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Subunit of I
B Kinase Functionally Associate to Induce NF-B Activation in Response to CD28 Engagement1
* Department of Cellular and Developmental Biology, University of Rome La Sapienza, Rome, Italy;
Laboratories of Cell Biology, Istituto Superiore di Sanità, Rome, Italy;
Fondazione Andrea Cesalpino, Policlinico Umberto I, University of Rome La Sapienza, Rome, Italy; and
Program in Signal Transduction Research, The Burnham Institute, La Jolla, CA 92037
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Abstract |
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B kinase (IKK) complex and, consequently, to NF-B activation, and we identified Vav-1 as an important mediator of this function. Here we report for the first time that Vav-1 constitutively associates with IKK
in both Jurkat and primary CD4+ T cells. Vav-1/IKK association is mediated by their helix-loop-helix domains, does not involve IKK
, and is functionally relevant in that Vav-1-associated IKK kinase activity is increased following CD28 engagement by B7. Moreover, we demonstrate that CD28-induced NF-B activation is augmented by both IKK and Vav-1, but not IKK. Confocal microscopy showed that endogenous Vav-1 and IKK, but not IKK, were recruited to the membrane and colocalized in response to CD28 stimulation. Taken together, these data evidence that Vav-1 plays a key role in the control of NF-B pathway by targeting IKK in the T cell membrane and favoring its activation in response to CD28 stimulation. |
Introduction |
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1 activation and Ca2+ response (3). The consequence of this action is an augmentation of early TCR-mediated signals (4) that facilitates reaching a threshold above which full activation occurs (5). In addition to activate specific signals integrating those delivered through TCR, CD28 may also act as an independent signaling unit able to furnish in trans costimulation (6). Indeed, CD28 stimulation can induce cytoskeletal rearrangements in T cells (7) or up-regulate IL-2/4 transcription independently of TCR (8). Despite these evidences, the molecular mechanisms as well as the targets of CD28 as an independent signaling unit remain unclear.
NF-B/Rel transcription factors are critical regulators of immune responses being positioned to integrate information from both innate and adaptive immune signaling pathways. NF-B activity is regulated by a protein kinase complex known as IB kinase (IKK) signalsome, which phosphorylates both IB and IB, thus leading to their proteolytic degradation and release of NF-B into the nucleus. The IKK complex contains two serine kinases, IKK and IKK, and a third subunit, IKK/NF-B essential modulator, with regulatory functions (9). The analysis of knockout mice indicates that IKK, rather than IKK, is critical for NF-B activation in response to inflammatory cytokines (10, 11). However, recent data show that IKK modulates IKK kinase activity to regulate NF-B (12) and that IKK can activate a second evolutionary conserved pathway leading to NF-B activation through the phosphorylation-dependent degradation of the NFKB2 molecule (13). Several upstream kinases including NF-B-inducing kinase, Cot-1 (14), mixed-lineage kinase 3 (15), and protein kinase C (PKC) 
(16) have been implicated in IKK activation by TCR/CD28, but their relevance and specific position in the signaling pathway that links the surface receptor to NF-B is still unclear. In this context, we have recently demonstrated that CD28 engagement by B7 can generate TCR-independent signals leading to IKK and NF-B activation (17) and that Vav-1 acts as the upstream regulator of this signaling pathway (18).
The protooncogene Vav-1 regulates both thymocyte development and activation of T lymphocytes (19). Vav-1 contains a helix-loop-helix (HLH) domain, a Dbl homology (DH) region characteristic of guanine nucleotide exchange factors for the Rho/Rac family of protein G (20), a pleckstrin homology domain, a single Src homology 2 domain, and two Src homology 3 domains, which mediate the formation of multimeric signaling complexes. Vav-1 is tyrosine-phosphorylated upon TCR and CD28 stimulation (21, 22). This event induces the up-regulation of Vav-1 exchange activity for Rac-1 (23) and appears to be important in regulating the capacity of Vav-1 to participate in the activation of several TCR- and CD28-mediated signaling pathways (24). Indeed, Vav-1-deficient T lymphocytes are defective in TCR-induced intracellular calcium fluxes and activation of extracellular signal-regulated kinase (ERK) and NF-B (25, 26). Recent data have shown that Vav-1 overexpression in Jurkat cells synergized with PKC to induce NF-B transcriptional activity (16) through the recruitment of PKC to the IS, thus favoring IKK activation (27). Despite its central role in CD28 costimulation, the mechanisms by which Vav-1 couples CD28 to IKK and NF-B activation remain still unknown.
In this study, we show that Vav-1 associates with IKK in both Jurkat and primary human CD4+ T cells. Vav-1/IKK association was mediated by their HLH domains observed and did not involve IKK. Moreover, Vav-1-associated IKK kinase activity was increased by CD28 stimulation with B7, and both Vav-1 and IKK cooperate to potentiate CD28-induced NF-B transcriptional activity. Finally, we also show that both Vav-1 and IKK translocate from cytoplasm to the membrane, and colocalize at the level of CD28 clusters induced by anti-CD28 Abs. Our results demonstrate that in T cells CD28 activates NF-B independently of TCR by cooperating with Vav-1 and IKK.
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Materials and Methods |
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Jurkat and its derivative 31.13, defective of TCR/CD3 expression (28), were maintained in RPMI 1640 supplemented with 10% FCS, L-glutamine, penicillin, and streptomycin (Life Technologies, Rockville, MD). The Vav-1-deficient cell line J.Vav1 (25) was maintained in RPMI 1640 supplemented with 10% FCS, and a J.Vav1 derivative cell line, J.Vav1.WT, stably re-expressing Vav1, was maintained as above with the addition of 0.5 mg/ml G418. The L cell transfectants expressing HLA-DRB1*0101 (5-3.1) and cotransfected with human B7.1 (5-3.1/B7) were previously described (4). Rabbit anti-hemagglutinin (HA) and mouse anti-c-myc (9E10) Abs were obtained from Boehringer Mannheim (Mannheim, Germany). Rabbit anti-Vav-1 Ab was obtained from Upstate Biotechnology (Lake Placid, NY). Mouse anti-human IKK and rabbit anti-human IKK Abs were obtained from BD Bioscience (Mountain View, CA). Anti-PKC mAb was purchased from Transduction Laboratories (Lexington, KY). Mouse anti-CD28 mAb (CD28.1) was kindly provided by D. Olive (Institut Paoli Calmettes, Marseille, France). The following fluorochrome-conjugated secondary Abs were used for indirect immunofluorescence examinations: Alexa fluor 488- and Alexa fluor 594-conjugated goat anti-rabbit IgG and Alexa fluor 488-conjugated goat anti-mouse IgG F(ab') 2, purchased from Molecular Probes (Eugene, OR).
Human peripheral blood CD4+ T cells, were sorted by negative selection using an indirect magnetic cell sorting kit (Miltenyi Biotec, Auburn, CA). In some experiments, cells were stimulated with polystirene latex microspheres (Polyscience, Warrington, PA) coated with anti-CD28 mAb.
Plasmids, cell transfection, and luciferase assays
The NF-B-luciferase reporter construct was kindly provided by J. F. Peyron (Faculté de Médicine Pasteur, Nice, France). HA-tagged IKK, IKK, and the double mutant IKK (L605R, F606P) defective of HLH (HLH-) expression vectors were kindly provided by M. Karin (University of California San Diego, La Jolla, CA). pEF-Bos expressing N-terminal myc-tagged Vav-1 was a kind gift of A. Weiss (University of California, San Francisco, CA). pEF-Bos expressing myc-tagged oncoVav (29) was kindly provided by A. Altman (La Jolla Institute for Allergy and Immunology, San Diego, CA). The pSV-gal vector (Promega, Madison, WI) contains the -galactosidase gene driven by the SV40 promoter/enhancer. FLAG-tagged human Vav1 expression construct was provided by Dan D. Billadeau (Mayo Clinic, Rochester, MN).
Transient transfections were performed by electroporating (at 260 V, 960 µF) 107 Jurkat cells or 31.13 cells in 0.5 ml RPMI 1640 supplemented with 20% FCS with the NF-B-luciferase (2 µg) reporter construct together with 20 µg of pSV-gal plasmid and the indicated expression vectors, keeping the total amount of DNA constant (50 µg) with empty vector. Twenty-four hours after transfection, cells were left unstimulated or stimulated at 37°C for 8 h with the indicated stimuli. -galactosidase and luciferase assays were performed according to the manufacturer's instruction (Promega). Luciferase activity determined in triplicate samples was expressed as fold induction over the basal activity of cells cultured with medium alone, after normalization to the -galactosidase values.
J.Vav1 and J.Vav1.WT cells were transfected as previously described (25) with 10 µg NF-B-luciferase construct, with or without 5 µg human FLAG-Vav1, together with a pRL-TK reporter plasmid (Promega) to control for variations in transfection efficiency. After 18 h in culture, cells were unstimulated or stimulated at 37°C for 8 h with adherent 5-3.1/B7 cells in 24-well plates. Firefly and pRL-TK-derived Renilla luciferase activities were measured in each sample with the dual luciferase assay kit (Promega). All reporter gene assays were repeated a minimum of three times, and representative results from a single assay are shown.
Cell stimulation, immunoprecipitation, in vitro kinase assays, and immunoblotting
Jurkat and primary CD4+ T cells were washed twice, resuspended in medium (108/ml), and incubated in suspension for 15 min at 37°C with 5-3.1/B7 cells (108/ml). At the end of incubation, cells were harvested and lysed for 30 min on ice in 1% Nonidet P-40 lysis buffer in the presence of inhibitors of proteases and phosphatases. Vav-1 and IKK were immunoprecipitated, and the kinase activity of coprecipitated IKKs was assayed at 30°C for 30 min in 20 µl of kinase buffer in the presence of 10 µM ATP, 10 µCi of [-32P]ATP (10 Ci/mmol) using GST-IB (aa 1–54) as substrate. The proteins were resolved by 10% SDS-PAGE, blotted onto polyvinylidene difluoride membrane and autoradiographed. Radioactivity in the phosphorylated proteins was quantitated by a phosphorimager.
For immunoblotting, proteins were resolved by 10% SDS-PAGE and blotted onto nitrocellulose membranes. Blots were incubated with the indicated primary Abs, extensively washed and after incubation with horseradish peroxidase-labeled goat anti-rabbit or goat anti-mouse (Amersham, Arlington Heights, IL), developed with the ECL detection system (Amersham).
Confocal laser-scanning microscopy (CLSM) analyses
For CLSM analyses, Jurkat and primary CD4+ T cells were activated for 15 min at 37°C with polystirene latex microspheres (0.5 bead/cell) coated with anti-CD28 mAb. Cells were then seeded on poly-L-lysine hydrobromide-coated cover glass, fixed by paraformaldehyde 3% and permeabilized by Triton X-100, as previously described (30). Vav-1 was stained using a rabbit anti-Vav-1 (10 µg/ml) primary Ab followed by Alexa fluor 594-conjugated goat anti-rabbit serum. IKK was stained using a mouse anti-IKK mAb (10 µg/ml) followed by Alexa fluor 488-conjugated goat anti-mouse serum. IKK was stained using a rabbit anti-IKK Ab (10 µg/ml) followed by Alexa fluor 488-conjugated goat anti-rabbit serum. Actin was stained by using Alexa fluor 488- or Alexa fluor 594-conjugated phalloidin. CLSM observations were performed using a Leica TCS 4D apparatus (Leica, Deerfield, IL) equipped with an argon-krypton laser, double-dichroic splitters (488/568 nm), 520-nm barrier filter for Alexa 488 (green), and 590-nm barrier filter for Alexa 594 (red) observations. Image acquisition and processing were conducted by using the SCANware and Multicolor Analysis (Leica Lasertechnik, Heidelberg, Germany) and Adobe Photoshop (Adobe Systems, Mountain View, CA) software programs. Signals from different fluorescent probes were taken in parallel. Several cells were analyzed for each labeling condition, and representative results are presented.
Nuclear extract preparation and EMSA
Jurkat cells were stimulated with adherent 5-3.1/B7 cells for 4 h and then subjected to nuclear extract preparation, as previously described (17). EMSA was performed by incubating the nuclear extract with 32P-radiolabeled NF-B oligonucleotide probe (5'-AGTTGAGGGGACTTTCCCAGGC-3') for 20 min at room temperature and then resolving the DNA-protein complexes on native 5% polyacrilamide gels. For the supershift analysis, normal serum, anti-p50, anto-p65, or anti-p52 Abs (Santa Cruz Biotechnology, Santa Cruz, CA), were preincubated with the nuclear exctracts for 20 min at room temperature before the binding reaction.
RNA interference
Oligonuclotide small interference RNA (siRNA) were designed on the sequence of a N-terminal region of human vav-1 cDNA that is not present in vav-2 and vav-3 (5'-AAGGUCAUGUACACCCUGUCUG-3'). Antisense siRNA oligonucleotides with 3'-overhangs UU were synthesized and annealed by Dharmacon Research (Lafayette, CO). Jurkat cells were transfected with 2, 5, or 10 µg of Vav-1 siRNA. Unrelated siRNA specific for green fluorescent protein (5 µg) were used as control (Xeragon, Huntsville, AL). After 24 h, cells were subjected to gradient centrifugations to eliminate dead cells, and Vav-1 expression was evaluated by flow cytometry.
Flow Cytometric Analysis
Cells were fixed with PBS containing 2% paraformaldehyde and subsequently permeabilysed in PBS containing 0.5% BSA, 0.02% sodium azide, and 0.5% saponin. Then cells were incubated for 15 min at room temperature with rabbit anti-human Vav-1 Abs or isotype-matched rabbit IgG Abs (Sigma, St. Louis, MO), washed and stained with FITC-labeled anti-rabbit IgG (Sigma). Flow cytometric analysis was performed on a Becton Dickinson FACSCalibur flow cytometer (BD Bioscience, Mountain View, CA).
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in unstimulated Jurkat and primary human CD4+ T cells
We have recently demonstrated in Jurkat cells that CD28 stimulation alone was able to induce IKK kinase activity and the nuclear translocation of NF-B (18). To analyze the protein compositions of the NF-B complexes binding to the NF-B site, nuclear extracts from CD28-stimulated Jurkat cells were treated with Abs that specifically recognize NF-B members. Fig. 1A shows that the NF-B complex induced by CD28-stimulation was supershifted by both anti-p50 and anti-p65 Abs but not by anti-p52 Ab. Accordingly with the data obtained in Jurkat cells, CD28 stimulation of primary CD4+ T cells also induced a significant increase of IKK kinase activity (Fig. 1B). The ability of CD28 to stimulate NF-B transcriptional activity in a TCR-independent manner was confirmed by data obtained in the TCR-negative Jurkat T cell line, 31.13. As shown in Fig. 1C, CD28 stimulation strongly up-regulated NF-B activity in both Jurkat and 31.13 cells. The stronger increase of NF-B observed in 31.13 cells was related to a higher expression of CD28 when compared with Jurkat cells (data not shown). Moreover, NF-B activity was not induced by other costimulatory molecules such as LFA-1 and CD2 (data not shown), thus indicating the specificity of CD28 effects. The CD28 signaling pathway leading to both IKK and NF-B activation was regulated by Vav-1 as demonstrated by the potentiation of both IKK and NF-B transcriptional activities observed in Vav-1-overexpressing Jurkat cells (18). To further demonstrate the requirement of Vav-1 for NF-B activation by CD28, we selectively knocked out Vav-1 transcript using siRNA. RNA interference has been successfully used to prevent gene expression in cultured mammalian cells (31). Jurkat cells were transfected with NF-B-luciferase reporter construct together with oligonucleotide siRNA duplexes directed against the human vav-1 gene. After 24 h, both Vav-1 expression and luciferase activity were measured. As shown in Fig. 2, siRNA duplexes specifically inhibited Vav-1 expression in transfected cells (30–40%) without affecting the levels of endogenous IKK. Unrelated siRNA (ctr) did not exert any significant effect (Fig. 2A). The inhibition of Vav-1 expression was associated with a reduction of CD28-induced NF-B activity (Fig. 2B), thus demonstrating that Vav-1 was required for CD28 signaling to NF-B. To clearly assess the role of Vav-1 in this response, we transfected a Vav-null Jurkat T cell clone (J.Vav1) with NF-B-luciferase reporter construct and analyzed the effect of CD28 stimulation. As shown in Fig. 2C, J.Vav1 cells showed a significant inhibition of CD28-induced NF-B activity that was largely restored following both transient and stable (J.Vav1.WT) transfection of Vav-1 expression construct. Similar results were obtained when CD28-induced IB degradation in both Jurkat and J.Vav1 cells was compared (data not shown)
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or IKK. Interestingly enough, we found that Vav-1 associated with IKK but not with IKK in unstimulated cells (Fig. 3A, upper panel). Vav-1/IKK association did not involve IKK as demonstrated by the observation that IKK coprecipitated with Vav also in the IKK-negative Jurkat cell line JM4.5.2 (data not shown). In a reverse protocol, anti-IKK Abs coprecipitated Vav-1 in unstimulated Jurkat cells (Fig. 3C). As shown in Fig. 3B, Vav-1 was constitutively bound to IKK also in primary human CD4+ T cells, indicating that this association was not peculiar of Jurkat cells (upper panel). Because IKK and IKK mostly associate to form heterodimeric complex (32, 33), we sought to determine whether Vav-1-coprecipitated IKK complexes also contained IKK. Lysates from unstimulated Jurkat cells were subjected to at least five cycles of precipitation with protein A-Sepharose beads alone or protein A-Sepharose beads preadsorbed with anti-IKK Abs. As shown in Fig. 3D, the content of IKK in total lysates was reduced at least 80% after the last cycle of IKK depletion. Consistent with the notion of IKK/ heterodimers being more abundant and stable than / homodimers in normal conditions, IKK content was also reduced by 80% in IKK-depleted lysates (Fig. 3D, left). In contrast, neither Vav-1 content nor Vav-1-associated IKK amounts were changed following IKK depletion (Fig. 3D, right).
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monomers or homodimers but not with IKK/IKK heterodimers.
Vav-1 and IKK functionally cooperate to activate NF-B in response to CD28 engagement
We have recently shown that Vav-1 potentiates CD28-mediated signals, which lead to NF-B activation by enhancing the activity of the IKK/ complex (18). Thus, we examined whether Vav-1/IKK association could play a role in CD28-mediated signals to NF-B. Jurkat cells were stimulated for 15 min with adherent 5-3.1/B7 cells, Vav-1 was immunoprecipitated, and the activity of coprecipitating IKK was measured in an in vitro kinase assay by using GST-IB as substrate. As shown in Fig. 4A, CD28 engagement by B7 induced a 2-fold activation of Vav-1-associated IKK kinase activity (upper panel), without affecting the amount of coprecipitated IKK (lower panel). Similar results were obtained in primary CD4+ T cells stimulated for 15 min with anti-CD28 mAb (Fig. 4B). These experiments were repeated five times with similar results.
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potentiated CD28-induced NF-B transcriptional activity. To this end, Jurkat cells were transiently transfected with a NF-B-luciferase reporter construct together with expression vectors encoding for myc-tagged Vav-1 and/or HA-tagged IKK and . Luciferase activity was measured after stimulation with 5-3.1/B7 cells. As we have recently shown (18), CD28 engagement by B7.1 induced NF-B transcriptional activity, which was strongly augmented by Vav-1 overexpression (Fig. 5). Overexpression of both IKK and IKK increased NF-B basal activity, but only IKK was able to potentiate CD28-induced NF-B transcriptional activation. Moreover, coexpression of IKK and Vav-1 potently activated NF-B-dependent luciferase activity and augmented that induced by CD28. No augmentation of either basal or CD28-induced NF-B activity was seen upon coexpression of Vav-1 with IKK. On the contrary, overexpression of IKK inhibited Vav-1-induced potentiation of CD28-mediated NF-B activation. Because IKK binds more readily to IKK than IKK itself (32), exogenously transfected IKK may sequester Vav-1-associated IKK, thus explaining the inhibitory effect observed on CD28-stimulated cells. The expression level of each transfected protein was verified by immunoblotting with anti-HA or anti-myc Abs (Fig. 5, lower panels).
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association plays an important role for NF-B activation in response to CD28.
Vav-1/IKK association is mediated the interaction of their HLH domains
Both IKK and Vav-1 contain HLH domains that are involved in protein-protein interactions through their hydrophobic surfaces. The HLH domain of IKK has been involved in interactions with regulatory proteins (33). The HLH region of Vav is located at its N-terminal (position 27–68 aa), but less is known about the involvement of the HLH domain of Vav-1 in interactions with other proteins. We used point mutants of IKK disrupting its HLH region (HLH-) (33) and an oncogenic form of Vav-1 lacking the first 68 aa containing the HLH domain (oncoVav) (29) to map this interaction. As shown in Fig. 6A, the IKK HLH- mutant was unable to bind wild-type Vav-1. In a parallel experiment, we observed also that oncoVav was deficient in binding IKK (Fig. 6B), indicating that the interaction between these two proteins is mediated by their HLH domains. Accordingly with the functional role of Vav/IKK association for NF-B activation in response to CD28, IKK HLH- did not cooperate with Vav in potentiating CD28-induced NF-B activity (Fig. 6C).
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relocate and colocalize in the membrane upon CD28 stimulation
Vav-1 is a key regulator of the actin cytoskeleton rearrangements that are necessary to the accumulation of signaling molecules at the APC/T cell interface (34, 35). Moreover, engagement of CD28 costimulatory molecule also regulates the movement of actin cytoskeleton, thus triggering the accumulation of molecules at the T/APC interface (2). Because several data suggest that Vav-1 may be the mediator of many CD28 functions (18, 24, 36), we monitored actin cytoskeleton reorganization as well as Vav-1 and IKK movements in response to CD28 stimulation by confocal microscopy. As shown in Fig. 7A, CD28 crosslinking for 15 min induced a highly polarized accumulation of actin in the contact zone between T cells and anti-CD28-coated microbeads. These data are consistent with previous results from Kaga et al. showing that CD28 stimulation by mAbs or B7 was able to induce rearrangement of the actin cytoskeleton in the absence of TCR (7). In unstimulated cells, Vav-1 was mostly expressed in the cytoplasm. Similar results were obtained with mouse Ig-coated beads as control (data not shown). CD28 stimulation induced clustering and translocation of Vav-1, which accumulated and colocalized with actin in the T/bead contact zone. Similarly to Vav-1, IKK was cytoplasmic in unstimulated cells, but translocated to the membrane and colocalized with Vav-1 following CD28 stimulation (Fig. 7B). On the contrary, IKK remained localized in the cytoplasm and was not recruited to the membrane in response to CD28 (Fig. 7C). As shown in Fig. 8, CD28 stimulation induced the membrane recruitment of Vav-1 and IKK also in primary CD4+ T cells (Fig. 8, A and B). Thus, Vav-1/IKK-containing complexes are selectively recruited to the membrane following CD28 stimulation.
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Discussion |
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B in both Jurkat and primary T cells. We also show that Vav-1 cooperates with CD28 to activate NF-B through a constitutive association with IKK. Coexpression of Vav-1 and IKK potentiated CD28-induced signals to NF-B. By contrast, IKK that has been associated to the activation of NF-B by TCR failed to cooperate with Vav-1 in the CD28-independent signals to NF-B. Lastly, CD28 induced actin cytoskeleton reorganization and the recruitment of both Vav-1 and IKK at the point of contact with anti-CD28 microbeads.
The existence of autonomous CD28 signaling has been recently suggested by data showing the ability of CD28 to promote cytoskeletal reorganization (7) or NF-AT nuclear translocation and IL-2/4 gene transcription independently of TCR ligation (8). Although Vav-1 and SLP-76 have been implicated in this independent CD28 signaling, most of the data have been obtained in overexpression experiments (8). Indeed, NF-AT activation was not induced by CD28 stimulation alone (8) and, as previously demonstrated by Michel et al., CD28/Vav-1-induced NF-AT increase was dramatically reduced in the absence of TCR (24). In this scenario, it is more plausible that CD28 uses Vav-1 to integrate and potentiate TCR-delivered signals to NF-AT than activates a signaling pathway independently of TCR. By contrast, our data demonstrate that in normal T cells CD28-mediated signals to NF-B do not require TCR stimulation.
NF-B has been termed the central mediator of immune responses. Indeed, the NF-B family promotes the expression of over 100 target genes encoding cytokines and chemokines, receptors required for immune recognition and Ag presentation, and adhesion receptors involved in transmigration across blood vessels walls (38). CD28 costimulation has been described to be necessary to allow IKKs and NF-B activation by TCR. Here we provide the first evidence that CD28 itself may activate a transcription factor involved not only in adaptive but also in innate immunity. Clearly, to allow NF-B activation the initial condition is that a T lymphocyte encounters an APC with a sufficient density of B7 on the cell surface. In normal conditions, CD28 on naive T cells does not support adhesion to physiological amounts of B7.1 (39). However, data from Michel et al. (3) clearly indicate that when a similar density of B7.1 and class II were expressed on the APC, CD28/B7 interaction, and not TCR/staphylococcal enterotoxin B/MHC complexes, promotes and augments conjugate formation. Indeed, comparable affinities have been estimated for CD28 interaction with B7 and for TCR and peptide/MHC (40). Thus, when an APC displays much higher density of B7 (e.g., mature dendritic cells) than potential TCR ligands, the stoichiometry of binding may be in favor of CD28/B7. In these conditions, the encounter of primary T cells with mature dendritic cells may favor sufficient CD28/B7 interactions to activate the CD28-dependent signals leading to NF-B and to the transcription of the proinflammatory genes normally regulated by this family of transcription factors. Recent data have evidenced that the production of macrophage inflammatory protein 1 and 1 as well as up-regulation of RANTES promoter activity, which are all NF-B target genes (13, 41), were dependent on CD28 costimulation (42, 43). Moreover, TCR-independent activation of NF-B by CD28 may also regulate T cell survival through the up-regulation of Bcl-xL (44, 45, 46, 47). In this context, it is interesting to note that similar to CD28 stimulation Tax activates NF-B, which also leads to Bcl-xL expression, by stimulating the kinase activity of IKK and functionally cooperating with it (48, 49).
Recent studies in mice lacking either Ikk or Ikk genes have suggested that IKK is required for NF-B activation in response to proinflammatory cytokine, whereas IKK is dispensable (10, 11). Despite the different functions exerted by IKK and IKK in many cell types, in vitro both subunits exhibit IB kinase activity (33). Moreover, recent data from O’Mahony et al. (12) have evidenced that the activation of the heterodimeric IKK complexes by different inducers is indeed dependent on the kinase activity of IKK to activate IKK. Thus, it is conceivable that CD28-mediated induction of IKK complex activity (17, 18) may be mediated by IKK and that Vav-1 may play an important role by favoring IKK recruitment and activation in response to CD28. Unfortunately, given the requirement of Vav-1 in both cytoskeletal rearrangements of lymphocytes (50, 51) and TCR signaling (19, 52, 53), knockout mice have been limited in providing information on its role in CD28-mediated costimulation. Our data clearly demonstrate that CD28 stimulation is able to induce actin cytoskeleton reorganization and the accumulation of both Vav-1 and IKK at the T cell/bead contact zone in the absence of TCR. Because Vav-1 is a central regulator of costimulation-controlled cytoskeletal rearrangemets during T cell activation (35), it is conceivable that it may also mediate CD28-induced cytoskeleton reorganization and IKK recruitment independently of TCR.
A mechanism by which Vav-1 has been suggested to regulate NF-B activation relies on Vav-1 ability to promote a selective translocation of PKC and IKK to the membrane in response to CD3/CD28 stimulation (27, 54). However, the observation that CD28 stimulation alone induces the dissociation of Vav-1 and PKC suggests that these two proteins may regulate NF-B activation by targeting different subsets of IKK complexes and acting on separate routes (16). Our demonstration of a physical association between Vav-1 and IKK as well as of an active recruitment of Vav-1/IKK-containing complexes to the cellular membrane upon CD28 stimulation indicate the existence of a new pathway for NF-B activation in CD28-stimulated T cells. The activation of Vav-1/IKK-containing complexes by CD28 might also be relevant for the modulation of the spectrum of transcriptionally active NF-B complexes through the selective recruitment of NF-B2/p52 (13). It remains to be established whether Vav-1/IKK interaction is direct or involves additional components of a macromolecular complex.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Loretta Tuosto, Department of Cellular and Developmental Biology, University of Rome La Sapienza, Rome, Italy. E-mail address: tuosto{at}uniroma1.it
3 Abbreviations used in this paper: IS, immunological synapse; IKK, IB kinase; ERK, extracellular signal-regulated kinase; PKC, protein kinase C; CLSM, confocal laser-scanning microscopy; siRNA, small interference RNA; HLH, helix-loop-helix; DH, Dbl homology; HA, hemagglutinin.
Received for publication June 28, 2002. Accepted for publication January 15, 2003.
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  C. Scotta, M. Soligo, C. Camperio, and E. Piccolella FOXP3 Induced by CD28/B7 Interaction Regulates CD25 and Anergic Phenotype in Human CD4+CD25- T Lymphocytes J. Immunol., July 15, 2008; 181(2): 1025 - 1033. [Abstract] [Full Text] [PDF]
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 M. Humar, H. Dohrmann, P. Stein, N. Andriopoulos, U. Goebel, M. Roesslein, R. Schmidt, C. I. Schwer, T. Loop, K. K. Geiger, et al. Thionamides Inhibit the Transcription Factor Nuclear Factor-{kappa}B by Suppression of Rac1 and Inhibitor of {kappa}B Kinase {alpha} J. Pharmacol. Exp. Ther., March 1, 2008; 324(3): 1037 - 1044. [Abstract] [Full Text] [PDF]
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K. M. Dennehy, F. Elias, S.-Y. Na, K.-D. Fischer, T. Hunig, and F. Luhder Mitogenic CD28 Signals Require the Exchange Factor Vav1 to Enhance TCR Signaling at the SLP-76-Vav-Itk Signalosome J. Immunol., February 1, 2007; 178(3): 1363 - 1371. [Abstract] [Full Text] [PDF]
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 J. E. Wood, H. Schneider, and C. E. Rudd TcR and TcR-CD28 Engagement of Protein Kinase B (PKB/AKT) and Glycogen Synthase Kinase-3 (GSK-3) Operates Independently of Guanine Nucleotide Exchange Factor VAV-1 J. Biol. Chem., October 27, 2006; 281(43): 32385 - 32394. [Abstract] [Full Text] [PDF]
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D. Poppe, I. Tiede, G. Fritz, C. Becker, B. Bartsch, S. Wirtz, D. Strand, S. Tanaka, P. R. Galle, X. R. Bustelo, et al. Azathioprine Suppresses Ezrin-Radixin-Moesin-Dependent T Cell-APC Conjugation through Inhibition of Vav Guanosine Exchange Activity on Rac Proteins J. Immunol., January 1, 2006; 176(1): 640 - 651. [Abstract] [Full Text] [PDF]
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B. Kovacs, R. V. Parry, Z. Ma, E. Fan, D. K. Shivers, B. A. Freiberg, A. K. Thomas, R. Rutherford, C. A. Rumbley, J. L. Riley, et al. Ligation of CD28 by Its Natural Ligand CD86 in the Absence of TCR Stimulation Induces Lipid Raft Polarization in Human CD4 T Cells J. Immunol., December 15, 2005; 175(12): 7848 - 7854. [Abstract] [Full Text] [PDF]
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J. A. Williams, K. S. Hathcock, D. Klug, Y. Harada, B. Choudhury, J. P. Allison, R. Abe, and R. J. Hodes Regulated Costimulation in the Thymus Is Critical for T Cell Development: Dysregulated CD28 Costimulation Can Bypass the Pre-TCR Checkpoint J. Immunol., October 1, 2005; 175(7): 4199 - 4207. [Abstract] [Full Text] [PDF]
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 E. Vigorito, L. Gambardella, F. Colucci, S. McAdam, and M. Turner Vav proteins regulate peripheral B-cell survival Blood, October 1, 2005; 106(7): 2391 - 2398. [Abstract] [Full Text] [PDF]
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R. Tavano, G. Gri, B. Molon, B. Marinari, C. E. Rudd, L. Tuosto, and A. Viola CD28 and Lipid Rafts Coordinate Recruitment of Lck to the Immunological Synapse of Human T Lymphocytes J. Immunol., November 1, 2004; 173(9): 5392 - 5397. [Abstract] [Full Text] [PDF]
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M. Quirling, S. Page, N. Jilg, K. Plenagl, D. Peus, C. Grubmuller, M. Weingartner, C. Fischer, D. Neumeier, and K. Brand Detection of IKK{beta}-IKK{gamma} Subcomplexes in Monocytic Cells and Characterization of Associated Signaling J. Biol. Chem., September 3, 2004; 279(36): 37452 - 37460. [Abstract] [Full Text] [PDF]
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 B. Marinari, A. Costanzo, V. Marzano, E. Piccolella, and L. Tuosto CD28 delivers a unique signal leading to the selective recruitment of RelA and p52 NF-{kappa}B subunits on IL-8 and Bcl-xL gene promoters PNAS, April 20, 2004; 101(16): 6098 - 6103. [Abstract] [Full Text] [PDF]
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K.-J. Park, R. B. Gaynor, and Y. T. Kwak Heat Shock Protein 27 Association with the I{kappa}B Kinase Complex Regulates Tumor Necrosis Factor {alpha}-induced NF-{kappa}B Activation J. Biol. Chem., September 12, 2003; 278(37): 35272 - 35278. [Abstract] [Full Text] [PDF]
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