INTRODUCTION

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T cell activation and IL-2¹ production is critically dependent on the transmission of signals derived from the cell surface to the nucleus in order to modulate changes in gene expression (1). It is now well established that activation and signaling through the T cell receptor (TCR) alone is not sufficient for IL-2 production or proliferation (2). Antigen-presenting cells achieve maximal activation of the antigen-reactive T cells by binding accessory molecules present on the T cell surface in addition to antigen presentation to the TCR via the context of major histocompatibility complex class II molecules. One of the most intensively studied accessory molecules, CD28, binds to the B7.1 and B7.2 molecules present on the surface of macrophages and dendritic cells (3). CD28 has been demonstrated to act as a costimulatory signal for T cells since IL-2 production and proliferation are enhanced when CD28 is engaged in addition to the TCR (4). CD28 also confers post-transcriptional mechanisms to enhance T cell activation by prolonging the half-life of IL-2 mRNA (5). However, engagement of CD28 alone has no measurable effect on T cell activation. The IL-2 gene promoter contains an enhancer known as the CD28 responsive element (CD28RE) which functions as an integrator of transcription factors activated through the TCR and CD28 and is essential for IL-2 transcription mediated through CD28 (6). The CD28RE enhancer also forms a composite element with a juxtaposed AP-1 binding site, and it has been shown that this composite element mediates CD28 responsiveness (7, 8). Studies by several laboratories suggest that members of the NF-B, AP-1, and ATF-CREB transcription factor families bind to the CD28RE/AP-1 composite element (7, 9).

The NF-B/Rel family of transcription factors is composed of a set of structurally related, evolutionarily conserved DNA-binding proteins consisting of p50, p52, p65, c-Rel, and RelB (10). The NF-B complexes are sequestered in the cytosolic compartment as latent complexes by members of the IB family, all of which have characteristic ankyrin repeat domains required for interactions with NF-B proteins (reviewed in Refs. 10 and 11). The two major IB proteins, IB and IB, both have two regulatory N-terminal serine residues that are phosphorylated in response to a wide array of signals (12-14). The phosphorylated IBs are then ubiquitinated and targeted to the proteasome for proteolytic degradation (15). Signals such as TNF- or mitogens such as PMA, which selectively induce the degradation of only IB, are associated with the transient activation of NF-B since the IB gene is positively regulated by NF-B factors (16-19). However, signals such as lipopolysaccharide, CD3 + CD28, IL-1, or the Tax protein of type I human T-cell leukemia virus-I induce a persistent NF-B activation, which appears to be due to both prolonged IB degradation (20, 21) and degradation of IB (22-24). The IB gene is presumably not under the control of NF-B since the protein is not rapidly replenished as is seen with IB (22).

Recently two serine kinases termed IKK and IKK, which are part of a large multiprotein complex known as the IKK signalsome have been cloned and demonstrated to phosphorylate both IB and IB in response to cytokines and other signals known to activate NF-B (25-29). An upstream kinase, NIK, has also been identified and shown to stimulate NF-B in response to distinct stimuli such as TNF- and IL-1 (30). The mechanism may be direct since NIK has been demonstrated to phosphorylate IKK on Ser¹⁷⁶ (31).

Although the CD28 signaling pathway has been shown to accelerate TCR-induced nuclear expression of various NF-B/Rel transcription factors, the underlying molecular mechanism remains elusive. We and others have previously demonstrated that ligation of CD28 initiates a potent costimulatory signal leading to the rapid and persistent degradation of IB and enhanced degradation of IB (20, 23). However, it is not known if CD28 is mediating enhanced IB kinase activity. We report here that CD28 potentiates the kinase activity of IKK and IKK, which are only weakly activated by mitogen or TCR signals alone.

	MATERIALS AND METHODS

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Cell Culture and Reagents-- Jurkat T cells (ATCC) and Jurkat cells expressing the SV40 large T antigen (Jurkat Tag) (32) were maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum, 2 mM L-glutamine, and antibiotics. Human peripheral blood T cells were prepared from lymphocyte-enriched human blood (Biological Specialty Corporation, Colmar, PA) with a Ficoll-Hypaque gradient (Amersham Pharmacia Biotech) followed by negative selection with human T cell enrichment immunocolumns (Biotex Laboratories Inc., Edmonton, Alberta, Canada). C305 (anti-clonotypic Jurkat TCR) was provided by Dr. Arthur Weiss (University of California, San Francisco) and used at a 1:1000 dilution. The monoclonal antibody for human CD28 (clone 9.3) was provided by Bristol-Myers Squibb Pharmaceutical Research Institute and used at a 1:10,000 dilution (0.3 µg/ml). The antibody against the influenza hemagglutinin (HA) epitope tag (anti-HA) and protein A- agarose was obtained from Boehringer Mannheim. Anti-IKK (H744) and anti-IKK (H470) were purchased from Santa Cruz Biotechnology, Inc. Anti-IB antiserum was provided by Dr. Warner Greene.

Plasmid Constructs and Transient Transfection-- HA-IKK (K44M) and HA-IKK (K44A) were kindly provided by Dr. Michael Karin (University of California, San Diego). GST-IB 1-55 and GST-IB 1-55 A32/A36 was constructed by inserting a DNA fragment encoding the N-terminal 55 amino acids from the IB and IB A32/A36 cDNAs (33) into the pGEX-4T-3 vector (Amersham Pharmacia Biotech). GST-IB 1-82 was constructed by ligating a 300-base pair NaeI and BamHI fragment derived from HA-IB with pGEX-4T-3 digested with SmaI and BamHI. Jurkat Tag cells (5 × 10⁶) were transfected using DEAE-dextran (34) with 2 µg of HA-IB and the indicated amounts of HA-IKK (K44M) expression vector. Between 40 and 48 h post-transfection, the cells were incubated with PMA (10 ng/ml) and CD28 (1:10,000) for 30 min and then subjected to whole-extract preparation and immunoblotting analyses as described below.

In Vitro Kinase Assays-- In vitro kinase assays were done essentially as described previously (25). Briefly, cell lysates were incubated with specific antisera (IKK or IKK) for 1 h, and then 20 µl of protein A-agarose (Boehringer Mannheim) was added and incubated for an additional 3 h. The immunoprecipitates were washed three times with cell lysis buffer containing 1% Nonidet P-40, 20 mM Hepes, 250 mM NaCl, 20 mM -glycerophosphate, 1 mM EDTA, 0.1 mM sodium vanadate, 1 mM dithiothreitol, 20 mM p-nitrophenyl phosphate, 1 mM phenylmethylsulfonyl fluoride, and 1:100 of a protease inhibitor mixture. The immunoprecipitates were then washed once with cell lysis buffer + 8 M urea, and twice with kinase buffer (20 mM Hepes and 20 mM magnesium chloride). Kinase reactions were then performed at 30 °C for 30 min in the presence of [-³²P]ATP and either GST-IB 1-55 or GST-IB 1-82. The reactions were terminated upon addition of 5× sample buffer followed by SDS-polyacrylamide electrophoresis and autoradiography.

Immunoblotting-- Jurkat cells, or transiently transfected Jurkat-Tag cells were stimulated with the indicated inducers and then collected by centrifugation at 800 × g for 5 min. Whole cell and subcellular extracts were prepared as described previously (35, 36). For immunoblotting analyses, whole cell extracts (~15 µg) were fractionated by reducing 8.75% SDS-polyacrylamide gel electrophoresis, electrophoretically transferred to nitrocellulose membranes, and then analyzed for immunoreactivity with the indicated primary antibodies using an enhanced chemiluminescence detection system (ECL; Amersham Pharmacia Biotech).

Luciferase Reporter Gene Assays-- Jurkat cells were transiently transfected with DEAE-dextran with 2 µg of the CD28RE/AP-1 reporter and either empty vector or the indicated amounts of IKK K44M or IKK K44A. Transfectants were split into two and either left untreated or treated with PMA (10 ng/ml) or PMA and anti-CD28 (1:10,000 dilution) for 8 h. The extracts were harvested with reporter lysis buffer (Promega) and then measured for luciferase activity as described previously (23).

	RESULTS

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The CD28 Signal Potentiates IKK Activation in Both Jurkat and Primary Human T Cells-- We and others have previously demonstrated that IB degradation is enhanced by CD28 costimulation (20, 23), although the underlying mechanism has remained unclear. We examined whether CD28 potentiates mitogen-mediated IKK activation. We first performed in vitro kinase assays with Jurkat T cells utilizing GST-IB 1-55 as a substrate. The kinase activity of IKK was slightly induced by PMA treatment as described previously (26) (Fig. 1A, lane 2, upper middle panel). However, when cells were treated with CD28 antibody in addition to mitogen treatment, the kinase activity of IKK was significantly elevated (lane 3), although CD28 alone had no effect on the kinase activity (lane 4). Autophosphorylation of IKK was also strongly induced by PMA plus anti-CD28 treatment (lane 3, upper panel). It should be noted that the time of each stimulation for this and subsequent experiments was 7 min, which represented maximal kinase activity as exerted by each stimulus. In a time course experiment, the kinase activity was highest at 7 min, was sustained for at least 15 min, and finally subsided by 30 min (data not shown). Pretreatment of cells with TPCK, a chymotrypsin-like protease inhibitor, known to block IB phosphorylation by unknown mechanisms (37), abolished all kinase activity associated with PMA and anti-CD28 treatment (Fig. 1A, lane 5, upper middle panel). To ensure that IKK kinase activity was directed to the two N-terminal serine residues of IB (serine 32 and 36), we used a GST-IB protein with alanine residues substituted for the two serines as substrate. Importantly, this substrate was not phosphorylated by IKK in Jurkat cells treated with mitogen and anti-CD28 (lane 6). Immunoblotting of the immunoprecipitated IKK revealed equal amounts of IKK for all samples (Fig. 1A, lower middle panel). In addition to IKK kinase activity, we examined the fate of endogenous IB from the same extracts used for kinase assays. Immunoblots of IB revealed that PMA only slightly induced IB phosphorylation as assessed by a slower migrating band on SDS-polyacrylamide gel electrophoresis gels (Fig. 1A, lane 2, lower panel). When CD28 antibody was added in addition to PMA, a strong phosphorylated band was readily observed (lane 3), and as expected, CD28 alone did not induce IB phosphorylation (lane 4). TPCK also inhibited the inducible phosphorylation of IB (lane 5). Taken together, IKK kinase activity is enhanced by PMA and CD28 treatment and is well correlated with in vivo IB phosphorylation.

View larger version (40K): [in this window] [in a new window]   Fig. 1.   IKK kinase activity is enhanced by CD28 costimulation in Jurkat T cells and primary T cells. A, Jurkat cells were either untreated (lane 1) or treated with PMA (10 ng/ml; this concentration was used for all subsequent studies as well, lane 2), PMA plus anti-CD28 (1:10,000, lanes 3 and 6), or anti-CD28 (lane 4) for 7 min. Jurkat cells were also pretreated with 50 µM TPCK for 15 min and then incubated with PMA and anti-CD28 for 7 min (lane 5). An in vitro kinase assay was performed with Jurkat cell lysates using anti-IKK-specific antiserum and either GST-IB 1-55 (upper middle panel, lanes 1-5) or GST-IB 1-55 A32/A36 (lane 6) as a substrate. Autophosphorylation of IKK is displayed in the uppermost panel. After autoradiography, the membrane was used for immunoblotting with IKK-specific antiserum to ensure the presence of comparable levels of IKK protein between different samples (lower middle panel). The same extracts used for kinase assays were also subjected to immunoblotting analysis with IB-specific antiserum (lower panel). B, CD28 also potentiates IKK kinase activity in primary T cells. Primary T cells were isolated from peripheral blood by Ficoll-Hypaque followed by negative selection. T cells were either left unstimulated (lane 1), or treated with PMA (lane 2) or PMA plus anti-CD28 (lane 3). Whole cell lysates were subjected to in vitro kinase assays as above with the GST-IB 1-55 substrate. C, TCR and CD28 costimulation also augments IKK kinase activity. Jurkat T cells were either left untreated (lane 1) or treated with C305 (1:1000, lane 2), C305 plus anti-CD28 (lane 3), or anti-CD28 alone (lane 4). Lysates were used for in vitro kinase assays with GST-IB 1-55 (upper panel). The membrane was subjected to immunoblotting with anti-IKK to ensure similar protein level (lower panel).

View larger version (17K): [in this window] [in a new window]   Fig. 2.   IB is a substrate for CD28-induced IKK kinase activity. Jurkat cells were either left untreated (lane 1) or incubated for 7 min with PMA, (lane 2), PMA and anti-CD28 (lane 3), or anti-CD28 alone (lane 4). Whole cell lysates were used for in vitro kinase assays with anti-IKK antiserum and GST-IB 1-82 as a substrate.

CD28 Also Mediates Enhanced IKK Phosphorylation of IB and IB-- We next focused on the other cytokine-responsive IB kinase IKK to determine if it had a similar activation response to CD28 costimulation. First, we examined the effects of endogenous IKK on GST-IB. In untreated Jurkat cells, there was no IKK activity toward IB (Fig. 3A, lane 1). Upon treatment with PMA, there was slight IKK activation (lane 2), which was subsequently elevated with CD28 treatment (lane 3). As expected, CD28 alone had no effect on IKK kinase activity (lane 4). These results suggest that IKK activation is reminiscent of IKK as shown in Fig. 1. We also tested the kinase activity of IKK toward IB. Similarly, PMA alone induces a degree of phosphorylation (Fig. 3B, lane 2), which is augmented by CD28 (lane 3). We conclude that IKK, in addition to IKK, is responsive to the CD28 costimulatory signal.

View larger version (29K): [in this window] [in a new window]   Fig. 3.   IKK kinase activity is potentiated by the CD28 costimulatory signal. A and B, Jurkat cells were treated exactly as described in Fig. 2, except that anti-IKK was used for the immunoprecipitation. Cell lysates were subjected to in vitro kinase assays with GST-IB 1-55 in A and GST-IB 1-82 as substrate in B.

A Catalytically Inactive IKK Mutant Blocks CD28-mediated in Vivo IB Phosphorylation-- CD28 synergizes with TCR signaling to accelerate IB phosphorylation and degradation (20, 23). To determine the role played by IKK in the in vivo phosphorylation of IB mediated by CD28 we utilized a catalytically inactive IKK mutant. This mutant, which has been previously demonstrated to inhibit TNF-- induced RelA nuclear translocation (28), has a methionine substituted for a lysine at position 44, resulting in defective ATP binding. We transiently transfected Jurkat-Tag cells with an HA-tagged IB construct, and split the transfection into two, leaving one sample untreated and treating the other sample with a combination of PMA and anti-CD28. As expected, we observed an unphosphorylated IB by immunoblotting when the cells were not treated (Fig. 4, lane 2). When the cells were treated with PMA/CD28, two bands were readily detected, with the slower migrating band representing the phosphorylated form (lane 3). Interestingly, when the catalytically inactive form of IKK was cotransfected with IB, the phosphorylation induced by PMA and CD28 was completely blocked (lanes 5 and 7). This result suggests that IKK is required for CD28-mediated IB phosphorylation.

View larger version (53K): [in this window] [in a new window]   Fig. 4.   A catalytically inactive IKK mutant blocks CD28-mediated in vivo IB phosphorylation. Jurkat Tag cells (5 × 106) were transfected with 2 µg of an HA-tagged IB cDNA together with either an empty vector (lanes 1-3), or the indicated amounts of HA-tagged IKK K44M (lanes 4-7). Approximately 40 h later, transfectants were split into two and either left untreated (lanes 1, 2, 4, and 6) or treated with PMA and anti-CD28 for 30 min (lanes 3, 5, and 7). Whole cell lysates were collected and subjected to immunoblotting with an anti-HA antibody.

Catalytically Inactive Forms of IKK and IKK Inhibit CD28-mediated CD28RE/AP-1 Transactivation-- It has been previously reported that CD28 responsiveness within the IL-2 promoter is mediated by a CD28RE/AP-1 composite element (7, 8). We transfected a luciferase reporter construct driven by this composite element into Jurkat cells and either treated with PMA alone or PMA together with anti-CD28. PMA alone was unable to substantially activate this reporter gene (Fig. 5A, lane 2), but when CD28 was added in conjunction with PMA, reporter activity rose to approximately 12-fold above that observed with untreated cells (lane 3 versus lane 1). We next cotransfected the catalytically inactive IKK with the CD28RE/AP-1 reporter and treated it with PMA and CD28. Interestingly, transfection of a moderate amount (150 ng) of IKK K44M cDNA reduced the reporter activity by approximately 50% (Fig. 5B, lane 3). With a higher dose (300 ng) transfection of this IKK mutant, more pronounced inhibition was observed (lane 4). It should be noted that this catalytically inactive mutant was previously demonstrated to block TNF--induced IKK activation by about 2- or 3-fold (28), suggesting the role of other compensatory factors to account for the activity observed. The catalytically inactive IKK K44A also inhibited reporter gene activity by about 50% at the first dose tested (100 ng) (Fig. 5C, lane 3). However, a higher dose of IKK K44A (200 ng) acted as an even more potent inhibitor of CD28-mediated reporter gene activation, reducing the activity to about 25% of the control (lane 4 versus lane 2). When both IKK K44M and IKK K44A were cotransfected, the inhibition observed was only slightly greater than the inhibition seen with a high dose of IKK K44A alone (data not shown). The lack of complete inhibition of reporter gene activity by the dominant negative IKKs is likely due to the high sensitivity of this assay, but we cannot preclude the involvement of additional IB kinases in CD28-mediated NF-B activation. From these experiments we conclude that both IKK and IKK are required for CD28-mediated transactivation of a CD28RE/AP-1 composite element.

View larger version (20K): [in this window] [in a new window]   Fig. 5.   CD28-mediated CD28RE/AP-1 transactivation is attenuated by catalytically inactive IKK mutants. A, Jurkat T cells (5 × 106) were transfected with 2 µg of a CD28RE/AP-1 reporter gene. Approximately 40 h later, transfectants were split into three and either left untreated (lane 1), or treated with PMA (lane 2) or PMA plus anti-CD28 (lane 3) for 8 h. Cells were then lysed and subjected to luciferase assays. Results shown are presented as the fold induction compared with untreated cells (approximately 30,000 cpm). The error bars represent the standard error of the mean for three independent sets of transfectants. B, Jurkat T cells were transfected with 2 µg of a CD28RE/AP-1 reporter gene and either empty vector (lanes 1 and 2) or 150 (lane 3) or 300 ng (lane 4) of IKK K44M. The cells were either untreated (lane 1) or treated with PMA and anti-CD28 (lanes 2-4). Lysates were subjected to luciferase assays with three independent experiments used to calculate the fold induction and the standard error of the mean. C, Jurkat T cells were transfected, treated, and subjected to luciferase as in B, except that 100 (lane 3) and 200 ng (lane 4) of IKK K44A were used in lieu of IKK K44M.

	DISCUSSION

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The NF-B transcription factors are activated by a diverse array of signals that target the inhibitory proteins IB and IB for phosphorylation and proteasome-mediated degradation (10). The T cell auxiliary molecule, CD28, which provides a costimulatory signal for T cell activation and IL-2 production, is a potent inducer of NF-B (23, 39). Although CD28 promotes the rapid degradation of both IB and IB, the mechanism has not been elucidated to date. Possibilities include synergy of IB kinase activity, activation of an IB kinase unique to the CD28 pathway, or perhaps enhanced ubiquitination and/or direct degradation. In this study, we report that CD28 specifically enhances the kinase activity of IKK and IKK, two recently identified cytokine-responsive IB kinases. Specifically, we found that the kinase activities of IKK and IKK were elevated for both IB and IB when T cells were treated with mitogen and anti-CD28. In addition, a catalytically inactive mutant of IKK effectively inhibited in vivo CD28-mediated IB phosphorylation. Inactive forms of IKK and IKK also attenuated CD28RE/AP-1 luciferase gene reporter activity induced by PMA and CD28. This study provides strong evidence that signaling through the TCR and CD28 converge at or upstream of IKK and IKK, resulting in enhanced kinase activity and NF-B activation.

Where could the TCR and CD28 pathways possibly converge? Kinases upstream of IKK include the mitogen-activated protein kinase kinase-related molecule, NIK, and the mitogen-activated protein kinase/ERK kinase kinase 1, MEKK1 (40). A recent report suggests that MEKK1 preferentially activates IKK, while NIK activates IKK and IKK equally well (41). Previous studies suggest that MEKK1 is a downstream target of CD28 signaling, and that its kinase activity is up-regulated when stimulated with both anti-CD3 and anti-CD28 (42, 43). It is not likely that MEKK1 is solely responsible for the CD28-mediated up-regulation of IKK kinase activity since we consistently observed stronger kinase activity associated with IKK rather than IKK. NIK is a potential candidate to mediate the signal integration between the TCR and CD28 if the signals do in fact converge upstream of IKK. NIK is already known to integrate signals from pathways initiated by IL-1 and TNF- to activate NF-B (30). We are currently investigating any potential role NIK may play in CD28 mediated NF-B activation.

Besides MEKK1, several other kinases have been identified which have up-regulated kinase activity when T cells are stimulated with anti-CD3 and anti-CD28. Full activation of JNK in T cells is dependent on integration of the two signals (44). As expected, kinases within the JNK pathway such as p21-activated kinase (43), SEK (45), and MKK7 (45) are similarly dependent on T cell costimulation for full activation. The transactivation capacity of the c-Jun protein, one of the AP-1 components, is activated as a result of signaling through the JNK pathway (46). The vital role that AP-1 plays in IL-2 transcriptional regulation is underscored by the finding that mice deficient in SEK1, a direct activator of JNK, are impaired in CD28-mediated IL-2 production (47). However, it is also known that NF-B is a critical regulator of the IL-2 gene as demonstrated by gene targeting of the c-rel gene (48). To date, kinases solely within the NF-B pathway have not been identified to be targets of CD28 mediated activation. Our finding that IKK and IKK have enhanced kinase activity when T cells are costimulated is the first such demonstration. It is likely that other kinases within the NF-B pathway are also targeted by CD28.

We have observed an excellent correlation between in vitro kinase activities of IKK and IKK and in vivo phosphorylation of IB mediated by CD28 (see Figs. 1 and 3). IB is also strongly phosphorylated in vitro by both IKK and IKK due to CD28 (Figs. 2 and 3). With regard to the in vivo phosphorylation of IB, it is more difficult to address this question, since a band shift is not apparent after cellular stimulations. Rather, IB is degraded partially within 15 min and completely in 30 min in response to mitogen and CD28 treatment (23). It is possible that IB is not phosphorylated as well in vivo due to the folding of the protein or perhaps due to the binding of other proteins that may interfere with the accessibility of IKKs to the two N-terminal serine residues. Further studies with in vivo ³²P-labeling of IB will more directly answer this question.

In conclusion, we have determined the mechanism of CD28-mediated NF-B activation to be at the level of enhanced IB kinase activity. It appears that CD28 targets the two cytokine-responsive IB kinases, IKK and IKK, which are able to respond to multiple signals. It is therefore not likely that CD28 induces an IB kinase distinct from IKK or IKK, which is unique to the CD28 pathway. We also consider it unlikely that CD28 directly enhances the ubiquitination or degradation of IB and IB. Studies are in progress to further delineate the TCR and CD28 pathways and to pinpoint the convergence of the two pathways in NF-B activation.