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*
Autoimmunity/Diabetes Group, The John P. Robarts Research Institute, and
Departments of Microbiology, Immunology, and Medicine, University of Western Ontario, London, Ontario, Canada
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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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How do upstream TCR- and CD28-proximal signaling events regulate
downstream signaling along the extracellular signal-regulated kinase
(ERK)-, JNK-, and p38 MAPK-mediated pathways in T cells? An essential
step in the activation of small GTPases is the phospho-tyrosine
(p-Tyr)-dependent recruitment of GDP releasing factors (GRFs) to the
membrane and the assembly of GTPase/GRF complexes. GRFs activate small
GTPases by promoting the conversion of GDP-bound GTPases to the active
GTP-bound state. Vav, a GRF expressed exclusively in cells of the
hematopoietic lineage, is critical for Ag receptor-triggered T and B
cell activation and thymocyte development (10, 11). In the
absence of Vav, IL-2 production by T cells is reduced considerably,
possibly due to a disruption of either the TCR or CD28 signaling
pathways (11). Indeed, Vav is Tyr phosphorylated in
response to TCR and CD28 triggering (12). When Vav is
overexpressed in Jurkat T cells, Vav elicits the activation of nuclear
factors that control IL-2 expression, including NF-AT
(13). Vav may also function as a GRF for Rho-related
proteins, including Rac-1 (14), Cdc42, and RhoA
(15), and the Lck protein Tyr kinase (PTK) activates the
GRF and transforming activity of Vav (15). The Tyr
phosphorylation of Vav is required to exchange GDP for GTP on Rac-1
(14, 15), and this Vav Tyr phosphorylation links some
receptors (e.g., Fc
RI) to activation of the Rac-1-JNK pathway
(16). Moreover, the binding of Vav to the TCR
-chain-associated PTK, ZAP-70, via residue Tyr315, plays
a critical role in Ag receptor-mediated signal transduction (17, 18). These observations prompted us to investigate whether TCR
and CD28 signaling are linked to MAPK pathways by Vav. Our objectives
were: 1) to analyze whether signaling pathways activated by TCR and
CD28 converge at the level of ZAP-70 and/or Vav/Rac-1 interactions, and
2) to determine whether ZAP-70 controls the catalytic activity of
multiple downstream effectors of Rac-1 involved in TCR and/or CD28
stimulation.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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BALB/cJ mice were purchased from Taconic (Germantown, NY), and were used at 6–8 wk of age.
Reagents
The mAbs used were: biotin-conjugated anti-mouse TCRß
(H57-597), CD4 (L3T4), CD28 (37.51), anti-human CD28 (CD28.2) (all
from PharMingen, San Diego, CA); CD3 (OKT3; American Type
Culture Collection (ATCC), Manassas, VA); anti-v-H-Ras (Oncogene
Research Products, Cambridge, MA), anti-Vav, anti-Rac-1 and
4G10 anti-p-Tyr (Upstate Biotechnology, Lake Placid, NY);
anti-ZAP-70 and anti-Grb2 (Transduction Laboratories,
Lexington, KY). The following polyclonal rabbit Abs were supplied by
Santa Cruz Biotechnology (Santa Cruz, CA): anti-Vav,
anti-Rac-1, anti-SLP-76, anti-TCR, anti-JNK,
anti-ERK-1, anti-ERK-2, anti-p38 MAPK, and anti-PAK-1.
Purified GST-Rac-1 was obtained from Upstate Biotechnology. Rabbit
polyclonal anti-LAT (linker for activation of T cells) serum was
kindly provided by Dr. L. Samelson (National Institutes of Health,
Bethesda, MD). The expression plasmid for the Rac-1 GST-fusion protein
was provided by Dr. M. J. Hart (Onyx Pharmaceuticals, Richmond,
CA), and this GST-fusion protein was expressed in Escherichia
coli and purified by glutathione-agarose affinity chromatography
(4).
Cell lines
The Lck-negative JCaM1.6 and parental human Jurkat leukemic cell line, E6.1, were obtained from ATCC, and have been described previously (19). The P116 ZAP-70-deficient cell line, a variant of the Jurkat E6.1 T cell line (20), was kindly provided by Dr. R. T. Abraham (Department of Immunology, Mayo Clinic, Rochester, MN). The level of CD28 and TCR surface expression is very similar in all of the Jurkat cell lines studied (data not shown). The EL4 mouse T cell line was purchased from ATCC. Chinese hamster ovary (CHO) cells, which were transfected with and express B7-2 on their surface (21), were a generous gift from Dr. T. Watts (Department of Immunology, University of Toronto, Toronto, ON, Canada). Cells were maintained in RPMI 1640 (Life Technologies, Burlington, ON) medium supplemented with 10% heat-inactivated FCS (Sigma, St. Louis, MO).
Cell activation and lysis
BALB/cJ peripheral splenic T cells were purified on T
cell-enrichment columns (R&D Systems, Minneapolis, MN) (purity >95%).
T cells were washed twice in RPMI 1640 medium supplemented with 2 mM
HEPES before stimulation. If not otherwise indicated, T cells were
stimulated at 37°C with 1 µg/107 cells of the
biotin-conjugated anti-TCR or anti-CD3 mAb either alone or
together with the biotin-conjugated anti-CD4 mAb or anti-CD28
mAb. Cross-linking of mAbs was accomplished using streptavidin (Sigma)
for various times at a 4:1 w/w ratio. The anti-human CD28 (CD28.2)
and CD3 (OKT3) mAbs were cross-linked using AffiniPure
F(ab')2 fragments of a donkey anti-mouse IgG
Ab (Jackson ImmunoResearch, West Grove, PA). Cells were lysed in
ice-cold 50 mM Tris (pH 8.0), 150 mM NaCl, 5 mM EDTA lysis buffer
containing 1% Brij 97 or 1% Nonidet P-40 , 5% glycerol and
supplemented with a mixture of protease and phosphatase inhibitors (100
µM p-nitrophenyl guanidinobenzoate, 1 mM PMSF, 10 µg/ml
aprotinin, 10 µg/ml leupeptin, 5 µg/ml pepstatin, 2 mM
Na3VO4, and 10 mM NaF) (all
obtained from Sigma). The Nonidet P-40-containing lysis buffer was used
for the kinase assays and guanine nucleotide exchange assays. All
subsequent steps were performed at 4°C. Lysates were clarified of
detergent-insoluble material by centrifugation (10 min at 14,000 rpm)
and precleared with rabbit Ig coupled to protein A-Sepharose CL-4B
(Pharmacia, Baie d’Urfe, Canada) for 30 min at 4°C.
Immunoprecipitation, gel electrophoresis, and immunoblotting
Precleared postnuclear cell lysates were normalized for protein concentration levels, immunoprecipitated (3 h, 4°C) with the specific polyclonal Abs or control isotype-matched preimmune Ig precoupled to 25 µl of protein A-Sepharose CL-4B, and the precipitates were washed four times with ice-cold lysis buffer. Precipitated proteins were solubilized in 2x Laemmli sample buffer, resolved by SDS-PAGE (8–16% gradient gel; Novex, San Diego, CA) under reducing conditions, transferred to nitrocellulose (Schleicher & Schuell, Keene, NH) membranes, and immunoblotted with the indicated Abs, as described (22). Signal intensities were quantified using a Molecular Imager System and Molecular Analyst imaging software (Bio-Rad, Hercules, CA).
Guanine nucleotide exchange assay
GST-Rac-1 fusion protein (5–10 pmol/time point) was loaded (60 min, 32°C) with [8-3H]5'-GDP (25 pmol, 13.3 Ci/mmol; Amersham, Arlington Heights, IL) in 40 µl exchange buffer (20 mM Tris-HCl (pH 7.5), 100 mM NaCl, 1 mM MgCl2, 1 mM DTT, and 50 µg/ml BSA). Vav immune complexes immunoprecipitated by rabbit anti-Vav Abs were washed five times with exchange buffer. Reactions (performed in duplicate) were started by addition of [8-3H]5'-GDP-loaded GST-Rac-1 in exchange buffer containing nonradioactive GTP (500 mM). At various times (0, 10, and 30 min), aliquots (40 µl) of reaction mix were removed, centrifuged, and GST-Rac-1 was immobilized on a solid support by using glutathione Sepharose, as described (14, 23). Bound radioactivity was quantitated by liquid scintillation.
Incorporation of [32P]-labeled guanine nucleotides by Rac-1
T cells (2 x 107) were cultured for 4 h in phosphate-free RPMI medium containing dialyzed 10% heat-inactivated FCS and labeled with [32P]orthophosphate (0.2 mCi/ml) for 4 h at 37°C in phosphate-free RPMI/HEPES (2 mM). Mouse primary T cells were permeabilized by addition of 0.4 U/ml of streptolysin O (Wellcome Diagnostics, Greenville, NC) before labeling, as described (24). Cells were left unstimulated (0 min) or were treated with anti-TCR ± anti-CD28 for 15 min. Precleared postnuclear lysates were immunoprecipitated by protein G agarose precoupled to either an anti-Rac-1 (6 µg) or anti-Ras (15 µg) Ab in 50 mM Tris-HCl (pH 7.5), lysis buffer containing 20 mM MgCl2, 350 mM NaCl, 1.5% Nonidet P-40, 0.01% SDS, 1 mM PMSF, 10 µl/ml pepstatin, 10 µl/ml aprotinin, 10 µl/ml leupeptin, 1 mM NaF, and 1 mM Na3V. [32P]-labeled guanine nucleotides bound to Rac-1 and Ras were eluted by heating in 20 mM EDTA, 2 mM DTT, 0.2% SDS, 0.5 mM GDP, and 0.5 mM GTP, and fractionated using polyethyleneimine TLC plates (J. T. Baker, Phillipsburg, NJ). Positions of the [32P]-labeled guanine nucleotides were determined according to the mobility of the unlabeled GDP and GTP markers visualized under ultraviolet light.
Anti-sense oligodeoxynucleotide (oligo-DN) treatment
The following oligo-DNs were used to block Vav expression in T cells: (5'-AAGGCACAGGAACTGGGA-3') anti-sense and (5'-AGCTCGAAAGACAGGGGA-3') control (scrambled) oligo-DN. The sequences of these oligo-DNs are based on the human proto-Vav cDNA sequence (25). T cells were cultured in the presence of 20 µg/ml oligo-DNs for 48 h.
Kinase assays
Jurkat or EL4 T cells were incubated at 37°C for 3–4 h in
FCS-free RPMI 1640 medium before stimulation to reduce background
kinase activities to workable levels. PAK-1 kinase activity in Jurkat T
cells was assayed after an overnight incubation in serum-free medium.
Primary T cells were used without additional preincubation in
serum-free medium. T cells were washed twice in RPMI 1640 medium
supplemented with 2 mM HEPES before stimulation. Proteins
immunoprecipitated from precleared postnuclear lysates were assayed for
associated in vitro kinase activity after washing the beads in kinase
buffer (25 mM HEPES (pH 7.4), 5 mM MnCl2) by
incubating (30 min, 30°C) with [
-32P]ATP
(15 µCi; New England Nuclear, Boston, MA) in 25 µl kinase buffer
containing 1.5 µg GST-c-Jun (Santa Cruz Biotechnology; JNK kinase
assay), 1.5 µg of the cAMP response element binding protein, ATF-2
(Santa Cruz Biotechnology; p38 MAPK assay), 3 µg of myelin basic
protein (MBP; Upstate Biotechnology; ERK-1, ERK-2, and PAK-1 kinase
assays) or 0.6 µg of the cytoplasmic fragment of human erythrocyte
band 3 (cfb3; kindly provided by Dr. A. Veillette, McGill University,
Montreal, Canada) as substrates. Reactions were stopped by boiling with
gel sample buffer. GST-c-Jun, ATF-2, MBP, and cfb3 were resolved by
SDS-PAGE and their phosphorylation was visualized using a phosphoimager
(Bio-Rad). Immunoblotting showed that equal amounts of the JNK, p38,
ERK-1, ERK-2, and PAK-1 proteins were precipitated before and after
stimulation of all Jurkat T cell variants.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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TCR triggering increases the Tyr phosphorylation of Vav
(12), which may be critical in the activation of nuclear
transcription factors that control the expression of IL-2, including
NF-AT (13). Anti-p-Tyr Western blots of Vav
immunoprecipitates showed that, while only weak basal Tyr
phosphorylation of Vav occurs in unstimulated BALB/c T cells (Fig. 1
A), Vav Tyr phosphorylation
is increased substantially following stimulation (5 min) by
anti-TCR, anti-TCR plus anti-CD4, anti-CD28, and
anti-TCR plus anti-CD28 mAbs, respectively. Vav Tyr
phosphorylation was rapidly increased within 1.5 min after TCR, CD28,
or TCR/CD28 cross-linking, and this increase is sustained at 20 min
after TCR and 60 min after CD28 or TCR/CD28 stimulation, respectively
(Fig. 1B). Similarly, Vav Tyr phosphorylation was induced
after stimulation of T cells with CHO cells expressing surface B7-2
(Fig. 1C), the natural ligand for CD28, as previously
reported (12). Note that both TCR and CD28 signaling
induce similar amounts of Vav Tyr phosphorylation. Moreover, TCR/CD28
coligation significantly increases the levels of Vav Tyr
phosphorylation relative to that of T cells stimulated with TCR or CD28
alone (Figs. 1 A–C). Thus, the costimulatory effects of
CD28 may result from an additive and sustained increase of Vav Tyr
phosphorylation leading to the activation of Vav downstream
effectors.
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A) and
primary mouse T cells (Fig. 2B), the majority of Rac-1
exists in the GDP-bound form. This result supports the notion that the
high intrinsic GTPase activity of Rho family proteins precludes the
detection of GTP incorporated by small GTPases (26). Under
these conditions, the level of 32P-labeled GDP
bound to Rac-1 is the only indicator of Rac-1 nucleotide exchange. The
absence of coimmunoprecipitation of other small GTPases with Rac-1 by
the anti-Rac-1 mAb used confirmed the specificity of this mAb (Fig. 2C). Neither Ras nor Rho or Cdc42 were detected in Rac-1
immuprecipitates (data not shown). Essentially no labeled GDP bound to
Rac-1 was observed in either control precipitates formed using
isotype-matched preimmune Ig or unstimulated T cells, suggesting that,
under these experimental conditions, Rac undergoes minimal nucleotide
exchange (Figs. 2, A and B). In contrast, TCR,
CD28, and TCR/CD28 cross-linking yielded 
5-, 4-, and 10-fold
increases, respectively, in the amounts of GDP-bound Rac-1 in Jurkat T
cells. Similarly, an acceleration of Rac-1 GDP/GTP exchange was evident
in primary mouse T cells after TCR, CD28, and TCR/CD28 stimulation (5-,
3-, and 7.5-fold increases, respectively) (Fig. 2B). Neither
TCR nor CD28 cross-linking affected the levels of
32P-labeled GTP incorporated by Rac-1, confirming
the high intrinsic GTPase activity of Rac-1. Moreover, CD28 triggering
by B7-2 accelerated Rac-1 GDP/GTP exchange in T cells (Fig. 2A).
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D). This finding was confirmed by analysis of the kinetics
of Ras GDP/GTP exchange in T cells at different time points of CD28
stimulation or costimulation (data not shown). Moreover, TCR-stimulated
ERK-1 and ERK-2 activity did not increase further following CD28
co-cross-linking, suggesting that ERK-1/2 do not require a
costimulatory signal for maximal activation (Fig. 2E).
Therefore, the observed small increase in Ras GDP/GTP exchange that
occurs in CD28-stimulated T cells is insufficient to elicit ERK-1 and
ERK-2 activation. These results reveal the differential activation of
SOS-Ras-ERK- and Vav-Rac-1-mediated signaling in TCR- and
CD28-stimulated T cells, respectively, and suggest that coupling of the
CD28 and Rac signaling pathways does not result from Ras activation
by SOS. TCR/CD28-induced Vav GRF activity requires the activity of ZAP-70
Using the P116 ZAP-70-deficient (20) Jurkat T cell
line, we tested whether TCR/CD28 coligation induces Vav Tyr
phosphorylation and GRF activity for Rac-1 by the activation of ZAP-70.
Both the basal and TCR/CD28-induced Tyr phosphorylation of Vav
were markedly increased in parental Jurkat T cells relative to that of
the P116 ZAP-70- T cells (Fig. 3A). In contrast to the
enhanced TCR/CD28-induced GTP/GDP exchange activity of Vav in parental
Jurkat T cells, significantly lower TCR/CD28-induced increases in
Vav GRF activity were detected in ZAP-70-deficient P116 T cells (Fig. 3B). Moreover, TCR and/or CD28 costimulation did not
significantly increase Rac-1 GDP/GTP exchange in ZAP-70-deficient
Jurkat T cells in vivo, as analyzed by polyethyleneimine TLC of Rac-1
immunoprecipitates (our unpublished data). Thus, ZAP-70 appears to
control Vav-mediated guanine nucleotide exchange on Rac-1 by means of
Vav Tyr phosphorylation stimulated by TCR and/or CD28 ligation.
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Based on the ability of TCR/CD28 costimulation to synergistically
activate the Vav-Rac-1 and JNK pathways in T cells (6, 9),
we analyzed the effect of anti-sense oligo-DN-mediated inhibition
of Vav expression on TCR- and CD28-induced JNK activity in T cells.
Pretreatment of Jurkat T cells with Vav anti-sense oligo-DN
significantly, albeit not completely, inhibited the TCR/CD28
stimulation of JNK activity for its c-Jun substrate in an in vitro
kinase assay. As shown in Fig. 3C, the anti-sense
treatment caused a 2.5-fold inhibition of the TCR/CD28-induced JNK
activity. The level of JNK expression was not significantly reduced in
Jurkat T cells following the Vav anti-sense oligo-DN treatment.
These results suggest that, in T cells, 1) TCR and CD28 regulate
downstream signaling along the JNK pathways by increasing
Vav-dependent Rac GDP/GTP exchange, and 2) JNK may be
activated by a Vav-independent pathway(s).
Requirements for TCR- and CD28-induced synergistic activation of PAK-1, JNK, and p38 MAPK: critical role of ZAP-70
Considering that the induction of Tyr phosphorylation and GRF
activity of Vav both require ZAP-70, we determined whether ZAP-70
controls the catalytic activity of downstream effectors of Rac-1
involved in TCR and CD28 costimulation. The activities of JNK and p38
MAPK were assayed, as they mediate an important role for Rho-family
GTPases in cell cycle progression through G1 and
IL-2 transcription stimulated by TCR and CD28 ligation (2, 6, 7, 8, 9). In wild-type Jurkat T cells, JNK activity for c-Jun was
significantly enhanced upon TCR or CD28 stimulation both before (Fig. 4A) and after (Fig. 4B) cross-linking of these receptors. An 4-fold increase
in the JNK activity was observed upon TCR ligation, and JNK activity
was not further enhanced (3.4-fold stimulation) after TCR
cross-linking. As expected, coligation of TCR and CD28 synergistically
activated JNK (22-fold enhancement). Interestingly, a 4.5-fold increase
in JNK activity was obtained upon CD28 activation, and this activity
was further enhanced upon CD28 cross-linking (8.5-fold increase) (Fig. 4, A and B). These findings agree closely with
previous reports that a TCR signal alone stimulates JNK activity and
that a CD28 signal synergistically amplifies TCR signaling (6, 7, 9, 27). Further, these results demonstrate that the strength of
CD28 signaling, as reflected by the stimulation of JNK activity, can be
further increased by CD28 cross-linking.
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D shows that no JNK activity was observed in either
unactivated T cells or in T cells stimulated by control untransfected
CHO cells. CD28 stimulation by B7-2+ CHO cells
induced a 2.5- to 3-fold increase in JNK activity that was sustained
for 
30 min, indicating that CD28 engagement itself is sufficient to
stimulate JNK activity. TCR/CD28 coligation yielded 8- to 10-fold more
JNK activity than that observed only upon B7-2 engagement of CD28. This
observation is compatible with the ability of a CD28 signal to
stimulate the Tyr phosphorylation and GRF-activity of Vav and
Vav-dependent Rac GDP/GTP exchange, and confirms that CD28 stimulation
augments JNK activity (6).
Ab-mediated stimulation of TCR, but not CD28, elicited a 2-fold rise in
p38 MAPK activity (Fig. 4C). Similarly, ligation of CD28 by
B7-2 also did not activate p38 MAPK (Fig. 4E). The greatest
elevation (4-fold) of p38 MAPK activity was observed after TCR/CD28
coligation, using either anti-CD28 (Fig. 4C) or B7-2
(Fig. 4E) to engage CD28. Thus, despite the inability of
CD28 stimulation to activate p38 MAPK, a CD28-dependent pathway
enhances TCR-mediated activation of p38 MAPK. Moreover, B7-2- and
anti-TCR/B7-2-mediated increase of JNK and p38 MAPK activities
could be detected over an extended period of time, since these
activities persisted even after 30 min of stimulation (Fig. 4, D and E). In contrast, TCR-induced kinase
activities of JNK and p38 were more transient with a tendency to
decline somewhat after 20 min of TCR ligation. Thus, the signals
generated by CD28 costimulation translate into a longer duration of
both Tyr phosphorylation of Vav and downstream signaling events,
including the activation of JNK and p38.
Both basal and TCR-, CD28-, and TCR/CD28-induced JNK and p38 MAPK
activities were reduced substantially in P116 ZAP-70-deficient Jurkat T
cells relative to that of wild-type Jurkat T cells, particularly if
total kinase activity is taken into account (Fig. 4, A–C).
The absence of ZAP-70 therefore affects TCR- and CD28-mediated signals
and results in the down-regulation of both the JNK and p38 MAPK
pathways. However, we noted a reduction in basal JNK activity in P116 T
cells when compared with wild-type Jurkat T cells, and therefore
analyzed JNK activation relative to the basal JNK activity in each cell
type. In contrast to the 4.3-, 4.5-, and 21.8–fold increases of JNK
activity seen in wild-type Jurkat T cells, TCR-, CD28-, and
CD28/TCR-ligation resulted in 2.5-, 3.2-, and 4-fold increases
(quantitation not shown) of JNK activity in ZAP-70-deficient P116
Jurkat T cells. Thus, despite the reduced total JNK activity in
ZAP-70-deficient T cells, these cells retained the ability to
up-regulate JNK activity following CD28 stimulation (3.2-fold increase
in P116 Jurkat T cells vs a 4.5-fold increase in wild-type Jurkat T
cells).
Accordingly, the regulation of CD28-mediated JNK activity cannot be
ascribed solely to the ZAP-70-dependent pathway and another pathway may
control the amplitude of CD28-induced JNK kinase activity. Consistent
with the partial dependence of JNK activity upon ZAP-70, secondary CD28
cross-linking yielded an 2-fold difference in stimulation of JNK
activity between wild-type (8.5-fold increase) and ZAP-70-deficient
(4.2-fold increase) Jurkat T cells. Conversely, the differences in JNK
activity between ZAP-70-deficient and wild-type Jurkat T cells were
evident upon TCR stimulation (4.3-fold vs 2.5-fold) and were
significantly more pronounced upon TCR/CD28 ligation (21.8-fold vs
4-fold). It is evident, therefore, that ZAP-70 plays an important role
in the TCR/CD28 costimulation-induced amplification of JNK
activity.
To further examine whether a CD28 costimulatory signal ("signal 2")
requires an intact TCR signal ("signal 1"), we bypassed the
requirement for ZAP-70-dependent events by substituting the
Ras-dependent "signal 1" with the mitogenic tumor promoter PMA,
which activates Ras via the protein kinase C pathway (1, 6). Phorbol esters are capable of sensitizing JNK to other
signals (6), and combined stimulation of T cells with PMA
plus CD28 mimics the TCR/CD28 synergistic effect on JNK activation in
Jurkat T cells (6, 9). Fig. 5A shows that PMA/CD28
costimulation elevates JNK activity 3-fold less in ZAP-70-deficient
Jurkat T cells than in wild-type Jurkat T cells. Moreover, PMA/CD28
costimulation did not bypass the requirement of ZAP-70 for the
stimulation of p38 MAPK activity (Fig. 5B). Our results
demonstrate that ZAP-70 is indeed required for the TCR/CD28
costimulation-induced synergistic activation of JNK and p38 MAPK, and
that PMA can substitute only partially for TCR in this type of
costimulation.
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C)
and primary mouse T cells, including thymocytes and peripheral splenic
T cells (Fig. 5D). The highest stimulation of PAK-1 kinase
activity was observed after TCR/CD28 coligation (Fig. 5, C
and D). Interestingly, in contrast to the 2.9- to 9.4-fold
increases seen in wild-type Jurkat T cells, neither TCR, CD28, nor
TCR/CD28 ligation elevated PAK-1 activity in ZAP-70-deficient P116
Jurkat T cells (Fig. 5C). These results confirm that PAK-1
is activated upon TCR, CD28, or TCR/CD28 stimulation, demonstrate the
activation of PAK-1 in primary T cells, and, more significantly,
illustrate that ZAP-70 regulates both TCR- and CD28-induced activation
of PAK-1. CD28 amplifies TCR-induced ZAP-70 activity and association of Vav with ZAP-70 and LAT
We have shown that ZAP-70 activity is required for the
Tyr-phosphorylation of Vav following TCR and CD28 ligation. To
investigate whether TCR and CD28 signals converge at the level of
association of ZAP-70 with Vav, we quantitated the amount of ZAP-70 in
Vav immunoprecipitates of unstimulated, TCR- and/or CD28-stimulated
Jurkat T cell lysates. At 7 min after stimulation, TCR and CD28
ligation each induced an 5-fold increase in the amount of ZAP-70
bound to Vav, while the amount of Vav-associated ZAP-70 was increased
by 16-fold after TCR/CD28 stimulation (Fig. 6A). ZAP-70 was also induced
to associate with Vav upon B7-2 stimulation of CD28 in the
presence or absence of TCR costimulation (Fig. 6B).
TCR/CD28 costimulation, but not stimulation by TCR or CD28 alone,
induced the association of Vav with TCR and LAT, a pp36 Tyr
phosphoprotein substrate of ZAP-70 (31).
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A). In marked contrast, TCR and/or CD28 stimulation of
ZAP-70-deficent Jurkat T cells failed to induce association of Grb2,
LAT, and TCR with Vav (data not shown).
While TCR and TCR/CD28 ligation elicited similar levels of ZAP-70 Tyr
phosphorylation, CD28 stimulation did not significantly increase ZAP-70
Tyr phosphorylation (Fig. 6C). Tyr phosphorylation of ZAP-70
occurs at both positive and negative regulatory sites of activation.
The Tyr-292-negative regulatory phosphorylation site of ZAP-70 binds to
the Cbl phospho-Tyr-binding domain (PTB-domain) following T cell
activation, which may result in the negative regulation of ZAP-70
(32). Since ZAP-70 activity is increased 3- to 4-fold by
TCR stimulation (17), we analyzed whether CD28 stimulation
modulates ZAP-70 kinase activity. The cytoplasmic fragment of human
erythrocyte band 3 was used as a specific substrate of ZAP-70
(33). The increase (8-fold) in ZAP-70 kinase activity,
evident after TCR/CD28 costimulation of EL4 T cells, was double that
(4-fold increase) activated by TCR alone (Fig. 6D).
Moreover, thymocytes and peripheral T cells displayed a similar
significant augmentation of ZAP-70 kinase activity following TCR/CD28
coligation (Fig. 6E). Anti-Syk immunoblotting
confirmed that phosphorylation of erythrocyte band 3 in this assay was
mediated by ZAP-70 and not by the Syk PTK (data not shown), which is
also capable of phosphorylating this substrate.
The relevance of the results obtained on ZAP-70 kinase activity in Fig. 6E was confirmed by examining the ZAP-70-mediated Tyr
phosphorylation of SLP-76, a natural in vivo substrate of ZAP-70
(34), which is involved in the regulation of Vav-mediated
PAK-1 activation and cytoskeleton organization (35). We
observed a significant increase in Tyr phosphorylation of SLP-76
following TCR/CD28 coligation in splenic T cells (Fig. 6F).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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1 and IL-2 promoter-driven and NFAT-dependent
transcription (20). Based on our results, we propose that ZAP-70 regulates the TCR/CD28-induced transcriptional activation of ATF-2- and c-Jun via the enhancement of Vav GRF activity. Our observations favor a model in which: 1) TCR/CD28-mediated signaling is coupled via ZAP-70 to the activation of PAK-1, JNK, and p38 MAPK by interactions between components of the CD28 and TCR signaling pathways; and 2) replacement of TCR proximal signals ("signal 1") by stimulation of protein kinase C by PMA in ZAP-70-deficient T cells does not fully restore the costimulatory potential of CD28 triggering ("signal 2") with respect to the activation of JNK and p38 MAPK. These data substantiate a recent report that CD28 potentiates the response to PMA more weakly in Lck-deficient than in wild-type Jurkat T cells (9).
This model provides a basis for the ability of CD28/B7-2-mediated
costimulation to prevent the induction of anergy. Indeed, ligation of
CD28 by B7-2 leading to productive immunity induces the Tyr
phosphorylation of TCR and CD3 chains and facilitates the
association of TCR with Lck following the recruitment of ZAP-70
to this complex (36). The ability of B7-2-mediated
costimulation to elevate ZAP-70 activity, as well as the
association of Vav with ZAP-70, TCR, and Tyr-phosphorylated LAT,
may be important for efficient signaling along the Vav/Rac-1-regulated
PAK-1 and p38 MAPK pathways required for T cell proliferation and
IL-2 production (6, 8, 9, 10, 11). The fact that this
Vav/ZAP-70/TCR complex contains LAT, which is localized mainly to
sphingolipid-cholesterol-rich microdomains that cluster critical
signaling molecules (e.g., Ras- and Rho-like GTPases) in the plasma
membrane of activated T cells (31, 37), suggests that
ZAP-70-dependent phosphorylation of LAT plays a critical role in the
assembly of Vav-LAT signaling complexes and its translocation in the
vicinity of Vav downstream effectors, including Rac-1. Our observations
demonstrate that CD28 signaling may potentiate ZAP-70 kinase activity
induced by TCR stimulation, and support the model proposed above in
which Tyr phosphorylation of SLP-76 bridges the TCR-associated
ZAP-70 with downstream effectors, such as Vav and PAK-1
(35). These findings also agree with the result that T
cells stimulated in the absence of CD28 ligation display diminished Tyr
phosphorylation of SLP-76 and LAT (38).
Vav Tyr phosphorylation links some receptors (e.g., FcRI) to
activation of the Rac-1-JNK pathway (16). Additionally,
Syk and Vav-1 cooperatively induce activation of JNK in
fibrinogen-adherent cells (39). Recent data obtained
using Vav-/- mice indicate that
Vav-/- thymocytes and T cells exhibit normal
phosphorylation of the c-Jun-GST fusion protein by a kinase(s)
(presumably JNK) in response to TCR/CD28 costimulation
(40). It follows that regulation of CD28-mediated JNK
activity cannot solely be ascribed to the ZAP-70/Vav pathway. In
agreement with this notion, we found that pretreatment of Jurkat T
cells with a Vav anti-sense oligo-DN resulted in the incomplete
inhibition of the TCR/CD28 stimulation of JNK activity for its c-Jun
substrate. This suggests that a ZAP-70–and Vav–independent pathway(s)
of CD28-mediated JNK activation exists in parallel to the
ZAP-70/Vav-dependent pathway of JNK activation. Moreover, our Vav
anti-sense study (Fig. 3C) was performed on continuously
cycling human Jurkat T cells, rather than primary T cells. Thus, TCR
signaling in preactivated, continuously cycling vs primary
naïve T cells may have different requirements for JNK
activation.
In summary, our findings are consistent with a role for CD28 as a costimulatory receptor that potentiates TCR signals. Signaling components that contribute to the synergistic effects provided by CD28 costimulation are presumed to be shared by the TCR and CD28 pathways (41, 42). As CD28 signals enhance TCR signaling during the early stages of T cell:APC interaction (37, 43), signals generated by CD28 do not appear to function independently of TCR signals. Rather, CD28 signals function mainly to augment and/or modify the activation of several TCR-signaling intermediates, such as PAK-1, p38 MAPK, and JNK.
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Acknowledgments |
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Footnotes |
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2 K.V.S. and J.Z. contributed equally to this work.
3 Address correspondence and reprint requests to Dr. Terry L. Delovitch, Autoimmunity/Diabetes Group, The John P. Robarts Research Institute, 1400 Western Road, London, Ontario, Canada N6G 2V4. E-mail address:
4 Abbreviations used in this paper: MAPK, mitogen-activated protein kinase; ATF, activating transcription factor; CHO, chinese hamster ovary; oligo-DN, oligodeoxynucleotide; ERK, extracellular signal-regulated kinase; LAT, linker for activation of T cells; GRF, GDP releasing factor; JNK, c-Jun N-terminal kinase; MBP, myelin basic protein; p-Tyr, phosphotyrosine; PTK, protein tyrosine kinase; SOS, son of sevenless.
Received for publication September 1, 1998. Accepted for publication May 10, 1999.
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-Sense
OligoDN) mediated inhibition of Vav expression on TCR- and CD28-induced
JNK activity in T cells. Jurkat T cells (3 x 106)
were cultured in the presence of 20 µg/ml Vav anti-sense or
control oligo-DNs (Cntrl OligoDN) for 48 h, activated for 10 min
with anti-TCR plus anti-CD28, and assayed for associated JNK in
vitro kinase activity using GST-c-Jun as substrate (upper
panel). Immunoblotting with anti-Vav and anti-JNK mAbs
confirmed that the amount of Vav, but not JNK, is markedly reduced
after treatment of T cells with the Vav anti-sense oligo-DN
(lower panels).
