From the Molecular Immunology Unit, Department of
Immunology, Institut Pasteur, 25 Rue du Docteur Roux, 75724 Paris
Cedex 15, France and the ¶ Department of Biology, Pharmacia & Upjohn, Viale Pasteur 10, 20014 Nerviano (Milan), Italy
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ABSTRACT |
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Following T cell antigen receptor (TCR) engagement, the protein tyrosine kinase (PTK) ZAP-70 is rapidly phosphorylated on several tyrosine residues, presumably by two mechanisms: an autophosphorylation and a trans-phosphorylation by the Src-family PTK Lck. These events have been implicated in both positive and negative regulation of ZAP-70 activity and in coupling this PTK to downstream signaling pathways in T cells. We show here that Tyr315 and Tyr319 in the interdomain B of ZAP-70 are autophosphorylated in vitro and become phosphorylated in vivo upon TCR triggering. Moreover, by mutational analysis, we demonstrate that phosphorylation of Tyr319 is required for the positive regulation of ZAP-70 function. Indeed, overexpression in Jurkat cells and in a murine T cell hybridoma of a ZAP-70 mutant in which Tyr319 was replaced by phenylalanine (ZAP-70-Y319F) dramatically impaired anti-TCR-induced activation of the nuclear factor of activated T cells and interleukin-2 production, respectively. Surprisingly, an analogous mutation of Tyr315 had little or no effect. The inhibitory effect of ZAP-70-Y319F correlated with a substantial loss of its activation-induced tyrosine phosphorylation and up-regulation of catalytic activity, as well as with a decreased in vivo capacity to phosphorylate known ZAP-70 substrates, such as SLP-76 and LAT.
Collectively, our data reveal the pivotal role of Tyr319
phosphorylation in the positive regulation of ZAP-70 and in
TCR-mediated signaling.
The signaling ability of the
TCR1 depends upon its
coordinated interaction with protein tyrosine kinases (PTKs) belonging
to the Src and Syk families (1). Several members of these PTK families
are expressed in T cells, including Lck, Fyn, Syk, and ZAP-70. Genetic
evidence has indicated that among the Syk-PTKs, ZAP-70 plays a major
role in both T lymphocyte development and functional activation.
Analysis of patients suffering from a severe immunodeficiency due to
the lack of this PTK showed that only CD4+ T cells develop,
although they are unresponsive to TCR stimulation (2-4). Moreover,
development of both CD4+ and CD8+ single
positive thymocytes is impaired in ZAP-70-null mice (5) but not
affected by Syk gene disruption (6, 7). According to current
models of the early activation steps following TCR triggering, the
Src-PTK Lck phosphorylates the tyrosine residues in the ITAMs of the
receptor invariant chains (CD3 and So far, tyrosine residues of ZAP-70 that have been shown to be
phosphorylated in vivo following TCR engagement include
Tyr292, Tyr492, and Tyr493 (19).
Mutational analysis of Tyr292 indicated that this residue
negatively regulates ZAP-70 function without affecting its catalytic
activity (20, 21). This negative effect could be mediated by Cbl
binding to phosphorylated Tyr292 (22, 23). Phosphorylation
of Tyr492 appears also to have a negative regulatory role,
as its mutation to phenylalanine results in increased ZAP-70 catalytic
activity (20, 21, 24). In contrast, up-regulation of ZAP-70 catalytic activity appears to depend on the phosphorylation of Tyr493
by Lck, as indicated by co-expression experiments in heterologous cell
systems (12, 24). Phosphorylation of this residue is essential for
connecting ZAP-70 to downstream signaling pathways (12), presumably
because it enables this PTK to phosphorylate its substrates (25).
Nonetheless, several pieces of evidence have suggested the existence of
other phosphorylation sites in ZAP-70. We have previously shown that
when Tyr492 and Tyr493 are simultaneously
mutated to phenylalanine, ZAP-70 is still phosphorylated after TCR
triggering and can still interact with Lck via an SH2-mediated
interaction (25). Moreover, although not proven to be phosphorylated,
Tyr315 is thought to be the binding site for the SH2 domain
of Vav (15) and has been shown to play a critical role in antigen
receptor signaling (26). Finally, recent reports have suggested a
regulatory role in TCR signaling for other tyrosine residues in ZAP-70
(27, 28), raising the possibility that other phosphorylation sites exist.
In an attempt to gain additional clues on the regulatory role of ZAP-70
tyrosine phosphorylation, we focused on conserved residues found in the
linker region between the C-terminal SH2 domain and the catalytic
domain of this PTK (called interdomain B; Ref. 10), namely
Tyr315 and Tyr319. We demonstrate here that
both tyrosines are phosphorylated in vitro and in
vivo and that Tyr319 plays a critical role in the
regulation of ZAP-70. Overexpression of ZAP-70 bearing a Y319F mutation
(ZAP-70-Y319F) impaired TCR-induced events, such as activation of NFAT
and IL-2 production. Mutation of Tyr319 substantially
reduced the activation-dependent tyrosine phosphorylation of ZAP-70 and the up-regulation of its catalytic activity. Moreover, we
show that ZAP-70-Y319F impaired TCR-mediated phosphorylation of known
ZAP-70 substrates, such as SLP-76 and LAT. In contrast to a previous
report (26), we found that a ZAP-70-Y315F had only minimal inhibitory
effects on TCR signaling. Thus, our results indicate that
phosphorylation of Tyr319 is a critical event for
up-regulation of ZAP-70 activity and highlight the pivotal role of this
residue in TCR signaling.
Cell Lines and Antibodies--
The human leukemia Jurkat T cell
line was maintained in RPMI 1640 medium supplemented with 10% fetal
calf serum, 10 mM Hepes, 2 mM
L-glutamine, 100 units/ml penicillin, and 100 µg/ml
streptomycin (Life Technologies, Cergy Pontoise, France) (complete
medium). Murine T cell hybridoma T.AL8.1 (T8.1), expressing human CD4
and TCR specific for a tetanus toxin peptide (tt830-843;
QYIKANSKFIGITE) (29) restricted for HLA-DRB1*1102 was maintained in
complete Dulbecco's modified Eagle's medium supplemented with 400 nM methotrexate, 1 mg/ml G418 and 50 µM
2- Constructions and Plasmids--
The GST-( Cell Transfection and NFAT Activity Assays--
Jurkat cells
(107) were transiently transfected by electroporation (25)
with the indicated amounts of pSR In Vitro and in Vivo Phosphorylation Analysis--
The
GST-(
For automated Edman degradation, gel-separated 32P-peptides
were transferred onto a polyvinylidene difluoride membrane, excised, and eluted in 40% trifluoroacetic acid, 0.1% acetonitrile at
70 °C. They were covalently coupled onto a SequelonTM-AA
filter (Millipore) and sequenced in an Applied Biosystems 494 sequencer
(36). ATZ amino acid derivatives were collected and monitored by liquid
scintillation counting.
Metabolic labeling was performed by incubating Jurkat cells (3.3 × 106/ml) for 4 h in phosphate-free medium containing
0.3 mCi/ml 32P (ICN). Cells were then stimulated by
anti-CD3 antibody (VIT3) at 1:200 dilution of ascities for 3 min or
pervanadate for 5 min.
Immunoprecipitation and Immunoblotting--
Jurkat cell
transfectants, stimulated with anti-CD3 mAb (1:200 dilution of ascites)
for 3 min at 37 °C or pervanadate for 5 min, were lysed in 1%
Nonidet P-40-containing buffer and immunoprecipitated with the relevant
antibody (25). Immunoprecipitation, immunoblotting, and detection of
proteins by enhanced chemiluminescence (Amersham Pharmacia Biotech)
were performed as described previously (14). 125I-Protein A
detection followed by PhosphorImager scanning of anti-ZAP-70 immunoblots were used to quantify the relative expression of exogenous versus endogenous ZAP-70 proteins in stable transfectants as
described (25).
Measurement of in Vitro Kinase Activity--
ZAP-70 catalytic
activity was assessed in vitro by incubating anti-tag
immunoprecipitates for 5 min at room temperature in 25 mM
MES, pH 6.5, 10 mM MnCl2, 5 µM
ATP, containing 10 µCi of [ Antigen Stimulation and IL-2 Production Measurement--
T cell
hybridomas (1 × 105 cells) were incubated overnight
at 37 °C with L625.7 fibroblast (2 × 104)
prepulsed or not with increasing concentration of tt830-843 (0.05-5
µg/ml) as described previously (29). IL-2 released in the
supernatants was then measured by using the DuoSeT enzyme-linked immunosorbent assay kit (Genzyme, Cambridge, MA) according to the
manufacturer's instructions. Analysis of protein phosphorylation in
total cell lysates was performed by anti-phosphotyrosine immunoblotting as described previously (38).
Residues Tyr315 and Tyr319 in ZAP-70 Are
Phosphorylated in Vitro and in Vivo--
In order to establish whether
Tyr315 and Tyr319 are phosphoacceptor sites, we
first made use of a GST-ZAP-70 fusion protein lacking the SH2 domains
(GST-(
According to the human ZAP-70 sequence (39), CNBr cleavage at
Met310 and Met359 would produce a peptide
containing Tyr315 and Tyr319 and having a
Mr of about 6000. Indeed, when
GST-(
Peptides I and II were further digested by trypsin and subjected to
two-dimensional peptide mapping. As shown in Fig. 2C, these
peptides gave an identical phosphopeptide pattern, indicating that
peptide II was a partial cleavage product including peptide I. As no
trypsin cleavage site exists between Tyr315 and
Tyr319, the multiplicity of spots is due to partial
digestion products (as predictable from peptide I sequence, see above).
Calculation of the electrophoretic and chromatographic mobilities for
the expected tryptic peptides (35) indicated that
Tyr315/Tyr319 doubly phosphorylated peptides
would migrate more toward the anode (Fig. 2C, right) and
have a decreased mobility in the chromatographic dimension compared
with their mono-phosphorylated counterparts. This prediction was
confirmed by Edman degradation, as the three peptides indicated by
arrowheads (Fig. 2, C and D) contained
phosphorylated amino acids at both cycles 5 and 9. Moreover, these
peptides selectively disappeared in tryptic peptide maps of peptide I
from GST-(
As peptide III was not visible in all digestions, and peptides IV
(Mr ~23,000) and V (Mr
~26,000-28,000) migrated with an Mr
In vivo phosphorylation of Tyr315 and
Tyr319 upon cell activation was demonstrated by comparing
CNBr and trypsin cleavage products of in vitro
autophosphorylated GST-(
Collectively, these results demonstrate that both Tyr315
and Tyr319 are phosphorylated in vivo following
TCR engagement, and the in vitro data suggest that this
event may be the consequence of ZAP-70 autophosphorylation.
Dominant Negative Effect of ZAP-70-Y319F on TCR-stimulated NFAT
Activity--
To investigate the possible role of the phosphorylation
on Tyr315 and/or Tyr319 in the signaling
ability of ZAP-70, a tagged ZAP-70-Y315F or ZAP-70-Y319F mutant was
first transiently overexpressed in Jurkat T cells. As a readout for
cellular activation, TCR-induced stimulation of NFAT, an essential
factor required for IL-2 gene transcription (40), was used.
Transfection of ZAP-70-Y319F-encoding plasmid resulted in a
dose-dependent inhibition of the TCR-induced NFAT activity
compared with the empty vector (Fig. 3A). We have previously shown that a similar inhibition was exerted by a kinase-defective mutant of ZAP-70 (ZAP-70KD) or by a ZAP-70 mutant carrying a double mutation Y492F/Y493F, whereas transfection of ZAP-70WT had no effect in
this system (25). On the other hand, only a modest inhibition of NFAT
activity was observed when transfecting a vector encoding ZAP-70-Y315F
(Fig. 3A). This mild effect on TCR-induced signaling, which
was also observed in another T cell model system (see below and Ref.
38), contrasts with a previous report that showed that the same
mutation strongly impaired the ability of ZAP-70 to reconstitute
antigen receptor signaling in a Syk-deficient chicken B cell line (26).
These discrepancies are possibly due to the different experimental
model used.
Mutation of Tyr319 Results in Decreased
Activation-induced ZAP-70 Tyrosine Phosphorylation and Kinase Activity
Up-regulation--
The molecular basis of the signaling defect due to
the Y319F mutation was analyzed in Jurkat cells stably overexpressing
ZAP-70-Y319F. The presence of the C-terminal epitope tag increased the
electrophoretic mobility of the transfected construct compared with
endogenous ZAP-70 and allowed us to measure the relative expression
levels of the exogenous versus endogenous ZAP-70 in each
cell line by anti-ZAP-70 immunoblotting, as described previously (25).
Two representative independent transfectants, named 1.40 and 1.60, expressing ZAP-70-Y319F at levels 3-4-fold over the endogenous ZAP-70
were used in most experiments, as well as two cell lines expressing the
ZAP-70-Y315F mutant (2.21/2 and 2.21/14). Jurkat cells transfected with
an empty vector (J05.2) or a representative cell line (15.8) stably
expressing a ~2-fold excess of tagged ZAP-70 (ZAP-70WT) (Ref. 25 and
data not shown) were utilized as controls. The relative expression
levels of the exogenous ZAP-70 constructs (collectively designated
ZAP-70tag) in these transfectants are shown in Fig. 3C. All
of these cell lines expressed comparable levels of TCR/CD3 and Lck
(data not shown).
Fig. 3B shows that the dominant negative effect of
ZAP-70-Y319F, but not that of ZAP-70-Y315F, could be confirmed in these stable transfectants. Again, TCR-induced NFAT activation was strongly reduced (by ~80%) in both cell lines expressing ZAP-70-Y319F, whereas only a limited inhibition was noted in cells expressing ZAP-70-Y315F (37 and 12% in 2.21/2 and 2.21/14 cell lines,
respectively), despite higher (2-3-fold) expression levels of this
mutant (Fig. 3C) compared with ZAP-70-Y319F. Collectively,
data shown in Fig. 3, A and B, demonstrate that
the Y319F mutation has a dramatic effect on TCR-induced signaling,
whereas the Y315F mutation has only a modest inhibitory effect (see
also below).
Based on the results described above, we focused our biochemical
analysis on ZAP-70-Y319F. In order to evaluate the impact of the Y319F
mutation on ZAP-70 phosphorylation level in vivo, ZAP-70-Y319F and ZAP-70WT were immunoprecipitated using an anti-tag antiserum and analyzed by SDS-PAGE and anti-phosphotyrosine
immunoblotting. These experiments revealed a strong reduction of
ZAP-70-Y319F tyrosine phosphorylation compared with ZAP-70WT (Fig.
4A, upper panel) after CD3
cross-linking, despite similar amounts of immunoprecipitated protein
(Fig. 4A, lower panel). These experiments also showed that
ZAP-70-Y319F, like ZAP-70WT, co-precipitated with the
tyrosine-phosphorylated
Considering that at least five tyrosine residues of ZAP-70 are known to
be phosphorylated upon TCR triggering (Ref. 19 and this work), the
absence of Tyr319 cannot in itself explain the considerably
decreased phosphorylation of ZAP-70-Y319F and suggests that other
phosphorylation sites can be indirectly affected. Thus, we asked
whether the catalytic activity of ZAP-70 was impaired by the mutation.
To this aim, ZAP-70WT and ZAP-70-Y319F were immunoprecipitated by an
anti-tag antiserum from Jurkat cells unstimulated or stimulated with
the tyrosine phosphatase inhibitor pervanadate, and their ability to
phosphorylate an exogenous substrate in vitro was assessed. Pervanadate was routinely used in these experiments as it reproducibly gave a higher increase in ZAP-70 kinase activity over the basal level
compared with anti-CD3 stimulation. As the in vivo
phosphorylation pattern of ZAP-70 was superimposable after pervanadate
or anti-CD3 antibody stimulation of Jurkat cells (data not shown; see
also Ref. 12) and because the induction of ZAP-70 tyrosine kinase activity correlates with its tyrosine phosphorylation (12), these two
stimulatory agents can be considered equivalent from a qualitative
point of view. Moreover, because the expression levels of the ZAP-70tag
proteins were slightly dissimilar in the different cell lines,
32P incorporation in the substrate was normalized in each
sample for the relative amount of immunoprecipitated protein measured by anti-tag immunoblot. As shown in Fig. 4B, these
experiments revealed that the ability of unstimulated ZAP-70-Y319F to
phosphorylate the cytosolic fragment of cfb3 was comparable to that of
ZAP-70WT. However, the increase in the kinase activity of ZAP-70-Y319F
after pervanadate treatment of both 1.40 and 1.60 cell lines was
markedly reduced compared with ZAP-70WT. Thus, ZAP-70-Y319F appeared to be defective in the activation-dependent up-regulation of
its catalytic activity. A possible explanation for this phenotype would
be that mutation of Tyr319 induced a structural change in
ZAP-70 that impaired the increase in its kinase activity following TCR
triggering. Alternatively, as this residue is phosphorylated in
vivo, it could serve as a docking site for other molecules
required for up-regulating ZAP-70 catalytic activity (see under
"Discussion").
Dominant Negative Effect of ZAP-70-Y319F on Antigen-induced IL-2
Production--
We then wanted to confirm the dominant negative effect
of ZAP-70-Y319F in a different T cell model and to evaluate the effect of this mutant on antigen-induced signaling. Thus, ZAP-70-Y319F was
stably overexpressed in the murine T cell hybridoma T8.1, expressing a
human TCR specific for the tetanus toxin peptide tt830-843 (29).
ZAP-70-Y315F and ZAP-70KD were also overexpressed in these cells as a
control. As shown in Fig. 5A,
the expression of these ZAP-70 constructs in a number of representative
clones was comparable and at levels over 10-fold higher than the
endogenous ZAP-70, as measured by PhosphorImager scanning of
anti-ZAP-70 immunoblot. Hybridomas were stimulated using as APCs the
human class II MHC-expressing murine fibroblasts L625.7 (29) prepulsed with increasing amounts of antigenic peptide. Overexpression of ZAP-70-Y319F markedly decreased IL-2 production in response to antigen
(Fig. 5B). Indeed, this mutant induced a shift of the dose-response curve of about 1 order of magnitude compared with untransfected T8.1 cells. A similar effect was found in cells overexpressing comparable levels of ZAP-70KD, whereas no effect of
ZAP-70-Y315F overexpression on IL-2 production was detectable (Fig.
5B). Moreover, no effect either was seen in T8.1 cells
overexpressing similar levels of ZAP-70 wild-type (data not shown).
Collectively, these results indicate that the inhibitory effect of
ZAP-70-Y319F on TCR-induced signaling cannot be overcome by using more
physiological stimulatory conditions (i.e. antigen
presentation by an APC able to deliver appropriate co-stimulatory
signals (38)) and underline the critical function of Tyr319
of ZAP-70 in TCR-mediated T cell activation.
Effects of ZAP-70-Y319F Overexpression on Tyrosine Phosphorylation
of ZAP-70 Substrates--
Because we have shown that ZAP-70-Y319F was
defective in the activation-dependent up-regulation of its
catalytic activity, it was expected that phosphorylation of ZAP-70
substrates would be impaired in cells overexpressing this mutant. We
took advantage of the higher expression levels of ZAP-70-Y319F obtained
in the T8.1 hybridoma, compared with Jurkat transfectants, to analyze the effects of this mutant on the pattern of tyrosine phosphorylation induced by antigen stimulation. Moreover, we were also able to compare
it to ZAP-70KD-expressing cells. As shown in Fig. 5C, anti-phosphotyrosine immunoblotting on total cell lysates revealed an
increased tyrosine phosphorylation of several proteins of 120-130, 95, 76, 66, and 36-38 kDa in antigen-stimulated T8.1 cells compared with
unstimulated cells. In particular, two phosphoproteins of about 76 and
36-38 kDa appeared to be the major substrates upon antigen
stimulation. The 76-kDa phosphoprotein, migrating as a doublet, was
previously identified as being SLP-76 (41, 42), whereas the
36-38-kDa-migrating phosphorylated band is likely to represent LAT
(43, 44). Both of these proteins are thought to play important roles in
connecting TCR-activated PTKs to downstream signaling pathways and have
been recently shown to be phosphorylated by ZAP-70 (44-46).
Antigen-induced tyrosine phosphorylation of both SLP-76 and LAT
appeared to be markedly reduced in cells overexpressing ZAP-70-Y319F.
Similarly to our previous observation about IL-2 production (see Fig.
5B), this effect was comparable to that exerted by the
kinase-defective mutant ZAP-70KD (Fig. 5C). Thus, the
inhibition of tyrosine phosphorylation of known ZAP-70 substrates in
ZAP-70-Y319F-expressing hybridomas can be accounted for by the lack of
activation-induced increase in kinase activity of this mutant and may
explain the impairment of antigen-induced IL-2 production.
In this work, we demonstrated that both Tyr315 and
Tyr319, located in the interdomain B of ZAP-70, are
phosphorylated in vivo upon TCR signaling. Moreover, we
provide multiple pieces of evidence that Tyr319, but not
Tyr315, is a critical positive regulatory site for ZAP-70
function and TCR-mediated signaling.
Other groups had failed in identifying Tyr315 and
Tyr319 as phosphorylation sites (19). This discrepancy
could be accounted for, at least in part, by the different protein
cleavage (i.e. CNBr instead of trypsin digestion) and
phosphopeptide analysis methods that we employed. Indeed, although
chemical digestion of ZAP-70 by CNBr was not complete (see Fig.
2A), it could have facilitated the recovery and thus the
identification of phosphopeptides containing Tyr315 and
Tyr319, as trypsin digestion was predicted to generate
multiple partial digestion products containing those residues.
Comparison of in vivo and in vitro labeled
peptide I shows that this peptide is almost exclusively phosphorylated
at both Tyr315 and Tyr319 in vivo,
whereas the monophosphorylated isoform of this peptide can be readily
detected after in vitro labeling (Fig. 2E). This difference suggests that phosphorylation of both tyrosines is most
efficient in vivo, possibly due to the proximity and/or
correct orientation of ZAP-70 molecules bound to the ITAMs of engaged TCR/CD3 complexes, compared with the phosphorylation of molecules in
solution obtained during in vitro labeling experiments.
However, it cannot be ruled out that this difference is accounted for
by the particular GST-( Previous works have demonstrated that the corresponding residues of the
homologous PTK Syk (Tyr348 and Tyr352) are
autophosphorylation sites in vitro (47). Our data showing that GST-( Several pieces of evidence underscore the functional importance of the
interdomain B of Syk-PTKs: in particular, this region contains several
phosphorylatable tyrosine residues in both ZAP-70 (Ref. 19 and this
work) and Syk (47). For example, Tyr292,
Tyr315, and Tyr319 in ZAP-70 are phosphorylated
upon TCR triggering. Tyr292 is thought to be a negative
regulatory site, as its mutation to phenylalanine increases
TCR-mediated signaling (20, 21). On the other hand, we show now that an
analogous mutation of Tyr319 results in a strong impairment
of TCR-dependent T cell activation (Figs. 3 and 5), thus
indicating a positive regulatory role for this residue. The role of
phosphorylation at Tyr315 remains so far unclear. It has
been proposed that this residue is the binding site for the SH2-domain
of Vav (15). Moreover, Wu et al. (26) have shown that the
Y315F mutation profoundly altered the ability of ZAP-70 to reconstitute
antigen receptor signaling in a Syk-negative chicken B cell line, in
contrast with the modest effect of ZAP-70-Y315F on TCR-induced NFAT
activation in Jurkat cells (Fig. 3) and on antigen-stimulated IL-2
production in the T8.1 hybridoma (Fig. 5). We have also previously
shown that ZAP-70-Y315F overexpression in the T8.1 hybridoma did not have a noticeable effect on TCR-induced tyrosine phosphorylation of Vav
and SLP-76 (38). These discrepancies may be ascribed to the different
cellular systems used. Nonetheless, our data on the functional role of
Tyr319 further underscore the view of interdomain B as a
critical regulatory region for ZAP-70 function.
It should be noted, however, that data from Zhao and Weiss (20)
indicated that expression of a ZAP-70 mutant in which residues 265-331
(spanning about 80% of the interdomain B) were deleted did not
significantly affect TCR signaling, in contrast with the evidence on
the functional importance of Tyr292 and Tyr319
cited above. Although further investigations are required to clarify
these discrepancies, the simultaneous elimination of negative and
positive regulatory mechanisms depending on Tyr292 and
Tyr319, respectively, could account for the apparent
neutral effect of the interdomain B deletion.
Our data show that TCR-induced tyrosine phosphorylation of ZAP-70-Y319F
is dramatically reduced compared with the wild-type molecule (Fig.
4A). This finding is not explained by an alteration of the
intrinsic kinase activity of the mutant, as its ability to
phosphorylate cfb3 in vitro is comparable to that of the
wild-type molecule when both are immunoprecipitated from unstimulated
cells (Fig. 4B). Moreover, this mutation did not alter the
ability of ZAP-70 to bind to phosphorylated ITAMs (Fig. 4A),
a defect previously observed for a naturally occurring deletion in the
interdomain B of Syk (48). These results argue against a structural
alteration of the PTK induced by the mutation of Tyr319. On
the other hand, ZAP-70-Y319F appears to be defective in the activation-induced up-regulation of its kinase activity, a finding that
correlates with the lack of in vivo phosphorylation of known ZAP-70 substrates, i.e. SLP-76 and LAT (see Fig.
5C). As these proteins have been shown to couple ZAP-70 to
the activation of downstream signaling pathways leading to NFAT
activation (44-46), our data indicate that the inability of
ZAP-70-Y319F to phosphorylate SLP-76 and LAT in vivo is
responsible for the inhibition of TCR-stimulated cellular activation.
However, the molecular mechanism underlying the inhibitory effect of
the Y319F mutation remains to be fully ascertained. The simultaneous
mutation of Syk residues Tyr348 and Tyr352
(homologous to ZAP-70 residues Tyr315 and
Tyr319, respectively) has been shown to affect the
SH2-mediated binding of PLC
INTRODUCTION
Top
Abstract
Introduction
References
) (8, 9) and allows ZAP-70
recruitment, via its tandem SH2 domains, to the ITAMs (8, 10).
Thereafter, ZAP-70 becomes tyrosine-phosphorylated as a result of both
autophosphorylation and trans-phosphorylation by Lck (11, 12). Tyrosine
phosphorylation of ZAP-70 correlates with its increased kinase activity
(13) and is thought to generate docking sites for several SH2 or PTB
domain-containing enzymes or adapters, including Lck (14), Abl,
Ras-GTPase-activating protein (11), Vav (15), Cbl (16), Shc (17), and
SH2-containing phosphatase-1 (18). These proteins may be involved in
regulation of ZAP-70 activity and/or coupling to downstream signaling pathways.
EXPERIMENTAL PROCEDURES
-mercaptoethanol. Mouse L625.7 fibroblasts expressing
HLA-DRB1*1102 (30) were kindly provided by Dr. R. W. Karr
(Monsanto Chemical Co., St. Louis, MO) and grown in complete minimum
Eagle's medium containing 250 µg/ml G418. Mouse monoclonal antibodies (mAbs) were as follows: anti-human TCR V
8 101.5.2 (IgM,
provided by E. L. Reinherz, Dana-Farber Cancer Institute, Boston,
MA), anti-CD3 VIT3 (IgM, provided by W. Knapp, University of Vienna,
Austria), anti-phosphotyrosine 4G10 (Upstate Biotechnology, Inc., Lake
Placid, NY), and anti-vesicular stomatitis virus-protein G epitope tag
P5D4 (IgG1; hybridoma kindly provided by Dr. T.E. Kreis,
University of Geneva, Switzerland) (31). Rabbit polyclonal antibodies
were as follows: affinity-purified anti-Lck antibody 2102 (Santa Cruz
Biotechnology, Santa Cruz, CA), anti-ZAP-70 antisera 4.06 (25) and
ZAP-4 (a gift of S. C. Ley) (32), and anti-vesicular stomatitis
virus-protein G epitope tag antiserum (provided by M. Arpin, Institut
Curie, Paris, France) (31).
SH2)ZAP-70 fusion
protein (containing residues 255 through 619 of human ZAP-70) was
obtained by polymerase chain reaction using oligonucleotide-directed
mutagenesis and confirmed by nucleotide sequencing. This construct was
expressed in COS-1 cells and purified by glutathione affinity
chromatography.2 The ZAP-70WT
construct bearing a C-terminal vesicular stomatitis virus-protein G
epitope tag was described previously (25). ZAP-70-Y315F and -Y319F
mutants were both derived from this construct by polymerase chain
reaction: the same 5' primer (base pairs 713-734) was used, encompassing the MluI unique site. The 3' primers, encoding
either the Y315F or the Y319F mutation, included base pairs 1147-1184 or 1157-1184, respectively, and both contained a SacI site.
The MluI-SacI-digested polymerase chain reaction
products were ligated with both a SacI-NsiI
fragment (base pairs 1179-1736) and a 3.8-kilobase pair ZAP-70WT pBS
fragment restricted with MluI and NsiI. ZAP-70 constructs were finally subcloned into the
EcoRI-XbaI sites of the pSR
-puro expression
vector (a gift of R. P. Sekaly, Institut de Recherches Cliniques,
Montreal, Quebec, Canada) (25). Mutations were verified by nucleotide sequencing.
-puro vector empty or containing
ZAP-70WT or ZAP-70 mutant cDNAs together with the NFAT-luciferase
(10 µg) and pSV-
-galactosidase (30 µg) reporter plasmids (25).
24 h after transfection, cells were left unstimulated or
stimulated at 37 °C for 8 h with 101.5.2 anti-TCR mAb precoated to wells at 1:1000 dilution of ascites or with phorbol 12-myristate 13-acetate (50 ng/ml) and the calcium ionophore A23187 (2 µg/ml) (Sigma).
-Galactosidase and luciferase assays were performed by
using the specific assay systems (Promega). Luciferase activities, determined in duplicate samples, were normalized to the
-galactosidase values to correct for transfection efficiency. Stable
transfectants were obtained by electroporating Jurkat cells with 30 µg of plasmid DNA and selected with puromycin as described (25).
Stable transfectants were routinely grown in medium containing 10 µg/ml puromycin. Clones positive for tagged protein expression were
analyzed by fluorescence-activated cell sorting for CD3 expression
levels (not shown). Stable T8.1 transfectants were obtained by
electroporation under the same conditions and were then selected in
complete Dulbecco's modified Eagle's medium containing methothrexate
(400 nM), G418 (1 mg/ml), and puromycin (1 µg/ml).
SH2)ZAP-70 fusion protein was autophosphorylated for 30 min at
room temperature in 1 mM Tris buffer, pH 7.4, 7.5 mM NaCl, 25 mM Hepes, 10 mM
MnCl2, 0.05% Nonidet P-40, containing 10 µCi of
[
-32P]ATP and, after SDS-PAGE, transferred onto
nitrocellulose membranes. 32P-Labeled bands were excised
and incubated with 0.5 M CNBr (Fluka) in 70% formic acid
for 1.5 h at room temperature (33). Cleavage products were
separated on Tris-tricine gels (16.5% total acrylamide concentration,
3% cross-linker) as described (34). For further tryptic digestion,
gel-separated 32P-peptides were transferred onto
nitrocellulose and digested in situ by adding 3 µg of
trypsin (Worthington) in 50 mM
NH4HCO3 overnight at 37 °C. Peptides were
routinely oxidized in performic acid and separated by thin layer
electrophoresis in pH 1.9 buffer for 15 min at 1000 V, followed by thin
layer chromatography for 10 h in phosphochromatography buffer
(35). Phosphorylated peptides were visualized by autoradiography.
-32P]ATP and 0.3 µg of
the cytoplasmic fragment of the erythrocyte band 3 protein (cfb3) as a
substrate (24, 37).
RESULTS
SH2)ZAP-70, containing residues 255-619 of human ZAP-70).
Although trypsin digestion has been previously employed to identify
phosphorylated tyrosines in ZAP-70 (19), inspection of the amino acid
sequence surrounding Tyr315 and Tyr319 (Fig.
1) indicated the presence of multiple
peptide bonds known to be cleaved with low efficiency or not cleaved at
all by trypsin (e.g. Arg306-Pro307,
Lys304-Pro305, and
Lys328-Lys329; see Ref. 35). Thus, in order to
avoid complexity of peptide maps due to partial digestion products, we
initially chose to perform phosphopeptide mapping after protein
cleavage by CNBr. This approach had also the advantage of allowing the
isolation of relatively large-sized peptides, analyzable by
one-dimensional gel electrophoresis.
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Fig. 1.
Structure of ZAP-70 and amino acid sequence
of the interdomain B. Schematic representation of the overall
structure of ZAP-70. The sequence of the portion of interdomain B
encompassing tyrosines Tyr292, Tyr315, and
Tyr319 is also shown.
SH2)ZAP-70 was autophosphorylated in vitro with
[
-32P]ATP and cleaved with CNBr, five phosphorylated
peptides could be detected (Fig.
2A), which included a major
product (peptide I) migrating at a Mr of about
6000. Edman degradation of peptide I (which, as expected, contained
only phosphotyrosines; data not shown) showed two
32P-labeled amino acids at cycles 5 and 9, as predicted for
Tyr315 and Tyr319 (Fig. 2B). Because
GST-(
SH2)ZAP-70 contains only these two tyrosines separated by four
residues, we concluded that Tyr315 and Tyr319
are autophosphorylated under these conditions. Although the amount of
radioactivity recovered at cycle 5 was usually higher compared with
cycle 9, suggesting a possible preferential phosphorylation at
Tyr315, this is likely not to be the case, as recovery of
radioactivity at cycle 9 (Tyr319) suffers from a low yield
due to poor cleavage at P318, as we verified by sequencing
a synthetic peptide encompassing Tyr315 and
Tyr319 (not shown).
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Fig. 2.
In vitro and in vivo
phosphorylation of residues Tyr315 and
Tyr319 of ZAP-70. A, analysis of
phosphopeptides from CNBr-cleaved 32P-labeled
GST-( SH2)ZAP-70 and ZAP-70. GST-(
SH2)ZAP-70 was
autophosphorylated in vitro in the presence of
[
-32P]ATP and subjected to CNBr cleavage. Peptides
were then separated on a Tris-tricine gel and transferred onto
nitrocellulose membrane. 32P-Labeled ZAP-70, isolated by
immunoprecipitation with the ZAP-4 antibody from metabolically labeled,
anti-CD3-stimulated Jurkat cells, was cleaved and analyzed likewise.
Phosphopeptides were visualized by autoradiography. Different exposure
times are shown for GST-(
SH2)ZAP-70 (6 h) or in vivo
labeled ZAP-70 (42 h). Arrows I-V indicate the major
phosphopeptide derived from GST-(
SH2)ZAP-70. Migration of molecular
weight markers is indicated on the right. Peptides likely to contain
phosphorylated Tyr292 or
Tyr492/Tyr493 are indicated by
filled and open arrowheads, respectively.
B, 32P-labeled peptides from CNBr-cleaved
GST-(
SH2)ZAP-70 were separated on a Tris-tricine gel and transferred
to polyvinylidene difluoride membrane. Peptide I was excised and eluted
as described under "Experimental Procedures," covalently coupled to
Sequelon-AA filter, and subjected to automated Edman degradation.
Derivatized amino acids from each cycle were monitored by liquid
scintillation counting. The expected residue for each cycle is given
according to ZAP-70 sequence overlapping the predicted N-terminal
portion of peptide I. Arrow indicates the CNBr cleavage
site. C, CNBr cleavage-derived phosphopeptides were
transferred onto nitrocellulose, and bands containing peptide I and II
were excised and further digested by trypsin. After lyophilization and
oxidation by performic acid, equal amounts of radioactivity from either
sample (panels I and II) or their mixture
(panel I + II) were analyzed by two-dimensional peptide
mapping on cellulose plates. + indicates the migration origin.
Arrowheads indicate putative doubly phosphorylated peptide
isoforms. D, putative doubly phosphorylated peptides
(indicated by arrowheads) were recovered from cellulose
plates after two-dimensional separation of trypsin-digested peptide I
and subjected to Edman degradation as outlined in B.
E, peptide I from in vivo labeled ZAP-70 or
in vitro labeled GST-(
SH2)ZAP-70 was analyzed by
two-dimensional tryptic peptide mapping as described in C.
In vivo and in vitro labeled material was mixed
and analyzed likewise for identifying shared peptides. Migration origin
and position of doubly phosphorylated peptides are indicated as in
C and D.
SH2)ZAP-70-Y319F or -Y315F
mutants.3 Thus, these data
show that peptides indicated by arrowheads (Fig. 2,
C and D) are actually isoforms phosphorylated on
both Tyr315 and Tyr319, whereas the additional
phosphopeptides observed in tryptic digest of peptide I (in the upper
part of the plate, Fig. 2, C and D) are likely to
represent peptides phosphorylated at either residue.
of the entire GST-(
SH2)ZAP-70 (Mr
~67,000) (Fig. 2A), these peptides are likely to be
partial cleavage products also containing Tyr(P)315 and/or
Tyr(P)319. Phosphorylation of ZAP-70 at residue
Tyr292, previously identified as an autophosphorylation
site (Ref. 19), was not evident with this construct.
SH2)ZAP-70 with those derived from
metabolically 32P-labeled ZAP-70 immunoprecipitated from
anti-CD3-treated Jurkat cells. CNBr cleavage of in vivo
phosphorylated ZAP-70 generated two peptides co-migrating with peptides
I and II (Fig. 2A). Analysis of tryptic maps of peptide I
from in vivo and in vitro phosphorylated ZAP-70
and their mixture revealed that both samples shared three peptides
migrating as Tyr315/Tyr319 doubly
phosphorylated forms (Fig. 2E). Singly phosphorylated peptides migrating in the upper part of the plates (see above) were
only barely detectable in the in vivo labeled peptide I. Moreover, an extra phosphopeptide appeared in this sample that was not
observed in the in vitro labeled protein. Identification of
this additional phosphopeptide was precluded by the very low radioactivity recovered after in vivo labeling. Similar
tryptic peptide patterns were obtained with ZAP-70 from
pervanadate-activated Jurkat (not shown). Preliminary experiments using
peptide-specific antibodies and ZAP-70 mutants suggest that peptides
from in vivo labeled ZAP-70 (Fig.
A, filled and open
arrowheads) contain phosphorylated Tyr292 and
Tyr492/Tyr493, respectively.3
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Fig. 3.
Dominant negative effect of the ZAP-70-Y319F
on TCR-induced NFAT activity. A, Jurkat cells were
co-transfected by electroporation with 0.2 µg (black
bars), 1 µg (open bars), or 5 µg (hatched
bars) of pSR -puro vector, either empty (vector) or
containing Y315F or Y319F mutant cDNAs, together with the NFAT-luc
and pSV-
-galactosidase reporter plasmids. Transfected cells were
stimulated for 8 h with either anti-TCR mAb or phorbol
12-myristate 13-acetate + calcium ionophore A23187. Luciferase
activities are corrected for transfection efficiency and expressed as a
percentage of the maximal luciferase activity measured after
stimulation with phorbol 12-myristate 13-acetate (PMA) + A23187. Comparable expression of both mutants was confirmed by anti-tag
immunoblot (not shown). Data are mean + S.E. from 2-4 independent
experiments. B, Jurkat cells stably expressing ZAP-70-Y319F
(1.40 and 1.60), ZAP-70-Y315F (2.21/2 and 2.21/14), or the empty
pSR
-puro vector (J05.2) were co-transfected with NFAT-luc and
pSV-
-galactosidase plasmids and left unstimulated (open
bars) or stimulated with anti-TCR mAb for 8 h (black
bars). Luciferase activities (mean + S.E. from 2-6 independent
experiments for each clone) were normalized and expressed as described
above. C, PhosphorImager scan showing the relative
expression levels of exogenous ZAP-70 proteins (WT or mutants) in
stably transfected Jurkat cells. Equal amount of proteins (as assessed
by Bradford assay) from the indicated cell line were subjected to
SDS-PAGE and immunoblotting with the anti-tag antiserum. Detection was
performed by 125I-labeled protein A, followed by
PhosphorImager scanning of the membrane.
-chain (Fig. 4A, upper panel),
thus indicating that the mutation did not affect the ability of ZAP-70
to bind the ITAMs.
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Fig. 4.
Effect of Y319F mutation on
activation-induced tyrosine phosphorylation and catalytic activity of
ZAP-70. A, unstimulated ( ) or anti-CD3-stimulated (+)
cells from the indicated transfectants were lysed, immunoprecipitated
with anti-tag antiserum, and analyzed by SDS-PAGE followed by
anti-phosphotyrosine immunoblotting (upper panel). Migration
of the molecular weight markers is indicated on the right;
positions of ZAP-70tag proteins (WT or Y319F), immunoprecipitating
antibody heavy chain (IgH), and phosphorylated
-chain
(
-PO4) are indicated on the left. The same blot
was stripped and reprobed with an anti-tag monoclonal antibody
(lower panel). Blots were revealed by enhanced
chemiluminescence. B, relative catalytic activities of
ZAP-70WT or ZAP-70-Y319F in anti-tag immunoprecipitates from
unstimulated (open bars) or pervanadate-treated (black
bars) transfectants were measured by an in vitro kinase
assay using cfb3 as substrate. Kinase reaction products were separated
by SDS-PAGE and then transferred onto polyvinylidene difluoride
membranes. In order to normalize the phosphorylation of cfb3 for the
amount of ZAP-70 immunoprecipitated in each sample, the upper part of
the membrane (Mr >55,000) was cut and subjected
to anti-tag immunoblot and detection by 125I-labeled
protein A. Both 32P-labeled cfb3 (Mr
~45,000) and 125I associated to ZAP-70tag band were
quantified by PhosphorImager scanning. Relative catalytic activities of
ZAP-70WT or ZAP-70-Y319F before and after activation were obtained by
normalizing 32P-cfb3 band volumes for the respective
protein 125I-band volume. The experiment shown is
representative of five independent determinations.
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Fig. 5.
Overexpression of ZAP-70-Y319F inhibits
antigen-induced IL-2 production and tyrosine phosphorylation of ZAP-70
substrates. A, PhosphorImager scan showing the
expression levels of endogenous wild-type ZAP-70 (ZAP-70endo)
versus exogenous ZAP-70 mutants (ZAP-70tag) in T8.1
hybridomas stably expressing ZAP-70KD, -Y315F, and -Y319F. Equal
amounts of proteins (as assessed by Bradford assay) from the T8.1
hybridoma or the indicated transfectant were analyzed by SDS-PAGE and
immunoblot with the 4.06 anti-ZAP-70 antiserum. The transfected
constructs (ZAP-70tag) have a decreased electrophoretic mobility
compared with the endogenous form (ZAP-70endo) due to the presence of
the C-terminal epitope tag. Detection and analysis were performed as
outlined in Fig. 3C. B, T8.1 T cell hybridoma or
transfectants stably expressing the indicated mutant (KD, Y315F, or
Y319F) were stimulated by APC prepulsed with increasing amount of
tt830-843 peptide antigen. IL-2 released in the supernatant was
measured by enzyme-linked immunosorbent assay. Each point
represents the average of triplicate samples. C, T8.1 T cell
hybridoma or transfectants stably expressing ZAP-70-Y319F (clone 11;
cf. Fig. 3B) or ZAP-70KD (clone 3.3) were
stimulated by APC prepulsed with 10 µg of tt830-843 peptide antigen.
Cells were then lysed, and the phosphorylation of cellular proteins was
assessed by SDS-PAGE and anti-phosphotyrosine immunoblotting. Similar
results were also obtained with two other independent transfectants
expressing ZAP-70-Y319F and another two expressing ZAP-70KD.
DISCUSSION
SH2)ZAP-70 construct used for in
vitro labeling. Also, we suspect that the direct fusion of GST
protein to the interdomain B might have precluded in vitro
phosphorylation of Tyr292 (19), perhaps as a consequence of
steric hindrance. Our experiments also show that in vivo
labeled peptide I contained an extra spot that could not be identified.
This spot, containing only phosphotyrosine residues,3
likely derives from a CNBr-cleaved peptide co-migrating with peptide I
and including a tyrosine that cannot be autophosphorylated by ZAP-70
in vitro.
SH2)ZAP-70 autophosphorylates in vitro on both
Tyr315 and Tyr319 are in agreement with that
report and suggest that in vivo phosphorylation of
Tyr315 and Tyr319 could similarly be achieved
by autophosphorylation of ZAP-70, although contribution of other PTKs
(e.g. Lck) cannot be ruled out. Indeed, as shown by
Neumeister et al. (11), trans-phosphorylation between
adjacent ZAP-70 molecules on multiple tyrosine residues occurs
following their binding to phosphorylated ITAMs on the TCR-
chain.
This mechanism, which would not require phosphorylation of ZAP-70 by
Src-PTKs (see below), generates binding sites for multiple substrates,
effectors, or other regulatory molecules on ZAP-70 and could lead to
phosphorylation of Tyr315 and Tyr319.
1 to Syk and the tyrosine phosphorylation
of the former by Syk (49). Although we cannot rule out the possibility
that Tyr319 is similarly involved in the binding of PLC
1
to ZAP-70, it would be difficult to explain how the mutation of
Tyr319 and the consequent lack of PLC
1 binding could
dramatically impair activation-induced tyrosine phosphorylation and
catalytic activity of ZAP-70 (see Fig. 4), as well as phosphorylation
of known substrates of this PTK (see Fig. 5). In this context, it is
worth noting that the strong inhibitory effect of ZAP-70-Y319F
contrasts with those seen associated with the mutation of
Tyr292 (20, 21) and, more recently, with
Tyr597/Tyr598 (28), both resulting in a
gain-of-function phenotype, or with the mutation of Tyr315
and Tyr474 (this work and Ref. 27), which have a mild
inhibitory effect in T cell signaling. Rather, the phenotype observed
for ZAP-70-Y319F is reminiscent of that caused by mutation of tyrosines
present in the activation loop of ZAP-70, Y493F or Y492F/Y493F. Indeed, we and others have previously shown that these mutations result in
impaired calcium mobilization and NFAT activation after antigen receptor stimulation (12, 25) and in decreased pp36-38 phosphorylation and extracellular signal-regulated kinase activation (25). These effects could be explained if Tyr319 and its neighboring
residues were the docking site for other molecules directly involved in
the regulation of ZAP-70 catalytic activity. A possible candidate is
the Src-PTK Lck, as this enzyme is responsible for the phosphorylaton
of Tyr493 (12), an event that has been shown to up-regulate
ZAP-70 catalytic activity and to be required for this PTK to
phosphorylate its substrates (25). Several biochemical and functional
pieces of evidence recently obtained in our laboratory support this
hypothesis: we have found that Tyr319, but neither
Tyr315 nor other in vivo phosphorylated
tyrosines of ZAP-70
(25),4
specifically binds the SH2 domain of Lck. Consistent with these data,
replacement by mutagenesis of the natural sequence 319YSDP
of ZAP-70 by an optimal binding motif for the SH2 domain of Src-PTKs
(sequence YEEI; Ref. 50) results in strong gain-of-function mutant
inducing augmentation of NFAT transcriptional activity in Jurkat cells
at levels >10-fold higher compared with the wild-type molecule and a
more efficient ZAP-70 phosphorylation.5 Binding of Lck to
ZAP-70 to up-regulate its catalytic activity bears an analogy with the
model proposed for the FAK PTK, the activation of which depends on an
SH2-mediated association with Src and/or Fyn PTKs (36, 51). Thus, the
SH2-mediated binding of Lck to ZAP-70 would ensure, by a relatively
stable interaction, persistent phosphorylation/activation of the latter
and downstream signaling while TCR is engaged with the antigen.
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ACKNOWLEDGEMENTS |
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We thank Drs. C. T. Baldari, M. Arpin, W. Knapp, R. P. Sekaly, E. L. Reinherz, R. Wange, A. Weiss, and S. C. Ley for antibodies and plasmids; J. D'Alayer and M. Davi for peptide sequencing; S. Pellegrini, B. Malissen, A. Alcover, and R. Weil for critical reading the manuscript and suggestions; and W. Houssin for excellent secretarial assistance.
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FOOTNOTES |
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* This work was supported by grants from the Institut Pasteur, the Association pour la Recherche sur le Cancer, the CNRS, and the Human Frontier Science Program.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.
§ Past recipient of an Association pour la Recherche sur le Cancer fellowship and presently supported by Human Frontier Science Program.
To whom correspondence should be addressed. Tel.:
33-1-4568-8637; Fax: 33-1-4061-3204; E-mail: oacuto{at}pasteur.fr.
2 M. Magistrelli, R. Bosotti, B. Valsassina, C. Visco, R. Perego, S. Toma, O. Acuto, and A. Isacchi, submitted for publication.
3 V. Di Bartolo and O. Acuto, unpublished results.
4 M. Pelosi, V. Di Bartolo, V. Mounier, D. Mège, J.-M. Pascussi, E. Dufour, A. Blondel, and O. Acuto, submitted for publication.
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ABBREVIATIONS |
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The abbreviations used are: TCR, T-cell antigen receptor; PTK, protein tyrosine kinase; SH2, Src homology 2; ITAM, immunoreceptor tyrosine-based activation motif; NFAT, nuclear factor of activated T cells; IL, interleukin; mAb, monoclonal antibody; cfb3, cytoplasmic fragment band 3; KD, kinase-defective; MES, 2-(N-morpholino)ethanesulfonic acid; WT, wild-type; PAGE, polyacrylamide gel electrophoresis; GST, glutathione S-transferase.
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REFERENCES |
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