From the Department of Evolutionary Biology, University of Siena, Via Mattioli 4, 53100 Siena, Italy and the § Basel Institute for Immunology, Grenzacherstrasse 437, CH-4005 Basel, Switzerland
Received for publication, September 20, 2000, and in revised form, October 31, 2000
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ABSTRACT |
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A key role in the communication between the
Engagement of the T cell antigen receptor
(TCR)1 complex by cognate
peptide antigen presented by major histocompatibility complex molecules is a central requirement for T cell development and activation (1, 2). Following interaction with its ligand, the TCR
triggers a tyrosine kinase (PTK) cascade orchestrated by Src and Syk
family PTKs (2, 3). As opposed to receptor PTKs, the TCR splits the
dual function of ligand binding and signal transduction between a
recognition module, the The evolution of a signaling strategy based on an effector module
composed of multiple elements, each of which can be involved in protein
recruitment to the activated receptor, has profound implications in
terms of signal output, both in terms of quality and quantity. This is
an essential feature for a receptor which must evaluate the quality of
the ligand and translate it into a signal which can lead to activation,
anergy, or apoptosis. The drawback of a receptor which has distinct
ligand binding and signaling modules is the requirement of a mechanism
whereby the TCR Several lines of evidence implicate Fyn in the activation and
modulation of calcium flux. Overexpression in T cells of wild-type Fyn,
or expression of a constitutively active mutant, results in enhanced
calcium mobilization and overcomes the requirement for a calcium
ionophore in a pharmacological model of NF-AT activation (7, 8). In
addition, Cells, Antibodies, and Plasmids--
The T cell hybridomas
expressing the SEB-specific TCR 3BBM74 and the TCR
Polyclonal rabbit antisera against PLC
NF-AT/luc contains a trimer of the NF-AT-binding site of the IL-2
promoter upstream of the gene encoding firefly luciferase (16). The
expression constructs encoding constitutively active mutants of Fyn,
Lck, and Ha-Ras were described previously (17-19). The cDNA
encoding F528Fyn was subcloned first into pGEM-3Z (Promega, Madison,
WI), then recovered as a BamHI fragment and cloned into the
BamHI site of the retroviral vector LXSH (20).
Transfections, Luciferase Assays, and Flow
Cytometry--
Transfections were carried out as described using a
modification of the DEAE-dextran procedure (16). To minimize
variability among samples, activation assays were carried out on
duplicate aliquots of a single pool of transfected cells. Cells were
allowed to recover for 22 h prior to activation and activated
either with SEB as described (5) or with a combination of phorbol
12-myristate 13-acetate (100 ng/ml) and A23187 (500 ng/ml). Cells were
collected 8-10 h after activation and assayed for luciferase activity
using the protocol from Promega and a BioOrbit 1253 luminometer.
Luciferase activities in cells tranfected with a control vector were
barely above background in the absence of stimulation (0.005-0.020
relative luciferase units). Although transfection with the expression
vectors encoding F528Fyn or F505Lck significantly increased the level of basal luciferase activity, this was further enhanced following stimulation with antigen. To normalize the results and compare them
among different experiments, basal activities were subtracted from
induced activities. The resulting values were expressed as ratio of the
SEB-induced activity to maximal phorbol 12-myristate 13-acetate/A23187
induced activity. Cells were analyzed for TCR/CD3 surface expression
using a FacsScan flow cytometer (Becton-Dickinson, San Jose, CA) and
saturating concentrations of biotinylated B20.1 (anti-V Activations, Immunoblots, Immunoprecipitations, and Kinase
Assays--
Activations with mAb or SEB were carried out as described
(5, 21). The optimal stimulation time using SEB-loaded APCs was
determined by a time course analysis of protein tyrosine
phosphorylation. The ratio of T cells to APCs was 10:1. Cells
(2-5 × 107/sample) were lysed in 1% Nonidet P-40 in
20 mM Tris-HCl, pH 8, 150 mM NaCl (in the
presence of 0.2 mg/ml sodium orthovanadate, 1 µg/ml leupeptin, 1 µg/ml aproteinin, 1 µg/ml pepstatin, and 10 mM
phenylmethylsulfonyl fluoride) and postnuclear extracts were
immunoprecipitated using the appropriate antibodies and protein A-Sepharose (Amersham Pharmacia Biotech Italia) or agarose-conjugated anti-mouse antibodies (Sigma Italia srl, Milan). When required, the
milder detergents digitonin (1%) or CHAPS (10 mM) were
used for cell lysis instead of Nonidet P-40. Lysates for in
vitro autophosphorylation assays were obtained using 3% Nonidet
P-40 to disrupt pre-existing intermolecular interactions. Molecular
weight markers were purchased from Amersham Pharmacia Biotech Italia.
In vitro autophosphorylation assays of Fyn, Lck, or ZAP-70
specific immunoprecipitates were carried out in 20 µl of 20 mM Tris-HCl, pH 7.4, 10 mM MgCl2,
10 mM MnCl2, 5 µCi of
[ Generation of Retroviral Supernatants, Infection of T Cell
Hybridomas, and IL-2 Assays--
Retroviruses encoding F528Fyn were
generated by transfection of the packaging line GP+E-86 and subsequent
selection in 500 µg/ml hygromycin (Life Technologies Italia) as
described (5). Virus-containing supernatants from drug-resistant pools
of transfected GP+E-86 cells were collected from confluent cell layers
after 7-10 days. T hybridoma cells (5 × 105) were
infected with 1 ml of virus-containing supernatant in the presence of 4 µg/ml Polybrene. After 4 h incubation at 37 °C, the
supernatants were replaced with complete medium containing 500 µg/ml
hygromycin and kept under selection for 7-10 days. Determination of
IL-2 in the culture supernatants of hybridoma T cells activated with
increasing concentrations of SEB was carried out as described using the
HT-2 indicator cell line (5).
Defective Phosphorylation of PLC Defective Activation of Fyn and Interaction with
Tyrosine-phosphorylated Pyk2 in Response to SEB in a T Cell Hybridoma
Expressing the Mutant TCR--
Fyn has been implicated both directly
and indirectly in the tyrosine phosphorylation of PLC
To assess whether the defective activation of Fyn was caused by an
impairment in the association between Fyn and
The focal adhesion kinase family PTK Pyk2 is phosphorylated in response
to TCR signaling and plays a key role in T cell activation (29, 30).
Pyk2 is constitutively associated with Fyn, and as such associates,
albeit loosely, with the resting TCR complex. Following TCR engagement,
Pyk2 is phosphorylated by Fyn (29). We asked whether Pyk2 was
constitutively associated with Fyn in cells expressing the TCR
Tyrosine-phosphorylated PLC Rescue of Signaling from the TCR
To confirm this finding and analyze biochemically the effects of
F528Fyn expression we cloned the cDNA encoding F528Fyn in a
retroviral vector. Retroviral supernatants containing this construct were used to infect the T cell hybridomas expressing either the wild-type TCR or the TCR
To understand whether constitutively active Fyn could restore the IL-2
response to SEB in cells expressing the The Fyn has been shown to be an important component of the TCR signaling
machinery. Expression of constitutively active Fyn enhances T cell
responsiveness to TCR-mediated stimulation and contributes to NFAT
activation, while inhibitory effects are elicited by overexpression of
a kinase-dead Fyn mutant (7, 8, 17, 43). Furthermore, genetic ablation
of Fyn results in impaired TCR signaling in thymocytes and splenic T
cells (44, 45). Interestingly, Fyn appears specifically implicated in
both IP3-dependent and IP3-independent calcium responses. IP3-dependent targets of Fyn are PLC Mice expressing the TCR and the CD3/
complex is played by a specific motif within
the connecting peptide domain of the TCR
chain (
-CPM). T cell
hybridomas expressing an
-CPM-mutated TCR show a dramatic impairment
in antigen-driven interleukin-2 production. This defect can be
complemented by a calcium ionophore, indicating that activation of the
calcium pathway is impaired. Several lines of evidence implicate Fyn in
the regulation of calcium mobilization, at least in part through the
activation of phospholipase C
. Here we have investigated the
potential involvement of Fyn in the TCR
-CPM signaling defect. Using
T cell hybridomas expressing either a wild-type TCR or an
-CPM
mutant, we show that Fyn fails to be activated by the mutant receptor
following SEB binding and fails to generate tyrosine-phosphorylated
Pyk2, a member of the focal adhesion kinase family. This defect
correlated with an impairment in phospholipase C
phosphorylation.
Production of interlukin-2 and activation of the transcription factor
NF-AT in response to triggering of the TCR
-CPM mutant with SEB were fully restored in the presence of constitutively active Fyn. Hence the
signaling defect generated by the TCR
-CPM mutation results at least
in part from an impaired coupling of the TCR·CD3 complex to Fyn activation.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
heterodimer, which is responsible for
ligand binding, and an effector module, the CD3/
complex, which
interacts with the constant regions of the TCR
and is composed of
the
and
heterodimers and a more loosely bound
/
or
/
dimer (4). The intracellular domains of all the components in
the CD3/
complex contain specific motifs, termed ITAMs
(immunoreceptor tyrosine-based activation motif), which are present as
one copy in the
,
, and
chains and as three copies in the
chain. Upon TCR engagement these ITAMs become phosphorylated on their
dual tyrosine residues by the Src kinases Lck and Fyn and as such
become docking sites for SH2 domain containing proteins. This permits
recruitment to the activated receptor of a number of signaling
proteins, among which the central one is the Syk family PTK, ZAP-70.
ZAP-70 preferentially interacts with fully phosphorylated
, an event
which results in enhancement of its kinase activity and
autophosphorylation/transphosphorylation on a number of tyrosine
residues, which in turn permits recruitment of the next components in
the signaling cascade (2, 3).
can inform the CD3/
complex that contact with
the specific peptide ligand has occurred and that signaling must
initiate. An important role in this process has been recently
attributed to a conserved motif (
-CPM) within the
-chain
connecting peptide domain of the TCR
. Mutation of this motif,
while not affecting surface expression of the TCR·CD3 complex,
results in specific functional deficits (5). Most notably, thymocytes
in transgenic mice expressing an
-CPM-mutated TCR fail to undergo
positive selection (6). Furthermore, T cell hybridomas expressing this
mutated receptor exhibit a dramatic impairment in IL-2 production and
activation of the transcription factor NF-AT, despite a normal
phosphorylation of the
chain. Interestingly, this defect can be
complemented by a calcium ionophore, indicating that activation of the
calcium pathway is specifically impaired (5).
chain-mediated calcium mobilization can be reconstituted
by Fyn coexpression in a heterologous cell system (9). At least part of
this effect is achieved, either directly or indirectly, through
phosphorylation and subsequent activation of PLC
(10, 11), which
hydrolyses membrane phospholipids, resulting in increased levels of IP3
and release of calcium ions from intracellular stores. Furthermore, Fyn
has been shown regulate the function of the IP3 receptor by
phosphorylation, thereby promoting release of calcium from the
endoplasmic reticulum (12). Part of the effect of Fyn appears, however,
independent of phosphatidylinositol bisphosphate hydrolysis,
although the underlying mechanisms are not known (13). Here we have
investigated the potential involvement of Fyn in the signaling defect
displayed by a TCR harboring a mutant
-CPM.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-CPM mutant
IV/
III have been described previously (5). An Epstein-Barr
virus-transformed B cell line (a kind gift of A. Lanzavecchia) was used
as antigen presenting cell (APC). Transient transfection experiments
were carried out on the TCR negative Jurkat subline J31.13 (14). Cells
were grown in RPMI (Life Technologies Italia srl) supplemented with
7.5% fetal calf serum, 2 mM L-glutamine, and
100 IU/ml penicillin. The packaging line, GP+E-86 was grown in
Iscove's modified Dulbecco's medium supplemented as above.
, Fyn, Pyk2, and Lck, and
anti-phosphotyrosine mAb, were purchased from Upstate Biotechnology Inc. (Boston, MA). Anti-Fyn mAb was purchased from Transduction Laboratories (Mamhead, United Kingdom). Anti-ZAP-70 and anti-CD3
mAbs were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). A
mAb suitable for immunoprecipitation of tyrosine-phosphorylated CD3
was kindly provided by M. Baniyash. The anti-V
8.1 mAb F23.1 (15) was
purified from hybridoma culture supernatants using protein G (Amersham
Pharmacia Biotech Italia, Milan). Peroxidase-conjugated secondary
antibodies were purchased from Amersham Pharmacia Biotech Italia
(Milan). Secondary unlabeled antibodies were purchased from Cappel
(Durham, NC).
2.1 mAb) or
F23.1 (anti-V
8.1 mAb) and phycoerithrin-labeled streptavidin, or
fluoresceinated 145.2c11 (anti-CD3 mAb).
-32P]ATP, at room temperature for 16 and 4 min for
Fyn and Lck, respectively, and at 37 °C for 20 min for ZAP-70. The
reaction products were subjected to SDS-PAGE, transferred to
nitrocellulose, and exposed to a PhosphorImager (Molecular
Dynamics, Sunnyvale, CA). The filters were subsequently analyzed by
immunoblot with anti-Fyn, Lck, or ZAP-70 antibodies to check that
similar amounts of specific PTK were recovered in each
immunoprecipitate. Immunoblots were carried out using a
chemiluminescence detection system (Pierce, Rockford, IL).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
in Response to SEB in a T Cell
Hybridoma Expressing a TCR
-CPM Mutant--
We have previouly shown
that a T cell hybridoma expressing a chimeric staphylococcal
enterotoxin B (SEB)-specific TCR lacking the
chain CPM has a severe
impairment in IL-2 production in response to antigenic ligand. This
defect could be fully rescued by a calcium ionophore, suggesting that
the calcium pathway fails to be activated by the mutant TCR (5). A
central molecule in the induction of calcium mobilization is PLC
,
which is activated by phosphorylation on tyrosine residues following
TCR triggering and as such induces release of calcium from
intracellular stores by generating IP3 from membrane phospholipids.
PTKs of the Src, Syk, and Tec families have been implicated, either
directly or indirectly, in PLC
phosphorylation (10, 11, 22-24). We
assessed the capacity of the
-CPM TCR mutant to promote PLC
phosphorylation. PLC
-specific immunoprecipitates from lysates of T
cell hybridomas expressing either wild-type or mutant TCR and activated
with SEB were probed by immunoblot with anti-phosphotyrosine
antibodies. As shown in Fig. 1,
SEB-dependent phosphorylation of PLC
was completely
abolished in the hybridoma expressing the mutant TCR. Hence the defect
in the capacity of TCR
-CPM mutant to activate the calcium pathway
is due at least in part to the failure of this TCR to activate
PLC
.
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Fig. 1.
Defective phosphorylation of
PLC in response to SEB in a T cell hybridoma
expressing the TCR
-CPM mutant.
Anti-phosphotyrosine immunoblot of PLC
-specific immunoprecipitates
from lysates of T cell hybridomas expressing either a SEB-specific TCR
(
wt/
wt) or an
-CPM mutant of the same TCR (
IV/
III).
Cells were either not activated or activated by SEB and antigen
presenting cells. After stripping, the filter was reprobed with
anti-PLC
antibodies (bottom panel). The migration of
molecular mass markers is shown.
(10, 11). A
portion of the total cellular Fyn is found in a complex with the TCR,
where it interacts through its N-terminal unique region with the
,
, and
chain, independently of the activation state of the TCR (25, 26). Following TCR engagement, Fyn is activated and can in turn
phosphorylate a number of substrates, which include, in addition to
PLC
, the adaptors FYB/SLAP-130, SKAP-55, and the PTK Pyk2 (27-29).
To determine whether Fyn could be activated by the
-CPM-mutated TCR,
Fyn activity was determined using in vitro kinase assays of
Fyn-specific immunoprecipitates from lysates of the hybridomas
expressing either wild-type or mutant receptors and activated with SEB.
As shown in Fig. 2A, a
dramatic reduction in SEB-dependent Fyn activation was
observed in cells expressing the mutant TCR, while direct TCR
triggering with an anti-TCR mAb (which bypasses antigen presentation)
resulted in normal levels of Fyn activation. No significant difference
in the basal activity of Fyn was detectable in the hybridomas
expressing wild-type and mutant receptor and in the parental hybridoma
lacking the TCR
(data not shown).
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Fig. 2.
Defective activation of Fyn in response to
SEB in a T cell hybridoma expressing the TCR
-CPM mutant, but normal association with the
TCR
chain. A, quantitation of
Fyn autophosphorylation on in vitro kinase assays of
Fyn-specific immunoprecipitates from lysates of T cell hybridomas
expressing either a SEB-specific TCR (
wt/
wt) or an
-CPM mutant
of the same TCR (
IV/
III), or from the parental hybridoma lacking
TCR surface expression (
). Cells were
either not activated, or activated by SEB-loaded APCs or by anti-TCR
mAb cross-linking (F23). After the kinase reaction, immunoprecipitates
were subjected to SDS-PAGE, transferred to a nitrocellulose filter, and
exposed and analyzed using a PhosphorImager. The filter was
subsequently probed with anti-Fyn mAb. The levels of Fyn
immunoprecipitated were quantitated using by laser densitometry. The
levels of 32P incorporation by Fyn in each
immunoprecipitate were normalized to the respective levels of Fyn. The
data show the ratio of Fyn autophosphorylation in activated to resting
cells in each cell line. B, anti-Fyn immunoblot of
CD3
-specific immunoprecipitates from lysates of T cell hybridomas
expressing either a SEB-specific TCR (
wt/
wt) or an
-CPM mutant
of the same TCR (
IV/
III). After stripping, the filter was
reprobed with anti-CD3
mAb (bottom panel). The migration
of molecular mass markers is shown, as well as the migration of Fyn in
a total hybridoma T cell lysate separated on the same gel.
,
-specific immunoprecipitates from lysates of the hybridomas expressing either wild-type or mutant TCR were probed by immunoblot with anti-Fyn antibodies. As shown in Fig. 2B, similar levels of Fyn
co-precipitated with
in both hybridomas, ruling out a defective
association between Fyn and
. Similar results were obtained from
in vitro binding assays using a glutathione
S-transferase fusion protein containing the intracellular
domain of the
chain (data not shown).
-CPM
mutant, and whether it could be phosphorylated in response to TCR
engagement. Fyn-specific immunoprecipitates from lysates of the
hybridomas expressing either the wild-type or the mutant TCR and
stimulated with SEB were sequentially probed by immunoblot with
anti-phosphotyrosine and anti-Pyk2 antibodies. As a control, the same
experiment was carried out on cells stimulated with anti-TCR mAb. The
results are presented in Fig. 3.
Following SEB presentation, a phosphoprotein of about 115 kDa, which
was identified as Pyk2, co-precipitated with Fyn in cells expressing the wild-type, but not the mutant TCR (left panel). Similar
levels of phospho-Pyk2 co-precipitated with Fyn in both hybridomas when the TCR was directly triggered with an anti-TCR mAb (right
panel). Of note, the association of Pyk2 with Fyn was similar in
both hybridomas (middle panel). Also a p72-kDa
phosphoprotein, which normally co-precipitates with and is
phosphorylated by Fyn following TCR engagement (31), is absent from
Fyn-specific immunoprecipitates from the hybridoma expressing the
mutant TCR following SEB presentation (Fig. 3). Hence the TCR
-CPM
mutant is incapable of activating Fyn and promoting phosphorylation of
Fyn's downstream substrates.
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Fig. 3.
Defective phosphorylation of Fyn-associated
Pyk2 in response to SEB in a T cell hybridoma expressing the
-CPM-mutated TCR. Top,
anti-phosphotyrosine immunoblot of Fyn-specific immunoprecipitates from
lysates of T cell hybridomas expressing either a SEB-specific TCR
(
wt/
wt) or an
-CPM mutant of the same TCR (
IV/
III).
Cells were either not activated, or activated by antigen presentation
(SEB; left) or by anti-TCR mAb cross-linking
(F23; right). After stripping, the filter was sequentially
probed with anti-Pyk2 (middle) and anti-Fyn antibodies
(bottom panel). The migration of molecular mass markers is
shown, as well as the migration of Pyk2 and Fyn in a total hybridoma T
cell lysate separated on the same gel.
is found in a complex with ZAP-70, LAT,
and Grb2 following TCR engagement (32), suggesting a role for ZAP-70 in
PLC
phosphorylation. In turn ZAP-70 activation requires a Src kinase
at two sequential stages, first to generate appropriate docking sites
on the CD3/
ITAMs, then to phosphorylate a tyrosine residue on
ZAP-70 critical for its function (2, 3, 33). We asked therefore whether
Lck and ZAP-70 could be activated in response to SEB by the TCR
-CPM
mutant. The activity of both kinases was determined with in
vitro kinase assays of Lck or ZAP-70 specific immunoprecipitates
from lysates of the hybridomas expressing either the wild-type or the
mutant TCR and activated with SEB. As shown in Fig.
4, activation of both kinases was
impaired in cells expressing the mutant TCR.
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Fig. 4.
Defective activation of Lck and ZAP-70 in
response to SEB in a T cell hybridoma expressing the TCR
-CPM mutant. Quantitation of Lck and ZAP-70
autophosphorylation on in vitro kinase assays of Lck- or
ZAP-70-specific immunoprecipitates from lysates of T cell hybridomas
expressing either a SEB-specific TCR (
wt/
wt) or an
-CPM mutant
of the same TCR (
IV/
III). Cells were either not activated, or
activated by SEB-loaded APCs. After the kinase reaction,
immunoprecipitates were subjected to SDS-PAGE, transferred to a
nitrocellulose filter, and exposed and analyzed using a PhosphorImager.
The filter was subsequently probed with anti-Lck or anti-ZAP-70 mAb.
The levels of Lck or ZAP-70 immunoprecipitated were quantitated by
laser densitometry. The levels of 32P incorporation by Lck
or ZAP-70 in each immunoprecipitate were normalized to the respective
levels of immunoprecipitated protein. The data show the ratio of Lck or
ZAP-70 autophosphorylation in activated to resting cells in each cell
line.
-CPM Mutant by Constitutively
Active Fyn--
Although a reduction in ZAP-70 activity is predictable
if Src kinases are impaired, the failure of the mutant TCR to activate Lck suggests that both Fyn and Lck might be implicated in the signaling
defect observed with the mutant TCR. To assess this possibility, we
used a TCR defective Jurkat T cell model. Expression of the mutant TCR
in these cells shows a defect in SEB-dependent activation
of the transcription factor NF-AT, which could be rescued by a calcium
ionophore (5). We used this model to ask whether bypassing
antigen-specific TCR triggering by forced expression of constitutively
active forms of Fyn or Lck could restore NF-AT activation by the TCR
-CPM mutant. Cells were transiently co-transfected with expression
vectors encoding wild-type or chimeric TCR
, a NF-AT/luciferase
reporter, and either empty vector or an expression vector encoding the
constitutively active F528Fyn mutant lacking the negative regulatory
tyrosine C-terminal residue. After recovery, cells were activated with
SEB. Similar experiments were carried out using a vector encoding the
constitutively active F505Lck mutant. The results are shown in Fig.
5. Expression of F528Fyn restored the
levels of NF-AT activation induced by SEB in cells expressing the
mutant TCR to levels comparable to those induced by the wild-type TCR.
Expression of F528Fyn also resulted in an enhancement of
SEB-dependent NF-AT activation by the wild-type TCR (Fig.
5). No significant increase in the NF-AT response to SEB was observed
either in the presence of F505Lck (Fig. 5), or by a constitutively
active Ras mutant (data not shown). Hence the introduction of
constitutively active Fyn effectively complements the signaling defect
in the
-CPM mutant receptor.
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Fig. 5.
Rescue of NF-AT activation in response to SEB
in a T cell hybridoma expressing the TCR -CPM
mutant by constitutively active Fyn. Relative luciferase activity
in lysates of J31.13 cells transiently co-transfected with a
NF-AT/luciferase reporter, expression plasmids encoding the
and
chains of a SEB-specific TCR, or the corresponding
-CPM mutated TCR,
and either empty vector or an expression construct encoding a
constitutively active Fyn (or Lck) mutant. After recovery, cells were
either exposed to APC alone, or to APC loaded with SEB. Luciferase
activity induced by treatment with SEB was normalized to maximal
activity induced by a combination of phorbol 12-myristate 13-acetate
and A23187. Relative SEB dependent luciferase activities in J31.13
cells coexpressing wild-type TCR and constitutively active Fyn
(
wt/
wt+F528Fyn), or expressing the TCR
-CPM mutant, alone
(
IV/
III) or in combination with constitutively active Fyn
(
IV/
III + F528Fyn), are expressed as % of the value obtained in
cells expressing the wild-type TCR alone (
wt/
wt). The same
experiment was carried out with a constitutively active Lck mutant
(right). The results obtained on duplicate samples of two
representative experiments for each constitutively active mutant are
shown.
-CPM mutant. Stable transfectants were selected in the presence of hygromycin. Flow cytometric analysis revealed no differences in the levels of surface TCR in these cells as
compared with the respective parental lines (data not shown). As shown
in Fig. 6A, the levels of Fyn
in total cells lysates were significantly increased in the F528Fyn
hybridomas, which is expected due to the presence of a long terminal
repeat-driven F528Fyn expression construct. Probing the same
filter with anti-phosphotyrosine antibodies showed a considerably more
complex phosphoprotein pattern in the F528Fyn-transduced hybridomas
(Fig. 6A). Furthermore, in vitro kinase assays of
Fyn-specific immunoprecipitates from hydridomas expressing the
wild-type or mutant TCR, and from their respective F528Fyn
transfectants, showed a large increase in Fyn activity in the
F528Fyn-transfected cells (Fig. 6B). Immunoblot analysis of
similar Fyn-specific immunoprecipitates with anti-phosphotyrosine antibodies, followed by anti-Pyk2 antibodies (not shown), revealed higher levels of Fyn-associated, phospho-Pyk2 in the F528Fyn hybridomas (Fig. 6C). Furthermore, anti-phosphotyrosine immunoblots of
-specific immunoprecipitates showed constitutive
chain
phosphorylation in F528Fyn-expressing cells (data not shown).
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Fig. 6.
Generation and characterization of T cell
hybridomas expressing a constitutively active Fyn mutant.
A, anti-Fyn (top) or anti-phosphotyrosine
(bottom) immunoblot of total lysates from T cell hybridomas
expressing either a SEB-specific TCR ( wt/
wt) or an
-CPM mutant
of the same TCR (
IV/
III), or stable transductants infected with a
retroviral construct encoding a constitutively active Fyn mutant
(F528Fyn). Equal amounts of proteins were loaded in each lane.
B, quantitation of Fyn autophosphorylation on in
vitro kinase assays of Fyn-specific immunoprecipitates from
lysates of resting T cell hybridomas expressing either a SEB-specific
TCR (column 1) or an
-CPM mutant of the same TCR
(column 2), or stable transductants infected with a
retroviral construct encoding a constitutively active Fyn mutant
(F528Fyn; columns 3 and 4). After the kinase reaction,
immunoprecipitates were subjected to SDS-PAGE, transferred to a
nitrocellulose filter, and exposed and analyzed using a PhosphorImager.
The filter was subsequently probed with anti-Fyn mAb. The levels of Fyn
immunoprecipitated were quantitated by laser densitometry. The levels
of 32P incorporation by Fyn in each immunoprecipite were
normalized to the respective levels of Fyn. C,
anti-phosphotyrosine immunoblot of Fyn-specific immunoprecipitates from
lysates of resting T cell hybridomas expressing either a SEB-specific
TCR (
wt/
wt) or an
-CPM mutant of the same TCR (
IV/
III),
or the respective transductants expressing F528Fyn. After stripping,
the filter was sequentially probed with anti-Pyk2 (bottom)
and anti-Fyn antibodies (not shown). The migration of molecular mass
markers is shown, as well as the migration of Pyk2 and Fyn in a total
hybridoma T cell lysate separated on the same gel.
-CPM-mutated TCR, the
hybridomas expressing the wild-type or mutant TCR, and their respective
counterparts expressing F528Fyn, were assayed for the capacity to
produce IL-2 in response to SEB stimulation. As shown in Fig.
7, the amount of IL-2 detected in the
culture supernatant of the hybridoma expressing the mutant receptor and stimulated with SEB was close to background. On the other hand, the
IL-2 response to SEB induced by the mutant TCR was fully restored in
the presence of F528Fyn. Similar to the results obtained in the Jurkat
T cell model, an enhancement of the response elicited by wild-type TCR
in the presence of F528Fyn was also observed (Fig. 7). Collectively,
the results strongly suggest that the signaling dysfunction in the
-CPM mutant TCR is caused at least in part by a defective activation
of Fyn, resulting in abortive downstream signaling, and leading to an
impairment of the signals involved in calcium mobilization.
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Fig. 7.
Rescue of IL-2 production in response to SEB
in a T cell hybridoma expressing the
-CPM-mutated TCR by constitutively active Fyn.
Levels of IL-2 in supernatants of T cell hybridomas expressing either a
SEB-specific TCR (
wt/
wt) or an
-CPM mutant of the same TCR
(
IV/
III), or their respective transductants expressing F528Fyn.
Cells were either exposed to APC alone, or to APC loaded with
increasing concentrations of SEB.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-CPM within the
TCR has a specialized role during T
cell development in that TCRs with a mutated
-CPM are unable to
drive thymocytes to undergo positive selection (6, 34). Studies of
-CPM mutant receptors have shown that positive selection requires a
prolonged activation of the Erk pathway and recruitment of proximal
signaling components into glycolipid rafts within the plasma membrane
(34). In addition, structural characterization of the
-CPM-mutated
TCR used in this study has revealed that, although expressed at normal
levels at the cell surface, the mutant TCR associates only loosely with
both CD3
and -
(5, 6). Genetic ablation of CD3
or -
results
in defective thymocyte development (35-37). However, in the case of
CD3
, this effect appears related to its key role in TCR·CD3
complex assembly and expression at the cell surface, as deletion of its
cytoplasmic tail or of individual ITAMs does not affect TCR signaling
(38, 39). Similar results have been obtained in T cell models
expressing TCR·CD3 complexes with mutations or deletions in the
cytoplasmic domain of CD3
(40). Furthermore, although TCR signaling
is impaired in CD3
-deficient thymocytes (41), the cytoplasmic domain
appears dispensable for antigen-dependent effector
functions in a CTL clone (42), suggesting a partial redundancy among
the ITAMs. Nevertheless, preferential coupling of individual TCR/CD3 components with specific signaling pathways is likely, especially in
conditions where they are all expressed so that no "emergency" compensation is required. The data presented above show that the signaling defect in the T cell hybridoma expressing the
-CPM-mutated TCR is at least in part dependent on the failure of Fyn to become activated. A significant part of the total intracellular content of Fyn
is constitutively associated with the
chain, as well as with the
and
chains of the CD3 complex (25, 26). Interestingly, no
association with CD3
has been
detected.2 Since Fyn is
stably associated with
in the T cell hybridomas expressing either
the wild type or the
-CPM mutant TCR (Fig. 2), another mechanism is
required to explain the defective activation of Fyn by the mutant
receptor. The fact that the
chain associates poorly with the
-CPM-mutated TCR may explain the failure of this receptor to
activate Fyn. In fact, coupling the TCR·CD3 complex to Fyn activation
might require stable association of
with the TCR·CD3 complex.
and the IP3
receptor, which are both implicated in calcium release from
intracellular stores (10-12). A role for Fyn in calcium mobilization
independent of phosphatidylinositol bisphosphate hydrolysis has
also been demonstrated (13). Furthermore, expression of constitutively
active Fyn obviates the requirement for a calcium ionophore in a
pharmacological model of NF-AT activation (8). The property of
promoting TCR-dependent calcium flux is specific to the T
cell isoform of Fyn (FynT) and has been mapped to its catalytic domain
(46). Furthermore, coexpression of Fyn and
in a heterologous cell
model is sufficient for reconstitution of calcium mobilization (9). Of
note, these effects are specifically elicited by Fyn, and not by the
related kinase Lck. Hence, although Lck activation is also impaired in
the T cell hybridoma expressing the TCR
-CPM mutant, restoration of
defective TCR function by a calcium ionophore (5), as well as the
specific complementation of the defect by constitutively active Fyn,
suggests that the impairment of Lck activation might not be causal to
the mutant TCR dysfunction at least in the hybridoma. This might be due
to the unique features of superantigen binding to the TCR in this system, which neither requires nor involves CD4 (47). In support of
this possibility, triggering of TCR signaling with staphylococcal enterotoxins has been shown to be unaffected in Lck defective T cells
(48). Furthermore, T cells from Fyn
/
mice showed
reduced responses to SEB (44). It should be noted that, despite the
reduction in PTK activation in response to SEB, the PKC/Ras/MAP kinase
pathway is still operational, as shown by the restoration of NF-AT
activation by calcium ionophore alone in cells expressing the
-CPM-mutated TCR (5), suggesting that the requirements for Ras
activation may be less stringent, possibly because of the redundancy in
the TCR signaling components involved in recruitement of Grb2·Sos
complexes (49). In this regard, we should point out that
CD4+/CD8+ thymocytes expressing an
-CPM-mutated receptor are capable of activating Erk in response to a
negatively selecting ligand, but show a specific defect in Erk
activation in response to a positive selection ligand (34). For this
TCR, SEB is a negatively selecting ligand and it is therefore expected
that the mutant TCR can activate the Erk pathway. Furthermore, there
are likey other differences between a T cell hybridoma and a
CD4+/CD8+ thymocyte. In support of this
possibility, a significant basal level of Erk2 phosphorylation was
detected in the T cell hybridoma expressing the TCR
-CPM mutant
which was not observed in CD4+/CD8+
thymocytes.2
-CPM-mutated TCR harbor a severe defect in
positive selection (6). Although a defective activation of
mitogen-activated kinases, but not of stress-activated kinases, was
observed in thymocytes expressing the
-CPM mutant when exposed to a
positively selecting ligand, the primary defect could be traced to the
very first steps of TCR signaling, and specifically to recruitment and
phosphorylation of signaling proteins in lipid rafts (34). These
specialized subdomains of the plasma membrane enriched in cholesterol
and glycosphingolipids have been recently shown to be critically
involved in PTK-dependent signaling pathways (50). Lck and
Fyn have been shown to be interchangeable in terms of ITAM
phosphorylation in a heterologous cell model (51). Furthermore, the
TCR
chain is phosphorylated, albeit weakly, in Lck-defective peripheral T cells (52), suggesting a functional redundancy among Src
family PTKs. A severe block in thymocyte development was, however,
observed only in mice lacking Lck or expressing a dominant negative Lck
mutant (53, 54), while both thymocyte development and positive
selection are unaffected in mice lacking Fyn (44, 45). On the other
hand, a role for Fyn in thymocyte development is supported by the
markedly more severe phenotype of developing
Lck
/
Fyn
/
thymocytes as compared with
thymocytes lacking only Lck, with a very early block at the double
negative stage (55, 56). Furthermore, a constitutively active form of
Fyn could partially substitute for Lck in thymocyte development and
positive selection, suggesting a cooperation between the two PTKs (56).
Hence, although the defective functional coupling of Fyn with the TCR
-CPM domain might not be causal in the defect in positive selection
in thymocytes expressing this TCR, this possibility cannot be
completely ruled out. Alternatively, the same function might be
subserved by different Src kinases in thymocytes and T lymphocytes. In
this context, the failure of the mutant TCR to activate Lck might be
relevant to the defective phosphorylation of signaling proteins in
lipid rafts in positively selected thymocytes expressing the TCR
-CPM mutant.
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ACKNOWLEDGEMENTS |
---|
We thank Sonia Grassini and Barbara Hausmann for excellent technical assistance, Giancarlo Benocci for secretarial assistance, Antonio Lanzavecchia for the gift of APC, and John L. Telford for productive discussions and critical reading the manuscript.
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FOOTNOTES |
---|
* The work was supported in part by the Associazione Italiana per la Ricerca sul Canero (AIRC), Telethon Grant E.1161, and the University of Siena (PAR). The Basel Institute for Immunology was founded and is supported by Hoffmann-La Roche Ltd., Basel, Switzerland.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.
Recipient of a fellowship from the Federazione Italiana per la
Ricerca sul Canero (FIRC).
¶ To whom correspondence should be addressed: Dept. of Evolutionary Biology, University of Siena, Via Mattioli 4, 53100 Siena, Italy. Tel.: 39-0577-232873; Fax: 39-0577-232898; E-mail: baldari@unisi.it.
Published, JBC Papers in Press, October 31, 2000, DOI 10.1074/jbc.M008588200
2 C. Ulivieri and C. T. Baldari, unpublished data.
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ABBREVIATIONS |
---|
The abbreviations used are:
TCR, T cell
antigen receptor;
PTK, protein-tyrosine kinase;
ITAM, immunoreceptor
tyrosine-based activation motif;
IL-2, interleukin-2;
IP3, inositol 1,4,5-trisphosphate;
APC, antigen presenting cell;
PLC, phospholipase C
;
mAb, monoclonal antibody;
CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid;
PAGE, polyacrylamide gel electrophoresis;
SEB, staphylococcal enterotoxin
B.
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