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The Journal of Immunology, 2006, 176: 4666-4674.
Copyright © 2006 by The American Association of Immunologists

NF-{kappa}B-Inducing Kinase Is Involved in the Activation of the CD28 Responsive Element through Phosphorylation of c-Rel and Regulation of Its Transactivating Activity1

Carmen Sánchez-Valdepeñas*, Angel G. Martín*, Parameswaran Ramakrishnan{dagger}, David Wallach{dagger} and Manuel Fresno2,*

* Centro de Biología Molecular, Consejo Superior de Investigaciones Científicas, Universidad Autónoma de Madrid, Madrid, Spain; and {dagger} Department of Biological Chemistry, Weizmann Institute of Science, Rehovot, Israel


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Previous evidence suggested that NF-{kappa}B-inducing kinase (NIK) might regulate IL-2 synthesis. However, the molecular mechanism is not understood. In this study, we show that NIK is involved in CD3 plus CD28 activation of IL-2 transcription. Splenic T cells from aly/aly mice (that have a defective NIK protein) have a severe impairment in IL-2 and GM-CSF but not TNF secretion in response to CD3/CD28. This effect takes place at the transcriptional level as overexpression of alyNIK inhibits IL-2 promoter transcription. NIK activates the CD28 responsive element (CD28RE) of the IL-2 promoter and strongly synergizes with c-Rel in this activity. We found that NIK interacts with the N-terminal domain of c-Rel, mapping this interaction to aa 771–947 of NIK. Moreover, NIK phosphorylates the c-Rel C-terminal transactivation domain (TAD) and induces Gal4-c-Rel-transactivating activity. Anti-CD28 activated Gal4-c-Rel transactivation activity, and this effect was inhibited by a NIK-defective mutant. Deletion studies mapped the region of c-Rel responsive to NIK in aa 456–540. Mutation of several serines, including Ser471, in the TAD of c-Rel abrogated the NIK-enhancing activity of its transactivating activity. Interestingly, a Jurkat mutant cell line that expresses one of the mutations of c-Rel (Ser471Asn) has a severe defect in IL-2 and CD28RE-dependent transcription in response to CD3/CD28 or to NIK. Our results support that NIK may be controlling CD28RE-dependent transcription and T cell activation by modulating c-Rel phosphorylation of the TAD. This leads to more efficient transactivation of genes which are dependent on CD28RE sites where c-Rel binds such as the IL-2 promoter.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Activation of T cells through the TCR (CD3) is not sufficient to activate optimal cytokine production. Additional costimulatory signals are required that can be provided by the CD28 molecule (1, 2, 3). CD28 is constitutively expressed in naive T cells and upon engagement provides signals that are integrated with those of the TCR to produce full expression of some cytokines as IL-2, although it is accepted that CD28 stimulation does not have an appreciable effect by itself (2, 3, 4). In agreement with those results, CD28-deficient mice have a severe reduction in the immune response and a severe defect in IL-2 production (5). Despite considerable research on the importance of CD28 in T cell activation, the signaling pathway induced by CD28 that leads to enhanced IL-2 production is not yet completely understood. CD28 engagement in T cells provides a synergistic stimulation with the TCR resulting in the activation of NF-AT, NF-{kappa}B, and AP-1 transcription factors, required for cytokine production and cell proliferation. Several downstream molecules have been involved in CD28 signaling, as the PI3K/Akt, the inducible T cell kinase tyrosine kinase, Vav1, p38 MAPK, (cancer Osaka thyroid) oncogene/tumor progression locus 2 (Cot/tpl-2),3 and mixed lymphocyte kinase-3 among others (2, 3, 6, 7, 8, 9).

IL-2 synthesis is strongly regulated at the level of transcription and the regulatory sequences conferring its inducible expression in T cells are localized in a region of ~300 bp 5'of the transcription start site (10). Within this region, the existence of binding sites for different ubiquitous and cell-specific transcription factors (NF-{kappa}B, AP-1, NF-AT, and Oct1 among others) has been reported (reviewed in Ref.11). The CD28 responsive element (CD28RE)/AP-1 (–164/–145) (–268) site of the human IL-2 promoter is a very representative example of cooperative interaction between the c-Rel and members of AP-1 families in Ref.12 . CD28 costimulation specifically targets the CD28RE inducing the binding of c-Rel/NF-{kappa}B and AP-1 to this site, leading to a complete activation of the IL-2 promoter (2).

The NF-{kappa}B family of transcription factors is composed of homo- and heterodimers of a family of proteins which include the dorsal gene of Drosophila and the mammalian genes nf{kappa}b1 (p105/p50), nf{kappa}b2 (p100/p52), c-Rel, relA (p65), and relB (for review, see Refs.13, 14, 15, 16, 17). Deletions of each of those genes in mice suggest nonredundant functions. Although nf{kappa}b1, nf{kappa}b2, and relA are ubiquitously expressed, c-Rel and relB are mostly detected in hemopoietic cells. c-Rel, RelB, and RelA have a C-terminal transactivation domain (TAD) which strongly activates transcription from NF-{kappa}B sites.

NF-{kappa}B complexes are held in the cytoplasm of cells in an inactive state complexed with members of the I{kappa}B family. In response to different activators, I{kappa}B is phosphorylated by I{kappa}B kinases (IKKs), and subsequently degraded, liberating the active NF-{kappa}B complex which translocates to the nucleus and activates transcription (13, 14, 15, 16, 17). In addition to this conventional pathway, a noncanonical one activated by several TNF members, such as lymphotoxin beta (LTbeta), and involving signaling through NF- {kappa}B-inducing kinase (NIK) and IKK{alpha} which results in the processing of p100/RelB to mature p52/RelB has been described (18, 19). However, there is accumulating evidence suggesting that a different level of NF-{kappa}B regulation independent of I{kappa}B degradation exists (reviewed in Refs.20 and 21). This second level of regulation relies in the activation of the transcriptional activity of p65 and c-Rel. A number of kinases have been shown to phosphorylate p65 NF-{kappa}B including casein kinase II and protein kinase A (PKA) resulting in enhanced transcription activity (22, 23). Recent evidence indicates that IKKbeta also phosphorylates p65/RelA at Ser536 (24, 25). PKC{zeta} has been also shown to be essential for the transcriptional activity of p65/RelA. Thus, PKC{zeta} knockout mice have a defect on p65 phosphorylation and activation of the transactivation activity (26, 27). We have previously shown that c-Rel can be phosphorylated in T cells after TNF triggering, being that PKC{zeta} is involved in this activation, resulting in enhanced transactivation activity (28, 29).

NIK mutant mice have revealed an essential role of NIK-IKK{alpha} for the induction of NF- {kappa}B by signaling through LTbetaR, whereas this pathway is dispensable in TNFRI signaling (30, 31, 32). Moreover, this effect seems to be mostly due to an activation of the transactivation activity, not affecting nuclear translocation. Consistent with this, it has been described that NIK is involved in regulation of NF-{kappa}B p65 TAD, as well as phosphorylation of p65 at Ser536, which depends on LTbetaR signaling (32, 33). However, recent results indicate that induction of I{kappa}B degradation in lymphocytes by CD70, CD40L, and B lymphocyte stimulator/B cell activating factor does depend on NIK function (34).

Interestingly, T cells from alymphoplasia (aly/aly) mice that bear a NIK mutation show a defect in IL-2 secretion response to CD3/CD28 and reduced levels of c-Rel protein (35, 36). In this study, we addressed the role of NIK in anti-CD3/CD28-induced IL-2 transcription and we found that NIK is required for CD28RE activation and IL-2 production. Moreover, NIK associates with c-Rel, phosphorylates its TAD, and strongly potentiates its transactivation activity through CD28RE.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Cell cultures and Abs

Jurkat D6 is a mutant clone derived from the parental T lymphoblastic line Jurkat that bears a mutated form of c-Rel (Ser471 to Asn in the TAD) (28). The Jurkat human leukemic T cell line, and its derivative D6, was cultured in RPMI 1640 medium supplemented with 5% FBS, 2 mM L-glutamine, and antibiotics. COS-7 cells were obtained from the American Type Culture Collection and were maintained in complete DMEM supplemented with 10% FBS.

Sera from rabbits hyperimmunized with peptides derived form human c-Rel (no. 265), provided by Dr. N. Rice (National Cancer Institute-Frederick Cancer Research and Development Center, Frederick, MD) were used to detect the corresponding protein on Western blots, used at a dilution of 1/10,000. mAbs anti-Flag and anti-myc used for immunoprecipitation studies were purchased from Boehringer-Mannheim and Santa Cruz Biotechnology, respectively.

Stimulation with anti-CD3/CD28

Mice (aly/aly) and control littermates were purchased from Clea Japan and were maintained under pathogen-free conditions. Spleen cells from those mice were obtained as described (37) and were cultured in triplicate in flat-bottom 96-well plates at 2 x 105 cells/well (200 µl/well). They were stimulated with immobilized anti-CD3 mAb (7 µg/ml) and/or anti-CD28 (1 µg/ml) Abs (BD Pharmingen). The concentration of IL-2, GM-CSF, or TNF in the culture supernatants of splenic cells after 18 h of stimulation was measured by ELISA (TNF, Bender MedSystems; GM-CSF and IL-2, R&D Systems). Jurkat cells were also stimulated with anti-human CD3/CD28 Abs in similar conditions. In all cases, the mean of at least four independent experiments performed is shown.

Plasmids

Expression plasmids pcDNA3-NIK-Flag, encoding wild-type and mutant K429A/K430A (NIK-Flag), alyNIK-myc, NIK-myc, and deletion constructs of this protein 1-367, 1-821, 1-771, 1-640, 367-947, and 624-947 were previously described (38). p65/relA and c-Rel expression plasmids were also described (28).The reporter Gal4-luc contains five tandem repeats of the Gal4 element upstream of the luciferase reporter gene (28). Gal4-c-Rel 309–588 wild-type and Ser471Ala spontaneous mutants and those generated by Ser-Ala substitution–A3 (Ser454), A4 (Ser460), A6 (Ser470, Ser471 and Ser473), A8 (Ser491 and Ser494) were described previously (29). Gal4 DNA-binding domain (DBD) fusions with different c-Rel TAD, Gal4-c-Rel (309-318), Gal4-c-Rel (309-372), Gal4-c-Rel (309-421), Gal4-c-Rel (309-455), Gal4-c-Rel (309-497), Gal4-c-Rel- (422-588), Gal4-c-Rel (456-588), Gal4-c-Rel- (498-588), Gal4-c-Rel (541-588), Gal4-c-Rel (498-540), Gal4-c-Rel (456-497), and Gal4-c-Rel (422-455) deletion mutants were also described (29). The reporter plasmid pIL-2luc containing the sequences from –326 to +46 of the human IL-2 gene directing transcription of the firefly luciferase gene was a gift from Dr. G. Crabtree (Howard Hughes Medical Institute, Stanford University, Stanford, CA) and has already been described (10) and the pCD28RE/AP-1luc plasmid containing four copies of the oligonucleotide corresponding to the CD28RE/AP-1 site of the human IL-2 gene promoter was a gift from Dr. A. Weiss (Howard Hughes Medical Institute, University of California, San Francisco, CA) (12).

Transfection and luciferase assays

Transcriptional activity in Jurkat cells was measured using reporter gene assays after transient transfection of exponential growing cells (2 x 106 cells/ml in OPTIMEM medium), with the Lipofectamine PLUS reagent (Invitrogen Life Technologies). To evaluate transfection efficiency, pRL-tk-luc plasmid (Promega) was used. It contains the HSV thymidine kinase promoter to provide low to moderate levels of Renilla luciferase expression in cotransfected mammalian cells. The LipofectAMINE PLUS-plasmid mixtures were prepared in accordance with the manufacturer’s instructions. The total amount of DNA in each transfection was kept constant by using the corresponding empty expression vectors. After 4 h of incubation, RPMI 1640 medium containing 5% FBS was added to cells and the incubation was continued for 4 h to complete transfection. Cells were then resuspended in complete medium containing 5% FBS. Cells were harvested, lysed 16 h later, and luminescence was measured for 10 s in a luminometer following the instructions in the "Dual-luciferase Assay System kit" (Promega). Data are expressed in relative firefly luciferase units (RLUs) normalized by the relative Renilla luciferase units obtained in the control samples of every transfection and micrograms of protein.

Western blotting and immunoprecipitation

COS-7 cells were transfected at 60–80% of confluence with 0.5–1 µg of the corresponding expression plasmids using the Lipofectamine PLUS reagent. Plasmid DNA was removed 3 h later, and cells were incubated for 48 h in DMEM containing 10% FBS. Whole cell extracts (WCE) from transfected COS-7 cells were made using radioimmunoprecipitation lysis buffer (50 mM Tris-HCl (pH 7.4), 1% Triton X-100, 150 mM NaCl, 0.5% deoxycholate, 0.1% SDS, 10 mM NaF) while Jurkat cells were lysed with immunoprecipitation buffer (50 mM Tris-HCl (pH 7.4), 1% Triton X-100, 150 mM NaCl, 2 mM EDTA, 1 mM EGTA, 20 mM NaF). Both buffers were supplemented with protease inhibitors (2 µg/ml aprotinin, 2 µg/ml leupeptin, 1 µM pepstatin, and 1 mM PMSF) and the phosphatase inhibitor Na3VO4 (1 mM). WCE were immunoprecipitated with 1 µl of the corresponding specific Ab or antiserum. Immunocomplexes were then recovered with 50 µl of protein A-G-agarose (Santa Cruz Biotechnology), and after washing seven times with lysis buffer, they were separated by SDS-PAGE. Detection of endogenous c-Rel by Western blot was performed as described (28). For the detection of NIK-Flag or NIK-myc, 10% polyacrylamide gels were electrophoretically transferred to nitrocellulose filters (Bio-Rad). After blocking overnight with 5% nonfat dried milk in PBS, the membranes were washed twice in PBS containing 0.05% Tween 20 (PBS-T) and incubated with the corresponding antiserum diluted in PBS-T for 1 h at room temperature. Membranes were then washed two times for 10 min each in PBS-T and incubated for 60 min with rabbit anti-mouse IgG secondary Ab linked to HRP (Pierce) at a 1/10,000 dilution. After three washes with PBS-T, the stained bands were visualized with the ECL detection reagent (Amersham Biosciences).

Solid-phase in vitro phosphorylation assay

c-Rel TAD constructs from position 422 to 588 or 422–540 (using as template pRc-hc-Rel wt) were cloned into the BamH1-EcoR1 site of plasmid pGEX2T (Pharmacia) to express rGST-c-Rel fusion protein (28). These recombinant proteins were purified from Escherichia coli-induced cultures according to the manufacturer’s instructions. Anti-Flag precipitates from WCE from transfected COS-7 cells were incubated in the kinase buffer described before, containing 1 µg of soluble rGST-c-Rel (422–588). The reaction mixture (kinase buffer) contained 20 mM HEPES (pH 7.6), 20 mM MgCl2, 20 mM beta-glycerophosphate, 20 µM ATP, and 1 µCi [32P]ATP (specific activity 3000 Ci/mol). After 20 min at 30°C, the reaction was terminated by washing with TNT buffer. Phosphorylated protein was boiled in 25 µl of Laemmli sample buffer and resolved in 10% SDS-PAGE, followed by autoradiography.

GST pull-down assay

GST-c-Rel (422–588) and GST-c-Rel (422–540) fusion proteins were purified from E. coli-induced cultures by absorption with glutathione agarose beads, after induction of expression with 1 mM isopropyl-beta-D-thiogalactopyranoside. GST-c-Rel and GST polypeptides complexed with the beads were quantified by SDS-PAGE and Coomassie stain and comparison with different amounts of BSA. Equal amounts of fusion proteins were added to in vitro-translated pcDNA3-NIK plasmid labeled with [35S]methionine (Amersham Biosciences), (7 µl of rabbit reticulocyte lysate, TNT in vitro transcription translation; Promega). After shaking for 2 h at 4°C with NETN binding buffer (200 mM NaCl, 20 mM Tris-HCl (pH 8), 1 mM EDTA, and 0.5% Nonidet P-40), the beads were washed seven times in binding buffer. Bound proteins were eluted in SDS, resolved by SDS-PAGE, and visualized by autoradiography.

Statistical analysis

Experiments were repeated at least four times to guarantee the reproducibility of results. The experiments shown are a representative example or the mean ± SD. Differences were analyzed using nonparametric tests (Mann-Whitney "U").


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
NIK is required for IL-2 production

First, we tested the role of NIK on IL-2, by using NIK mutant spleen cells from aly/aly mice. In agreement with previous results (35), aly/aly spleen cells have a defect in T cell proliferation (data not shown) and in IL-2 production (Fig. 1). This defect on IL-2 was proportionately more pronounced in response to anti-CD3 than in response to anti-CD3/CD28. Similarly, GM-CSF secretion was also severely impaired in stimulated T cells from aly/aly mice. In contrast, TNF production, which was induced by anti-CD3, but was not further increased by anti-CD28 stimulation, was not impaired in aly/aly mice (Fig. 1). The small increase in TNF observed in aly/aly mice was not statistically significant.


Figure 1
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  		FIGURE 1. T cells from aly/aly mice have a defect on IL-2 and GM-CSF but not TNF synthesis. Spleen cells from wild-type or aly/aly mice were left unstimulated (–), or stimulated with anti-CD3 alone or anti-CD3 plus anti-CD28. Supernatants were collected 48 h later and TNF, GM-CSF, and IL-2 were evaluated by ELISA. Results are shown as the mean ± SD of the data from three mice. *, p < 0.05; differences from the wild-type animals.

 
Next, we tested whether NIK affects IL-2 transcription. For this, Jurkat cells were transfected with the human IL-2 promoter, together with NIK-Flag wild-type NIK-kinase deficient (KD)-Flag or aly NIK-myc. Anti-CD3 stimulation increased IL-2 transcription ~2- to 5-fold depending on the experiment, which was strongly potentiated by costimulation with anti-CD28 (up to 20-fold) (Fig. 2). Interestingly, those effects were sharply reduced by transfection of the aly mutant or the kinase-deficient NIK molecule (Fig. 2A). Again, the effect on anti-CD3 was proportionally more sensitive to mutant NIK molecules than the one of anti-CD3 plus CD28 (Fig. 2A). In contrast, as shown in Fig. 2B, overexpression of NIK significantly increased IL-2 promoter-driven transcription (by ~3- to 4-fold on average) in Jurkat T cells and potentiated the effect of anti-CD3 or anti-CD3 plus CD28.


Figure 2
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  		FIGURE 2. NIK affects IL-2 transcription. A, Jurkat cells were transfected with IL-2 reporter together with alyNIK-myc, NIK-KD-Flag, or identical amount of empty DNA plasmid as indicated. The cells were then left in medium (basal) or stimulated with anti-CD3 alone or plus anti-CD28 as indicated. Only the optimal dose of NIK is shown. B, Jurkat cells were transfected with IL-2 reporter together with wild-type or aly NIK or identical amount of empty DNA plasmid and stimulated with CD3 plus CD28. The amount of NIK-transfected proteins is shown by Western blot (inset). Transfection efficiency was evaluated with the pRL-tk-Renilla luciferase plasmid. Data are expressed in RLUs normalized by the relative Renilla luciferase units obtained in the control samples of every transfection and by microgram of protein. Results shown are the mean ± SD of the four experiment performed. *, p < 0.05 differences from the pcDNA3-transfected control cells.

 
IL-2 transcription is dependent on the binding of transcription factors to various sites in the promoter region. Among those, NF-{kappa}B, NF-AT, and CD28RE sites play a predominant role (11). Because aly/aly mice seem to have reduced responses of two C28RE-dependent genes, GM-CSF and IL-2, (2) but not of TNF that depends on AP-1, NF-AT, and NF-{kappa}B but not on CD28RE (39, 40), we tested whether NIK affects CD28RE-mediated responses. As shown in Fig. 3, transfection of wild-type NIK strongly up-regulated (5- to 6-fold) the transcription mediated by this response element. As expected, c-Rel overexpression was able to up-regulate up to 2000-fold the transcription of this reporter in Jurkat cells. More interestingly, NIK synergistically potentiated the ability of c-Rel producing up to 6000-fold increase on average (Fig. 3). Interestingly, although p65/relA overexpression was also able to increase this CD28RE reporter, NIK did not significantly potentiate its effect (Fig. 3).


Figure 3
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  		FIGURE 3. NIK affects CD28RE-dependent transcription. Jurkat cells were transfected with CD28RE/AP-1 reporter gene with wild-type NIK-Flag and/or c-Rel or p65/RelA or identical amount of empty DNA plasmid. Transfection efficiency was evaluated with the pRL-tk Renilla luciferase plasmid. Only the optimal dose of NIK-Flag and c-Rel is shown. Data are expressed in RLUs normalized by the relative Renilla luciferase units obtained in the control samples of every transfection and by microgram of protein. Results shown are representative of the four experiments performed. Numbers on the top of each bar represent the fold induction over basal pcDNA3-transfected cells.

 
NIK interacts with c-Rel and phosphorylates it

Next, we tested whether NIK interacts with c-Rel. For this, COS-7 cells were transfected with c-Rel, NIK-Flag, and NIK-myc. As shown in Fig. 4A, NIK-Flag was coimmunoprecipitated with endogenous c-Rel in COS-7 cells, but not in control cells transfected with control empty plasmid. In addition, transfection of PKC{zeta} did not bring down c-Rel (data not shown). To confirm those results, we did the opposite experiment immunoprecipitating with anti-myc, coimmunoprecipitated c-Rel (Fig. 4B). Moreover, a GST fusion protein with the aa 422–588 and 422–540 of human c-Rel, that contains the TAD of c-Rel, but not control GST, specifically pulled-down NIK (Fig. 4C). There are two regions in the TAD of c-Rel (41); one is inducible and the other is not. Deletion of the constitutive TAD2 did not affect NIK pull-down, suggesting that NIK interacts with the phosphorylation-inducible TAD1.


Figure 4
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  		FIGURE 4. Phosphorylation and association of c-Rel and with NIK. Interaction of NIK with c-Rel. COS-7 cells were transfected with 500 ng of the c-Rel and plasmids expressing NIK-Flag (A), NIKmyc (B), or pcDNA3 as indicated. A total of 20 µl of WCE (500 µl) were assayed to analyze the expression of the transfected c-Rel and NIK proteins by Western blot. Those WCE were immunoprecipitated with c-Rel serum or anti-myc and fractionated by SDS-PAGE. The presence of NIK and c-Rel in the immunocomplexes was analyzed by immunoblotting with an anti-Flag (A) or anti-c-Rel (B) Ab. C, In vitro-translated pcDNA3-NIK-Flag plasmid was incubated with GST or GST-c-Rel (422-588) or GST c-Rel (422-540). D, NIK phosphorylates the TAD region of c-Rel. WCE from Jurkat cells transfected with NIK-Flag or pcDNA3 were immunoprecipitated with anti-Flag Ab and the immunocomplexes assayed in an in vitro kinase assay using the rGST-c-Rel (422-588) protein as substrate or GST as control. Phosphorylated proteins were resolved by SDS-PAGE and detected by autoradiography.

 
Once we established the existence of an interaction between NIK and c-Rel, we next tried to assess whether NIK could phosphorylate c-Rel, testing its kinase activity in an in vitro kinase assay with the rGST fusion protein comprising of region 422-588 of c-Rel TAD as substrate. NIK was immunoprecipitated from transfected COS-7 with an anti-Flag Ab and the immunocomplexes were incubated with GST-c-Rel in an "in vitro" kinase assay. Interestingly, this resulted in the phosphorylation of the TAD of this protein (Fig. 4D). PKC{zeta} or Cot/tpl-2 kinases did not phosphorylate this construct (data not shown).

Mapping of the NIK domain which interacts with the TAD of c-Rel

The above results indicate that NIK and c-Rel interact. Thus, we found it interesting to map the region of NIK involved in its interaction with c-Rel. For this purpose, various NIK deletion constructs were passed through a column of GST fused to the TAD of c-Rel (GST-c-Rel (422-588)). Only fragments 367-947 and 624-947 and weakly 1-821 were significantly and specifically retained by the GST-c-Rel (422-588) column. In contrast, no significant interaction was detected with NIK 1-367, 1-640, 1-720, and 1-771 (Fig. 5). Those results mapped NIK interaction with c-Rel to the region encompassing aa 771–947.


Figure 5
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  		FIGURE 5. Mapping of the interacting region of NIK with the c-Rel TAD domain. A, Schematic representation of NIK deletion mutants used. B, NIK deletion proteins were labeled with [35S]methionine. NIK deletions were pulled-down with GST-c-Rel (422–588) or GST as control. B, The Coomassie blue staining of input of the material is shown. C, Proteins bound by GST or GST c-Rel (422–558) resolved by SDS-PAGE.

 
NIK potentiates the transactivating function of c-Rel

The above results indicated that NIK phosphorylates c-Rel TAD. Moreover, we had previously found that the phosphorylation of the Rel TAD is important for signal-induced transcription (29). Thus, we tested whether NIK potentiates the intrinsic transactivation activity of c-Rel providing a system where transcriptional activity of this protein could be assayed without interference of I{kappa}B association and/or degradation. For this, we transfected NIK together with Gal4-c-Rel (422-588). These constructs were transfected into Jurkat T cells along with a 5xGal4-luc reporter plasmid and luciferase activity was recorded. However, as shown in Fig. 6A, NIK up-regulated c-Rel transactivating activity by ~4-fold. Overexpression of a related MAP3K, such as MEKK-1 or glycogen synthase kinase 3, was ineffective in this assay (data not shown). NIK did not affect the basal transactivation activity of the Gal4-DBD-negative control (see Fig. 7). Moreover, stimulation through CD28 stimulation in the absence of CD3 was sufficient to induce Gal4-c-Rel transactivation activity. Interestingly, this CD28-mediated enhancement was abolished by overexpression of NIK-KD (Fig. 6B).


Figure 6
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  		FIGURE 6. NIK is involved in anti-CD28 stimulation of c-Rel-transactivating activity. Jurkat cells were transfected with Gal4-c-Rel (422-588), Gal4-luc reporter plasmid and cotransfected with NIKwt, kinase defective mutant, or an identical amount of empty pcDNA3 plasmid and normalized for transfection efficiency as described in Materials and Methods. A, Effect of NIK overexpression. B, Cells were stimulated with anti-CD28 for 16 h. Results are shown as the mean ± SD of the three experiment performed. (*), p < 0.05 and (ns), nonsignificant differences from the pcDNA3-transfected control cells.

 

Figure 7
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  		FIGURE 7. Effect of c-Rel deletion mutants on NIK-induced transcriptional activity. Jurkat cells were transiently transfected with the reporter plasmid Gal4-luc (250 ng) along with 7 ng of the various Gal4-c-Rel fusion deletion mutant constructs. Cultures of Jurkat wild-type cells were also cotransfected with 200 ng of the expression plasmid for wild-type NIK or with control pcDNA3 where indicated. Cells were cultured for 4 h and luciferase activity was evaluated and normalized for transfection efficiency as described in Materials and Methods. A, Schematic representation of the c-Rel protein showing the Gal4-c-Rel (309–588) deletion mutants used. B, Effect of NIK overexpression on the transactivation activity of the various Gal4-c-Rel deletion mutants. A summary of the NIK effects is indicated by + or – symbols in the part A of C. The amount of the different Gal-4 c-Rel constructs expressed is shown. Results are shown as the mean ± SD of the three experiments performed

 
To map the region responsible for the transcriptional activation of human c-Rel by NIK, we tested its enhancing effect on several c-Rel deletion mutants from positions 309 to 588 (Fig. 7A). Although the basal transcription varied among the various constructs as reported (29), those spanning the c-Rel region from 309 to 455 were unresponsive to NIK (Fig. 7B). By contrast, Gal4-c-Rel (309-540) responded to NIK stimulation. In addition, progressive deletions toward the C terminus Gal4-c-Rel (422-588), Gal4-c-Rel (456-588), and Gal4-c-Rel (498-588) also remained responsive to NIK. Although the region 540-588 is required for basal transcriptional activity (29), it did not respond to NIK stimulation, suggesting that this C-terminal region was necessary for the basal transcriptional activity of c-Rel but was not involved in NIK stimulation (Fig. 7B). This is in agreement with the fact that this region is dispensable for NIK pull-down. The above differences cannot be attributed to the differences in the expression levels of the various deletion mutants (Fig. 7C).

In contrast, we had previously identified some Ser residues in the c-Rel TAD required for its signal-induced transcriptional activities (29). Thus, we tested whether those mutants can be activated by NIK overexpression. For this, we cotransfected NIK kinase with various Gal4-c-Rel constructs in which some serines were mutated to alanine and tested the effect of the transactivation activity (Fig. 8A). Basal transcription was not statistically different in all those mutants. Interestingly, the A4 and A8 mutants that we had described previously and were important for TNF-induced transactivation activity (22) have a decreased ability to be activated by NIK overexpression (Fig. 8B). In addition, NIK-enhancing activity was severely reduced in the A6 mutant and in the spontaneous TNF-unresponsive mutant Ser471Asn (28). In contrast, A3 mutation which is outside the 456–540 region had no effect on the enhancing effect of NIK. Again, those differences cannot be attributed to the differences in the expression levels of the various deletion mutants (Fig. 7C).


Figure 8
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  		FIGURE 8. Effect of c-Rel Ser mutants on NIK-induced transcriptional activity. A, Amino acid sequence of the c-Rel transactivation domain (422-588), with the indicated Ser to Ala substitution mutants (A3, A4, A6, and A8 and Ser471Asn) used. B, Effect of NIK overexpression on c-Rel transactivation of the various Gal4-c-Rel Ser mutants. Jurkat cells were transiently transfected with the reporter plasmid Gal4-luc (250 ng) along with 7 ng of the various Gal4-c-Rel fusion constructs (Ser to Ala Gal4 mutants or the Ser471/Asn mutant). Cultures of Jurkat wild-type cells were also cotransfected with 200 ng of the expression plasmid for wild-type NIK or with control pcDNA3 where indicated. Cells were cultured for 16 h and luciferase activity was evaluated and normalized for transfection efficiency as described in Materials and Methods. C, Similar amounts of the different constructions were expressed in transfected cells as detected by Western blot assays (data not shown). Results are shown as the mean ± SD of at least four independent experiments. *, p < 0.05 and ns, nonsignificant differences from the Gal4-DBD-transfected control cells.

 
To get some insights into the functional role of those phosphorylated serines, we used a previously described cell line, Jurkat D6, that has a mutation in c-Rel Ser471 (28). This cell line has also severely impaired its ability to synthesize IL-2 in response to CD3 or CD3 plus CD28 as compared with Jurkat wild type (Fig. 9A). Similarly, IL-2 promoter-driven transcription induced by anti-CD3 plus CD28 was impaired (Fig. 9B). Interestingly, NIK overexpression did not significantly increase CD28RE-dependent transcription in those mutant D6 cells in contrast to Jurkat wild-type cells (Fig. 9C), strongly suggesting that NIK is acting on IL-2 transcription through phosphorylation of that Ser residue of c-Rel. NIK inducibility of the full IL-2 promoter was also decreased in Jurkat D6 (Fig. 9D).


Figure 9
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  		FIGURE 9. Anti-CD3 plus anti-CD28 stimulation and NIK-induced IL-2 transcription is impaired in Jurkat D6 (c-Rel Ser471Asn) mutant cells. A, Jurkat wild-type or mutant D6 cells were left unstimulated (–), or stimulated with anti-CD3 alone or plus anti-CD28 and IL-2 production evaluated in the supernatants 24 h later. B, Jurkat cells were transfected with IL-2 reporter. They were then stimulated with anti CD3 alone or plus anti-CD28 for 18 h. Results shown in A and B are representative of the four experiments performed. Data are expressed in B in RLUs normalized by the relative Renilla luciferase units obtained in the control samples of every transfection and by micrograms of protein. C and D, Jurkat cells were transfected with CD28RE (C) or IL-2 (D) reporter together with wild-type NIK-Flag or identical amount of empty DNA plasmid. Only the optimal dose of NIK-Flag is shown. Results are shown as the mean ± SD of the fold induction over cells transfected with control empty plasmid of four independent experiments. *, p < 0.05, differences from the pcDNA3-transfected control cells.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
It is well-known that TCR signaling is not enough to provide a full stimulation of T cells. Other costimulatory molecules are required. Among those, CD28 plays a very prominent role. However, despite the numerous studies the signal transduction by which CD28 acts is still far from settled. IL-2 transcription is a paradigm of coordinated regulation by several transcription factors that includes members of NF{kappa}B, AP-1, and NF-AT families (11). Those factors bind to (response elements) in the promoters. Among those, CD28RE plays a very important role in IL-2 transcription. In this study, we show that NIK is involved in regulating CD28RE. This leads to the up-regulation of CD28RE responsive cytokines such as IL-2. This is supported by experimental evidence. First, aly/aly NIK mutant mice have a defect in IL-2 production in response to TCR stimulation, in agreement with previous results (35). We also found that the synthesis of GM-CSF, another cytokine strongly dependent on a CD28RE site for its transcription (2), was also similarly impaired in those mice. The fact that IL-2 but not GM-CSF synthesis was less severely decreased in response to CD3 than to CD3 plus CD28 may have several explanations. First, CD3 also induces CD28RE although to a lesser extent than in the presence of costimulatory CD28 (2, 3). The prevailing view is that differences between CD3 and CD3/CD28 are quantitative rather that qualitative. Thus, a stronger signal may be less affected than a weaker one. Second, CD28 gives other signals different from those dependent on NIK to the IL-2 promoter. Those signals may partially compensate for the lack of the ones supplied through NIK to CD28RE. In this regard, IL-2 promoter transcription requires the coordinated action of many transcription factors (11). Therefore, the loss of one pathway combined with a stronger stimulus may result in less inhibition. In addition, some of those extra signals for IL-2 may not be needed for GM-CSF promoter transcription, which seems to be more dependent on CD28RE than IL-2. So, in this case a similar inhibition was observed. However, TNF transcription that depends on AP-1, NF-AT, and NF-{kappa}B, but not on CD28RE sites (39, 40), was not impaired in T cells from aly/aly mice. Those results pointed to the CD28RE element as the main element affected in T cells from aly/aly mice. In agreement with that hypothesis, we found that CD28RE-dependent transcription was enhanced by NIK. CD28RE is a preferred binding site for c-Rel (2) and thus c-Rel transfection greatly enhanced CD28RE-dependent transcription. Interestingly, our results show that NIK synergizes with c-Rel but not with p65/RelA in promoting CD28RE-dependent transcription activity producing a very strong up-regulation (up to 2000-fold). Biological responses controlled by several extracellular stimuli may require the convergence of signaling pathways mostly at the level of the transcription factors (3). The TCR signal may activate NF-{kappa}B (p65/relA and c-Rel), whereas CD28 via NIK may induce phosphorylation of the TAD of c-Rel.

Taken together, our results suggest that NIK may play an important role in T cell activation through CD28RE elements. Besides, T cells from aly/aly mice have a normal early NF-{kappa}B activation phase but a severely impaired late phase (35). In this regard, we have previously found, both in humans and mice, that the early phase of T cell activation is dependent on p65/relA whereas the second phase of NF-{kappa}B activation depends mostly on c-Rel (28, 42). Moreover, the Jurkat D6 mutant cell line, bearing a Ser to Asn mutation in position 471 of human c-Rel, has a similar phenotype regarding NF-{kappa}B activation. Thus, this mutation affects the second phase of NF-{kappa}B activation, being only the late c-Rel-dependent activation affected (28). Moreover, we showed here that D6 cells did not produce IL-2 in response to CD3/CD28. This mutation also impairs c-Rel transactivation and both CD28RE reporter and IL-2 promoter activity induced by NIK transfection. Moreover, we describe for the first time that CD28 induces Gal4-c-Rel transactivation activity independently of CD3 and this effect is blocked by NIK-KD.

In this regard, it is interesting to note that CD28 costimulation is probably the best stimulus to induce high IL-2 production and that c-Rel is required for IL-2 secretion as demonstrated in c-Rel-deficient mice (43). Moreover, CD28 costimulation sustains IL-2 promoter activation largely due to the activation of c-Rel binding to the CD28RE (44), although some other factors bind to this site, such as p65 and NF-AT (45). Of note, aly/aly mice also have a severe defect in IL-2 (Ref.35 and this manuscript) and GM-CSF secretion. Those cytokines are strongly dependent on CD28RE (or CD28 stimulation) and are also two of the cytokines more severely impaired in c-Rel-deficient mice (43, 46). Thus, the similar behavior of T cells harboring c-Rel mutations (Jurkat D6 and c-Rel-deficient mice) or the aly/aly mutation strongly argues in favor of a connection between NIK and c-Rel at least in T cell activation of CD28RE-dependent cytokines. Moreover, aly/aly mice also have lower levels of c-Rel bound to the DNA (36).

It is well-established that IKK activation, and subsequent I{kappa}B degradation, is a prerequisite for NF-{kappa}B activation in response to several stimuli (13, 14, 15, 16). However, posttranscriptional modifications are increasingly recognized as an important way to modulate NF-{kappa}B activity. This regulation involves the signal-dependent phosphorylation and activation of the TAD of p65 and c-Rel by various kinases although the exact mechanism has not yet been defined. Among those protein kinases, PKA, PKC{zeta}, and IKK have been implicated in this process (reviewed in Refs.20 and 21). In this study, we show that NIK, which has been previously shown to activate NF-{kappa}B and induce I{kappa}B{alpha} degradation, at least by a subset of ligands (34), and also induces NF-{kappa}B2/p100 processing (18, 19), is also involved in regulating the transactivation function of c-Rel. Moreover, our results suggest that direct phosphorylation of c-Rel by NIK might contribute to the activation of its transactivating activity. NIK was one of the first kinases to be involved in the activation upstream of NF-{kappa}B (38). However, studies in NIK knockout and aly/aly mice have shown that NIK is not a common upstream kinase that activates IKKs (31, 32). Rather, it seems to act in a receptor-specific manner through LTbetaR to promote the transcriptional activity. Thus, in those mice neither I{kappa}B{alpha} degradation nor NF-{kappa}B translocation or DNA binding seems to be affected, although the NF-{kappa}B-dependent reporter transcription is decreased in response to LTbeta. Those results strongly suggest that NIK may act after nuclear translocation and are in agreement with our results indicating that NIK is affecting c-Rel NF-{kappa}B transactivation.

Moreover, we have mapped the interaction with c-Rel and NIK to aa 422–540 (TAD-1) of c-Rel and to the region 771–949 of NIK. This region acts as dominant negative of the TAK1/TAB1 activation pathway (47), and those kinases have also been involved in NIK activation and in the alternative pathway of NF-{kappa}B activation (48). It remains unclear whether c-Rel is phosphorylated in the nucleus or in the cytoplasm. Because Gal4-DBD-fusion proteins are normally located in the nucleus, it seems likely that this phosphorylation takes place in the nucleus. In this regard, it has been shown that NIK undergoes shuttling between the cytoplasm and the nucleus, where it may perform its function not only as an IKK activator (49), but also leading to Ser phosphorylation in c-Rel and increasing transactivating activity, as our results suggest.

Our previous work delimited the location of c-Rel TAD between positions 422-588 of the C terminus. This region is composed of two domains: TAD-2 (540-588) that has basal-transactivating activity and TAD-1 (422-540) with stimulus-inducible transactivating activity (29); these results were later confirmed by other authors (41). In the present study, we have mapped the region 455-540 as the most important in NIK activation of Gal4-c-Rel transactivation domain. Moreover, deletion of 540-588 did not impair c-Rel association with NIK. Although the region 540-588 (TAD2) was required for basal transactivation, only region 422-540 (TAD1) of c-Rel was phosphorylated and activated by stimulation by TNF and TCR stimuli (28). Thus, the activation of this region requires extracellular signal-dependent phosphorylation. Our studies with deletion mutants indicated that regions 455-497 and 498-540 could be stimulated by NIK. However, these fragments individually bound to Gal4 do not respond to stimulation. We think that the adjacent regions are important in the control of the conformation of the region with capacity of response. Moreover, the substitution of the Ser by Ala residues at position 454 (mutant A3), 460 (mutant A4), at Ser470, Ser471, and Ser473 (mutant A6) and at Ser491 and Ser494 (mutant A8) completely prevented the activation of this domain by TNF (29). NIK-inducing activity was also affected by A4, A6, A8, and Ser471Asn mutations, which are included inside the region 455-540 but not by A3 which is outside this region.

In contrast, the molecular mechanism by which NIK becomes activated by CD28 remains unknown. The signal transduction pathways elicited by CD28 costimulation have been extensively studied (2, 3, 4). From those studies, it is clear that CD28 operates via multiple molecular pathways. However, few unique signaling components and/or genes activated only by CD28 have been elucidated (50). Activation of Cot (Tpl2) kinase (9) and PI3K/Akt (51) pathways have been suggested among the putative ones stimulated mainly by CD28. Besides, both Cot (52) and PI3K/Akt (53) have been involved in the control of NF-{kappa}B activity independent of nuclear translocation and I{kappa}beta{alpha} degradation and PI3K/Akt activates Cot kinase in T cells (54). Moreover, we also found that PI3K is also involved in regulating the c-Rel transactivation through the phosphorylation of the same serines (mostly Ser491 and Ser494) (29) that are also targets for NIK, as observed here. In the case of p65/relA, a recent report indicated that the TCR induction of serine 536 phosphorylation and transactivating activity is regulated by the Cot (Tpl2), receptor-interacting protein), PKC{theta}, and NIK kinases, but is independent from the PI3K/Akt signaling pathway (52).

In summary, our results support that NIK may be controlling IL-2 transcription and T cell activation by modulating c-Rel phosphorylation in specific serines of the TAD. This leads to a more efficient transactivation of genes which are dependent on CD28RE sites where c-Rel binds.


   Acknowledgments
 
We especially thank Dr. M. A. Iñiguez for helpful discussion and critical reading of the manuscript. We also thank Hugo Salgado, María Chorro, María Cazorla, and Gloria Escribano for their excellent technical assistance.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
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 with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported in part by grants from Programa Nacional de Salud of Spain Fondo de Investigaciones Sanitarias, Recava Cardiovascular Network (C03/01), Red investigación en SIDA Network (G03/173), Main Network of Excellence and EICOSANOX Integrated Project (6th European Union Framework Programme), Laboratorios del Dr. Esteve, Comunidad Autónoma de Madrid, and Fundación Ramón Areces. Back

2 Address correspondence and reprint requests to Dr. Manuel Fresno, Centro de Biología Molecular, Consejo Superior de Investigaciones Científicas, Universidad Autónoma de Madrid, Cantoblanco, E-28049 Madrid, Spain. E-mail address: mfresno{at}cbm.uam.es Back

3 Abbreviations used in this paper: Cot/tpl-2, (cancer Osaka thyroid) oncogene/tumor progression locus 2; CD28RE, CD28 responsive element; TAD, transactivation domain; IKK, I{kappa}B kinase; LTbeta, lymphotoxin beta; NIK, NF-{kappa}B-inducing kinase; PKA, protein kinase A; PKC, protein kinase C; DBD, DNA-binding domain; WCE, whole cell extracts; RLU, relative luciferase unit; PBS-T, PBS containing 0.05% Tween 20; KD, deficient kinase. Back

Received for publication July 21, 2005. Accepted for publication February 6, 2006.


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 Introduction
 Materials and Methods
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 Discussion
 Disclosures
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D. P. Young, I. Kushner, and D. Samols
Binding of C/EBP{beta} to the C-Reactive Protein (CRP) Promoter in Hep3B Cells Is Associated with Transcription of CRP mRNA
J. Immunol., August 15, 2008; 181(4): 2420 - 2427.
[Abstract] [Full Text] [PDF]

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K. E. King, R. M. Ponnamperuma, C. Allen, H. Lu, P. Duggal, Z. Chen, C. Van Waes, and W. C. Weinberg
The p53 Homologue {Delta}Np63{alpha} Interacts with the Nuclear Factor-{kappa}B Pathway to Modulate Epithelial Cell Growth
Cancer Res., July 1, 2008; 68(13): 5122 - 5131.
[Abstract] [Full Text] [PDF]