From the Division of Oncology Research and Department
of Immunology, Mayo Clinic, Rochester, Minnesota 55905 and the
¶ Department of Metabolic Medicine, Imperial College School of
Medicine, Hammersmith Hospital Campus, Du Cane Road,
London W12 0NN, United Kingdom
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ABSTRACT |
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Stimulation of the T cell antigen receptor (TCR)
triggers a complex series of signaling events that culminate in T cell
activation and proliferation. The complex structure of the TCR has
hindered efforts to link specific signaling events induced by TCR
cross-linkage to downstream activation responses, such as interleukin-2
(IL-2) gene transcription. Previous studies have shown that the
polyomavirus-derived oncoprotein, middle T antigen (mT), transforms
rodent fibroblasts by interacting with and activating several
cytoplasmic signaling proteins (Src kinases, phospholipase C
(PLC)-1, Shc, and phosphoinositide 3-kinase (PI3-K) implicated in
cell growth control. In this study, we demonstrate that expression of
mT activates Jurkat T cells, as measured by increases in IL-2 promoter-
and NFAT (nuclear factor of activated T cells)-dependent
reporter gene transcription. The transcriptional response provoked by
mT was blocked by the immunosuppressive drug FK506, a potent inhibitor
of TCR-mediated IL-2 gene expression. Mutations that disrupted the
binding of mT to Src kinases or PLC-
1 abrogated the ability of mT to
deliver the signals needed for IL-2 promoter activation. In contrast, a
mT mutant that failed to bind PI3-K induced a markedly elevated
transcriptional response in Jurkat cells, whereas mutation of the Shc
binding site in mT had little effect on the transactivating potential
of this viral oncoprotein. Additional studies demonstrated that the
association of mT with PLC-
1 was necessary and sufficient to
activate both Ca2+- and Ras-dependent
signaling cascades in Jurkat cells. These results indicate that
PLC-
1 activation plays pivotal and pleiotropic roles in the
stimulation of IL-2 gene expression, whereas activation of PI3-K
negatively modulates this response in Jurkat T cells.
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INTRODUCTION |
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Engagement of the multi-subunit T cell antigen receptor
(TCR)1 complex triggers a
cascade of intracellular signaling events that culminate in the
activation and proliferation of T lymphocytes (1, 2). The proximate
biochemical response to TCR stimulation is the phosphorylation of
several intracellular proteins on tyrosyl residues. A critical target
for the TCR-activated protein-tyrosine kinases (PTKs) is the
immunoreceptor tyrosine-based activation motif (ITAM), which is present
in multiple copies in the and CD3 subunits of the receptor itself
(3-6). The current model posits that TCR engagement leads to the
sequential activation of members of the Src and ZAP-70/Syk families of
PTKs (1, 2). The Src family members, Lck and Fyn, initiate TCR
signaling by phosphorylating tyrosine residues located within the
ITAMs, which, in turn, serve as docking sites for the tandem SH2
domains of ZAP-70 or Syk. Although interaction with a phospho-ITAM may
be sufficient for activation of Syk, ZAP-70 activation requires an additional tyrosine phosphorylation event catalyzed by Lck or Fyn
(6-9). The clustered Src family and ZAP-70/Syk PTKs mediate the
phosphorylation of multiple intracellular substrates, including the
adapter proteins, SLP-76, Cbl, and LAT (1, 2, 10). Tyrosine
phosphorylation of these adapter proteins provides additional binding
sites for SH2 domain-containing proteins, effectively drawing them into
the vicinity of the TCR-associated PTKs. Thus, as is the case for
polypeptide growth factor receptors, TCR ligation triggers the assembly
of multimolecular signaling complexes in T cells.
Steady progress has been made with respect to the identification of
downstream substrates for the PTKs activated in response to TCR
occupancy. A critical target for these PTKs is phospholipase C
(PLC)-1, which itself becomes activated as a consequence of tyrosine
phosphorylation (11-13). PLC-
1 is responsible for the generation of
second messengers that activate protein kinase C and mobilize
intracellular calcium, both of which are crucial events for the
transcriptional activation of the IL-2 and other cytokine genes in
antigen-stimulated T cells. Furthermore, PLC-
1 contains SH2, SH3,
and pleckstrin homology domains (14), which suggests that this enzyme
may also function as an adapter protein during TCR signaling.
Recent studies suggest that the components of the TCR-linked signaling machinery overlap extensively with those engaged by receptors for polypeptide growth factors. For example, the Ras-MAP kinase cascade, which has been widely implicated in the initiation of cell growth responses, is also activated during ligation of the TCR. Although the mechanism by which the TCR couples to Ras remains uncertain, the recruitment of a complex containing the adapter protein, Grb2, and the Ras-specific guanine nucleotide exchange factor, mSOS, to the plasma membrane may be an important step (15, 16). Another adapter protein, Shc, binds with low affinity to phospho-ITAMs, and it has been proposed that tyrosine-phosphorylated Shc supplies a membrane docking site for the Grb2-mSOS complex during TCR stimulation (17). One well documented role of the Ras-MAP kinase pathway is the activation of AP1, which, in addition to its own transactivating functions, is a component of the nuclear factor of activated T cells (NFAT), a pivotal transcription factor for the induction of IL-2 gene expression in response to antigenic stimuli (18). A related signaling pathway induced by TCR ligation, as well as coligation of the TCR with CD28, involves the activation PI3-K (19-20). Although PI3-K participates in mitogenic signaling through many growth factor receptors, its function with respect to TCR signaling is poorly understood.
The polyomavirus-derived oncoprotein, middle T antigen (mT), offers a
useful tool for understanding the roles of various signaling molecules
in cell growth and transformation (21, 22). Like the TCR, mT is
localized to the plasma membrane and lacks intrinsic protein kinase
activity. The growth-promoting and oncogenic activities of mT are
explained by its ability to nucleate a multimolecular signaling complex
at the inner leaflet of the plasma membrane. Membrane-associated mT
binds to and activates certain members of the Src kinase family,
notably Src and Fyn. These PTKs, in turn, phosphorylate mT on several
tyrosine residues, including Tyr250, Tyr315,
and Tyr322. Mutagenesis studies indicate that tyrosine
phosphorylation of mT generates binding sites for the SH2 domains of
Shc, PI3-K, and PLC-1, respectively (23, 27). Association with the
mT-Src PTK complex results in the tyrosine phosphorylation and
functional activation of all three signaling proteins. A Tyr
Phe
substitution at residues 250 or 315 severely impairs the transforming
activity of mT in fibroblasts (23, 24, 27), whereas a mT mutant
containing a Tyr322
Phe substitution displays a less
dramatic defect in fibroblast transformation assays (25). Hence,
studies with transformation-defective mT mutants supply strong
supporting evidence for positive roles of Src PTKs, PI3-K, and Shc in
cell growth and carcinogenesis.
The nature of the signaling complex assembled by mT invites some
obvious comparisons to the signaling machinery engaged by both cytokine
receptors and the TCR in lymphoid cells. Indeed, we have reported that
expression of wild-type mT in factor-dependent T cells
partially replaces the survival- and growth-promoting signals normally
supplied by the ligand-bound IL-2 receptor (28). These earlier results
prompted speculation that mT might also serve as a "surrogate" for
the activated TCR complex in Jurkat T cells. In the present study, we
show that transient expression of mT in Jurkat cells stimulates both
NFAT- and IL-2 promoter-driven transcription. Studies with a panel of
mT mutants indicate that association of mT with a Src PTK and PLC-1
is necessary and sufficient to activate IL-2 gene expression in Jurkat
T cells. On the other hand, deletion of the PI3-K binding site in mT
markedly enhanced these transcriptional responses, suggesting that
PI-3K delivers a negative regulatory signal during the activation
process. These findings suggest that mT is a novel molecular probe for
studies of the signaling pathways that initiate and modulate the
activation program in T lymphocytes.
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EXPERIMENTAL PROCEDURES |
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Cell Lines-- The human T cell leukemia line, Jurkat (clone E6-1), and the Jurkat-derived subclones, J.RT3-T3.5 (JRT-3), and J.CaM1.6 (JCAM) were obtained from the American Type Culture Collection, Rockville, MD. The ZAP-70-deficient Jurkat subclone, P116, has been described previously (29). All cell lines were maintained at cell densities below 8 × 105 cells/ml in RPMI 1640 medium supplemented with 5% (v/v) fetal bovine serum, 5% (v/v) bovine calf serum, 2 mM L-glutamine, and 10 mM HEPES to pH 7.2.
Plasmids-- The pIL2-luciferase (Luc) reporter plasmid was generously provided by Dr. Ellen Rothenberg (30). The pNFAT-Luc reporter (31) was kindly supplied by Dr. David J. McKean (Mayo Clinic). The plasmid pPyMT1 (32), which encodes mouse polyomavirus mT, was obtained from Dr. Ed Leof (Mayo Clinic). The wild-type polyomavirus mT coding sequence was amplified from pPyMT1 by the polymerase chain reaction, using 5'-AGATACTCGTCTAGACATCATGGATAGAGTTCTGAGC-3' as the 5' primer and 5'-CAGATCAGTCTCGAGCTAGAAATGCCGGGAACGTTT-3' as the 3' primer. These primers introduced XbaI and XhoI sites at the 5' and the 3' ends, respectively, of the polymerase chain reaction product. The mT coding sequence was subcloned into the XbaI and XhoI sites of the pMH-Neo expression vector (33). mT mutant constructs were created by introducing point mutations using the TransformerTM site-directed mutagenesis kit (CLONTECH).
The strategy for the preparation of expression plasmids encoding wild-type PLC-Transfections-- Unless indicated otherwise, transient transfections were done by electroporation of 1 × 107 cells with a total of 30 µg of DNA. Prior to electroporation, the DNA was ethanol-precipitated and resuspended in 50 µl of RPMI 1640 medium containing 10 mM HEPES, pH 7.2. The dissolved DNA was mixed with 250 µl of cell suspension (4 × 107 cells/ml) in standard growth medium, and the mixture was incubated for 10 min at room temperature. The samples were transferred to 4-mm gap cuvettes, and electroporation was performed with an Electro Cell Manipulator model 600 system (BTX, San Diego, CA) at settings of 350 V and 960 microfarads. After electroporation, the cells were incubated for an additional 10 min at room temperature, and then diluted into 13 ml of standard growth medium. Two ml of the diluted cell suspension were then aliquoted into each well of a six-well tissue culture plate, and then 5 ml of growth medium were added to each well. The cultures were incubated for 6 h at 37 °C prior to the addition of pharmacologic agents.
The transfected cells were stimulated with 20 ng/ml phorbol myristate acetate (PMA; Sigma), diluted from a concentrated stock solution in ethanol, or with 2 µM ionomycin (Calbiochem), diluted from a stock solution in dimethyl sulfoxide (Me2SO). The appropriate volumes of drug vehicle (maximum solvent concentration never exceeded 0.05%, v/v) only were added to unstimulated control samples. Where indicated, the cells were pretreated for 15 min with 100 nM wortmannin, added from a 10 mM stock solution in Me2SO. The immunosuppresive agent FK506 was generously supplied by Dr. G. Wiederrecht (Merck Research Laboratories, Rahway, NJ), and concentrated stock solutions were prepared in ethanol. The cells were pretreated with 10 nM FK506 for 30 min prior to stimulation with PMA. After addition of stimuli or inhibitors, the transfected cells were cultured for an additional 18 h at 37 °C, at which time cell lysates were prepared for reporter gene assays and immunoblot analyses. The cells in each well were pelleted and lysed with 60 µl of 1× cell culture lysis reagent (Promega) for 20 min at room temperature. The cell lysates were centrifuged to remove insoluble cellular debris, and 10 µl of the cleared extracts were assayed for luciferase activity with the luciferase assay reagent (Promega) and a Berthold LUMAT LB 9501 luminometer.Anti-mT mAb Immunoblotting-- To determine the levels of expression of mT proteins in transiently transfected cells, the cells from one tissue culture well (see above) were harvested and lysed for 10 min on ice in 300 µl of TNE buffer (20 mM Tris-HCl, pH 7.4, 40 mM NaCl, 5 mM EDTA), containing 1% Triton X-100, 30 mM Na4P2O7, 50 mM NaF, and a protease inhibitor mixture (10 µg/ml leupeptin, 5 µg/ml pepstatin A, 5 µg/ml aprotinin, and 2 mM phenylmethylsulfonyl fluoride). The cell lysate was cleared by centrifugation, and 30 µl of the extract was mixed with 10 µl of 4× sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer (248 mM Tris-HCl, pH 6.8, 40% glycerol, 9.2% SDS, 20% 2-mercaptoethanol, and 0.04% bromphenol blue). After heating for 5 min at 95 °C, the solubilized proteins were resolved by SDS-PAGE though a 10% gel, and were transferred for 1 h at 150 volts to an Immobilon-P membrane (Millipore, Bedford, MA). The membrane was blocked overnight at room temperature in 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, and 0.2% Tween 20 (TBST) containing 2% bovine serum albumin (BSA). The blotted proteins were probed with a 1:10 dilution in TBST plus 0.1% BSA of hybridoma-conditioned culture medium containing the mT-specific mAb, Pab 750 (34). After 1 h, the membrane was washed three times by agitation in TBST (10 min/wash cycle). The membrane was then incubated for 1 h with a 1:10,000 dilution of rabbit anti-mouse immunoglobulin G (IgG) (Pierce) in TBST plus 2% BSA. After three washes in TBST, the blot was incubated for 1 h with horseradish peroxidase-linked protein A (Amersham Pharmacia Biotech) at a 1:10,000 dilution in TBST plus 2% BSA. The blot was then washed as described above, and immunoreactive proteins were revealed with the enhanced chemiluminescence (ECL) detection system (Amersham Pharmacia Biotech).
MEK Kinase Assay and Immunoblotting--
The MEK kinase assay
was performed essentially as described (35) with minor modifications.
Wild-type Jurkat cells were transfected with wild-type or mutant mT
constructs, together with 10 µg of Myc-tagged MEK (mycMEK)-encoding
reporter plasmid, as described above. After electroporation, the cells
were diluted into 20 ml of standard growth medium and cultured for
18 h in 100-mm tissue culture dishes. The cells were harvested and
lysed in 1 ml of MEK lysis buffer (20 mM Tris-HCl, pH 7.4, 40 mM Na4P2O7, 50 mM NaF, 10 mM EGTA, 5 mM
MgCl2, 1% Triton X-100, 0.25% sodium deoxycholate, 1 mM Na3VO4, 20 µg/ml leupeptin, 20 µg/ml aprotinin, 10 µg/ml pepstatin A, 40 nM
microcystin, 600 µM phenylmethylsulfonyl fluoride, 50 mM para-nitrophenyl phosphate, 100 mM -glycerophosphate, pH 7.4, and 100 µM
each of phosphoserine, phosphotyrosine, and phosphothreonine). The
lysate was cleared by centrifugation, and 100 µl of the lysate were
set aside for immunoblotting (see below). The remainder of the lysate
was incubated for 30 min on ice with 15 µg of protein G-purified 9E10
mAb, which recognizes a Myc-derived peptide epitope (MEQKLISEEDLN)
(36). The resulting immune complexes were immunoprecipitated for 30 min
at 4 °C on ice with 15 µg of rabbit anti-mouse IgG prebound to 15 µl of packed protein A-Sepharose beads. The immunoprecipitates were
washed three times in MEK wash buffer (20 mM Tris-HCl, pH 7.4, 40 mM Na4P2O7, 50 mM NaF, 5 mM MgCl2, 10 mM EGTA, 1% Triton X-100, 0.25% sodium deoxycholate, 1 mM Na3VO4, 20 mM
para-nitrophenyl phosphate, 100 mM
-glycerophosphate, pH 7.4, and 0.1% SDS), two times in TE wash
buffer (50 mM Tris-HCl, pH 7.4, 0.1 mM EGTA, 55 mM
-glycerophosphate, pH 7.4, 5 mM
octyl-
-glucopyranoside, and 1 mM dithiothreitol), and
two times in kinase wash buffer (30 mM Tris acetate, pH
7.4, 0.1 mM EGTA, 10 mM magnesium acetate, 1 mM
-glycerophosphate, pH 7.4, 0.5 mM
octyl-
-glucopyranoside, and 1 mM dithiothreitol). The
substrate for the MEK kinase assays was a bacterially expressed and
purified, catalytically inactive ERK1 mutant fused to glutathione
S-transferase (GST-ERK1 kinase-inactive). The kinase
reactions were initiated with 20 µl of reaction mixture (kinase wash
buffer containing 10 µM adenosine 5'-triphosphate (ATP),
10 µCi of [
-32P]ATP, and 0.5 µg of GST-ERK1
kinase-inactive). The reactions were incubated for 10 min at 30 °C
and terminated with 8 µl of 4× SDS-PAGE sample buffer. The samples
were heated for 5 min at 95 °C, the beads were removed by
centrifugation, and the solubilized proteins were resolved by SDS-PAGE
in a 10% gel. The samples were transferred electrophoretically (50 V
for 15 min, followed by 150 V for 1 h) to an Immobilon-P membrane
(Millipore), and radiolabeled GST-ERK1-KD was visualized by
autoradiography.
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RESULTS |
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Activation of IL-2 Promoter-driven Transcription by mT-- Efforts to understand the intracellular signaling pathways engaged by growth factor receptors, such as the platelet-derived growth factor receptor, have been greatly facilitated by the availability of mutant receptors that fail to interact with defined cytoplasmic signaling proteins (37-40). Due to the structural complexity of the TCR, it has not been possible to apply a similar genetic approach to identify the roles of downstream signaling proteins in the induction of specific T cell activation responses though this receptor. However, we noted that the viral oncoprotein, mT, contains defined binding sites for a number of intracellular proteins previously identified as components of the signaling machinery engaged by the ligand-stimulated TCR. In initial studies, we examined the possibility that ectopic expression of mT would deliver signals required for the transcriptional activation of the IL-2 gene in Jurkat T cells.
Jurkat cells were transiently transfected with a plasmid encoding the wild-type mT protein, together with the pIL2-Luc reporter plasmid, which contains the luciferase coding sequence under the transcriptional control of the IL-2 promoter. Control cells were electroporated with the empty expression vector (pMH-Neo) and the pIL2-Luc reporter plasmid. As reported previously (41), activation of transcription from the IL-2 promoter in Jurkat cells requires two synergistic signals, one of which can be supplied by TCR cross-linkage, and the other by PMA (Fig. 1). Jurkat cells transfected with the mT expression vector displayed a strong increase in IL-2 promoter-mediated transcription, and this response was synergistically increased by addition of either PMA (Fig. 1) or ionomycin (data not shown) as a costimulus. These results demonstrate that expression of wild-type mT triggers the full complement of signaling events required for the stimulation of IL-2 gene transcription in Jurkat cells.
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Activation of Mutant T Cell Lines by mT--
To further
characterize the mechanism of cellular activation by mT, we took
advantage of a previously described panel of Jurkat T cell-derived
somatic mutants that lack different components of the TCR-associated
signaling machinery. A potential explanation for the activating effects
of mT in Jurkat cells is that this protein induces the phosphorylation
of ITAMs within the TCR by activating Fyn or Lck. This event might
trigger the association of ZAP-70 with the ITAMs, which, in turn, could
initiate the signaling cascade normally provoked by TCR ligation. To
examine this possibility, we determined whether mT could activate the
JRT3 subclone (42), which expresses no TCR complex, and hence no ITAMs,
at the cell surface. Transient expression of mT in JRT-3 cells
stimulated an increase in IL-2 promoter-driven transcription that was
virtually indistinguishable from that observed in the TCR-positive,
parental cell line (Fig. 2). These
results indicate that the activating effects of mT in Jurkat cells are
not simply due to a surreptitious interaction between mT and the CD3
and subunits of the TCR at the inner leaflet of the plasma
membrane.
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Role of mT-associated Proteins in IL-2 Promoter
Activation--
Tyrosine phosphorylation of mT by Src kinases creates
binding sites for several SH2 domain-containing proteins, including PI3-K, Shc, and PLC-1 (21-23). To examine the roles of these
signaling proteins in the activating effects of mT in Jurkat T cells,
we transiently expressed a series of mT mutants containing Tyr (Y)
Phe (F) substitutions at the binding sites for each of these proteins
(Fig. 3A). Previous studies
have shown that these substitutions specifically block the interaction
of mT with the appropriate target protein and alter the transforming
activity of mT in rodent fibroblasts (21-27). As described above,
Jurkat cells were cotransfected with the pIL2-Luc reporter plasmid, and
luciferase activity was measured after cellular stimulation with PMA or
with the drug vehicle only. The mT(Y250F) mutant fails to bind the Shc
adapter protein, which couples certain receptors to the Ras signaling pathway (26, 27). Unexpectedly, expression of mT(Y250F) led to a
moderate increase in IL-2 promoter-driven luciferase activity in Jurkat
cells. An even more striking gain of activity was observed when Jurkat
cells were transfected with the mT(Y315F) mutant, which fails to bind
or activate PI3-K (24). Relative to wild-type mT, expression of
mT(Y315F) elevated luciferase activity by 4-fold in the absence of PMA,
and by 6-fold when the cells were costimulated with PMA. Thus,
interaction of mT with either PI3-K or Shc is not required for the
stimulation of transcription from the IL-2 promoter. Rather, these
results suggest that PI3-K activation actually delivers a net negative
signal for IL-2 gene transcription in Jurkat T cells.
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Effect of Wortmannin on IL-2 Promoter Activation-- The striking increase in luciferase expression obtained with the mT(Y315F) mutant suggests that PI3-K delivers a negative signal during the assembly of the transactivating complex on the IL-2 promoter. To confirm this possibility, we examined the effect of wortmannin, a pharmacologic inhibitor of PI3-Ks (45), on transcriptional activation induced by wild-type mT. Jurkat cells were cotransfected with wild-type mT plus either the pIL2-Luc or the pNFAT-Luc reporter plasmid. The cells were treated with 100 nM wortmannin for 16 h prior to cell harvest for measurements of luciferase activity. At this drug concentration, wortmannin nearly abolishes intracellular p85-associated PI3-K activity in T cells (46). Wortmannin treatment increased IL-2 promoter and NFAT-dependent luciferase activity in mT-transfected Jurkat cells by approximately 20- and 10-fold, respectively (Fig. 4). These pharmacologic results corroborate those obtained with the mT(Y315F) mutant, and support the conclusion that PI3-K activation negatively modulates a signaling pathway(s) involved in IL-2 gene expression in Jurkat T cells.
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Cellular Activation by mT Single, Double, and Triple
Mutants--
The mechanism by which mT activates IL-2 gene
transcription in Jurkat cells was explored further by studying a
broader panel of mT mutants. Previous studies showed that several
single amino acid substitutions in mT (Trp180 Arg,
Cys150
Ala, and Cys150
Tyr) disrupt the
interaction of mT with Src family PTKs, and consequently abrogate the
transforming activity of mT (23). In T cells, FynT is thought to be the
principal Src family member that associates with mT (28, 47-49). To
determine whether the association of mT with Src kinases is required
for the activation of Jurkat cells, transient transfection
experiments were performed with the mT(W180R), mT(C150A), and
mT(C150Y) mutants (Fig. 5). These mT
mutants uniformly failed to deliver the signals needed for IL-2
promoter-dependent transcription, indicating that the interaction of mT with FynT or Lck is required for initiation of the
activation program in Jurkat T cells.
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Sensitivity of TCR-dependent NFAT Activation to FK506-- The immunosuppressive drug, FK506, interferes with TCR-dependent IL-2 production in T cells by inhibiting Ca2+-dependent activation of the serine-threonine phosphatase, calcineurin (53). A principal target of calcineurin during T cell activation is the cytoplasmic subunit of NFAT. To determine whether mT-induced NFAT activation was similarly dependent on calcineurin, Jurkat cells were cotransfected with wild-type mT or mT mutants and pNFAT-Luc, and parallel samples were treated with 10 nM FK506 or with drug vehicle only (Fig. 6). The activation of NFAT by mT, in the presence or absence of PMA, was uniformly sensitive to inhibition by treatment of the transfected cells with FK506. Thus, mT interacts with the signaling cascade leading to NFAT activation at a level upstream of calcineurin.
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Inhibition of mT-induced IL-2 Promoter Activation by
Dominant-negative PLC-1--
The results presented above
demonstrate that the phosphorylated Tyr322 residue is
crucial for the activation of Jurkat T cells by mT. Although
Tyr322 lies within the target binding site for PLC-
1, it
remained possible that this site binds an undefined signaling protein
whose association with mT was essential for the activation of the IL-2
promoter. An alternative approach to determine the role of PLC-
1 in
mT-dependent transactivation is to overexpress a
catalytically inactive form of PLC-
1 in Jurkat cells transfected
with the wild-type mT expression plasmid. The PLC-
1(H335Q) mutant
contains a single amino acid substitution in the "X" region of the
catalytic domain. This mutation has been shown to ablate the lipase
activity of PLC-
1 (54).2
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Role of PLC-1 in Activation of the MAP Kinase Cascade--
In
addition to supplying the triggering signal for the increase in
intracellular free Ca2+ concentration, activation of
PLC-
1 may stimulate various components of the Ras to MAP kinase
pathway in response to TCR ligation (40, 55). To determine whether
PLC-
1 carries out similar functions in mT-expressing Jurkat cells,
transient transfections were performed with wild-type or the various
mutant mTs, together with a reporter plasmid encoding a Myc
epitope-tagged version of MEK (mycMEK), the protein kinase that lies
immediately upstream of MAP kinase (Fig.
8). Transient expression of wild-type mT
strongly stimulated MEK activity in Jurkat cells, as did expression of
the mT(Y250F) and mT(Y315F) mutants, indicating that binding of mT to
Shc or PI3-K was not required for the activation of this downstream
component of the Ras signaling pathway. In contrast, mT mutants that
fail to interact with either a Src family kinase (mT(W180R)) or
PLC-
1 (mT(Y322F) were clearly defective as MEK activators,
indicating that both proteins critical roles in the activation of MEK
and, in turn, MAP/Erk kinase in Jurkat T cells.
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DISCUSSION |
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Cellular transformation by mT depends on the assembly of a
multi-protein signaling complex at the inner leaflet of the plasma membrane. In this report, we demonstrate that the membrane-proximal signals generated as a consequence of mT expression elicit the full
complement of nuclear responses needed for transcriptional activation
of the IL-2 gene in Jurkat T cells. This finding led to the hypothesis
that mT might serve as a surrogate TCR for genetic studies of the
signaling machinery that controls IL-2 gene expression and other
cellular responses normally associated with TCR ligation. The
availability of a detailed map of the regions of mT that mediate association with specific signaling molecules permitted the use of a
panel of mT mutants as probes for participation of these interacting
proteins in the control of IL-2 gene transcription. The present results
point toward a central role of PLC-1 in the orchestration of the
cytoplasmic signaling events required for IL-2 gene expression in
activated T cells.
The mechanism whereby ectopically expressed mT induces the activation of Jurkat cells bears a number of similarities to the activation response triggered by TCR stimulation. Both stimuli activate NFAT- and IL-2 promoter-dependent transcription in a FK506-sensitive fashion, indicating that both the TCR and mT transduce signals though the Ca2+-activated phosphatase, calcineurin. The signaling pathways engaged by both the TCR and mT synergize with those stimulated by the phorbol ester, PMA. Moreover, studies performed in Jurkat somatic mutant subclones lacking either Lck or ZAP-70 indicated that both PTKs play important roles in mT-induced cellular activation. These mutant subclones also display profound defects in TCR-mediated signaling (29, 43).
A notable observation was that Lck-deficient J.CaM1 cells displayed no increase in IL-2 promoter-dependent transcription when transfected with wild-type mT. Previous studies suggest that mT associates directly with FynT, but not Lck, in T cells (27, 45, 47). Nonetheless, the nonresponsiveness of J.CaM1 cells to ectopically expressed mT indicates that Lck is necessary for activation of Jurkat T cells by mT. The essential function of Lck in mT-expressing Jurkat cells cannot be to phosphorylate ITAMs, as mT readily activates the Jurkat-derived J.RT3 subclone, which fails to express a TCR, and therefore ITAMs, at the cell surface. These findings add to previous evidence that Lck expression is crucial, and under certain circumstances, sufficient for the phosphorylation of downstream substrates during T cell activation (29, 56).
While the present studies were in progress, Brizuela et al.
(49) reported that introduction of hamster mT into EL4 thymoma cells
also induced transcription of NFAT-, NFB-, or IL-2 promoter-linked reporter genes. These authors found that the hamster
polyomavirus-derived mT was significantly more active than the mouse
polyomavirus mT used in the present study. The difference in activity
in EL4 cells was attributed to the almost exclusive association of
hamster mT with FynT, whereas mouse mT associates preferentially with Src and Yes. Although we have not compared the activities of hamster and mouse mTs in Jurkat cells, we observed that the level of IL-2 promoter-dependent reporter gene expression induced by mT
is comparable to that provoked by TCR ligation.3 The
dramatic difference in the activities of mouse versus
hamster mT in EL4 cells may be attributed in part to the unusual
signaling requirements for activation of these cells, which are capable of responding strongly to PMA alone in the absence of a
Ca2+-mobilizing costimulus (57).
The finding that ectopic expression of mT can circumvent the requirement for TCR ligation in Jurkat cells allowed us to take advantage of a previously described series of mT mutants that lack binding sites for cytoplasmic proteins implicated in the normal pathway of TCR signaling. Of particular interest was the mT(Y315F) mutant, which fails to associate with the regulatory p85 subunit of the PI3-K heterodimer. In T cells, coligation of the TCR with CD28 provokes a robust increase in PI3-K activity, although the function of this response is poorly understood (58). Nonetheless, based on the numerous parallels between TCR and growth factor receptor signaling, it seemed likely that PI3-K plays a positive role in the induction of the T cell activation program, and, consequently, that the mT(Y315F) mutant would fail to stimulate IL-2 promoter-dependent transcription. Instead, we found that disruption of the PI3-K binding site in mT led to a 4-6-fold increase in the stimulation of both NFAT-and IL-2 promoter-dependent transcription in Jurkat cells. Treatment of wild-type mT-expressing cells with the PI3-K inhibitor, wortmannin, provoked even more dramatic increases in reporter gene expression. Wortmannin treatment also enhanced the transcription of these reporter genes in untransfected Jurkat cells stimulated with anti-TCR antibodies plus PMA.3 Hence, it appears that PI3-K activation transmits a down-modulatory signal for the activation of NFAT and possibly other IL-2 promoter-linked transcription factors in Jurkat cells. The enhanced activity of the mT(Y315F) mutant in the Jurkat T cell activation model contrasted sharply with the dramatically impaired growth-promoting activity of this mT mutant in the FDC-P1 myelomonocytic progenitor cell line.3
While these studies were in progress, a report by Reif et al. (59) supplied more direct genetic evidence supporting a negative regulatory role for PI3-K activity during TCR signal transduction. These investigators transfected Jurkat cells with a constitutively active form of PI3-K, and observed a significant repression of the NFAT-dependent transcriptional response induced by anti-TCR antibody stimulation. Interestingly, two prominent targets of PI3-K, the protein serine-threonine kinase, Akt, and the small GTPase, Rac, were ruled out as downstream transducers of the negative signal delivered by PI3-K (59). Thus, the mechanism of negative signaling though PI3-K remains an important area for further investigation. The potential immunologic relevance of these findings is supported by the fact that CTLA4, an important inhibitory coreceptor on T cells, associates with PI3-K (58, 60). Although the outcome (positive or negative) of PI3-K activation will likely vary with the context in which the T cells are activated and the type of response under investigation, one function may be to down-regulate the transmission of stimulatory signals for the expression of IL-2 and other cytokine genes.
Like PI3-K, the adapter protein, Shc, participates in signaling pathways that promote cell survival and growth in cells stimulated with polypeptide growth factors. In cytokine-stimulated hematopoietic cells, Shc undergoes rapid phosphorylation on tyrosine residues and subsequently associates with the Ras-activating Grb2-SOS complex (61, 62). The notion that Shc positively regulates cell growth via Ras and/or other molecular interactions (62-67) is again supported by findings that the mT(Y250F) mutant is transformation-defective in rodent fibroblasts and non-mitogenic in hematopoietic cells (69).3 A recent report provided evidence that these growth-promoting activities of Shc are due to the mT-induced phosphorylation of Tyr239 and Tyr240 in Shc, which drives the recruitment of Grb2 into the mT signaling complex (69). Thus, the mT(Y250F) mutant should be defective in terms of coupling to the Ras-MAP kinase cascade. In the present study, we again obtained a paradoxical result when we examined the ability of mT(Y250F) to support an NFAT- or IL-2 promoter-dependent transcriptional response in Jurkat T cells. The disruption of the Shc binding site moderately increased the capacity of mT to stimulate the transcription of an IL-2 promoter-linked reporter gene in transiently transfected Jurkat cells. These results indicate that the formation of the Shc-Grb2-Sos complex is not essential for transcriptional activation of the IL-2 gene by mT, and, by inference, TCR ligation in these cells.
The impact of mutations in the binding sites for PI3-K and Shc on the
activation of Jurkat cells by mT contrasts sharply with the drastic
loss of function resulting from mutation of the binding site for PLC-
1. Once again, this outcome would not have been anticipated based on
the behavior of the mT(Y322F) mutant in fibroblast transformation
assays. In culture medium containing 10% serum, mT(Y322F) transformed
BALB/3T3 fibroblasts as efficiently as wild-type mT. A transformation
defect for mT(Y322F) only became apparent when focus formation was
examined under low (3%) serum conditions (25). Similarly, we have
found that mT(Y322F) is virtually indistinguishable from wild-type mT
in terms of supporting the survival and growth of
factor-dependent FDC-P1 cells cultured in optimal amounts
of serum.3 Thus, whereas the activation of PLC-
1 is,
under normal conditions, dispensable for the generation of
pro-mitogenic signals by mT, the interaction of mT with PLC-
1 is
absolutely essential for the induction of IL-2 gene transcription in
Jurkat cells.
The studies performed with mT double and triple mutants support the
conclusion that activation of PLC-1 is not only necessary, but may
also be sufficient to activate the IL-2 promoter in Jurkat cells. In
particular, the dramatic increase in reporter gene expression induced
by the mT(Y250F/Y315F) double mutant indicated that interaction of mT
with a Src kinase and PLC-
1 initiated the complete set of signaling
events required for IL-2 promoter activation in these cells. Of course,
our experiments do not rule out the possibility that other known
(protein phosphatase 2A, 14-3-3 proteins; Ref. 22) or yet to be
described mT-interacting proteins contribute to the observed
transactivation response. However, a central role for PLC-
1 in the
activation process is underscored by the observation that coexpression
of a lipase-inactive form of PLC-
1 completely suppressed the
activation of the IL-2 promoter by wild-type mT in Jurkat cells.
The multi-domain structure of PLC-1 raises the possibility that this
protein functions as both an adapter and a lipid-metabolizing enzyme
during T cell activation. The notion that PLC-
1 carries out
additional signaling functions is supported by our observations that
ionomycin stimulation fails to reverse the defect in IL-2 promoter-dependent transcription caused by mutation of the
PLC-
1 binding site in mT. The nature of the additional signal(s)
delivered by PLC-
1 in T cells is not fully understood; however, one
candidate is activation of the MAP/Erk kinases. In addition to
intracellular Ca2+ mobilization, PLC-
1 activation
triggers the production of the protein kinase C-activating second
messenger, diacylglycerol. In T cells, protein kinase C may stimulate
MAP/Erk kinases via activation of Ras, Raf-1, or MEK (55, 70-73).
Whatever the actual mechanism, our results reinforce the possibility
that, in addition to its role in the regulation of intracellular free
Ca2+, PLC-
1 may serve as an upstream activator of the
Ras to MAP kinase cascade in response to TCR engagement.
Previous studies have shown that the signaling machinery engaged by the
TCR resembles in many respects that coupled to receptors for
polypeptide growth factors. Given the complex structure of the TCR, the
assignment of specific signaling pathways to the generation of nominal
downstream responses remains a highly challenging undertaking. The use
of mT mutants as molecular probes in Jurkat T cells allowed us to
uncover some striking differences between the signaling requirements
for T cell activation, as measured by IL-2 gene expression, and those
for cell survival, growth, and transformation. Our results highlight
the pleiotropic roles of PLC-1 as a transducer of
TCR-dependent activating signals in T cells. On the other
hand, PI3-K activation may function to dampen TCR-mediated cytokine
production in these cells. Further studies of with mT may offer
additional insights into the signaling pathways leading to both T cell
activation and anergy.
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ACKNOWLEDGEMENTS |
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We thank the members of our laboratories for helpful discussions and Kathy Jensen for excellent secretarial assistance with the preparation of this manuscript. We gratefully acknowledge Dr. David McKean for providing the pIL2-Luc and pNFAT-Luc reporter plasmids.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant GM-47286 (to R. T. A.), by a Scholar Award from the Leukemia Society of America (to R. T. A.), by the Mayo Foundation, and by a grant from the Cancer Research Campaign (to S. M. D.).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.
§ Current address: Dept. of Cardiology, Children's Hospital, Boston, MA 02115.
To whom correspondence should be addressed: Div. of Oncology
Research, Rm. 1342, Guggenheim Bldg., Mayo Clinic, Rochester, MN 55905. Tel.: 507-284-4095; Fax: 507-266-5146; E-mail:
abraham.robert{at}mayo.edu.
1 The abbreviations used are: TCR, T cell antigen receptor; mAb, monoclonal antibody; PLC, phospholipase C; PI3-K, phosphoinositide 3-kinase; mT, polyomavirus middle T antigen; PTK, protein-tyrosine kinase; NFAT, nuclear factor of activated T cells; PMA, phorbol 12-myristate 13-acetate; PAGE, polyacrylamide gel electrophoresis; BSA, bovine serum albumin; GST, glutathione S-transferase; ITAM, immunoreceptor tyrosine-based activation motif; SH, Src homology; MAP, mitogen-activated protein; MEK, MAP kinase kinase; TBST, Tris-buffered saline with Tween 20.
2 B. J. Irvin, A. E. Nilson, and R. T. Abraham, manuscript in preparation.
3
A. Sekuli, A. P. Kennedy, and
R. T. Abraham, unpublished observations.
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REFERENCES |
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