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Rosalind Russell Arthritis Research Laboratory, Department of Medicine, San Francisco General Hospital, San Francisco, CA 94110, and University of California, San Francisco, CA 94143
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Mitogen-activated protein kinases (MAPKs)6 are a group of serine/threonine-specific protein kinases thought to play major roles in the regulation of gene transcription in response to a wide variety of extracellular stimuli, including TCR and CD28 signaling. Several distinct MAPKs have been identified, and these participate in only partially overlapping kinase cascades, thereby creating signaling networks of considerable complexity (reviewed in Refs. 12, 13, 14). Stimulation of the TCR alone activates a subfamily of MAPKs, extracellular signal-regulated kinases 1 and 2 (ERK1, ERK2) (15). In contrast, activation of another MAPK subfamily, the Jun-N-terminal kinases (JNKs), depends upon simultaneous stimulation of the TCR and CD28 (15). The observation that perturbation of the TCR and CD28 together, but neither receptor individually, activates JNKs suggests that this MAPK subfamily plays an important role in the signal integration process during costimulation.
The transcription factor AP-1 is a major target of the JNK pathway and requires both TCR-mediated and costimulatory signals for its activation in primary T cells (16). AP-1, which is composed of members of the Jun family in the form of either homodimers or heterodimers with members of the Fos family, plays a direct role in the activation of T cell-specific genes, such as the gene encoding IL-2, and participates in the formation of other transcription factors, such as nuclear factor-AT and nuclear factor-IL-2 (17, 18, 19, 20). Activation of JNK leads to the phosphorylation of c-Jun in its N-terminal activation domain on serine 63 and serine 73, thereby potentiating its ability to activate transcription as either a homodimer or a heterodimer with c-Fos (17, 18, 19, 20, 21, 22, 23, 24, 25). JNK activation further up-regulates AP-1 activity by increasing expression of c-Jun and, possibly, c-Fos (26). Of interest, anergic T cells, which are no longer able to transcribe the IL-2 gene, show reduced ERK activity and only weak JNK activity after costimulation through CD3 and CD28 (27).
The upstream signaling events initiated by stimulation of CD28 are incompletely understood. The cytoplasmic domain of CD28, which is highly conserved between different species, is necessary and sufficient for delivery of a costimulus that enhances IL-2 production (28). Engagement by either B7 molecules or mAbs induces the tyrosine phosphorylation of CD28, which has four cytoplasmic tyrosines (29, 30). Phosphorylation of Tyr170 (murine sequence) recruits phosphatidylinositol-3-kinase (PI3-K) (31, 32, 33). This site may also recruit Grb-2 but there is not yet uniform agreement on this point (34, 35). Mutation of Tyr170 to Phe impairs CD28 signaling in some systems but not others (36, 37, 38, 39). In the human T cell line Jurkat, for example, the Phe170 mutation does not affect the ability of CD28 to promote IL-2 production (38, 39). Instead, Tyr188 appears critical for the delivery of a costimulus in this system, but the biochemical basis for the Tyr188-dependent signal has not yet been identified (40).
Because activation of JNK depends on simultaneous stimulation of the TCR complex and CD28, it is likely that this MAPK subfamily plays a role in the costimulation of T cells. The CD28 signals that regulate coupling to JNK and the relative importance of JNK in mediating cellular responses to CD28 ligation, however, are not known. For these reasons we studied the ability of a series of CD28 cytoplasmic domain mutants to deliver costimuli for JNK activation and IL-2 production.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Murine anti-human CD3 mAbs were purchased from PharMingen
(San Diego, CA). Murine anti-human CD28 mAbs (9.3) were a gift from
Dr. Jeffrey Ledbetter (Bristol-Meyers Squibb Pharmaceutical Research
Institute, Seattle, WA). Hamster anti-murine CD28 mAbs were a gift
from Dr. James Allison (University of California, Berkeley, CA). Goat
anti-mouse and goat anti-hamster polyclonal Abs were purchased
from Cappel (Organon Teknika, Durham, NC). Wortmannin was purchased
from Sigma (St. Louis, MO). Adenosine-5'-triphosphate
(
-32P) was purchased from ICN Pharmaceuticals
(Irvine, CA).
Cell culture
Jurkat cells stably transfected with mutant murine CD28 cDNAs have been described previously (38) and were passaged in RPMI 1640 medium supplemented with 10% FBS and 2 mg/ml Geneticin (Life Technologies, Gaithersburg, MD). For JNK assays as well as for IL-2 assays, cells were pelleted and resuspended in RPMI 1640 with 10% FBS at a density of 0.5 million cells/ml.
Preparation of glutathione-S-transferase (GST)-Jun fusion protein
The plasmid encoding GST-Jun (1-79) fusion protein was a gift from Dr. Roger Davis (University of Massachusetts Medical School, Worchester, MA) and was transformed into XL1-Blue Escherichia coli. Bacterial cultures were grown in CircleGrowth medium (BIO 101, Vista, CA) with 100 µg/ml ampicillin. Expression of the GST-Jun fusion protein was induced at OD0.4 with 0.25 mg/ml isopropyl-ß-D-thiogalactopyranoside (Boehringer Mannheim, Indianapolis, IN). Cultures were harvested after 2 h of induction. The bacteria were pelleted, resuspended in PBS, pH 7.3, and lysed by sonication. Triton X-100 (1%) was added to the lysate. After an additional spin, the supernatant containing GST-Jun fusion protein was stored at -80°C. For purification of GST-Jun fusion protein, glutathione-Sepharose beads (Pharmacia, Uppsala, Sweden) were equilibrated with PBS, pH 7.3, and incubated with the bacterial lysate for 1 h at 4°C. The beads were washed twice with PBS, pH 7.3, and four times with lysis buffer (see below) before use.
Cell stimulation and JNK assays
A total of 5 million cells were used per sample and resuspended
in 200 µl of RPMI 1640 medium with 10% FBS. mAbs to CD3, human CD28
(hCD28), and murine CD28 (mCD28) were added at a concentration of 10
µg/ml. Samples were incubated at 37°C for 5 min. After addition of
goat anti-mouse or goat anti-hamster Abs at 150 µg/ml,
samples were incubated for another 15 min at 37°C. Cells were
pelleted and lysed in 1 ml lysis buffer per sample (25 mM HEPES, pH
7.7, 300 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, pH 8.0, 0.1%
Triton X-100, 0.5 mM DTT, 20 mM disodium ß-glycerophosphate, 0.1 mM
sodium vanadate, 2 µg/ml leupeptin, 100 µg/ml phenylmethylsulfonyl
chloride) rotating for 30 min at 4°C. Lysates were cleared by
centrifugation for 30 min at 4°C. Cleared lysates were combined with
GST-Jun coupled to glutathione-Sepharose beads and rotated for 3 h
at 4°C. Subsequently, the beads were washed three times in lysis
buffer and incubated in 30 µl of kinase buffer (20 mM HEPES, pH 7.7,
20 mM MgCl2, 25 mM disodium-ß-glycerophosphate, 100 mM
sodium vanadate, 2 mM DTT, and 20 mM ATP) containing 1 µCi
[-32P]ATP per sample for 30 min at 30°C. Beads were
washed twice in lysis buffer and resuspended in sample buffer. The
samples were boiled and separated by SDS-PAGE. Gels were analyzed using
a Molecular Imager System GS-363 (Bio-Rad, Hercules, CA).
Cell stimulation and IL-2 assays
A total of 0.1 million cells were used per sample and resuspended in 200 µl of RPMI 1640 medium with 10% FBS. mAbs to CD3, hCD28, and mCD28 were added at a concentration of 10 µg/ml. Samples were incubated at 37°C for 5 min. Goat anti-mouse or goat anti-hamster Abs were added at 100 µg/ml. Cells were incubated at 37°C. After 24 h of incubation, supernatants were harvested and analyzed for IL-2 production by ELISA (Biosource International, Camarillo, CA).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Recent data implicate JNK in signal integration during
costimulation of T lymphocytes (15). In an effort to
define the region of the cytoplasmic tail of CD28 required for JNK
activation, we studied clones of Jurkat that had been stably
transfected with cDNAs encoding wild-type mCD28, mCD28 with cytoplasmic
domain truncations, or mCD28 in which cytoplasmic Tyr residues had been
mutated to Phe (Fig. 1
) (38, 40). The different forms of mCD28 are expressed at comparable
levels with the exception of the ALL F mCD28 mutant, whose level of
surface expression is approximately threefold lower than the other
mCD28s studied (Fig. 2). The Jurkat
clones express the CD3/TCR complex and the endogenous hCD28 (data not
shown), which can serve as an internal positive control for CD28
function in individual clones. We have shown that hCD28 and mCD28 do
not form detectable heterodimers when coexpressed in Jurkat
(38).
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A, compare lanes
5 and 7) and sixfold in comparison with stimulation via
hCD28 only (Fig. 3A, compare lanes 5 and
7). The activation of JNK by the combination anti-CD3
and anti-hCD28 can be substantially enhanced by the addition of a
secondary cross-linking Ab (Fig. 3A, lane 8).
Wild-type mCD28 expressed in Jurkat also can deliver signals that act
in synergy with those of the TCR to activate JNK. When cross-linked by
a second-step Ab, the combination of mAbs to CD3 and mCD28 triggers a
substantial increase of JNK activity in Jurkat cells that express
wild-type mCD28 (Fig. 3B). The level of JNK activity
achieved by cross-linking CD3 with wild-type mCD28 ranges between 50
and 110% of that induced by the cross-linking of CD3 and the
endogenous hCD28 (data not shown).
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The cross-linking of both anti-CD3 and anti-hCD28 triggers a
substantial increase in IL-2 production over that observed with
cross-linking anti-CD3 alone (Fig.
4A). A similar effect on
CD3-mediated IL-2 production is observed when mAbs to CD3 and to
wild-type mCD28 are cross-linked (Fig. 4B). The IL-2
response elicited by cross-linking CD3 and mCD28 mAbs ranges from 30 to
70% of that observed when CD3 is cross-linked with the endogenous
hCD28 (Figs. 4 and 7). Therefore, wild-type mCD28 can deliver a
costimulus for both the activation of JNK and IL-2 production.
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To define a minimal cytoplasmic domain capable of delivering
a costimulus for JNK activation, we used truncation mutants of mCD28
stably expressed in Jurkat cells. Cross-linking CD3 with either the T1
or T2 mCD28 mutant induces JNK activation to a level that is comparable
with that achieved upon stimulation of CD3 and the endogenous hCD28
(Figs. 5 and 6). The
more proximal of these truncations, the T2 mutation, occurs after amino
acid 182 and removes the carboxyl-terminal 17 amino acids (Fig. 1
).
Therefore, these residues, which include the distal three tyrosine
residues (Tyr185, Tyr188, and
Tyr197), are not necessary for mCD28 to synergize with CD3
to activate JNK. Further truncation to amino acid 174 (the T3
truncation) or deletion of all but the first four amino acids of the
cytoplasmic domain (the TL mutant), however, abrogates the ability of
mCD28 to deliver a costimulus for JNK activation (Figs. 5 and 6). The
inability of the T3 and TL mutants to promote JNK activation is not due
to distal defects in the signals that lead to JNK activation as
evidenced by the intact costimulatory ability of the endogenous hCD28
in these clones (Fig. 5). Impaired coupling to JNK was observed with
three independently derived clones expressing the T3 mutant (Fig. 6).
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To examine the effects of cytoplasmic domain truncations on costimulation of IL-2 production, we determined the abilities of mCD28 and mutants to boost IL-2 production in response to CD3 cross-linking. The boost in IL-2 mediated by the endogenous hCD28 in each clone was used as an internal standard.
When cross-linked with CD3, the two T1 truncation mutants deliver a
costimulus for IL-2 production comparable with (T1D6), or greater than
(T1A1), wild-type mCD28 (Fig. 7). When
all experiments (n = 4-6) were analyzed, the difference
between wild-type mCD28 and T1D6 did not quite reach significance
(two-tailed p value = 0.06), but the boost delivered by
T1A1 was significantly greater than wild-type (p <
0.01). Therefore, the carboxyl-terminal 5 amino acids, which
include Tyr197, are not essential for costimulation of IL-2
production and possibly may down-regulate the ability to promote IL-2
production. The T2 truncation mutants also can promote IL-2 production
when cross-linked with CD3, but their ability to do so is impaired
relative to the wild-type and to the T1 mutants (Fig. 7). The boost in
IL-2 delivered by the wild-type mCD28 is significantly greater than the
boosts delivered by either T2C2 or T2D5 (p < 0.01
and < 0.05, respectively; n = 5-6). Similarly,
the boost delivered by either of the T1 mutants is significantly
greater than that delivered by either T2 mutant (p <
0.001 for T2C2 and < 0.01 for T2D5; n =
4-7). Because both T2 mutants can costimulate JNK activity (Fig. 6), the diminished ability of T2 to enhance IL-2 production likely
reflects the loss of JNK-independent signals when the mCD28 cytoplasmic
domain is truncated from amino acids 194 to 182. The costimulatory
ability of the T3A4 mutant (Fig. 7), other T3 mutants (data not shown),
and TL mutant (Fig. 7), in contrast, is severely disrupted. The loss of
costimulatory ability when the cytoplasmic domain is truncated from
amino acid 182 to 174 (i.e., from T2 to T3) is of interest because this
truncation also abrogates the ability of CD28 to augment JNK activity
(Figs. 5 and 6).
Wortmannin does not inhibit JNK activation following stimulation of CD3 and CD28
The T2 truncation mutant retains Tyr 170 which, when
phosphorylated, serves as a binding site for the Src homology 2 (SH2)
domains of the p85 regulatory subunit of PI3-K (Fig. 1). We have shown
that the T2 truncation does not alter recruitment of PI3-K to mCD28
(40). Tyr170 is also present in the T3 mutant,
but this more severe truncation impairs the recruitment of PI3-K p85
(40). Because the ability to costimulate JNK activity also
is lost when the mCD28 cytoplasmic domain is truncated from T2 to T3,
we considered the possibility that PI3-K might couple CD28 to the
activation of JNK. However, wortmannin, a potent inhibitor of PI3-K,
enhances the ability of cross-linked CD3 and mCD28 to promote JNK
activity (Fig. 8). Because 1000 nM
wortmannin completely inhibits PI3-K enzymatic activity and blocks
CD28-mediated production of phosphatidylinositol-3,4,5-trisphosphate
when added to Jurkat (38), we conclude that PI3-K activity
is not required for JNK activation in this system. Interestingly,
wortmannin also enhances the ability of CD28 to promote IL-2 production
by Jurkat (38).
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To address the possibility that tyrosine phosphorylation of CD28
is critical for coupling to the activation of JNK, we studied a mCD28
mutant in which all four Tyr residues have been mutated to Phe (ALL F
mutant; Fig. 1). These mutations eliminate the PI3-K- and Grb-2-binding
sites at Tyr170 as well as a proposed distal secondary
binding site for PI3-K (41). We have previously shown that
the ALL F mutant fails to recruit PI3-K. Cross-linking CD3 and the ALL
F mutant reproducibly boosts JNK activity relative to CD3 stimulation
alone (Figs. 6 and 9), demonstrating that
this signaling event can be Tyr independent. Back mutation to Tyr at
position 170 on the ALL F background (Y170 mutant) restores the ability
of mCD28 to recruit PI3-K (38), but does not alter its
ability to synergize with CD3 in the activation of JNK (Figs. 6 and 9).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The CD28 cytoplasmic domain contains four tyrosine residues, at
least two of which, Tyr170 and Tyr188 (murine
sequence), appear to be involved in CD28-mediated signaling. The
ability of CD28 to couple to JNK activation, however, appears to be
independent of tyrosine-based interactions of signaling molecules.
Deletion of the carboxyl-terminal three tyrosines does not alter the
ability of mCD28 to deliver a costimulus for JNK activation (T2 mutant;
Figs. 5 and 6), and a mCD28 mutant in which all four cytoplasmic
tyrosine residues have been mutated to phenylalanine delivers a potent
costimulus for the activation of JNK (ALL F mutant, Figs. 6 and 9).
Studies with truncation mutants indicate that the membrane-proximal 24
amino acids of the cytoplasmic domain are sufficient for mCD28 to
deliver a costimulus for JNK activation. Deletion of an additional 8
amino acids, however, abrogates coupling to JNK activation. Therefore,
these 8 amino acids are either directly involved in generating a signal
for JNK activation or are required for the structural integrity of the
remaining cytoplasmic domain. The deleted 8 amino acids contain a
Pro-Xaa-Xaa-Pro motif, which represents the minimal binding element for
SH3-mediated associations and which is conserved in hCD28 (Fig. 1)
(42). Therefore, one possibility, is that an interaction
with SH3-domain containing molecule couples CD28 to activation of the
JNK-signaling cascade.
The best-characterized early CD28-mediated signaling event is the
recruitment of PI3-K. This event is triggered by the phosphorylation of
Tyr170, which lies within a consensus binding motif
(Tyr-Met-Asn-Met) for the SH2 domains of the p85 subunit of PI3-K
(33, 34, 35). Activation of PI3-K has been implicated in a
range of cellular events, including the mitogenic responses to growth
factors, cytoskeleton organization, receptor trafficking, inhibition of
apoptosis, and the regulation of integrin function (43).
Attempts to link PI3-K to CD28-mediated costimulation of IL-2
production, however, have led to contradictory results, with mutation
of the PI3-K-binding site abrogating costimulation in certain systems
and having no effect in others (36, 37, 38, 39). Based on the
studies with the ALL F mutant and wortmannin presented here, it is
highly unlikely that CD28-activated PI3-K delivers the costimulus for
JNK activation. Interestingly, wortmannin augments JNK activation in
response to CD3 and CD28 cross-linking, raising the possibility that a
wortmannin-sensitive target down-regulates JNK activation. Because
restoration of the PI3-K-binding site on the ALL F mutant background
(Y170 mutant, Figs. 6 and 9) does not affect costimulation of JNK, it
is unlikely that wortmannin augments the activation of JNK through
inhibition of the PI3-K recruited to CD28. Rather, the coupling of CD28
to PI3-K and to JNK appear to be independent events.
The membrane-proximal 24 amino acids of the mCD28 cytoplasmic domain
can support activation of JNK and delivery of an impaired, but readily
detectable, costimulus for IL-2 production. Thus the cross-linking of
mAbs to CD3 and to mCD28 activates JNK in Jurkat cells expressing the
T2 truncation mutant and triggers the production of IL-2 (
Figs. 5–7).
Truncation of an additional 8 amino acids, however, abrogates these
responses (T3 mutant,
Figs. 5–7). This concomitant loss of
costimulation of JNK activation and IL-2 production is consistent with
the possibility that coupling to JNK plays a role in CD28-mediated
enhancement of IL-2 production. However, it is also possible that the
inability of the T3 mutant to costimulate IL-2 production reflects loss
of signaling pathways unrelated to JNK activation.
Taken together with our previously published studies of mCD28 mutants,
the data presented here demonstrate that the optimal CD28 costimulus
for IL-2 production must involve signals in addition to those for JNK
activation and the recruitment of PI3-K. The ability to costimulate
IL-2 production is clearly diminished when the mCD28 cytoplasmic domain
is truncated from residue 194 to 182 (i.e., from the T1 to T2
truncation mutants), (Fig. 7) but this truncation does not affect the
ability of mCD28 to couple to JNK activation (Figs. 5 and 6) or to
recruit PI3-K (40). The diminished ability to deliver a
costimulus for IL-2 production may reflect loss of a signaling motif
based on Tyr188 (40). Mutation of all four Tyr
residues to Phe impairs the ability of CD28 to augment IL-2 production
in response to the combination of ionomycin and PMA (40).
Restoration of Tyr at position 188, but not at any of the other mutated
sites, restores this costimulatory ability (40). The
biochemical basis for the Tyr188-dependent signal is not
known.
The impaired but still detectable ability of the T2 mutant to
costimulate IL-2 production when cross-linked with CD3 (Fig. 7)
contrasts with our earlier demonstration that a mAb to this mutant
cannot augment IL-2 production in response to the combination of
ionomycin and PMA (40). Interestingly, ionomycin and PMA
act in synergy to activate JNK and thus may circumvent any requirements
for CD28-mediated costimulation of JNK (15). The ability
of CD28 to promote IL-2 production in response to this combination,
therefore, may depend largely on signals other than costimulation of
JNK activity, and these signals are abrogated by the T2 truncation.
Taken together with earlier studies of CD28 mutants, the results presented here indicate that there are at least three discrete structural elements involved in mCD28 signaling: Tyr170, whose phosphorylation recruits PI3-K; the Tyr188-dependent signal; and a tyrosine-independent signal for JNK activation, which maps to the membrane-proximal 24 amino acids of the mCD28 cytoplasmic domain.
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Footnotes |
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2 Current address: Max von Pettenkofer-Institut für Hygiene und Mikrobiologie der Ludwig-Maximilians-Universität München, Germany.
3 Current address: Medizinische Klinik III, Institut für Klinische Immunolgie, Friedrich-Alexander-Universität Erlangen-Nürnberg, Germany.
4 Current address: Merck Research Laboratories, 126 East Lincoln Avenue, P.O. Box 2000, RY32-645, Rahway, NJ 07065.
5 Address correspondence and reprint requests to Dr. John B. Imboden, Box 0868, University of California, San Francisco, CA 94143. E-mail address:
6 Abbreviations used in this paper: MAPK, mitogen-activated protein kinase; ERK, extracellular signal regulated kinase; GST, glutathione-S-transferase; hCD28, human CD28; JNK, Jun-N-terminal kinase; mCD28, murine CD28; PI3-K, phosphatidylinositol-3-kinase; SH2, SH3, Src homology 2 and 3.
Received for publication May 6, 1997. Accepted for publication July 16, 1998.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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