optimal for each inhibitor.

Taken together, these results demonstrate that although multiplepathways are involved in CREB activation and its subsequentbinding on the IL-2 promoter, PKC and especially theu isoformplay a critical role.

Rottlerin prevents the formation of p300/p-CREB complexes

CBP and p300 are known to bind p-CREB, resulting in the for-mation of heteromeric activator complexes that contribute to effi-cient initiation of transcription. As shown above, PKC-u has acentral role in CREB phosphorylation. To examine the role ofPKC-u in p-CREB/CBP-p300 complex formation, we performedimmunoprecipitation experiments in stimulated T cells treatedwith or without rottlerin. First, we immunoprecipitated nuclear ex-tracts from unstimulated T cells with anti-p300 and anti-CBP Abs,but we did not detect any p-CREB/p300 complexes, as expected.In contrast, these complexes were abundant in immunoprecipitatesof nuclear extracts from T cells stimulated with PMA and iono-mycin (Fig. 7). The presence of rottlerin (Fig. 7, Table I) or cal-phostin C (data not shown) inhibited completely the formation ofp-CREB/p300 complexes. Similarly, rottlerin and calphostin C in-hibited the formation of p-CREB/p300 complexes in nuclear ex-tracts from T cells stimulated with anti-CD3 and anti-D28 Abs(data not shown). When T cells were treated with PD98059,SB203580, W7, or KT5720 before stimulation with PMA andionomycin, or with anti-CD3 and anti-CD28 Abs, the p-CREB/p300 complexes could be detected, but densitometric analysis re-vealed that diminished by;20% (data not shown). Thus, PKC-uplays an important role in CREB phosphorylation and activationand the subsequent formation of p-CREB-CBP/p300 complexesthat can affect transcription.

The2180 site of the IL-2 promoter is important in thetranscriptional regulation of the IL-2 gene, and PKC-uenhances the2180 site-driven reporter activity

To prove that the2180 site of the IL-2 promoter is functional andPKC-u regulated in normal human T cells, we transiently trans-fected fresh normal human T cells with a luciferase-reported con-struct driven by two tandem2180 sites (pGL2-(2180)32) (32).As shown in Fig. 8A, stimulation of pGL2-(2180)32-transfectednormal T cells with PMA and ionomycin for 6 h induced a mean3-fold increase in luciferase activity compared with stimulatedcells transfected with empty pGL2 vector. Normal T cells that were

FIGURE 6. PKC-u participates in the activation of CREB and its sub-sequent binding on the2180 site of the IL-2 promoter.A, Normal T cellswere treated with inhibitors for pathways known to activate CREB, beforestimulation and preparation of the nuclear extracts. Calphostin C, a PKCinhibitor, diminished p-CREB binding, whereas rottlerin, a specific PKC-uinhibitor, decreased this binding by 80% (mean values of all experiments;n 5 20). The presence of SB203580, the specific MAPKAP-K2 inhibitor,or PD98059, a MEK inhibitor, decreased the p-CREB binding by 25%,respectively. Inhibition of the PKA pathway resulted in 50% decrease ofp-CREB binding. W7, a Ca21/calmodulin inhibitor, decreased p-CREBbinding also by 25%. The data shown and the mean percentage of inhibi-tion are representative of 20 experiments.B, Normal T cells were stimu-lated with PMA alone in the presence of inhibitors. The presence of cal-phostin C prevented p-CREB binding, whereas rottlerin decreased thisbinding by 70%. The presence of SB203580, PD98059, or W7 did not havea significant effect on the observed p-CREB binding.

FIGURE 7. Rottlerin prevents the formation of p300/p-CREB com-plexes. We immunoprecipitated nuclear extracts from unstimulated T cellswith anti-p300 and anti-CBP Abs, but we did not detect any p-CREB/p300complexes. In contrast, these complexes were abundant in immunoprecipi-tates of nuclear extracts from T cells stimulated with PMA and ionomycin.The presence of rottlerin inhibited completely the formation of p-CREB/p300 complexes (n5 5). Left margin, Molecular size marker migration.Ionom, ionomycin.

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cotransfected with pGL2-(2180)32 construct and a PKC-u-con-taining vector showed 2-fold increase in luciferase activity com-pared with cells transfected with the pGL2-(2180)32 constructalone (p , 0.001). When T cells were cotransfected with pGL2-(2180)32 and plasmids encoding PKC-a or PKC-e, we did notobserve a significant increase in luciferase activity, compared withcells transfected with the pGL2-(2180)32 construct alone. Fur-thermore, T cells were cotransfected with the pGL2-(2180)32 andCREMa construct in the presence or absence of the plasmid en-coding PKC-u. The presence of CREMa construct caused 70%suppression of the2180-driven luciferase activity, which was lim-ited to 50% when a PKC-u construct was also present (Fig. 8A).

Because the2180 site represents only a small portion of theIL-2 promoter, we conducted experiments using a reporter geneconstruct driven by the2575/157 region of the IL-2 promoter/enhancer, which simulates the function of the intact IL-2 promoter.Normal T cells were transfected with a luciferase reporter con-struct driven by the2575/157 region of the IL-2 promoter/en-hancer alone or in the presence of a plasmid expressing PKC-u,PKC-a, PKC-e, CREMa, or PKC-u and CREMa. Stimulation ofthe cells that were transfected with the intact IL-2 promoter withPMA and ionomycin caused a 23-fold (mean) increase in lucif-erase activity compared with unstimulated cells. Cotransfectionwith PKC-a and PKC-e constructs caused a 2.4- and 0.5-fold in-crease in luciferase activity compared with stimulated T cells thatwere transfected with the whole IL-2 promoter alone. Cotransfec-tion with a PKC-u construct revealed a 4-fold increase in the re-porter gene activity, indicating the important role of PKC-u in IL-2production. To examine whether CREM can affect the activity ofthe IL-2 promoter, we cotransfected T cells with a CREMa con-struct in the presence or absence of a plasmid encoding PKC-u.The presence of the CREMa construct suppressed the IL-2 pro-moter-driven luciferase activity by 65%, which was limited to 50%when PKC-u was present (Fig. 8B).

Finally, we transfected normal T cells with the2575/157 IL-2promoter constructs in which the2180 site had been mutated (M1and M2). Normal T cells that were transfected with the M1 or theM2 construct displayed 60% and 85% decrease in luciferase ac-tivity, respectively, compared with the cells that were transfectedwith the wild-type IL-2 promoter construct, following stimulationwith PMA and ionomycin for 6 h. The presence of PKC-u con-struct did not restore the IL-2 promoter-driven luciferase activity(Fig. 8c). The transfection experiments described above were alsoexamined using the same constructs in Jurkat cells. As shown inFig. 9, the pattern obtained after transfecting Jurkat cells was sim-ilar to that observed in normal T cells. Specifically, stimulation ofpGL2-(2180)32-transfected Jurkat cells with PMA and ionomy-cin for 6 h induced a 3.5-fold increase in luciferase activity com-pared with stimulated cells transfected with empty pGL2 vector.Jurkat cells that were cotransfected with the pGL2-(2180)32 con-struct and plasmids encoding PKC-u showed a 2-fold increase inluciferase activity compared with cells transfected with the pGL2-(2180)32 construct alone. The presence of PKC-e or PKC-a plas-mids in the presence of pGL2-(2180)32 construct did not affectthe luciferase activity compared with Jurkat cells that were trans-

fected with the pGL2-(2180)32 construct alone. The presence ofCREMa construct caused a 75% decrease in luciferase activity,which was limited by 50% when a PKC-u construct was alsopresent (Fig. 9). Jurkat cells were also examined in transfectionexperiments using a construct driven by the2575/157 region ofthe IL-2 promoter/enhancer alone or in the presence of plasmidsexpressing PKC-u, PKC-e, PKC-a, CREMa, or PKC-u andCREMa. Stimulation of the cells that were transfected with theintact IL-2 promoter with PMA and ionomycin caused a 10-foldincrease in luciferase activity compared with unstimulated cells.Cotransfection with PKC-a and PKC-e constructs caused a 1- and0.5-fold increase in luciferase activity compared with Jurkat cellsthat were transfected with the whole IL-2 promoter alone (Fig. 9).Cotransfection with a PKC-u construct resulted in a 10-fold in-crease in the reporter gene activity, confirming the results obtainedfrom transfecting normal T cells with the same constructs andfurther supporting the important role of PKC-u on IL-2 gene tran-scription (Fig. 9). The presence of the CREMa construct sup-pressed the IL-2 promoter-driven luciferase activity by 50%. Co-transfection of the PKC-u and the CREMa construct caused an80% decrease in luciferase activity (Fig. 9). Thus, the2180 site ofthe IL-2 promoter/enhancer is critical in the transcriptional regu-lation of IL-2, and PKC-u is involved in the phosphorylation andsubsequent binding of CREB to this site.

DiscussionIt has been suspected that the2180 site of the IL-2 promoter mayrepresent a distal AP-1 binding site, despite the fact that it does notshare precise homology with the consensus AP-1 sequence (2, 4)and binding ofjun/fosheterodimers has never been clearly shown(3–5, 35). Recently, it was reported that anergic T cell clones dis-play increased binding of p-CREB/p-CREM to the2180 site ofthe IL-2 promoter (5). The2180 site is located in a minor grooveof the DNA, and the use of poly(dIz dC) may prevent any bindingof nuclear proteins to it. Indeed, we verified (Fig. 1) that no bind-ing can be detected to the2180 site-defined oligonucleotide ifpoly(dI z dC) is used as a nonspecific competitor. Instead, we usedpoly(dG z dC) as a nonspecific inhibitor in performing EMSA ex-periments and we observed that nuclear extracts from fresh humanT cells treated with anti-CD3 and anti-CD28 Abs, or PMA alone,or PMA and ionomycin bind to the2180 site-defined oligonucle-otide. In determining the specificity of this binding, 1) we wereable to eliminate it in the presence of sufficient amounts of excessunlabeled oligonucleotide; 2) we were able to eliminate the band inpresence of anti-p-CREB Ab; and 3) the presence of Abs directedagainst the components of AP-1 failed to change the intensity ofthe band significantly. Therefore, we concluded that the2180 siteof the IL-2 promoter represents a p-CREB binding site. Our ex-periments have also established that the2180 site is functionalbecause a luciferase reporter construct driven by two tandem2180sites increased its activity when transfected into normal T cells thatwere stimulated with PMA and ionomycin. The importance of the2180 site in the transcription of the IL-2 gene in human T cells isfurther underscored by the fact that mutation of two bases within

Table I. Rottlerin prevents the formation of p300/p-CREB complexes

Cytoplasmic Extracts p-CREB(mean OD6 SEM)

Nuclear Extracts p-CREB (meanOD 6 SEM)

Unstimulated 726 2 90 6 2PMA 1 ionomycin 806 1 1636 1PMA 1 ionomycin1 rottlerin 836 2 91 6 2

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this region limited the ability of the whole IL-2 promoter to drivethe expression of the reporter gene.

Different signals delivered by hormones, synaptic activity,growth factors, stress, and inflammatory cytokines can enhancecascades that lead to CREB phosphorylation (6–8). Recent reportshave shown that in T cells PKC-u is involved in the activation of

AP-1 (18, 33) and NF-kB (19, 20), two transcription factors that liedown- and upstream of the2180 site of the IL-2 promoter/en-hancer and influence the regulation of the IL-2 gene transcription(32–34). Our experiments, besides establishing the binding ofCREB to the2180 site of the IL-2 promoter, have demonstratedthat PKC-u is involved in the phosphorylation and activation of

FIGURE 8. The2180 site of the IL-2 promoteris important for the transcriptional regulation ofIL-2 in fresh human T cells. PKC-u enhances the2180 site-driven reporter activity.A, Stimulationof transfected (pGL2-(2180)32) normal T cellswith PMA and ionomycin for 6 h induced a mean3-fold increase in luciferase activity comparedwith stimulated cells transfected with the emptypGL2 vector. Cotransfection with a PKC-u plas-mid displayed a 2-fold increase in luciferase ac-tivity (p , 0.001), whereas cotransfection withPKC-a or PKC-e constructs did not affect lucif-erase activity. Normal T cells were cotransfectedwith the pGL2-(2180)32 and the CREMa con-structs in the presence or absence of a plasmidencoding PKC-u. The presence of CREMa causeda 70% suppression to the2180-driven luciferaseactivity, which was limited to 50% when thePKC-u construct was present.B, Stimulation ofIL-2 promoter (IL-2: 2575/157)-driven reporterconstruct caused a 23-fold increase in luciferaseactivity. Cotransfection with PKC-a, PKC-e, andPKC-u constructs caused a 2.4-, 0.5-, and 4-foldincrease in luciferase activity, respectively. Thepresence of the CREMa construct suppressed theIL-2-promoter-driven and the pGL2-(2180)32-driven luciferase activity by 65%, which was lim-ited to 50% when PKC-u was present.C, The ac-tivity of luciferase was limited significantly whentwo bases within the2180 site of the IL-2 pro-moter were mutated (M1 and M2 constructs), andit did not increase when T cells were cotransfectedwith the PKC-u construct. The data are represen-tative of 10 experiments.

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CREB. This conclusion is supported by several facts: first,p-CREB protein levels diminished in the presence of calphostin C,a PKC inhibitor, and in the presence of rottlerin, a specific PKC-uinhibitor, but they did not change significantly in the presence ofinhibitors directed against other kinases important in T cell sig-naling; second, the binding of p-CREB to the2180 oligonucleo-tide diminished in T cells previously treated with specific PKC-uinhibitors; third, transfection of T cells with a PKC-u constructincreased the ability of the2180 site to drive the transcription ofthe reporter gene, whereas transfection of T cells with other PKCisoforms, including PKC-e and PKC-a, either failed or onlyslightly affected the transcription of the2180 site reporter con-struct. The behavior of the2180 site reporter construct was mim-icked by a whole (2575/157 region) IL-2 promoter/enhancer-driven reporter construct. Specifically, its activity increased whenthe PKC-u construct was cotransfected. In addition, mutation oftwo bases in the2180 region limited its ability to drive transcrip-tion, and the presence of a PKC-u plasmid did not restore its ac-tivity. The central importance of the2180 site in the IL-2 genetranscription should be noted in view of the reports that have pro-posed that PKC-u participates in the activation of AP-1 and NF-kBthat bind to the2150 and2200 sites of the IL-2 promoter, re-

spectively (18–20). Our experiments also showed that MAP-KAP-K2 and PKA inhibitors affected moderately the binding ofp-CREB to the2180 oligonucleotide, suggesting that other ki-nases, besides PKC-u, may be involved in the phosphorylation ofCREB. Whether phosphorylation by PKA influences the DNA-binding function of CREB remains a controversial point (6). PKAcan activate CREB in normal human T lymphocytes because for-skolin, a PKA activator, and KT5720, a specific PKA inhibitor,affected moderately p-CREB protein levels and p-CREB/DNAbinding to the IL-2 promoter/enhancer. Although our findings donot allow us to pinpoint the cascade that leads to CREB phosphor-ylation upon TCR engagement, we propose that PKC leads to theactivation of Ras-Raf-1-MEK-mitogen-activated protein kinase ina Ca21-independent manner (31). In this pathway, PKC-u seems tohave an important role, although it is possible that other isoformsmay participate. In contrast, the observed nuclear protein bindingto the2180 site after stimulation with PMA and ionomycin, andthe decrease of this binding when T cells were treated with W7, acalcium/calmodulin inhibitor, suggest that a Ca21-dependent path-way may also participate.

It has been established that CREB associates with the coactiva-tors CBP/p300, resulting in the formation of heteromeric activatorcomplexes, which interact with the general transcription apparatus.In this communication, we have shown that this interaction occursalso in fresh human T cells. CBP/p300 exhibits intrinsic histoneacetyltransferase activity, which facilitates transcription by di-rectly participating in chromatin remodeling at the level of induc-ible promoters (39, 40). We have shown that rottlerin, a specificPKC-u inhibitor, prevented the formation of p-CREB-CBP/p300complexes, whereas inhibitors of other pathways also known toactivate CREB only slightly diminished the formation of thesecomplexes. Therefore, PKC-u displays a major role in CREBphosphorylation and its subsequent activation and further engage-ment in the transcription initiation complex.

The 2180 site appears to have an important role in the regula-tion of IL-2 gene transcription as it relates to normal T cell func-tion, anergy, and the expression of human disease. Our experi-ments show that in fresh human T cells and in Jurkat cells,p-CREB binds to this site and enhances the transcription of theIL-2 gene. Powell et al. (5) have shown that nuclear proteins fromanergic T cell clones contain CREB/CREM heterodimers that bindto the 2180 site of the IL-2 promoter and may suppress IL-2transcription (5). Transgenic mice expressing a dominant-negativeform of CREB display defective thymocyte proliferation and IL-2production (22). Recently, we showed that T cells from patientswith systemic lupus erythematosus, a systemic autoimmune dis-ease characterized by decreased IL-2 production in vitro, expressincreased levels of p-CREM protein that binds to the2180 site ofthe IL-2 promoter. CREM was shown to suppress IL-2 gene tran-scription (32). Finally, Bodor et al. (41) have shown that repressorICER can suppress the transcription of the IL-2 gene.

In conclusion, we have presented evidence that PKC-u partici-pates in the phosphorylation of CREB in human T cells and pro-motes IL-2 transcription. Additional studies are needed to inves-tigate how p-CREB assumes a central role in the transcription ofthe IL-2 gene in view of the fact that the IL-2 promoter definesadditional sites for well-established transcription factors such asAP-1, NF-AT, and NF-kB (42). Apparently, each factor contrib-utes variable degrees of transcriptional activity depending on theincoming signal and probably through interaction with other tran-scription factors and coactivators. Because IL-2 has a central rolein the function of T cells from normal individuals and patients with

FIGURE 9. The 2180 site of the IL-2 promoter is important for thetranscriptional regulation of IL-2 in Jurkat T cells. Stimulation of trans-fected (pGL2-(2180)32) Jurkat cells with PMA and ionomycin for 6 hinduced a mean 3.5-fold increase in luciferase activity compared with stim-ulated cells transfected with the empty pGL2 vector. Cotransfection with aPKC-u plasmid displayed a 2-fold increase in luciferase activity, whereascotransfection with PKC-a or PKC-e constructs did not affect luciferaseactivity. Jurkat cells were cotransfected with the pGL2-(2180)32 andCREMa constructs in the presence or absence of a plasmid encodingPKC-u. The presence of CREMa caused a 75% suppression to the2180-driven luciferase activity, which was limited to 50% when the PKC-u con-struct was present. Stimulation of IL-2 promoter (IL-2:2575/157)-drivenreporter construct caused a 10-fold increase in luciferase activity. Cotrans-fection with PKC-a, PKC-e, and PKC-u constructs caused 1-, 0.5-, and10-fold increases in luciferase activity, respectively. The presence of theCREMa construct suppressed the IL-2 promoter-driven luciferase activityby 50%. Cotransfection with PKC-u and CREMa constructs caused 80%decrease in luciferase activity compared with that observed when Jurkatcells were transfected with the IL-2 promoter and PKC-u constructs.

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autoimmune diseases, further studies should characterize the crit-ical role of members of the CREB family of proteins in the tran-scriptional regulation of IL-2. It is probable that at any given timepoint, the rate of IL-2 gene transcription is determined by the lev-els of positive and/or negative isoforms of CREB/CREM/ICERpresent in the vicinity of the IL-2 promoter and the availability ofother transcription factors.

AcknowledgmentsWe thank all donors for kindly donating blood samples. We are grateful toDr. A. Rao for providing the IL-2 plasmid; Dr. P. Sassone-Corsi for pro-viding the CREM construct; and Drs. G. Baier, W. C. Greene, and A.Altman for providing the PKC plasmids. We thank our colleagues forcritically reviewing this manuscript and Lee Collins for help with the prep-aration of the figures.

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