From the Program in Molecular Immunology, Institute of Molecular Medicine and Genetics, Medical College of Georgia, Augusta, Georgia 30912-2600
Received for publication, October 23, 2002, and in revised form, February 12, 2003
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ABSTRACT |
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Engagement of the T cell antigen receptor (TCR)
rapidly induces multiple signal transduction pathways, including ERK
activation. Here, we report a critical role for ERK at a late stage of
T cell activation. Inhibition of the ERK pathway 2-6 h after the start of TCR stimulation significantly impaired interleukin-2 (IL-2) production, whereas the same treatment during the first 2 h had no
effect. ERK inhibition significantly impaired nuclear translocation of
c-Rel with a minimum reduction of NF-AT activity. Requirement for
sustained ERK activation was also confirmed using primary T cells. To
induce sustained activation of ERK, T cells required continuous
engagement of TCR. Stimulation of T cells with soluble anti-TCR
antibody resulted in activation of ERK lasting for 60 min, but failed
to induce IL-2 production. In contrast, plate-bound anti-TCR antibody
activated ERK over 4 h and induced IL-2. Furthermore, T cells
treated with soluble anti-TCR antibody produced IL-2 when phorbol
12-myristate 13-acetate, which activates ERK, was present in the
culture medium 2-6 h after the start of stimulation. Together, the
data demonstrate the presence of a novel activation process following
TCR stimulation that requires ERK-dependent regulation of
c-Rel, a member of the NF- The T cell receptor
(TCR)1 initiates signal
transduction through the intracellular regions of the CD3 and Downstream of ZAP-70, the MAPK family of kinases has been shown to play
critical roles in T cell activation and differentiation (reviewed in
Refs. 1 and 4). Biological outcomes that have been reported to be
controlled by ERK, a member of the MAPK family, include cytokine
production, apoptosis, proliferation, positive and negative selection,
and cytolysis. A major target of ERK has been postulated to be Elk1,
which in turn up-regulates expression of c-Fos (5). c-Fos, when
dimerized with the Jun family of transcription factors, forms AP-1. A
number of AP-1-binding sites have been identified in promoter regions
for genes induced by TCR stimulation, such as IL-2 (6). A function of
AP-1 in IL-2 gene activation is to form a complex with another
transcription factor, NF-AT (7, 8). Formation of this complex has been shown to play critical roles in IL-2 gene activation.
Recently, we identified a Jurkat T cell-derived mutant cell line
(J.SL1) that has lost expression of the adaptor molecule Shc (9). In
this cell line, TCR activation leads to impaired IL-2 production and
ERK activation, whereas AP-1 and NF-AT activation is unaffected.
Detailed analysis revealed that nuclear translocation of an NF- Although studies using c-rel knockout mice showed that c-Rel
is essential for IL-2 production (10, 11), it is not well understood
how TCR triggers c-Rel activation. Other studies have shown that
protein kinase C Here, we report evidence that the ERK signaling pathway is essential
for c-Rel nuclear translocation in the TCR signaling pathway. Most
interestingly, ERK activity is required for c-Rel activation 2-6 h
after the start of TCR stimulation. This late phase ERK activity is
also required for IL-2 production.
Cells--
Jurkat cells (a gift from Dr. A. Weiss, University
California, San Francisco, CA) and 2B4 and CHO cells (gifts from Dr.
M. M. Davis, Stanford University, Stanford, CA) were maintained in RPMI 1640 medium supplemented with 5% (for Jurkat cells) or 10% (for
2B4 and CHO cells) fetal calf serum, 100 IU/ml penicillin, 100 µg/ml
streptomycin, 2 mM L-glutamine, and 50 µM Antibodies and Reagents--
Anti-Jurkat TCR antibody (C305, a
kind gift from Dr. A. Weiss) was purified from mouse ascites using an
Immunopure IgM purification kit (Pierce). Anti-c-Rel and
anti-ERK polyclonal antibodies were purchased from Upstate
Biotechnology, Inc. (Lake Placid, NY). Anti-phospho-ERK polyclonal
antibody was from Promega. Anti-p65 NF- Stimulation and Inhibitor Treatment of Cells--
For
stimulation with plate-bound anti-TCR antibody, tissue culture plates
and polystyrene beads (Polyscience, Warrington, PA) were
incubated with antibody C305 (0.35 µg/ml for Jurkat cells) or with
antibody 2C11 (1 µg/ml) for 3 h at room temperature or overnight
at 4 °C. After removal of unbound antibody by washing with
phosphate-buffered saline, cell suspensions (5 × 105
cells/ml) were added.
For stimulation with soluble antibody, Jurkat cells were incubated with
antibody C305 at a final concentration of 0.35 µg/ml. 2B4 cells were
cultured with biotinylated antibody 2C11 (1 µg/ml) for 15 min at
37 °C, followed by culture with streptavidin (0.5 µg/ml). Mouse
splenic T cells were stimulated with plate-bound antibody 2C11 in the
presence or absence of soluble anti-CD28 antibody (1 µg/ml).
For stimulation with antigen peptide, CHO cells (2.5 × 104) were cultured with the agonist peptide (1 µM) (22) overnight and fixed with 1% paraformaldehyde.
2B4 cells (2.5 × 104) were added to the
antigen-presenting cells and cultured for 24 h with or without
estrogen (200 nM) or PD98059 (25 µM).
For experiments with inhibitor treatment, cells were preincubated with
inhibitors (30 min for PD98059 or 15 min for actinomycin D) or with
Me2SO as a solvent control. During the time course analysis
of inhibitor treatment, supernatants were replaced with medium
containing an inhibitor or Me2SO at the indicated time points.
ELISA and Fluorescence-activated Cell Sorter Analysis--
IL-2
concentration in the supernatants was determined using a human or mouse
IL-2 immunoassay kit from R&D Systems (Minneapolis, MN). For flow
cytometry analysis, cells were incubated with antibodies in Hanks'
balanced saline solution containing 1% bovine serum albumin and 0.05%
sodium azide (staining buffer). Intracellular staining was performed
using phycoerythrin-conjugated anti-IL-2 antibody
(Pharmingen) according to the manufacturer's directions. Samples
were analyzed by FACScan (BD Biosciences).
Cell Transfection and Luciferase Assay--
The NF-AT luciferase
reporter was obtained from Dr. G. Crabtree (Stanford University) and
used as previously described (23). Luciferase activity was determined
using the Dual-Luciferase assay kit (Promega) following the
manufacturer's directions.
Immunoblot Analysis--
The preparation of total cell lysates
and nuclear extracts and the conditions of immunoblot analysis have
been described previously (9). Protein concentrations of samples were
measured using a BCA kit (Pierce) or Coomassie Plus protein assay
reagent (Pierce), and the same amount of proteins for each sample was
applied to the gel. Quantitative analysis of the bands was performed by
densitometry (D-700, Bio-Rad) and with NIH Image.
In Vitro Kinase Assay--
Jurkat cells were preincubated with
either PD98059 at the concentrations indicated or Me2SO
(1:2000 dilution as a solvent control) for 30 min at 37 °C. Cells
(1 × 107/ml) were then stimulated with soluble
anti-TCR antibody (C305; 0.3 µg/ml) for 5 min at 37 °C and lysed
with ice-cold lysis buffer (10 mM Tris-HCl (pH 7.4), 1.0%
Triton X-100, 0.5% Nonidet P-40, 150 mM NaCl, 20 mM sodium fluoride, 0.2 mM sodium
orthovanadate, 1.0 mM EDTA, 1.0 mM EGTA, and
0.2 mM phenylmethylsulfonyl fluoride). Immunoprecipitation
of ERK and kinase assay were performed following the protocol obtained
from Pharmingen with minor modifications. In brief, the cell lysates
were precleared with rabbit nonimmune IgG·protein G-Sepharose complex
(Amersham Biosciences) for 15 min at 4 °C. Precleared cell lysate
(0.5 mg/ml) was incubated with anti-ERK polyclonal antibody·protein
G-Sepharose complex for 2 h at 4 °C and washed three times with
lysis buffer, followed by two washes with kinase buffer (10 mM Tris-HCl (pH 7.4), 150 mM NaCl, 10 mM MgCl2, and 0.5 mM
dithiothreitol). The precipitated immunocomplexes were incubated with
40 µl of kinase buffer containing 25 µM ATP, 2.5 µCi
of [ ERK Activation Is Required for c-Rel Nuclear
Translocation--
Because lack of Shc in Jurkat cells results in
partial loss of ERK activation and significant impairment of c-Rel
activation and IL-2 production, we hypothesized that a high level of
ERK activity is required for c-Rel nuclear translocation and IL-2 production. To test this, we examined the effect of ERK inhibition on
Jurkat cells stimulated with anti-TCR antibody in the presence of
varying amounts of a MEK-specific inhibitor, PD98059. As shown in Fig.
1A, treatment of Jurkat cells
with 25 µM PD98059 blocked IL-2 production by 80%, and 5 µM PD98059 still inhibited IL-2 production by 50%. In
contrast, NF-AT-dependent transcriptional activity was less
sensitive to PD98059 (Fig. 1A). 25 µM PD98059 inhibited activity by ~50% whereas 5 µM PD98059 had
little effect, if any.
To examine the effectiveness of treatment with PD98059, cell lysates
were isolated from samples to which differing doses of PD98059 were
added 30 min before the start of 5 min of TCR stimulation. As shown in
Fig. 1B, PD98059 blocked TCR-induced ERK activation in a
dose-dependent manner. Thus, the lack of NF-AT inhibition with low concentrations of PD98059 treatment was not due to ineffective ERK inhibition.
To examine whether other T cell responses are sensitive to inhibition
of ERK, we tested the effect of PD98059 on activation-induced expression of the cell-surface antigens CD69, CD154 (CD40 ligand), and
CD25 (IL-2 receptor
Next, we determined whether low doses of the MEK inhibitor affect c-Rel
activation. Cells were stimulated with anti-TCR antibody in the
presence of 2 or 25 µM PD98059, and their nuclear and
cytoplasmic proteins were analyzed by Western blotting with anti-c-Rel
antibody. As shown in Fig. 1D, treatment of Jurkat cells
with either 2 or 25 µM PD98059 reduced the amount of
nuclear c-Rel in a dose-dependent manner (upper
panel). No significant change was observed for cytoplasmic c-Rel
(lower panel). In contrast, treatment with 5 µM PD98059 had very little effect on RelA nuclear
localization as shown in Fig. 1E. Even with 25 µM, reduction of nuclear localization was limited to 50%
of untreated samples. It should be noted that RelA translocated to the
nucleus much earlier than c-Rel (see 2-h samples) and that the RelA
levels in the nucleus decreased after 4 h. These data suggest that
c-Rel and RelA are under the control of two different signaling
pathways and that ERK plays a more significant role in c-Rel activation.
c-Rel Activation Partially Rescues Inhibition of IL-2 Production by
PD98059--
To test whether loss of c-Rel nuclear translocation was
responsible for reduced IL-2 production following treatment with
PD98059, we established T cell lines constitutively expressing a
c-Rel/ER fusion protein. This fusion protein accumulated both in the
cytoplasm and nucleus in the presence of estrogen as shown in Fig.
2A. This increase in
cytoplasmic and nuclear c-Rel/ER was also observed in 2B4 mouse T cell
hybridoma cells in an estrogen dose-dependent manner (Fig.
2B). When Jurkat T cells expressing c-Rel/ER were stimulated
with anti-TCR antibody in the presence of 25 µM PD98059, IL-2 production was reduced by 50% compared with 70% for control Jurkat cells (Fig. 2C). Treatment with estrogen alone showed
almost no effect on IL-2 production in both Jurkat cells. This
shows that nuclear accumulation of c-Rel is not sufficient for IL-2 production. When estrogen was added along with PD98059,
c-Rel/ER-transfected Jurkat cells (but not parental Jurkat cells)
showed a significant increase in IL-2 production over cells treated
with only PD98059.
We also established transfectants of the 2B4 mouse T cell hybridoma
cells (specific for I-Ek plus moth cytochrome c)
(22) expressing c-Rel/ER. Transfected cells were stimulated with
antigen peptide presented by I-Ek-positive CHO cells. When
2B4 T cells were treated with PD98059, antigen-induced IL-2 production
was almost abrogated (Fig. 2D). However, when estrogen was
added to the culture along with PD98059, c-Rel/ER transductants
(but not parental 2B4 cells) showed significant levels of IL-2
production, as observed with control Jurkat cells. No IL-2 production
was observed with estrogen treatment alone (data not shown). The data
suggest that the reduced IL-2 production caused by inhibition of ERK is
partially due to the loss of c-Rel nuclear translocation.
c-Rel Activation and IL-2 Production Require Sustained ERK
Activation--
Because nuclear localization of c-Rel is a slow event
(peaks at 4 h after the start of TCR stimulation), we examined
which time point of ERK activity is most important for c-Rel activation and IL-2 production. We first determined the critical time point of ERK
activation in IL-2 production. Jurkat cells were stimulated with
plate-bound anti-TCR antibody, and the level of IL-2 production was
determined in untreated and PD98059-treated samples. When PD98059 was
added at the beginning of stimulation (with a pretreatment for 30 min)
and removed 2 or 4 h later, IL-2 production was not reduced (Fig.
3A). However, when PD98059 was
present for 6 h, a significant reduction was observed. To our
surprise, when the inhibitor was added 2 h after the start of
stimulation and present for up to 6 h, IL-2 production was almost
eliminated. Addition of the inhibitor even 4 h after the start of
stimulation caused a significant reduction in IL-2 production. NF-AT
activity was affected much less (maximum 30% reduction) by PD98059.
IL-2 production by antigen peptide-stimulated 2B4 cells showed similar
kinetic characteristics compared with Jurkat cells (Fig.
3B), although the amounts of IL-2 produced were much
higher.
The requirement for sustained ERK activation was also tested with
primary T cells. Purified CD4+-enriched mouse splenic T
cells (from TCR A1(M) transgenic mouse, >90% CD4+) (data
not shown) were stimulated with anti-CD3 antibody for 24 h (Fig.
3C). PD98059 was added to the culture at various time points. As shown, addition of PD98059 12 h after the start of stimulation still blocked IL-2 production as effectively as addition at
the start of culture.
To examine whether the effect of PD treatment was at the level of
synthesis or secretion, we next tested the level of IL-2 production by
intracellular staining (Fig. 3D). A1(M) splenic T cells were
stimulated with anti-CD3 and anti-CD28 antibodies and showed clear
expression of intracellular IL-2 (left panel). Addition of
PD98059 for the last 6 h of culture resulted in IL-2 production
similar to the levels generated by unstimulated samples. Culture
supernatants from cells treated in this manner did not contain IL-2
when examined by ELISA (data not shown). Thus, PD98059 blocks the
synthesis and not the secretion of IL-2. As observed with Jurkat cells,
CD25 and CD69 expression was at comparable levels in untreated and
PD98059-treated samples (data not shown).
Next, we examined the effect of the same treatment on nuclear
localization of c-Rel (Fig.
4A). Treatment of
TCR-activated Jurkat cells with PD98059 for the first 2 h (with 30 min of pretreatment) had only a minor effect on c-Rel nuclear
localization. However, when the inhibitor was present for 4 h,
nuclear localization of c-Rel was significantly reduced. Inhibition of
ERK 2 or 4 h after the start of stimulation was equally
effective.
It was previously reported that de novo synthesis of protein
is required for nuclear translocation of c-Rel (17). The ERK requirement in the late phase of activation raised the question of
whether ERK is required for de novo protein synthesis. To
test this, we examined whether the kinetics of de novo
protein synthesis required for c-Rel activation are the same as those
of ERK. RelA was also analyzed in this experiment to examine whether
the ERK requirement is specific for c-Rel. Jurkat cells were treated
with actinomycin D to inhibit mRNA/protein synthesis at different
points of activation, and nuclear localization of c-Rel was measured by
Western blotting. As shown in Fig. 4B, actinomycin D
treatment of Jurkat cells inhibited c-Rel nuclear translocation very
effectively when it was added to the culture for the first 2 h.
Treatment between the 4- and 6-h time points of stimulation also showed a significant inhibitory effect. This pattern differs from that of
PD98059 inhibition and indicates that protein synthesis required for
c-Rel activation at an early stage (0-2 h) is ERK-independent. In
contrast, inhibition of ERK showed very little effect on nuclear localization of RelA (Fig. 4B). Moreover, RelA nuclear
translocation was only mildly affected by 6 h of actinomycin D
treatment and even increased with limited treatment between 4 and
6 h. This increase may reflect the loss of I
To confirm the effect of ERK inhibition, the levels of phospho-ERK were
compared among samples treated with PD98059 at different time points.
As shown in Fig. 4 (C and D), when the level of
phospho-ERK in cells stimulated with anti-TCR antibody was taken as
100%, treatment with PD98059 for the first 2 h caused a slight
increase in ERK phosphorylation. This is likely due to inhibition of
ERK phosphatase induction (24). In contrast, treatment of cells 0-6 or
2-6 h after stimulation resulted in effective inhibition of ERK
activity at the 6-h time point.
Activation of ERK at Late Stages Can Restore IL-2 Production by
Soluble Anti-TCR Antibody--
It has been well documented that
stimulation with antibody against TCR in soluble forms does not induce
lymphokine production even though early biochemical events appear to
represent what occurs with full T cell activation (25, 26). Because we
found that sustained activation of ERK is essential for IL-2
production, we tested whether soluble anti-TCR antibody induces
sustained ERK activation. Jurkat T cells were stimulated with either
soluble or plate-bound anti-TCR antibody. When we compared the state of the activated form of ERK, soluble anti-TCR antibody induced robust activation of ERK (Fig. 5A,
left panels). This activation lasted for 30 min, but quickly
declined thereafter. In contrast, plate-bound antibody stimulation
induced ERK activation slowly (peaked at 30 min), but the level of
activation was sustained and remained high even 6 h after the
start of stimulation. A similar pattern was observed for activation of
MEK (right panels). NF-AT activity induced by soluble
antibody was ~30% of that induced by plate-bound antibody.2
To test whether activation of ERK could be the missing element required
for IL-2 production induced by soluble antibody treatment, we used a
pharmacological agent to stimulate ERK in a TCR-independent manner. PMA
induces strong activation of Ras and downstream molecules, including
ERK, in T cells (27, 28). When ERK was activated by addition of PMA to
the culture medium, soluble anti-TCR antibody induced a significant
amount of IL-2 (Fig. 5B). Stimulation by ionomycin together
with soluble anti-TCR antibody had no significant effect on IL-2
production. Treatment of Jurkat cells with PMA also induced rapid c-Rel
and RelA nuclear translocation (Fig. 5C). Interestingly,
soluble antibody alone induced an early (30 min), but not late (6 h),
increase in nuclear RelA.
Using this function of PMA, we determined the time period when PMA
treatment is required for IL-2 production. Jurkat cells were stimulated
with soluble anti-TCR antibody. PMA was added to the culture at the
start of stimulation and was removed at four different time points. As
shown in Fig. 5D, when PMA was removed as late as 4 h
after stimulation, the effect of PMA on IL-2 production was not
observed. However, if PMA was present in the culture for 6 h or
longer, soluble anti-TCR antibody stimulation induced IL-2 production
at a level comparable to that of plate-bound antibody.
Next, Jurkat cells were first stimulated with soluble anti-TCR
antibody, and then PMA was added later to the culture to test how long
PMA stimulation can be delayed for the induction of IL-2. As shown in
Fig. 5E, we observed no reduction of IL-2 production by
soluble anti-TCR antibody when PMA was added as late as 4 h after
the start of stimulation. When PMA was added 6 h after TCR stimulation, a slight decrease in IL-2 production was observed. All
these results indicate that activation of ERK at late time points (4-6
h) is essential for the production of IL-2.
To confirm that the function of PMA involves activation of ERK, we
added PD98059 to the cells that were stimulated with soluble anti-TCR
antibody and PMA. As shown in Fig. 5F, addition of PD98059 (25 µM) abrogated IL-2 production that could be induced
by soluble anti-TCR antibody plus PMA treatment. This strongly suggests
that ERK activation is an essential requirement for IL-2 production induced by PMA stimulation in this system.
The data presented here demonstrate that TCR-induced activation of
ERK must be sustained for the production of IL-2. Most strikingly,
treatment of both human and mouse T cells with an ERK inhibitor was not
effective in blocking subsequent IL-2 production during the initial
phase of TCR-induced activation, but rather exerted its effect in a
late phase (2-6 h). The loss of c-Rel translocation is, in part,
responsible for the loss of IL-2 production because activation of
c-Rel/ER partially counteracts the reduction in IL-2 production caused
by ERK inhibition.
This result indicates that there is a novel signaling pathway that
connects ERK and c-Rel at a late time point in T cell stimulation. As
an NF- A model regarding how ERK controls c-Rel nuclear translocation is that
ERK is involved in de novo synthesis of c-Rel. This model is
based on previous findings showing that de novo synthesized c-Rel translocates to the nucleus after TCR stimulation (31). However,
the period when ERK is most required for IL-2 production and c-Rel
activation does not coincide with the time when de novo RNA/protein synthesis is required. This raises another possibility that
ERK is required for regulation of newly synthesized molecules that are
involved in c-Rel activation. The target may be newly synthesized c-Rel
itself or its regulator. Indeed, c-Rel phosphorylation in Jurkat cells
has been reported recently (32). These two possibilities are not
mutually exclusive, and ERK may be required for both the synthesis and
phosphorylation of activating molecules.
Recently, a number of studies reported that sustained TCR engagement is
required for T cell activation (reviewed in Ref. 33). Formation of the
supramolecular activation clusters (SMAC)/immunological synapse
could provide the source of continued receptor engagement. Such
sustained receptor engagement could enable the prolonged ERK activation
that is required for T cell activation as presented here. In this
sense, ERK could play a role in determining the threshold for full
versus partial activation. Lack of sustained TCR engagement
may attenuate activation of ERK at the late stage and thus inhibit IL-2 production.
It is not yet clear how ERK activation can be sustained for several
hours after the start of stimulation. There are at least two pathways
known that can activate Ras and the MEK/ERK pathway: the
SOS-dependent pathway and the RasGRP pathway (34,
35). Recent analysis using LAT mutants showed that it is a
phospholipase C An alternative possibility for how ERK is activated at a later time
point is by the contribution of secreted lymphokines such as
macrophage migration inhibitory factor (39, 40). It has been
shown that macrophage migration inhibitory factor can activate ERK in a
sustained manner and plays a role in IL-2 production. Because secretion
of macrophage migration inhibitory factor occurs in a relatively early
phase of stimulation, it is possible that this lymphokine provides
sustained activation of ERK.
In summary, the data presented here show a novel function for ERK in
TCR signaling. Activation of ERK is essential for nuclear translocation
of c-Rel, and this role for ERK occurs during a late period of T cell
stimulation. The data also suggest the possibility that ERK activation
may contribute to determining the threshold between sustained agonistic
stimulation and temporary partial agonist stimulation.
B family.
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
molecules via the sequence referred to as the immunoreceptor
tyrosine-based activation motif (1). Antigenic stimulation induces Src
family kinase-mediated phosphorylation of both tyrosines in
immunoreceptor tyrosine-based activation motifs and creates a binding
site for the cytoplasmic protein-tyrosine kinase ZAP-70 (2, 3).
Following this recruitment, ZAP-70 is activated and initiates a
downstream signaling cascade.
B
transcription factor, c-Rel, is severely reduced in this cell line,
whereas RelA, another member of the NF-
B family, is activated
normally. Loss of IL-2 promoter activity in J.SL1 cells could be
rescued by activation of c-Rel using estrogen receptor fusion protein,
indicating that loss of IL-2 production in J.SL1 cells is partly due to
lack of c-Rel activation.
is required for TCR-induced NF-
B activation
(12-14). This function of protein kinase C
appears to be required
for activation of I
B kinase (15, 16). The activation of
protein kinase C
and I
B kinase takes place within minutes after
engagement of TCR, and nuclear translocation of RelA is observed within
15 min after stimulation. In contrast, nuclear translocation of c-Rel
takes place 3-4 h after stimulation and also requires de
novo synthesis of protein (6, 17, 18). This suggests that
TCR-induced c-Rel nuclear translocation requires a signaling pathway
distinct from the one that activates RelA.
MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
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-mercaptoethanol (2B4 and primary T cells). The 2B4
cell line, which stably expresses c-Rel/estrogen receptor (ER) fusion
protein (9), was established by retroviral transduction using the MIGR1
retroviral vector and BOSC23 packaging cell line (gifts from Dr.
W. S. Pear, University of Pennsylvania, Philadelphia, PA) as
described (19). Primary T cells were purified from splenocytes of
C57BL/6 mice (Jackson ImmunoResearch Laboratories, Inc., West Grove,
PA) or TCR A1(M) transgenic mice (transgenic for the TCR of an
I-Ek-restricted H-Y antigen-specific T cell clone) (20)
using nylon wool (Robbins Scientific Corp., Sunnyvale, CA) or goat
anti-mouse IgG antibody-based panning (21), respectively.
B (RelA; sc-8008) and
anti-estrogen receptor (sc-543) antibodies were from Santa Cruz
Biotechnology (Santa Cruz, CA). Fluorescein isothiocyanate-labeled
anti-CD69 and anti-CD154, biotinylated anti-CD3 (2C11), and
phycoerythrin-labeled anti-CD25 monoclonal antibodies were from
Pharmingen. Horseradish peroxidase-labeled anti-mouse and anti-rabbit
polyclonal antibodies and the MEK inhibitor PD98059 were
purchased from New England Biolabs Inc. (Beverly, MA). Actinomycin D
and estrogen (
-estradiol) were from Sigma. PMA and ionomycin were
from Calbiochem. Goat anti-mouse Ig polyclonal antibody was from
Jackson ImmunoResearch Laboratories, Inc.
-32P]ATP, and 40 µg of myelin basic protein
(Sigma) for 15 min at 37 °C. The samples were then separated by
12.5% SDS-PAGE.
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ABSTRACT
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MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Requirement for ERK activation by IL-2
production and c-Rel nuclear translocation. A,
inhibition of IL-2 production and NF-AT activity by PD98059. Jurkat
cells were stimulated with anti-TCR antibody in the presence of various
concentrations of PD98059. Percent inhibition of IL-2 secretion ( )
and NF-AT luciferase activity (
) is presented (samples with no
inhibitor were taken as 100%). B, ERK activation in
PD98059-treated cells. Jurkat cells were treated with PD98059 for 30 min prior to the start of stimulation. Cells were then stimulated in
the presence of PD98059 with soluble anti-TCR antibody. Cell lysates
were made 5 min after the start of stimulation and analyzed by Western
blotting with anti-phospho-ERK antibody (pERK; upper
panel) or by an in vitro kinase assay using myelin
basic protein as the substrate (lower panel).
S, stimulated; NS, not stimulated;
pMBP, phosphorylated myelin basic protein. C,
effect of PD98059 treatment on the induction of surface antigens.
Jurkat cells were stimulated with anti-TCR antibody in the absence or
presence of 25 µM PD98059 as indicated. Expression of
CD25, CD69, and CD154 was analyzed using a fluorescence-activated cell
sorter. The solid lines show the profiles of stimulated
cells, and the dashed lines those of unstimulated
cells. D, inhibition of c-Rel nuclear localization by
PD98059. Jurkat cells were stimulated with anti-TCR antibody for 4 or
6 h in the absence or presence of PD98059 (2 or 25 µM). Nuclear extracts (N; upper
panel) and cytoplasmic fractions (C; lower
panel) were isolated and analyzed by Western blotting using
anti-c-Rel antibody. The relative amount of each band was determined by
densitometry and is shown below each lane. The amount
detected at 4 h of stimulation with no PD98059 treatment was taken
as 100%. U, unstimulated. E, comparison of
PD98059 effect on c-Rel and RelA. Jurkat cells were stimulated with
anti-TCR antibody for 2, 4, or 6 h in the absence or presence of
PD98059 (5 or 25 µM). Nuclear extracts were isolated and
analyzed by Western blotting with anti-c-Rel (upper panel)
and anti-RelA (lower panel) antibodies. The relative amount
detected for each sample is shown below each lane. The
amount detected at 4 h of stimulation with no PD98059 treatment
was taken as 100%.
-chain). Jurkat cells were stimulated with
anti-TCR antibody in the presence of PD98059 (25 µM), and the surface expression of these molecules was determined. The expression patterns of CD69, CD154, and CD25 were only slightly impaired even though 25 µM PD98059 had been shown to
substantially inhibit IL-2 production (Fig. 1C).
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Fig. 2.
Nuclear accumulation of c-Rel restores IL-2
production by T cells treated with an MEK inhibitor. A,
shown is the nuclear accumulation of c-Rel/ER. Jurkat cells were
treated with anti-TCR antibody or estrogen as shown. Nuclear extracts
and cytoplasmic fractions were isolated and analyzed by Western
blotting with anti-ER antibody. B, shown is the increase in
c-Rel/ER in nuclear and cytoplasmic fractions in an
estrogen-dependent manner. 2B4 cells stably expressing
c-Rel/ER were treated with medium containing estrogen at various
concentrations. Nuclear and cytoplasmic fractions were analyzed by
anti-ER antibody. C, Jurkat cells transfected with c-Rel/ER
(black bars) and parental Jurkat cells (white
bars) were stimulated with anti-TCR antibody and treated with
PD98059 (PD; 25 µM), estrogen (es;
200 nM), or both (PD+es). IL-2 production was
determined by ELISA and normalized to samples from TCR-stimulated cells
with no PD98059 or estrogen taken as 100%. D, 2B4 mouse
hybridoma cells transfected with c-Rel/ER (black bars) and
parental 2B4 cells (white bars) were treated as described
for B, and relative amounts of IL-2 in each sample are
presented as described for B.
View larger version (39K):
[in a new window]
Fig. 3.
Kinetic characteristics of inhibition of IL-2
production by an MEK inhibitor. A, inhibition of IL-2
production and NF-AT activity by PD98059 (PD) treatment.
Jurkat cells were stimulated with plate-bound anti-TCR antibody.
PD98059 (25 µM) was added to the culture at various time
points as shown below each bar. 90% of the medium of each
sample was replaced with fresh medium 6 h after the initial
stimulation. After 12 h of stimulation, the supernatant was used
for ELISA analysis, and the amount of IL-2 was determined. NF-AT
activity was measured by luciferase assay and is shown as a percentage
of the activity detected in the anti-TCR antibody-stimulated samples
without PD98059 treatment. B, inhibition of
antigen-induced IL-2 production by PD98059 treatment of 2B4 cells. 2B4
cells were stimulated with antigen peptide presented by CHO cells.
Cells were treated as described for A. C,
inhibition of IL-2 production by PD98059 treatment of splenic T cells.
T cells from TCR A1(M) transgenic mice were stimulated with anti-CD3
antibody. Cells were treated with PD98059 (25 µM) during
the period shown under each bar. D, effect of
PD98059 treatment on IL-2 synthesis and expression of CD25 by primary T
cells. T cells were purified from C57BL/6 mice spleens and stimulated
with anti-CD3 and anti-CD28 antibodies. PD98059 (25 µM)
was added after 18 h of stimulation. Cells were harvested
at 24 h and analyzed for intracellular IL-2 (left
panel) and CD25 (right panel) by flow cytometry. The
solid lines show stimulated cells, and the dotted
lines show unstimulated samples. The filled areas show
the profiles from stimulated and PD98059-treated samples.
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[in a new window]
Fig. 4.
Requirement for late ERK activation by c-Rel
nuclear translocation. A, inhibition of c-Rel nuclear
translocation by PD98059. Jurkat cells were stimulated with anti-TCR
antibody. PD98059 (PD; 25 µM) was added to the
culture during the period shown above each lane. Nuclear extracts were
isolated from each sample 6 h after the start of stimulation. The
relative amount of nuclear c-Rel was determined by densitometry and is
shown below each lane. The sample from stimulated Jurkat
cells with no PD98059 treatment was taken as 100%. B,
comparison of c-Rel and RelA nuclear localization in response to
PD98059 and actinomycin D treatment. Jurkat cells were stimulated with
anti-TCR antibody. PD98059 (25 µM) or actinomycin D
(ActD; 0.1 µg/ml) was added to the culture during the
period shown above each lane. Nuclear extracts were isolated
6 h after the start of stimulation and analyzed by Western
blotting with anti-c-Rel (upper panel) and anti-RelA
(lower panel) antibodies. The relative amounts were
determined as described for B and are shown below each
lane. C, inhibition of ERK activation by PD98059
treatment. Jurkat cells were stimulated with plate-bound anti-TCR
antibody. PD98059 (25 mM) was added to the culture for the
period shown above each lane. Cells were harvested after
6 h of stimulation, and cell lysates were used for
anti-phospho-ERK blots. D, quantitation of phospho-ERK
(pERK). Phosphorylated p42 ( ) and p44 (
) of ERK shown
in C were quantified by NIH Image. The quantity detected in
stimulated samples without PD98059 was taken as 100%. U,
unstimulated.
B expression, which
plays a critical inhibitory role in nuclear localization of RelA.
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[in a new window]
Fig. 5.
Production of IL-2 by cells stimulated with
soluble anti-TCR antibody and PMA. A, kinetics of ERK
and MEK activation by soluble and plate-bound anti-TCR antibodies.
Jurkat cells were stimulated with soluble anti-TCR antibody
(S; upper panels) or plate-bound (for ERK) or
bead-bound (for MEK) anti-TCR antibody (F; lower
panels). Cells were lysed at various time points (indicated above
each lane) and analyzed by Western blotting with antibodies
against phospho-ERK (pERK; left panels) or
phospho-MEK (pMEK; right panels). B,
IL-2 production by Jurkat cells stimulated with soluble anti-TCR
antibody and PMA. Jurkat cells were stimulated with soluble
(Ab(s)) or plate-bound (Ab(f)) anti-TCR antibody.
PMA (10 ng/ml) or ionomycin (Iono; 1 µM) was
added to the culture. The levels of secreted IL-2 for each sample
24 h after the start of stimulation were determined by ELISA.
C, nuclear translocation of c-Rel and RelA in cells
stimulated with soluble anti-TCR antibody and PMA. Nuclear
translocation of c-Rel (upper panels) and RelA (lower
panels) was determined for Jurkat cells stimulated as described
for B. Nuclear (N) and cytoplasmic (C)
fractions were isolated 30 min (left panels) or 6 h
(right panels) after the start of stimulation. D,
IL-2 production by Jurkat cells stimulated by soluble anti-TCR antibody
with limited PMA treatment. Jurkat cells were stimulated with soluble
anti-TCR antibody, and PMA (10 ng/ml) was added to the culture for
limited periods as shown below each bar. After removal of
PMA-containing medium, cells were washed, and anti-TCR
antibody-containing medium was added back to the culture. 90% of the
medium of each sample was replaced with fresh medium 6 h after the
initial stimulation. IL-2 production was determined by ELISA using he supernatant of cells stimulated for 24 h.
E, IL-2 production by Jurkat cells stimulated with anti-TCR
antibody with delayed PMA treatment. Jurkat cells were stimulated with
soluble anti-TCR antibody, and PMA (10 ng/ml) was added for the period
shown below each bar. Both PMA and antibody were kept in the
medium until the end of the culture (24 h). IL-2 production was
determined by ELISA. F, inhibition of soluble
antibody/PMA-induced IL-2 production by PD98059. Jurkat cells were
stimulated with anti-TCR antibody and PMA. PD98059 (25 µM; white bars) or Me2SO alone
(DMSO; black bars) was added to the culture. The
supernatant was harvested 18 h later and subjected to ELISA for
IL-2 production. NS, not stimulated; PB,
stimulated with plate-bound anti-TCR antibody; Sol,
stimulated with soluble anti-TCR antibody.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
B family member, c-Rel is regulated by the I
B family of
proteins, and their regulation is controlled by the I
B kinase family
of kinases (29). Although MEKK1 (JNK kinase) has been shown to activate
I
B kinase, no involvement of MEK or ERK has been detected (30). Our
data show that RelA is much less sensitive to inhibition by MEK
inhibitors, indicating that the MEK/ERK pathway is specifically
involved in the regulation of c-Rel. The possibility of a
c-Rel-specific regulatory mechanism is also supported by the fact that
it takes 2-4 h for TCR stimulation to induce nuclear translocation of
c-Rel, whereas RelA translocates into the nucleus within 15 min after stimulation.
-1-binding site of LAT that mainly regulates ERK
activation in T cells (36, 37). This suggests that RasGRP may play a major role in Ras activation after TCR engagement downstream of LAT.
Recently, we found that Ras is activated weakly but in a sustained
manner in the absence of
LAT.3 Shc is clearly
tyrosine-phosphorylated in the absence of LAT and appears to play a
critical role in this pathway. Because Shc has been implicated in the
SOS activation pathway in many receptor systems (38), it is likely that
SOS-dependent Ras activation can be carried out via Shc.
Our recent study also showed that loss of Shc causes loss of c-Rel
activation and IL-2 production in Jurkat cells (9). Together, the data
imply that TCR utilizes two pathways to activate Ras via
LAT/phospholipase C
-1/RasGRP and Shc/Grb2/SOS and these two Ras
activation pathways may play essential roles in enabling early and
sustained activation of the Ras/MAPK pathway.
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ACKNOWLEDGEMENTS |
---|
We thank Drs. A. Weiss, G. Crabtree, W. S. Pear, M. M. Davis, and M. Zurovec for reagents; P. Koni, R. Markowitz, and A. Mellor for critical reading of this manuscript; and M. Keskintepe for fluorescence-activated cell sorter analysis.
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FOOTNOTES |
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* This work was supported in part by a grant from the National Institutes of Health (5R01AI47266) (to M. I.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Supported by the Japanese Ministry of Science and Education. On
sabbatical leave from the Department of Ophthalmology, School of
Medicine, Kochi Medical College, Kochi 783-8505, Japan.
§ Supported by an NIAID independent scientist award from the National Institutes of Health. To whom correspondence should be addressed: Program in Molecular Immunology, Inst. of Molecular Medicine and Genetics, Medical College of Georgia, CA2004, 1120 15th St., Augusta, GA 30912-2600. Tel.: 706-721-6226; Fax: 706-721-8732; E-mail: miwashima@mail.mcg.edu.
Published, JBC Papers in Press, February 20, 2003, DOI 10.1074/jbc.M210829200
2 T. Koike and M. Iwashima, unpublished data.
3 A. Fukushima, Y. Hatanaka, J.-w. Chang, M. Takamatsu, N. Singh, and M. Iwashima, manuscript in preparation.
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ABBREVIATIONS |
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The abbreviations used are: TCR, T cell antigen receptor; MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; IL-2, interleukin-2; CHO, Chinese hamster ovary; ER, estrogen receptor; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase; MEKK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase kinase; PMA, phorbol 12-myristate 13-acetate; ELISA, enzyme-linked immunosorbent assay; JNK, c-Jun N-terminal kinase.
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