From the Molecular Immunology Unit, Department of Immunology, Pasteur Institut, 25, Rue du Docteur Roux, 75724 Paris Cedex 15, France, and the § Howard Hughes Medical Institute, Univerisity of California, San Francisco, California 94143
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ABSTRACT |
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T cell receptor (TCR) triggering induces
association of the protein tyrosine kinase ZAP-70, via its two
src-homology 2 (SH2) domains, to di-phosphorylated
Immunoreceptor Tyrosine-based
Activation Motifs (2pY-ITAMs) present in the
intracellular tail of the TCR- chain. The crystal structure of the
SH2 domains complexed with a 2pY-ITAM peptide suggests that the
60-amino acid-long inter-SH2 spacer helps the SH2 domains to interact
with each other to create the binding site for the 2pY-ITAM. To
investigate whether the inter-SH2 spacer has additional roles in the
whole ZAP-70, we raised antibodies against two peptides of this region
and probed ZAP-70 structure under various conditions. We show that the
reactivity of antibodies directed at both sequences was dramatically
augmented toward the tandem SH2 domains alone compared with that of the entire ZAP-70. This indicates that the conformation of the inter-SH2 spacer is not maintained autonomously but is controlled by sequences C-terminal to the SH2 domains, namely, the linker region and/or the
kinase domain. Moreover, antibody binding to the same two determinants
was also inhibited when ZAP-70 or the SH2 domains bound to the
chain or to a 2pY-ITAM. Together, these two observations suggest a
model in which intramolecular contacts keep ZAP-70 in a closed
configuration with the two SH2 domains near to each other.
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INTRODUCTION |
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ZAP-70 is a protein tyrosine kinase (PTK)1 essential for the initiation of the signaling cascade activated by T cell antigen receptor triggering (1). Overall, ZAP-70 displays two structurally and functionally distinct moieties, an N-terminal one composed of two SH2 domains and a C-terminal kinase domain tethered by an ~80-amino acid-long linker (2) (also referred to as Interdomain B, (IB); Ref. 3).
So far, the only known function of the region comprising the two SH2
domains (hereafter indicated as (SH2)2) is to provide a
means to recruit ZAP-70 to the plasma membrane by those TCRs engaged
with the ligand (4, 5). This is achieved through the coordinated
anchorage of the SH2 domains to di-phosphorylated tyrosine-containing
motifs
(D/E)XXYXX(I/L)X6-8YXX (I/L)
called ITAMs (for Immunoreceptor Tyrosine-based
Activation Motifs) present within the
cytoplasmic tails of TCR subunits and
(6-8). Thereafter,
ZAP-70 undergoes tyrosine phosphorylation culminating in the
up-regulation of its catalytic activity which in turn is required for
phosphorylating cellular substrates (9-12).
The x-ray crystal structure of the (SH2)2 of human ZAP-70
complexed with a di-phosphorylated ITAM (2pY-ITAM) peptide (3) has
revealed an unsuspected structural complementarity and immediate contiguity of the two SH2 domains needed to create a high affinity binding site for the 2pY-ITAM. Thus, while the C-terminal SH2 possesses
a binding pocket for the first pY of the ITAM, the corresponding pocket
for the second pY in the N-terminal SH2 is contributed, in part, by
residues of the C-terminal SH2. Moreover, the 60 amino acids forming
the inter-SH2 spacer (hereafter referred to as Interdomain A (IA)) (3)
bulges out the SH2 domains and for the most part is structured as a
coiled-coil of two antiparallel -helices which assists in the
formation of an interface between the two SH2 domains. It has been
speculated that the IA may mediate additional intra- or inter-molecular
interactions required for regulating ZAP-70 (3). This idea stems from
several considerations. First, coiled-coils are often found to
intervene in protein-protein interactions (13, 14). Second, there is at
least one highly suggestive example involving the spacer connecting the
two SH2 domains of the p85 subunit of the phosphatidylinositol
3'-kinase (PI 3-kinase). This region, predicted to be a coiled-coil,
mediates the interaction with the catalytic p110 subunit (15, 16), and
occupancy of the two SH2 domains influences the enzymatic activity (17,
18). Moreover, binding of singly or doubly phosphorylated peptides to
the tandem SH2-containing SH-PTP-2 activates the phosphatase activity
(19, 20). Finally, it has been reported that the catalytic activity of
p72syk, a PTK homologue of ZAP-70, may be increased by binding
to a 2pY-ITAM (21, 22). The latter examples are suggestive of
allosteric regulation mediated by the SH2-containing region of the
protein.
To explore what could be the structural role of the IA in the entire
ZAP-70, anti-peptide antibodies directed at this region were generated.
The use of these antibodies revealed that binding of ZAP-70 or its
isolated (SH2)2 to a 2pY-ITAM or the TCR- chain influences the conformation of the IA. Additional experiments also
indicated that conformational constraints are imposed on the IA by the
regions of the protein downstream of the (SH2)2. These two
observations, in combination, suggest a structural model for the entire
ZAP-70.
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EXPERIMENTAL PROCEDURES |
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Antibodies, Cell Lines, and Vectors--
Anti-human ZAP-70 4.06 and 2.06 polyclonal antisera were produced by immunizing rabbits (a
total of four, two for each peptide) with the synthetic peptides
described in Table I coupled via their C- and N-terminal cysteines
(cysteine 117 of ZAP-70), respectively, to maleimide-activated keyhole
lympet hemocyanin as recommended (Pierce). A similar procedure was used
to generate an anti- antiserum (named zeta-N), which is directed at
the first 11 amino acid residues of the human
chain. The
anti-kinase domain polyclonal antiserum 21.11 has been previously
described (23). The ZAP-4 antibody (24) was kindly provided by S. Ley
(National Institute of Medical Research, Mill Hill, London. UK). The
anti-phosphotyrosine mAb 4G10 was purchased from Upstate Biotechnology,
Inc., (Lake Placid, NY). The anti-human TCR V
8 (101.5.2) mAb was
provided by E. L. Reinherz (Dana Farber Cancer Institute, Boston,
MA). Anti-VSV-G epitope antiserum (kindly provided by M. Arpin,
Institut Curie, Paris, France) reacts against an 11-amino acid
determinant derived from a vesicular stomatitis virus glycoprotein
(VSV-G) (25). WT15.8, a Jurkat cell line expressing a ZAP-70 containing
a VSV-Tag at the C terminus (12) was maintained in RPMI 1640 medium
supplemented with 10% fetal calf serum, L-glutamine,
penicillin, and streptomycin (Life Technologies Inc., France). The
generation of the cDNA construct that encodes the amino acids
1-276 of ZAP-70, comprising the SH2(N+C) plus 22 residues of the IB,
and the expression vector used (pBJ1) have been previously described
(26). The prokaryotic vector expressing the (SH2)2 of
ZAP-70 as a glutathione S-transferase fusion protein (a gift
from L. Samelson, National Institutes of Health, Bethesda, MD) and the
purification procedure of the protein have been reported (4). The
(SH2)2 molecule was cleaved from the glutathione
S-transferase with factor Xa on a glutathione-Sepharose column during the purification as recommended (Amersham Pharmacia Biotech). Gel-filtration analysis revealed the absence of aggregated protein.
Synthetic Peptides--
Peptides corresponding to human ZAP-70
sequences 106-117 and 117-130 and to amino acids 1 through 11 of the
human TCR chain (an additional cysteine was added to the C terminus
of this peptide for coupling to the carrier) purified by high
performance liquid chromatography were purchased from Neosystems
(Strasbourg, France). Peptides corresponding to the first ITAM of the
human TCR
chain (
1, residues 48-66) plus a 4-amino acid linker
at the N terminus (final sequence SGSGNQLYNELNLGRREEYDVLD) were
synthesized as mono- (on Tyr62) and di-phosphorylated (on
Tyr51 and Tyr62) forms (by F. Baleaux,
Dept. of Organic Chemistry, Institut Pasteur, Paris). Peptides
were purified by reverse-phase high performance liquid chromatography
and in part biotinylated at the N terminus by Biotin sulfo-NHS
(Pierce). The purity and the molecular weight of the peptides were
confirmed by ion electro-spray ionization mass spectroscopy.
Activation and Immunoprecipitation-- For activation, cells were stimulated with anti-TCR mAb 101.5.2 at 1:200 dilution of ascites for 2 min at 37 °C. Unstimulated or TCR-activated cells were solubilized at 108 cells/ml for 15 min in ice-cold lysis buffer containing 1% Nonidet P-40, 20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM MgCl2, and 1 mM EGTA in the presence of inhibitors of proteases and phosphatases (10 µg/ml leupeptin, 10 µg/ml aprotinin, 1 mM Pefabloc-sc, 50 mM NaF, 10 mM Na4P2O7, and 1 mM NaVO4). Precleared postnuclear lysates were incubated with or without the synthetic ITAM peptides at the indicated concentration for 90 min at 4 °C. After incubation, lysates were subjected to immunoprecipitation for 1-2 h with 1 to 5 µl of intact polyclonal antisera preadsorbed to protein A-Sepharose as described (12). In some experiments streptavidin-agarose beads (Amersham Pharmacia Biotech) were utilized to precipitate ITAM-bound ZAP-70. Similar procedures were employed when purified recombinant ZAP-70 (SH2)2, typically ~50 ng in 100 µl of lysis buffer containing 0.1 mg/ml of bovine serum albumin, were reacted with anti-IA antisera and ITAM peptides. After separation on SDS-polyacrylamide gel electrophoresis under reducing conditions and blotting, proteins were detected by enhanced chemiluminescence when the anti-phosphotyrosine antibody 4G10 was used or by 125I-labeled protein A (Amersham Pharmacia Biotech) in all the other experiments as described previously (12). Molecular weight markers, Mark12 MW standards were purchased from Novex. Quantitation of 125I-labeled proteins was performed using ImageQuant software after scanning in a PhosphorImager (Molecular Dynamics).
Transient Transfections-- Transfection of Jurkat cells was performed by electroporating (at 260 V, 960 microfarads) 107 Jurkat cells in 0.5 ml of RPMI 1640 medium supplemented with 20% fetal calf serum in a Gene Pulse cuvette (Bio-Rad) (12) with various amounts (5-30 µg) of pBJ1 vector expressing the (SH2)2 domains of ZAP-70. Forty h later, transfected cells were either left unstimulated or stimulated with anti-TCR mAb for 2 min at 37 °C, lysed, and subjected to immunoprecipitation with the indicated antisera.
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RESULTS |
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Antibodies Directed at the IA of ZAP-70 Do Not Recognize the
Protein Bound to the Chain--
Two antisera (named 4.06 and 2.06)
were raised against synthetic peptides corresponding to the amino acid
sequences 106-117 and 117-130 of ZAP-70, respectively (Table
I). The peptide segment 106-117 begins
shortly after the end of the N-terminal SH2 domain, and together, the
two peptides cover ~40% of the IA (2, 3). To investigate whether the
two antisera recognized native ZAP-70 and to compare their reactivity
with that of other anti-ZAP-70 antisera directed at different regions
of the molecule, immunoprecipitation experiments were carried out
from lysates of Jurkat cells unstimulated or stimulated with an
anti-TCR mAb (Fig. 1). Similarly to 21.11 and ZAP-4 anti-peptide antisera, specific for sequences contained within the kinase domain and the IB, respectively (Table I), 4.06 and
2.06 immunoprecipitated tyrosine-phosphorylated ZAP-70 (Fig. 1,
pY-ZAP-70 in lanes 1-8). In this as
well as in other experiments, 2.06 was found to be weaker than 4.06 (see also below). However, in striking contrast with the antisera 21.11 and ZAP-4, in TCR-activated Jurkat cells, 4.06 and 2.06 did not show
co-immunoprecipitation of phosphorylated
chain (cf.
pY-
in lanes 2 and 4 with lanes 6 and 8). The
chain was positively identified by an
anti-
antiserum (zeta-N, lane 9) which, as
expected, in activated Jurkat cells co-immunoprecipitated with ZAP-70.
The lack of detection of the
chain with both anti-IA antisera was
not due to a lower capacity to immunoprecipitate ZAP-70. Indeed, with
the anti-IA antisera,
was not visible even when the signal of
ZAP-70 was similar to that obtained with 21.11 and ZAP-4
(cf. lane 2 with lane 6 or lane
4 with lane 5). In addition, the
chain remained
undetectable after longer exposure times (not shown). Phosphorylated
ZAP-70, not associated to the
chain but observable with anti-IA
antisera is likely to represent a fraction of the molecules which
detached from
spontaneously or as a consequence of anti-IA Ab
binding. These results suggested that the epitopes recognized by the
anti-IA antisera are masked or structurally modified when ZAP-70 is
complexed with the
chain.
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Binding of 2pY-ITAM to ZAP-70 or to the Isolated (SH2)2
Domains Inhibits Recognition by Anti-IA Antibodies--
The lack of
recognition of ZAP-70· complexes by anti-IA Abs could be due to an
alteration of the corresponding epitopes consequent to the binding of
the SH2 domains to the ITAMs. To directly test this hypothesis, we
assessed whether a synthetic peptide corresponding to a 2pY-ITAM
inhibited recognition of ZAP-70 by anti-IA Abs. For these experiments,
we used a Jurkat cell line named WT15.8, stably expressing ZAP-70
tagged at its C terminus with a VSV sequence (12). The use of tagged
ZAP-70 was preferred since the anti-tag antiserum was found to be the
strongest, thus allowing sensitive detection of ZAP-70. Cell lysates
from unstimulated WT15.8 were incubated with increasing concentrations
of mono- (pY) or di- (2pY) phosphorylated ITAM peptides containing a
biotin molecule at the N terminus and then reacted with
steptavidin-agarose or with the anti-ZAP-70 antisera. In agreement with
previous reports (5), only the 2pY-ITAM was able to bind ZAP-70, as
shown by precipitation with streptavidin-agarose (Fig.
2, lanes 12 and 13). Moreover, the 2pY-ITAM, but not the pY-ITAM, inhibited
the immunoprecipitation of ZAP-70 with the 4.07 antibody in a
dose-dependent manner (Fig. 2, cf. lanes
4, 5 and 6 with lanes 1, 2, and
3). Quantitation of the ZAP-70 band allowed calculation to
an ~90% inhibition of ZAP-70 immunoprecipitation in the presence of
10 µM of 2pY-ITAM. Similar levels of inhibition were
obtained when untagged ZAP-70 was immunoprecipitated from Jurkat cells
or when non-biotinylated 2pY-ITAM was used. Moreover, similar results were reproduced with 2.06 antiserum (data not shown). This effect was
restricted to the anti-IA antibodies since no inhibition was detected
with the anti-kinase domain 21.11 (lanes 8 and 9)
or with anti-Tag antisera (lanes 10 and 11). From
these results, we conclude that the binding of the 2pY-ITAM to ZAP-70
is the event that determines the loss of immunoreactivity.
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The Epitopes Recognized by the Anti-IA Antibodies Are Conformationally Dependent on the IB and/or the Kinase Domain-- During the course of the experiments involving the expression of isolated (SH2)2 of ZAP-70 in Jurkat cells, we consistently noted that this molecule was immunoprecipitated by the anti-IA Abs more efficiently than the whole ZAP-70. Experiments were therefore set up to determine the magnitude of this effect and to exclude possible artifacts due to overexpression of the (SH2)2. Thus, Jurkat cells were transiently transfected with different amounts of the expression plasmid containing the (SH2)2 to obtain different (SH2)2/endogenous ZAP-70 ratios. Immunoprecipitations were then carried out with the 4.06 and 2.06 antisera, and the bands corresponding to ZAP-70 and (SH2)2 were quantitated by immunoblotting using 4.06. Before the immunoprecipitation step, an aliquot of the total lysate was used to estimate the relative expression of both molecules. In the experiment shown in Fig. 4A, endogenous ZAP-70 was expressed at approximately a 10-fold excess compared with the transfected (SH2)2 (see lane 1 and, for quantitation, Experiment I in Table II). If the reactivity of the antisera against the two proteins was the same, then the (SH2)2/ZAP-70 ratio should remain constant in the immunoprecipitate. However, after immunoprecipitation with both 4.06 and 2.06 antisera, a relative increase in the signal of the transfected (SH2)2 over ZAP-70 was clearly evident (Fig. 4A, lane 2 and 3). Thus, while the amount of (SH2)2 was 10-fold lower than ZAP-70, after immunoprecipitation with 4.06, the signal obtained for the two proteins was nearly the same. This higher reactivity toward the (SH2)2 was even more dramatic for the 2.06 antiserum. This reagent immunoprecipitated ZAP-70 inefficiently compared with 4.06 (cf. lanes 2 and 3) but immunoprecipitated the (SH2)2 as efficiently as 4.06. The magnitude of these modifications in reactivity toward (SH2)2 compared with ZAP-70 can be quantitatively appreciated by confronting the (SH2)2/ZAP-70 signal ratios in the cell lysate and in the immunoprecipitates of 4.06 and 2.06. These ratios are reported in Table II for the experiment shown in Fig. 4A (Experiment I) and for two additional ones in which higher amounts of (SH2)2 compared with ZAP-70 were expressed. Independently of the initial amounts of (SH2)2 and ZAP-70 present, there is an increase of the (SH2)2/ZAP-70 ratio in the immunoprecipitates. It is clear that on average for the epitope recognized by 4.06 Abs there is a gain of reactivity of ~5-fold, whereas such a change can be estimated to be >100-fold for 2.06 (with this antiserum only, <1% of the total intact ZAP-70 is detected after a single immunoprecipitation). Fig. 4B also shows, from one of these experiments, a control of the structural intactness of the (SH2)2 versus endogenous ZAP-70. Both proteins were able to bind with similar capacity to the 2pY-ITAM peptide as demonstrated by the fact that their ratio after binding is comparable with the one seen in the total lysate (lane 1). This result excludes that the anti-IA antisera recognize a grossly altered population of (SH2)2 molecules unable to bind to 2pY-ITAM.
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DISCUSSION |
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The major factors determining recognition of an intact protein by
anti-peptide Abs are accessibility and conformational flexibility of
the epitope (27, 28). Thus, differences in reactivity of site-specific
anti-peptide Abs observed in proteins after ligand binding or due to
changes in the milieu composition have been useful in revealing
conformational changes (29, 30). In this paper, we show that the
reactivity of anti-peptide Abs directed at sequences within the
inter-SH2 spacer of ZAP-70 is dramatically decreased in two apparently
distinct situations: the binding of the two SH2 domains to the 2pY-ITAM
or the presence of the C-terminal moiety of ZAP-70 which includes the
IB and the kinase domain. Antisera to two contiguous sequences of the
inter-SH2 spacer reacted very strongly and equally well with the
(SH2)2 alone, but moderate-to-low, depending on the epitope
(106-117 > 117-130), toward the intact ZAP-70. Moreover, Ab
binding to the (SH2)2 alone as well as to the entire ZAP-70
was inhibited by the interaction with a 2pY-ITAM peptide, and both
proteins could not be co-immunoprecipitated with these antisera when
complexed to the chain.
In the crystal structure of the isolated (SH2)2 complexed
with a di-phosphorylated 1-ITAM (the same used in our study) (3), the sequence corresponding to the peptide 106-117 begins after three
residues from the end of the N-terminal SH2 domain as part of a type II reverse turn followed by a long
-strand. The contiguous 117-130 sequence, instead, assumes an
-helix configuration and represents part of the stem, composed of two antiparallel
-helices wrapped around each other, protruding at ~90° with respect to the
SH2 domains axis. The positioning of the 2pY-ITAM peptide in the complex is on the opposite side and away from the two sequences, thus making highly unlikely the possibility that the peptide itself causes inhibition of Ab binding by steric hindrance. Moreover, since
2pY-ITAM-dependent inhibition was observed also with
recombinant bacterially expressed (SH2)2 (Fig.
3C), we can exclude that an unknown protein present in T
cells binds to the complex and hinders the sequences targeted by the
Abs. In addition, lack of Abs detection of (SH2)2
associated to the
chain was not the consequence of a
phosphorylation event (Fig. 3A) that might involve
Tyr126 in the inter-SH2 spacer (10). Thus, the most likely
explanation of our results is that in the isolated (SH2)2
the two determinants (and perhaps the entire IA) exist in a state of
high conformational flexibility that facilitates binding of the
anti-peptide antibodies (27, 28). In support of this, Hatada et
al. (3) noted from isoelectrofocussing experiments that the
uncomplexed (SH2)2 of ZAP-70 existed in multiple isoforms
that could be converted to a single one after binding to the 2pY-ITAM.
The 2pY-ITAM-dependent inhibition of antibody binding can
therefore be explained by assuming that the determinants in question
undergo a conformational change and/or they are partially or totally
masked as a consequence of global changes coinciding with a
stabilization of the entire (SH2)2 structure by the
2pY-ITAM. By inference, the same reasoning may apply to the intact
ZAP-70 where loss of anti-IA Ab reactivity was also induced by binding
to 2pY-ITAM or tyrosine-phosphorylated TCR-
. It appears, therefore,
that when ZAP-70 binds to the
chain, the (SH2)2 region
assumes a configuration that is not present in at least part of the
unbound molecules (see also below). A recent work has described
conformational changes of the ZAP-70 homologue p72syk when
bound to a 2pY-ITAM peptide detected with anti-peptide antibodies directed at its C-terminal end (31). These data together with ours
suggest that relatively important changes in the configuration of this
family of kinases take place upon binding to antigen receptors.
The second information gained from our studies is that in the intact ZAP-70 the IA assumes a particular configuration imposed by the protein portion downstream of the SH2 domains. Thus, the inability of the anti-IA Abs, especially those directed at the 117-130 sequence, to recognize a large proportion of intact ZAP-70 might be due to the IB and/or the kinase domain restricting the conformational freedom of the IA or causing a steric hindrance. Independently of these two possibilities, our observation indicates that an intramolecular interaction must exist that links the (SH2)2 to the IB and/or the kinase domain and suggests that ZAP-70 exists in a closed rather than in an extended configuration.
The observation that both inter- (e.g. 2pY-ITAM
binding) and intramolecular interactions of ZAP-70 lead to structural
changes identified by similar effects (e.g. inhibition/loss
of anti-IA Ab reactivity) is highly suggestive of comparable
configurations assumed by the (SH2)2 in both instances.
Thus, it is possible that the interaction of the (SH2)2
(perhaps through the IA) with a distal part of the molecule helps
maintain the (SH2)2 in a relatively stable configuration
which approaches the one assumed when bound to a 2pY-ITAM. Such a
configuration may favor a fast kinetics of ZAP-70 binding to the
2pY-ITAM (due to an entropic gain) as the SH2 domains would already be
oriented close to each other in such a way as to facilitate their
targeting. This is particularly relevant in light of the fact that the
constitution of a high affinity binding site for 2pY-ITAM necessitates
the immediate proximity of the N-terminal SH2 and C-terminal SH2 (3).
2pY-ITAM binding kinetics experiments with intact ZAP-70 and
(SH2)2 alone should help verify this hypothesis. However,
if the proposed model is correct, the closed configuration may be
metastable since both anti-IA antisera were able to recognize a
population of molecules that transit through, or are constitutively in,
an open configuration. Binding to the chain may then impose further
modification to the IA, independent of the regions downstream of the
(SH2)2.
A growing body of data suggests that inter-SH2 domain spacers may
be important for contributing toward establishing the orientation of
tandem SH2 domains and perhaps transmit allosteric signals to the
catalytic portion of the proteins when the SH2s bind to phosphorylated
peptides (15, 20, 22, 32-35). Among these examples and of particular
relevance for our studies is that p72syk binding to a 2pY-ITAM
is sufficient for inducing an initial up-regulation of its enzymatic
activity (21, 22). It is conceivable that, given the high structural
homology between p72syk and ZAP-70, also the IA of the former
is subjected to structural constraints by the C-terminal moiety of the
molecule. Interestingly, Brunati et al. (36) found that the
catalytic domain of rat p72syk exhibits a
kcat 6-fold higher than that of full-length
p72syk. This suggests that the (SH2)2 region may
repress the catalytic activity of p72syk by interacting with
the catalytic domain. However, so far, attempts to evidence an
up-regulation of ZAP-70 catalytic activity by 2pY-ITAMs-containing peptides have failed (37, 38), but interestingly enough, an increase of
ZAP-70 activity was noted after binding to a dimeric form of
phosphorylated chain and was presumably the consequence of a
trans-phosphorylation event (38).
In conclusion, anti-IA Abs have allowed us to define structural changes occurring in the ZAP-70 molecule after TCR engagement. Moreover, we provide for the first time evidence that intramolecular interactions exist between different regions of ZAP-70, a finding that should prompt additional studies to understand whether they have regulatory roles in the PTKs of the Syk family.
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ACKNOWLEDGEMENTS |
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We thank Drs. E. L. Reinherz, S. Ley, M. Arpin, and L. Tuosto for kindly providing antibodies; L. A. Samelson for ZAP-70 SH2 constructs, and F. Baleaux for help with peptide synthesis and purification. We also thank V. Di Bartolo, L. Tuosto, M. Pelosi, F. Colotta, A Pintar, and P. Alzari for critical reading of the manuscript and Ms. 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.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.
On leave of absence from Pharmacia-Upjohn (Nerviano, Italy).
¶ Investigator of the Howard Hughes Medical Institute.
To whom correspondence should be addressed: Molecular
Immunology Unit, Dept. of Immunology, Pasteur Institute, 25, rue du Dr.
Roux, 75724 Paris, Cedex 15, France. Tel.: 33-1-4568-8637; Fax:
33-1-4061-3204. E-mail address: oacuto{at}pasteur.fr.
1 The abbreviations used are: PTK, protein tyrosine kinase; Ab, antibody; mAb, monoclonal antibody; TCR, T-cell antigen receptor; ITAM, immunoreceptor tyrosine-based activation motif; SH2, Src homology domain 2; IA, interdomain A; IB, interdomain B; 2pY-ITAM, di-phosphorylated ITAM; VSV, vesicular stomatitis virus.
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REFERENCES |
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