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B Kinase Complexes: Role in Activation of the IL-2 Promoter1
*
Division of Clinical Immunology and Allergy, Department of Medicine, Center for Health Sciences, Los Angeles, CA 90095;
Signal Pharmaceuticals, Inc., San Diego, CA 92121; and
Abramson Family Cancer Research Institute at the University of Pennsylvania Cancer Center, Philadelphia, PA 19104
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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B transcription factors play an important role in the
activation of the IL-2 gene in response to TCR ligation. The release of
NF-B factors to the nucleus requires phosphorylation and degradation
of the inhibitory -B proteins (IBs). IB
and IBß
phosphorylation is dependent on dual signaling by the TCR and the CD28
accessory receptor. This pathway involves a multisubunit IB kinase
(IKK) complex, which includes the IKK (IKK-1) and IKKß (IKK-2)
kinases. We demonstrate that stimulation of primary human
CD4+ T cells by CD3/CD28 activates two distinct endogenous
IKK complexes, a heterodimeric IKK/ß and a homodimeric IKKß
complex. IKKß overexpression in a Jurkat cell line resulted in the
formation of a constitutively active IKK complex, which was CD3/CD28
inducible. In contrast, ectopic expression of IKK assembled into a
complex with negligible IB kinase activity. Moreover, IKKß, but
not IKK, overexpression enhanced transcriptional activation of the
CD28 response element in the IL-2 promoter. Conversely, only
kinase-inactive IKKß interfered in the activation of the IL-2
promoter. Sodium salicylate, an inhibitor of IKKß, but not IKK,
activity, inhibited IL-2 promoter activation as well as IL-2 secretion
and interfered in activation of both the heterodimeric as well as the
homodimeric IKK complexes in primary CD4+ T cells. Taken
together, these data demonstrate the presence of an IKKß-mediated
signaling pathway that is activated by TCR and CD28 coligation and
regulates IL-2 promoter activity. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B
transcription factors, which have cognate binding sites in the CD28RE
domain (9, 10, 11).
The NF-B pathway, in addition to its role in TCR activation, also
plays a role in T cell responsiveness to TNF-, IL-1, and phorbol
esters (12, 13). In resting cells, NF-B transcription
factors are sequestered in the cytoplasm by a group of inhibitory
proteins known as the IBs (12, 13, 14). Upon stimulation,
IB and/or IBß are phosphorylated on specific serine residues
and are subsequently degraded through the 26S proteosome
(12, 13, 14, 15, 16). This allows the release and nuclear localization
of NF-B transcription factors. The phosphorylation of IBs is
mediated by a recently identified multicomponent signalsome, known as
the IB kinase (IKK) complex (16, 17, 18, 19, 20, 21, 22). This complex
contains two serine kinase subunits, IKK (IKK-1) and IKKß (IKK-2),
with relative molecular masses of 85 and 87 kDa, respectively
(17, 18, 19, 20, 21, 22). IKK and IKKß genes have been cloned, and
their expressed proteins have been purified (23, 24, 25).
IKK and IKKß, which are nearly 50% homologous, are able to homo-
and heterodimerize in vitro and in vivo through their C-terminal
leucine zipper motifs (23, 24, 25). Although the active kinase
complex purified from TNF-- and IL-1-stimulated cells contains
IKK and IKKß heterodimers, homodimerized versions of each IKK are
capable of phosphorylating IB proteins in vitro
(23, 24, 25). Interestingly, purified IKKß has substantially
higher basal IB kinase activity than IKK (23, 25). Furthermore, in transient transfection studies, expression
of a kinase-inactive IKK mutant has minimal effects on the kinase
activity compared with the potent inhibitory effect of kinase-inactive
IKKß (19). These findings raise the possibility
that, in addition to IKK heterodimers, homodimers may contribute to
NF-B signaling in vivo. Mercurio et al. have recently demonstrated
the existence of heterodimeric IKK/IKKß as well as homodimeric
IKKß complexes that can be activated by TNF- stimulation in HeLa
cells (23). In contrast, SLB cells contained IKK/IKKß
heterodimers but no homodimers (23). This suggests the
existence of heterogeneous IKK complexes that may play differential
roles in NF-B activation in different cell types. The possible
existence of homo- or heterodimeric IKK complexes in T cells has not
been addressed.
Activation of the IKK complex is dependent on the action of upstream
kinases (9, 18, 19, 20, 21, 22, 25, 26, 27, 28). NF-B-inducing kinase (NIK)
and MEKK1, members of the mitogen-activating protein kinase kinase
kinase (MAP3K) family, have been shown to activate the IKK complex
(9, 20, 21, 25, 26, 27, 28, 29). NIK copurifies with the IKK complex
and has been shown to be involved in IKK activation by IL-1 and TNF-
(20, 21, 26, 27). Although overexpression of NIK leads to
stimulation of IKK and IKKß activities in vivo, NIK preferentially
phosphorylates IKK and has little activity on IKKß
(26). At least one study has suggested that NIK is
involved in CD28 costimulation (27). While MEKK1 also
copurifies with IKK complexes (18), its overexpression
leads to differential activation of IKKß in Jurkat and other cell
types (9, 29). HTLV-I tax protein also activates the
NF-B complex by interacting with MEKK1 and preferentially activating
IKKß kinase activity (30). However, in some cell types,
MEKK1 has been linked to both IKK and IKKß activation (25, 31). Taken together, it is possible that several MAP3Ks may be
involved in the activation of IKK complexes in T cells.
We were interested in determining whether signaling by TCR/CD3 and CD28
activates different IKK complexes in primary human T lymphocytes. In
addition, we were interested in whether there is a functional
relationship between IKKs and in the activation of the CD28RE in the
IL-2 promoter. Here we describe that CD3 and CD28 costimulation results
in the activation of IKK/IKKß heterodimeric and IKKß homodimeric
complexes in primary human CD4+ T cells. We also
provide evidence that IKKß, rather than IKK, is critical for
activation of the CD28RE in the IL-2 promoter.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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OKT3 (anti-CD3) was obtained from Ortho Pharmaceuticals
(Raritan, NJ), and the 9.3 mAb (anti-CD28) were provided by
Bristol-Meyer Squibb (Princeton, NJ). The primary stimulating Abs were
cross-linked with mAb 187.1 (Bristol-Meyer Squibb). For Western
blotting and immunoprecipitation experiments, polyclonal
anti-IKK, anti-IKKß, anti-IB, and
anti-IBß Abs were purchased from Santa Cruz Biotechnology
(Santa Cruz, CA). Anti-Flag (M2) Abs were obtained from Sigma (St.
Louis, MO). Anti-IKK
(IKAP1) Abs and
GST-IB1–54 have been described by us
(23). HRP conjugated to protein A was obtained from
Amersham (Arlington Heights, IL). Tosyl-activated magnetic beads and
M-450 anti-CD4 beads were purchased from Dynal (Great Neck, NY).
PMA and ionomycin were purchased from Sigma.
Cell lines
Jurkat T cells (clones BMS2, Jurkat-hIL-2-Luc) were grown in RPMI medium, supplemented with 10 mM HEPES (pH 7.4), 10% FCS, 2 mM glutamine, 100 U of penicillin, and 100 µg of streptomycin/ml. The Jurkat-hIL-2-luciferase cell line was provided by Dr. A. Weiss (Howard Hughes Medical Institute, University of California, San Francisco, CA), while Jurkat BMS2 was a gift from Dr. B. Mittler (Bristol Myers Squibb) (32).
Preparation of primary CD4+ T cells
Mononuclear cells were isolated from human peripheral blood by density centrifugation and depletion of adherent cells on plastic culture dishes. CD4+ T cells were positively selected with anti-CD4 Dynabeads according to the manufacturer’s instructions (Dynal). This yielded a T cell subset that was >98% positive for the CD4 marker as determined by dual-color CD4/CD8 flow cytometry. Isolated CD4+ T cells were stimulated with anti-CD3 and anti-CD28 mAb coupled to tosyl-activated magnetic beads as previously described (21). Cells were replenished with 40 U/ml rIL-2 (Chiron, Emeryville, CA) on day 3 and allowed to complete their growth cycle over the course of 12–13 days. At this point, IL-2 and beads were removed, and cells were allowed to return to their resting state over a 48-h period. Resting vs activated state was assessed by cell size analysis in a Coulter Counter (Hialeah, FL). Cells were subjected to flow cytometry for a second time to confirm that >95% of the cells remained CD4+.
Gene constructs and cellular transfection
IB kinase constructs were previously described and
characterized (21). Flag-tagged wild-type IKK and
IKKß and kinase-inactive mutants (K44A) cloned in pRK5 vector were
provided by Dr. M. Rothe (Tularik, San Francisco, CA)
(21). Full-length kinase-inactive MEKK1 (K1253 to M), a
gift from Dr. M. Karin (University of California, San Diego, CA)
(33), was cloned in pcDNA3 (Invitrogen, Carlsbad, CA). The
JNK-interacting protein 1 (JIP-1), cloned in CMV5 vector
(34), was a gift from Dr. R. Davis (Howard Hughes Medical
Institute, Worcester, MA) (33). The CD28RE/AP-1 luciferase
reporter and related mutants, provided by Dr. A. Weiss (Howard Hughes
Medical Institute) were cloned in pAEODLO vectors (8).
CD28RE/AP-1 Luc contains four tandem copies of the sequence
5'-tttaaagaaattccaaagagtcatca-3', which is situated 150–160 bp
upstream from the start site of the IL-2 gene (8). For
cellular transfection, we used the stated amounts of an individual or
combination of plasmids for electroporation (240 V, 950 µF) into
1 x 107 Jurkat cells as previously
described (9, 35).
Purification of heterogeneous IKK complexes by sequential immunoprecipitation and Western blot analysis
CD4+ T cells (2 x
107) were lysed in kinase lysis buffer and
cleared by centrifugation as previously described (23).
For immunoprecipitation, 200 µg of precleared lysate was treated with
2 µg of anti-IKK Abs, bound to protein A-Sepharose, and rocked
for 2 h at 4°C (9, 17, 23). To completely remove
all IKK, this step was repeated once. The remaining supernatants
were subjected to further immunoprecipitation using 2 µg of the
antisera to either IKKß or IKK. Immunoprecipitated complexes were
washed extensively and subjected to immunoblotting as previously
described (9, 23). IKK, IKKß, and IKK Abs were
used at a 1/1000 dilution for primary staining. Protein A-conjugated
HRP was used at a 1/2000 dilution, and blots were developed by enhanced
chemiluminescence. Western blotting of cellular lysates was performed
as described previously (9).
Immune complex kinase assays
Jurkat cells or CD4+ resting T cells
(1 x 107) in 1 ml of RPMI were left
unstimulated or were stimulated with 2 µg/ml anti-CD3 or a
combination of 2 µg/ml anti-CD3 and 2 µg/ml anti-CD28 mAb,
secondarily cross-linked with 10 µg/ml mAb 187.1. In another group of
experiments, primary human CD4+ T cells were
treated with 20 mM sodium salicylate (36) for 2 h
before stimulation as described above. All kinase assays were performed
as described previously (23). Briefly, after cell lysates
were precleared with protein A-Sepharose beads, 200 µg of lysate was
treated with 2 µg of the specific anti-IKK protein A-Sepharose
for 2 h. In a third variation, the immunoprecipitation was
performed sequentially, first by adding anti-IKK in two rounds,
and then adding either anti-IKKß or anti-IKK to the
remaining supernatants (23). Immune complexes were washed
and equilibrated in kinase buffer as previously described
(23). Kinase reactions were initiated by the addition of
10 µCi of [-32P]ATP and 3 µg
GST-IB1–54 substrate. The reaction was
conducted for 30 min at 30°C. Products were analyzed on SDS-PAGE and
detected by autoradiography.
Luciferase assays
Twelve micrograms of the indicated reporter gene constructs were transiently transfected into 107 Jurkat cells (9). The cells were rested for 24 h and then stimulated for 6 h with 2 µg/ml anti-CD3, a combination of 2 µg/ml anti-CD3 and 2 µg/ml anti-CD28 mAb, or a combination of 100 nM PMA and 1 µg/ml ionomycin (PMA+I). The cells were washed and lysed in luciferase buffer (Analytical Luminescence, Ann Arbor, MI), and luciferase activity was measured in 50 µg of lysate in a Monolight 2010 luminometer (Analytical Luminescence). Transfection efficiency was monitored by cotransfection of a ß-galactosidase plasmid (CMV-ß-gal); ß-galactosidase activity was used for adjusting luciferase values among cell populations transfected with different vector combinations (9).
IL-2 measurement
Jurkat T cells (1 x 106) in 2 ml of RPMI were treated with either anti-CD3 or anti-CD3 plus anti-CD28 mAb in the presence or the absence of 20 mM sodium salicylate for 24 h. Cells were removed by centrifugation, and the supernatants were collected. Triplicate aliquots were analyzed by ELISA for the presence of IL-2 (UMAB Cytokine Core Laboratory, Baltimore, MD).
Electrophoretic mobility shift assays
Nuclear protein was extracted from 107
Jurkat T cells, harvested at the indicated time to evaluate DNA binding
of NF-B as previously described (37). Briefly, after
washing, the cell pellet was suspended in 1 ml of buffer A (10 mM HEPES
(pH 7.9), 1.5 mM MgCl2, 10 mM KCl, and 1 mM DTT)
containing 0.1% Triton X-100. After incubating for 10 min on ice, the
lysates were centrifuged, and the nuclei were resuspended in 20–40
µl of buffer C (20 mM HEPES (pH 7.9), 25% glycerol, 420 mM NaCl, 1.5
mM MgCl2, 0.2 mM EDTA, and 1 mM DTT). This
suspension was incubated for 30 min on ice followed by centrifugation
at 10,000 x g for 20 min. Double-stranded
oligonucleotides containing a consensus NF-B sequence (Promega,
Madison, WI) were end labeled with T4 polynucleotide kinase in the
presence of [-32P]dATP (Amersham). The DNA
binding reaction was performed at room temperature for 30 min in a
final volume of 15 µl, which contained 3–5 µg of nuclear extract,
oligonucleotide probe (40 fmol), and binding buffer containing 100
µg/ml poly(dI-dC) as nonspecific competitor. Reactions were subjected
to electrophoresis on nondenaturing 5% polyacrylamide gels in 0.5x
TBE at 125 mA for 4 h at 4°C.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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CD28 is an important costimulatory receptor that regulates
cellular activation by the TCR/CD3 complex (1, 2, 3, 4, 5). While
CD28 can independently signal T cells (38), this receptor
also participates in signaling events that require TCR/CD3 coligation.
This has been best demonstrated in the activation of the N-terminal
c-Jun kinase (JNK) cascade, which regulates transcriptional activation
of AP-1 response elements in the IL-2 promoter (9, 35, 39). More recently, CD28 has been shown to contribute to the
activation of a composite CD28RE in the IL-2 promoter (8).
Since NF-B signaling is mediated through a multicomponent IB
kinase (IKK) complex, we wanted to determine whether
immunoprecipitation of IKK or IKKß shows dual receptor requirement
for the activation of IKK complexes. In vitro kinase assays using
GST-IB as a substrate showed CD3- and CD28-inducible kinase
activity in IKK and IKKß immunoprecipitates obtained from Jurkat T
cells (Fig. 1
A). In contrast,
ligation of CD3 alone had no effect on IKK activation, indicating that
CD28 cooperates with CD3 in IKK activation. P+I treatment had the same
effect as dual receptor ligation (Fig. 1A). Similar
cooperation between PMA and anti-CD28 mAb in IKK activation in
Jurkat T cells has been demonstrated by Harhaj and Sun
(11).
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B and IBß
(12, 13, 14, 15, 16), which, in turn, leads to ubiquitination and
proteolytic degradation of these proteins and release of NF-B
transcription factors into the nucleus (12, 13, 14, 15, 16). While
recombinant IB as well as IBß were substrates for CD3- and
CD28-inducible IKK complexes, the former substrate was more robustly
phosphorylated than the latter in vitro kinase assays (Fig. 1
B, top two panels). This finding agrees with
previous studies that showed that IB is a better substrate for
the IKKs than IBß (17). The kinetics of IB and
IBß phosphorylation were coincident with the degradation of
endogenous IB as well as the appearance of DNA-binding NF-B
complexes in the nucleus at approximately 15–30 min (Fig. 1B, bottom three panels). Although de novo
IB expression was detected at 60 min (due to the transcriptional
activation of its gene by NF-B), the intranuclear translocation of
NF-B factors still continues at this time point (Fig. 1B,
bottom panel). Since IBß degradation was still seen at
60 min, IBß probably plays a role in prolonged NF-B activation
in T cells. Taken together with the findings in Fig. 1A,
these data show cooperation between CD3 and CD28 in activating IKK
kinases and NF-B transcriptional activity in T cells.
To investigate IKK activation in primary human T lymphocytes, we
generated CD4+ T cell blasts, which were rested
for 48 h before restimulation. This was accomplished by using
magnetic bead separation of a CD4+ subset from
human peripheral blood, followed by expansion in tissue culture with
anti-CD3- and anti-CD28 (9.3 mAb)-conjugated beads
(32). After allowing these cells to return to the resting
state, CD3 and CD28 coligation or P+I stimulation induced IKK activity
(Fig. 2), which could be precipitated by
either anti-IKK or -IKKß Abs. These data confirm that
endogenous IKK complexes in primary T cells are activated by dual CD3
and CD28 coligation. While IKK activation was detected with both
anti-IKK and anti-IKKß immune complexes, it was not clear
from these assays whether CD3/CD28 coligation activated heterodimeric
or homodimeric IKK complexes.
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and IKKß composition, are induced by CD3 and CD28 coligation in
primary T cells
Recombinant IKK or IKKß form homo- and heterodimeric kinase
complexes in vitro, suggesting that heterogeneous IKK
com- plexes may exist in vivo (23, 24). In this
regard, Mercurio et al. have recently shown that HeLa cells contain two
distinct IKK complexes, one consisting of IKK/IKKß heterodimers,
and the other containing IKKß homodimers (23). Moreover,
both complex types could be activated by TNF-, with heterodimeric
complexes showing more abundant IKK activity than homodimeric complexes
(23). However, not all cell types contain heterogeneous
complexes (23). We were interested in determining the
composition of IKK complexes in primary human T cells. First, we
examined primary human CD4+ as well as Jurkat T
cell lysates for expression of different IKK subunits. Western blot
data indicated that IKKß and IKK were abundantly expressed in
Jurkat and primary T cells (Fig. 3A, lanes 1–4).
The relative amounts of IKK or IKKß messages were not affected by
cellular stimulation (data not shown). Secondly, we used a sequential
immunoprecipitation approach, previously used in HeLa cells
(23), to examine primary T cells for assembly of
IKK/IKKß heterodimers and homodimers (23).
This required preclearing of IKK-containing complexes from
CD4+ cell lysates with excess anti-IKK Abs
before precipitating any possible remaining IKKß from the
supernatant with IKKß-specific antiserum (23). These
immunoprecipitates were transferred to immunoblotting membranes, which
were sequentially overlaid with IKK and IKKß antisera. As
demonstrated in Fig. 3B (lanes 1 and
4), anti-IKK Abs precipitated both IKK and IKKß
from the cell lysates, indicating the presence of a heterodimeric
complex. The remaining supernatant was devoid of IKK
(lane 2), but still contained IKKß protein, which
could be precipitated by the anti-IKKß antiserum
(lane 6). These complexes were devoid of IKK
(lane 3). The residual amount of IKKß protein after
IKK clearance (lane 6) was more abundant than the
amount of IKKß that associates with IKK (lane
4). These results demonstrate the presence of a homodimeric IKKß
complex in primary human CD4+ T cells. No
evidence was obtained, however, for the existence of IKK homodimers
in experiments in which we attempted to immune precipitate IKK from
lysates that were precleared with anti-IKKß Abs (data not
shown).
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or
IKK-associated protein (IKKAP1) (23, 40). Using an
antiserum to IKK, we could demonstrate that Jurkat as well as
primary human CD4+ T cells, in addition to IKK
and IKKß, express IKK (Fig. 3A, lanes 5 and
6). Since IKK has selective affinity for IKKß
(40), we asked whether IKK is present in the
homodimeric IKKß complexes shown in Fig. 3B. In a further
sequential immunoprecipitation experiment, anti-IKK Abs were
used to immunoprecipitate this subunit from an IKK-depleted
CD4+ cell supernatant. Subsequent immunoblotting
showed that IKK interacts with IKKß complex in vivo in the absence
of IKK (Fig. 3C, lanes 4 and 8).
However, heterodimeric complexes also contained IKK
(40) (not shown). Therefore, IKK is a common component
of heterodimeric and homodimeric IKK complexes in T cells. Also notice
that immunoprecipitation of IKKß after IKK depletion (Fig. 3C, lanes 3 and 7), yielded positive
blotting for IKKß alone. This confirms the existence of IKKß
homodimers, as noted in Fig. 3B.
Since the data in Fig. 3, B and C, indicate the
existence of heterogeneous IKK complexes in primary human
CD4+ T cells, we asked whether one or both
complexes could be activated by CD3 and CD28 or P+I stimulation. This
experiment was conducted in exactly the same way as shown in Fig. 3C, except that the washed immune complexes were incubated
with GST-IB and [-32P]ATP. Our data
show that heterodimeric complexes, precipitated with anti-IKK
(Fig. 3D, lanes 1–4), and homodimeric complexes,
precipitated with either anti-IKKß (lanes 5–8)
or anti-IKK (lanes 9–12) after IKK/IKKß
heterodimer depletion, harbored CD3- and CD28-inducible as well as PMA-
and ionomycin-inducible IKK activities. Neither type of complex could
be activated by anti-CD3 alone (Fig. 3D, lanes 2,
6, and 10). These results demonstrate that two distinct
IKK complexes are activated upon CD3/CD28 and P+I costimulation in
primary human CD4+ T cells.
IKKß activation is required for transcriptional activation of the CD28RE in the IL-2 promoter
To study the biological significance of IKK activation by
TCR/CD28, we concentrated on the IL-2 gene, since its promoter contains
a composite c-Rel/AP-1 element, previously characterized as the CD28RE
(7, 8, 9). CD28RE plays a critical role in CD3- and
CD28-induced activation of the IL-2 gene (7, 8, 9). Moreover,
CD28RE is a key integrator of TCR/CD3 and CD28 signaling pathways
(7, 8, 9). In a Jurkat cell line transfected with a
CD28RE/AP-1 luciferase gene construct, IKK overexpression had no
independent stimulatory effect (Fig. 4A). In contrast, IKKß
overexpression dramatically enhanced CD3- and CD28-stimulated
reporter activity. Similar trends were seen in PMA- and
ionomycin-activated cells (Fig. 4A). It is interesting to
note that IKK and IKKß overexpression in Jurkat cells generate
anti-Flag-precipitable complexes with different levels of IKK
activity (Fig. 4B). While immunoprecipitation of exogenously
expressed IKKß yielded constitutively active complexes,
immunoprecipitation of Flag-tagged IKK yielded a complex with
negligible levels of kinase activity (Fig. 4B). These
differences were not due to different amounts of these kinases being
expressed, since anti-Flag immunoblotting showed roughly equal
amounts of the kinase proteins in the cellular lysate (Fig. 4C). Since overexpressed IKKß does not coprecipitate
significant amounts of endogenous IKK (not shown), the data in Fig. 4B probably reflect the formation of active IKKß
homodimers. The activity of these complexes could be further increased
by CD3 and CD28 or P+I stimulation (Fig. 4B, lanes
5 and 6). While overexpressed IKK was not
biochemically active in Jurkat cells, we have previously shown that
overexpression of Flag-tagged IKK in a macrophage cell line can
result in the formation of an LPS-inducible IKK complex
(41).
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and IKKß also yield
differential effects on the full-length IL-2 promoter, we compared the
effects of wild-type and kinase-inactive IKKs on a cotransfected IL-2
Luc promoter (Fig. 5). While
kinase-inactive IKK had a minor effect, kinase-inactive IKKß was a
potent (76% decrease) inhibitor of IL-2 Luc activity (Fig. 5).
Wild-type IKK and IKKß had minimal effects (Fig. 5). The effect of
kinase-inactive IKKß was comparable to the effect of kinase-inactive
MEKK1, which acts as a MAP3K in the JNK cascade (Fig. 5). We have
recently shown that the JNK cascade plays a critical role in regulating
the CD28RE/AP-1 element in the IL-2 (9). The role of JNK
in this activation of this promoter is also demonstrated by the
inhibitory effect of the JIP-1 (33), which sequesters
endogenous JNK in the cell (Fig. 5). Taken together, these results
indicate that MEKK1 and IKKß play critical roles in the activation of
the IL-2 promoter.
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,
activity in vivo and in vitro (36). At a dose (20 mM) that
was previously shown to inhibit IKKß in vivo (36),
sodium salicylate completely inhibited the activation of a full-length
IL-2 promoter luciferase construct stably transfected into a Jurkat
cell line (Fig. 6A).
Furthermore, sodium salicylate suppressed induction of IL-2 secretion
in Jurkat cells stimulated by CD3 and CD28 or P+I (Table I). This drug was also effective in
interfering in CD28RE/AP-1 activation during CD3 and CD28 or P+I
stimulation, even in the presence of overexpressed IKKß (Fig. 4A).
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D.
Prior salicylate treatment of intact lymphocytes almost completely
inhibited activation of the homodimeric complex (Fig. 6B,
lanes 9–12), but also had a significant inhibitory effect
on IKK/ß heterodimers (Fig. 6B, lanes 1–4),
although not to the same extent as the homodimeric complex. Similar to
Jurkat cells, sodium salicylate interfered in the induction of IL-2
secretion during CD4+ cell stimulation by CD3 and
CD28 (not shown). These data confirm that IKKß plays a critical role
in regulating NF-B transcription factors that leads to activation of
the IL-2 promoter. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B pathways synergize in the activation of the IL-2 gene in Jurkat
T cells (9). In the present study we extend those findings
by showing that IKK complexes play a critical role in primary T cells
stimulated via the CD3 and CD28 receptors (Figs. 1 and 2). Moreover,
biochemical analysis revealed the presence of at least two types of IKK
complexes in CD4+ T lymphocytes, i.e., a
heterodimeric complex containing equivalent amounts of IKK and
IKKß, and a homodimeric complex containing IKKß alone (Fig. 3).
Both complexes included IKK, a recently discovered non kinase
component of the IKK signalsome (Fig. 3A) (23, 40). These findings agree with previous demonstration of
heterogeneous IKK complexes in HeLa cells (23) and suggest
that different IKK complexes may contribute to differential NF-B
signaling in human primary CD4+ T cells. Although
the roles of different IKK complexes still need to be clarified, it is
interesting that kinase-inactive IKKß exerts a stronger effect on the
IL-2 promoter than IKK (Fig. 5A). While there may be a
number of explanations for this finding, our results suggest one of two
possibilities. The first is that IKKß homodimers rather than
IKK/ß heterodimers play the dominant role in regulating the IL-2
promoter in response to CD3/CD28 costimulation. The second possibility
is that homo- and heterodimers both contribute to the activation of the
IL-2 promoter, but the regulation of these complexes involves a
CD3/CD28-inducible component that primarily signals IKKß. IKK,
which binds IKKß selectively (40), may play a role in
this process.
Several lines of evidence support the idea that IKKß is the
predominant kinase regulated by CD3/CD28 coligation in primary
human CD4+ T cells. First, IKK depleted
lysates from CD4+ cells contained an active
kinase complex that could be precipitated by anti-IKKß alone
(Fig. 3). This finding is consistent with observations in other cell
types that ectopically expressed, kinase-inactive IKK minimally
interfered in IKK activation, while overexpressed, kinase-inactive
IKKß was a potent inhibitor of IKK activation (19, 42).
The contribution of IKKß to CD3/CD28 costimulation was further
demonstrated by pharmacological interference in IKK activation by a
specific IKKß inhibitor, sodium salicylate (36). Not
only did this drug inhibit IKK activation, but it also abrogated
transcriptional activation of the CD28RE as well as the full-length
IL-2 promoter (Fig. 6). Although heterodimeric IKK complexes were also
activated by CD3/CD28 costimulation, it is possible that initially this
event is triggered by IKKß. This will explain why sodium salicylate
also interfered in the activation of the heterodimeric complex (Fig. 6B). What role, if any, IKK plays in the phosphorylation
of the IB proteins remains to be determined. In fact, IKK has
been shown to phosphorylate IB at carboxyl-terminal residues that
are distinct from the serines
(S32/S36) that play a role
in proteolytic degradation (24, 25); the significance of
that phosphorylation event still needs to be determined. Recently,
several additional reports have appeared that indicate that IKKß has
a dominant effect in regulating IB kinase activity
(43, 44, 45, 46). Mutational alteration of the regulatory and
catalytic domains of IKK and IKKß showed that IKKß is the main
target for TNF- and IL-1 stimulation (43). Moreover,
embryonic fibroblasts obtained from IKK-deficient mice exhibit
reasonably good IKK activation, while embryonic cells from
IKKß-deficient mice do not express significant IKK activity
(44, 45, 46). These findings together with our observations
that kinase-inactive IKKß, but not kinase-inactive IKK, interfered
in the activation of the IL-2 promoter (Fig. 5) support our conclusions
that IKKß is the principal kinase involved in CD3/CD28-mediated
NF-B activation.
New information on the regulation of IKK complexes is emerging with
the discovery of new IKK subunits. One example is IKK (IKKAP-1), a
nonkinase subunit that was recently identified as an essential
component of the heterodimeric IKK complex (23, 40). We
show that IKK is also a component of the homodimeric IKKß
complexes in T cells (Fig. 3). This is probably due to the specific
binding affinity of IKK for IKKß (23, 40). Moreover,
an anti-IKK antiserum immunoprecipitated active IKK complexes
from IKK-depleted lysates in primary human T cells (Fig. 3). While
the role of IKK still needs to be clarified, one suggestion has been
that it relays signals to IKKß from afferent components in the IKK
complex (40). A second novel protein that influences
IKKß activity is IKAP (note that IKAP is distinct from
IKKAP-1/IKK), which appears to function as a scaffold protein
regulating the assembly of IKK complexes (42). While in
unstimulated cells most endogenous IKKß appears to be IKAP associated
(42), IL-1 stimulation results in the dissociation of
IKKß from IKAP (42). Similar to the role of JIP-1 in the
JNK cascade, overexpression of IKAP abrogates IKK activity
(42). While the role of IKAP in T cells still needs to be
clarified, one possibility is that IKAP may play a role in the assembly
of specific IKK complexes.
The apparent selectivity of IKKß in the activation of the IL-2
promoter is compatible with our recent finding that a second, but
interconnected, signaling cascade is activated in a synergistic fashion
by CD3/CD28 coligation. Activation of the Jun kinase cascade is
medicated by a MAP3K, MEKK1, that additionally activates IKKß
(9). This selectivity for IKKß, but not IKK, has also
been demonstrated by other investigators (29, 30). The
inhibitory effect of dominant-negative MEKK1 on the IL-2 promoter (Fig. 5) may therefore reflect a role of MEKK1 in both the JNK and NF-B
signaling pathways. That idea is compatible with the stimulatory
effects of dominant-active MEKK1 on both the AP-1 and c-Rel binding
sites in the CD28RE/AP-1 (9). While we do not know at this
stage whether MEKK1 selectively activates homo- or heterodimeric IKK
complexes, further studies to address that question are ongoing. The
idea that heterogeneous IKK complexes may be selectively activated
by different upstream kinases is further strengthened by the recent
demonstration that the proto-oncogene, Cot/Tp1, a MAP3K-related
serine-threonine kinase, also participates in IKK activation by
CD3/CD28 (27). Interestingly, Cot interacts directly with
IKK and NIK, and it has been proposed that Cot acts upstream of NIK
(27). Although ectopic expression of a kinase-inactive
version of NIK inhibits IKK and IKKß activities, NIK only
phosphorylates IKK in vitro and in vivo (26). Since
this implies that Cot may selectively signal IKK, it is possible
that this MAP3K may be regulating the activities of those complexes
that contain IKK, while MEKK1 may regulate the homodimeric complex.
We are in the process of testing that hypothesis.
Finally, the data presented in this paper indicate that the composition
of IKK signalsome may not be rigid, and its assembly is subject to
regulation by the afferent signal as well as the cell type. Such
regulation may be important for selective gene expression by the
NF-B pathway. We are currently pursuing the role of heterogeneous
IKK complexes in selective gene activation during CD3/CD28
costimulation of primary human CD4+ T cells.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Andre. E. Nel, Department of Medicine, Division of Clinical Immunology and Allergy, 10833 Le Conte Avenue, 52–175 Center for Health Sciences, Los Angeles, CA 90095. E-mail address:
3 Abbreviations used in this paper: CD28RE, CD28 response element; IKK, IB kinase; MAP3K, mitogen-activated protein kinase kinase kinase; MEKK1, mitogen-activated protein kinase kinase kinase 1; JNK, N-terminal Jun kinase; NIK, NF-B- inducing kinase; IKAP, IKK complex-associated protein; IKKAP-1, IKK-associated protein 1; JIP-1, JNK-interacting protein; DA, dominant active; DN, dominant negative; P+I, PMA plus ionomycin; Tpl, tumor progression locus; Cot, cancer Osaka thyroid; Luc, luciferase.
Received for publication June 17, 1999. Accepted for publication August 27, 1999.
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