From the The protein tyrosine kinase ZAP-70 plays a
central role in T-cell activation. Following receptor engagement,
ZAP-70 is recruited to the phosphorylated subunits of the T-cell
antigen receptor (TCR). This event results in ZAP-70 activation and in
association of ZAP-70 with a number of signaling proteins. Among these
is the Shc adaptor, which couples the activated TCR to Ras. Shc
interaction with ZAP-70 is mediated by the Shc PTB domain. The
inhibitory effect of a Shc mutant containing the isolated PTB domain
suggests that Shc interaction with ZAP-70 might be required for TCR
signaling. Here, we show that a point mutation (Phe474) of
the putative Shc binding site on ZAP-70, spanning tyrosine 474, prevented ZAP-70 interaction with Shc and the subsequent binding of Shc
to phospho- Since its identification as a tyrosine phosphoprotein associated
with the The interaction with Shc links ZAP-70 to the Ras activation pathway.
Ras activity is strictly controlled by a differential subcellular
localization of Ras itself and of nucleotide exchange factors, such as
Sos, responsible for guanine nucleotide exchange on Ras. Activation is
achieved through recruitment of Sos from the cytosol to the plasma
membrane, an event that permits its interaction with Ras (reviewed in
Ref. 20). Shc plays a key role in this process by binding either to
activated receptor PTKs or to other membrane localized PTKs, such as
Src. This interaction, which involves the SH2 domain of Shc, results in
the generation by these PTKs of two binding sites for Grb2, a second
adapter molecule, which in turn can recruit Sos to the plasma membrane through its double SH3 domain (reviewed in Ref. 21). Shc has been
implicated in both T-cell and B-cell antigen receptor signaling to Ras
(16, 22-24). Upon TCR triggering, Shc binds to the phosphorylated TCR
We have recently shown that Shc binds to ZAP-70 in response to TCR
triggering (16). This interaction is mediated by a second phosphotyrosine binding domain, termed PTB, which is located in the
amino-terminal region of Shc and which binds tyrosine-phosphorylated proteins with a different specificity as compared with the SH2 domain
(25). Overexpression of the isolated PTB domain inhibits downstream
events induced by TCR engagement, suggesting that Shc interaction with
ZAP-70 might be required for TCR signaling. We have identified a
putative binding site for the Shc PTB domain at tyrosine 474 of ZAP-70
(16). In this study, we used a point mutant of ZAP-70 to investigate
the role of Tyr474 in the interaction of ZAP-70 with Shc
and the relevance of this interaction to TCR signaling. We show that
mutation of Tyr474 impairs TCR-dependent gene
activation and prevents ZAP-70 interaction with Shc, suggesting a
crucial role for Tyr474 in ZAP-70 function and supporting
the relevance of ZAP-70 interaction with Shc in TCR signaling.
Furthermore, we provide evidence for a role of ZAP-70 in linking the
TCR to the Ras activation pathway through phosphorylation of the Grb2
binding sites of Shc.
Plasmids--
NF-AT/luc contains a trimer of the NF-AT binding
site of the interleukin-2 promoter upstream of the gene encoding
firefly luciferase (16). LTR/CAT, containing the chloramphenicol acetyl transferase (CAT) gene under the control of the HIV-1 long terminal repeat (26), was used as transfection control. A Shc deletion mutant
containing the isolated PTB domain under the control of the CMV
enhancer was described previously (16). A ZAP mutant containing a
tyrosine to phenylalanine substitution at position 474 (Phe474ZAP-70) was generated by M13-based,
oligonucleotide-directed, site-specific in vitro mutagenesis
of ZAP-70 cDNA using standard procedures. The sequence of the
mutagenized cDNA was checked by automated DNA sequencing. The
cDNA was subcloned as an EcoRI fragment into the
mammalian expression vectors pcDNAamp and RcCMV (Invitrogen, Leek,
Netherlands)
Antibodies, GST Fusion Proteins and Peptides--
IgG from OKT3
(American Type Culture Collection) hybridoma supernatants were purified
on Mabtrap (Amersham Pharmacia Biotech). The anti-TCR mAb BMA031 (27)
was kindly provided by R. Kurrle. Anti-Shc antibodies included rabbit
polyclonal antibodies raised against a Shc CH-GST fusion protein (28)
and a PTB-GST fusion protein (16) and a mouse mAb raised against a Shc
SH2-GST fusion protein (23). Anti-phosphotyrosine and anti-ZAP-70 mAbs
were purchased from Upstate Biotechnology Inc. Anti-ZAP-70 polyclonal antibodies and an anti- Transfections, Luciferase and CAT Assays, and Flow
Cytometry--
Transfections were carried out as described using a
modification of the DEAE-dextran procedure (30). To minimize
variability among samples, activations were carried out on aliquots of
a single pool of transfected cells. In addition, all samples were in
duplicate. A plasmid encoding bacterial CAT under the control of the
HIV long terminal repeat (0.4 µg/sample) was included in all
cotransfections as a control of transfection efficiency. Cells were
allowed to recover for 22 h before activation. Cells were
collected 8-10 h after activation and assayed for luciferase activity
using a modification of the Promega (Madison, WI) protocol (31).
Luciferase activity in the absence of stimulation was barely detectable
(0.005-0.010 relative luciferase units), whereas luciferase activities
in control samples activated with anti-TCR mAb ranged from 1.000 to
2.000 relative luciferase units. Test samples were compared with
control samples within the same experiment. CAT assays were carried out as described (32) using equal amounts of proteins for each sample. Thin
layer chromatograms were scanned, and chloramphenicol acetylation was
quantitated using a PhosphorImager (Molecular Dynamics, Sunnyvale, CA).
Luciferase values were adjusted to the protein concentration and
normalized to the CAT values to correct for variations in transfection
efficiency. Each experiment was repeated 3-5 times. The
RcCMV-Phe474ZAP vector was introduced into Jurkat cells by
electroporation, and stably transfected cells were selected in medium
containing 1 mg/ml G418 (Life Technologies, Inc.). Expression of
Phe474ZAP-70 was evaluated by sequential immunoblotting of
the cell lysates with anti-ZAP-70 and anti- Activations, Immunoprecipitations, Immunoblots, and Kinase
Assays--
Activations were carried out as described (16). Cells were
lysed in 1% (v/v) Triton X-100 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 aprotinin, 1 µg/ml
pepstatin, and 10 mM phenylmethylsulfonyl fluoride), and
postnuclear extracts (500-1000 µg/sample) were immunoprecipitated
using the appropriate polyclonal antibodies. To disrupt pre-existing
molecular interactions in competition experiments with ZAP-70 peptides,
cells were lysed in 3% (v/v) Triton X-100. Immunoblots were carried
out using a chemiluminescence detection kit (Pierce). Recovery of the
specific proteins in the immunoprecipitates was always checked by
immunoblotting the stripped filters with mAbs of the same specificity
as the polyclonal antiserum used for immunoprecipitation. Molecular
weight markers were purchased from Amersham. In vitro kinase
assays of either Shc-specific immunoprecipitates, glutathione-Sepharose-bound GST fusion proteins, or Affi-Gel-conjugated peptides were carried out in 20 µl of 20 mM Tris-HCl, pH
7.4, 10 mM MgCl2, 10 mM
MnCl2, 50 µM ATP, 5 µCi
[ Mutation of Tyr474 on ZAP-70 Results in Impaired
TCR-dependent Gene
Activation--
TCR-dependent gene activating signals can
be conveniently traced using a reporter construct under the control of
the NF-AT transcription factor (reviewed in Ref. 33). To assess the
relevance to TCR signaling of residue Tyr474 on ZAP-70, a
ZAP-70 mutant was generated by site-specific mutagenesis, carrying a
tyrosine to phenylalanine substitution at position 474 (Phe474ZAP-70). The mutant cDNA was subcloned in a
mammalian expression vector under the control of the CMV enhancer and
cotransfected in Jurkat cells together with a NF-AT/luciferase reporter
construct. As shown in Fig. 1, a
significant reduction of TCR-induced NF-AT activation was observed in
the presence of Phe474ZAP-70, suggesting a role for
Tyr474 of ZAP-70 in TCR signal transduction. As reported
(16) and as shown in Fig. 1, a similar inhibition was also detected
following expression of a dominant negative mutant encoding the
isolated Shc PTB domain, suggesting that the inhibitory effect of
Phe474ZAP-70 might result from a failure of this mutant to
interact with Shc. No significant NF-AT activation was detected in the absence of stimulation in cells transfected with either control vector
or the test expression constructs (see under "Materials and
Methods").
Department of Evolutionary Biology,
Pharmacia & Upjohn SpA,
ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
. Neither ZAP-70 catalytic activity nor the pattern of
protein phosphorylation induced by TCR triggering was affected by this
mutation. However expression of the Phe474 ZAP-70 mutant
resulted in impaired TCR-dependent gene activation. ZAP-70
could effectively phosphorylate Shc in vitro. Only the CH
domain, which contains the two Grb2 binding sites on Shc, was phosphorylated by ZAP-70. Both Grb2 binding sites were excellent substrates for ZAP-70. The data show that Tyr474 on ZAP-70
is required for TCR signaling and suggest that Shc association with
ZAP-70 and the resulting phosphorylation of Shc might be an obligatory
step in linking the activated TCR to the Ras pathway.
INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
chain of the activated T-cell antigen receptor (TCR),1 the protein tyrosine
kinase (PTK) ZAP-70 has emerged as a central molecule in the generation
and propagation of critical early signaling events initiated by TCR
triggering and culminating in T-cell proliferation and differentiation
(1-5). In contrast to Src family PTKs, ZAP-70 is found in resting
peripheral T-cells cells as a cytosolic protein. Activation of ZAP-70
requires plasma membrane localization, which is achieved through a
complex interaction of the tandem SH2 domains of ZAP-70 with specific
tyrosine-phosphorylated motifs, termed immunoreceptor tyrosine-based
activation motifs (ITAMs) (6), on the CD3-bound
chain (7, 8). The
importance of this step is highlighted by recent reports showing that
the SH2 domains become dispensable if ZAP-70 is artificially targeted
to the plasma membrane (9). A key role in ZAP-70 recruitment to the
activated TCR is played by Src family PTKs, the most prominent one
being Lck, which are responsible for
chain phosphorylation
(10-12). Lck is also implicated in the subsequent catalytic activation of ZAP-70 through phosphorylation of tyrosine 493, a site required for
TCR function (13). Activated ZAP-70 autophosphorylates a number of
tyrosine residues, which become potential docking sites for a number of
signaling proteins, including Lck itself, Fyn, Ras-GAP, abl, Shc, Cbl,
and Vav (14-18). However, with the exception of Vav (19), the
relevance of these interactions has as yet not been established.
chain through its carboxyl-terminal SH2 domain and becomes in turn
phosphorylated, an event that results in recruitment of Grb2/Sos (22,
23).
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
mAb were purchased from Santa Cruz
Biotechnology (Santa Cruz, CA). Soluble or Sepharose 4B-immobilized GST
fusion proteins containing either the SH2 domain, or the PTB domain or the CH domain of Shc were previously described (29). Furthermore, GST
fusion proteins containing a 20-amino acid region of Shc spanning residues 231-248 or 309-325, as well as similar constructs with tyrosine to phenylalanine substitutions at residues 317 or 239/240, were used. Lck kinase was purchased from Upstate Biotechnology Inc.
Recombinant
GST-ZAP-702 was
purified on GSH-Sepharose (Pharmacia) according to the manufacturer's instructions. Peptides were synthesized and purified by Chiron Mimotopes (Clayton, Victoria, Australia). Peptide coupling to Affi-Gel
(Bio-Rad srl, Milan, Italy) was carried out according to the
manufacturer's instructions.
tubulin mAb. Cells were
analyzed for TCR/CD3 surface expression using fluorescein
isothiocyanate-labeled anti-CD3 mAb and a FACScan flow cytometer
(Becton Dickinson, San Jose, CA).
-32P]ATP for 20 min at 37 °C using 10 units of Lck
(Upstate Biotechnology) or 125 ng of GST-ZAP-70 per reaction. The
reaction products were extensively washed in PBS/0.2 mg/ml sodium
orthovanadate before either scintillation counting or gel
electrophoresis. ZAP-70 autophosphorylation activity in
Phe474ZAP-70-expressing cells was tested under the same
experimental conditions on ZAP-70-specific immunoprecipitates from
cells lysed in 3% Triton X-100.
RESULTS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Fig. 1.
Inhibition of TCR-dependent NF-AT
activation by expression of Phe474ZAP-70. Relative
luciferase activity in Jurkat cells cotransfected with NF-AT/luc and
either control vector or the same vector expressing either
Phe474ZAP-70 (F474ZAP) or the isolated Shc PTB
domain. Cells were activated with anti-TCR mAb. The reporter plasmid
LTR/CAT was included in all transfections as a control of transfection
efficiency. Luciferase activities, adjusted to the protein
concentration of each sample, were normalized to CAT values and are
expressed as a percentage of the values obtained with cells transfected
with the empty vector and activated with anti-TCR mAb. Bars
represent the SD for multiple experiments (n = 3), each
with duplicate samples. Luciferase activity was below detection in the
absence of stimulation both in cells transfected with control vector
and in cells transfected with the Phe474ZAP-70 or the
Shc-PTB expression constructs.
Shc Interacts with Tyr474 of ZAP-70-- To address the role of Tyr474 in the interaction of ZAP-70 with Shc, we tested the capacity of Shc to bind activated ZAP-70 in the presence of either a phosphopeptide spanning ZAP-70 Tyr474 or its nonphosphorylated analogue. Shc was immunoprecipitated from lysates of nonactivated Jurkat T-cells lysed in 3% Triton X-100 to disrupt pre-existing complexes. The Shc-specific immunoprecipitates were then incubated with lysates of nonactivated or anti-CD3 mAb activated Jurkat cells and subsequently probed by immunoblot for the presence of ZAP-70. As shown in Fig. 2, ZAP-70 from activated cells, but not that from nonactivated cells, specifically bound to Shc. Incubation of a Shc-specific immunoprecipitate with a lysate of activated cells in the presence of the phosphorylated ZAP-70 peptide encompassing Tyr474 resulted in a significant reduction of ZAP-70 binding, whereas the nonphosphorylated peptide had little effect (Fig. 2, upper panel). Similar amounts of Shc were detected in all immunoprecipitates after immunoblotting with an anti-Shc mAb (Fig. 2, lower panel). These results show that ZAP-70 associates with Shc through Tyr474 in a phosphorylation-dependent manner.
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Mutation of ZAP-70 at Tyr474 Prevents the Interaction
with Shc without Affecting ZAP-70 Kinase Activity--
To analyze the
effect of Tyr474 substitution on the interaction of ZAP-70
with Shc, we generated a Jurkat T cell line stably expressing
Phe474ZAP-70. As shown in Fig.
3A, the level of the ZAP-70
immunoreactive band in this cell line is significantly higher than in
the parental line, indicating a level of 2-3-fold overexpression of
Phe474ZAP-70 as compared with endogenous ZAP-70. This
difference was confirmed by immunoblotting the same filter with
anti- tubulin mAb, which revealed similar amounts of tubulin in the
two lanes (data not shown). No significant difference could be detected in the level of surface TCR/CD3 expression, as evaluated by flow cytometry (MIF 26.4 for parental cells compared with 24.6 for Phe474ZAP-70 expressing cells). Comparison by immunoblot
with anti-phosphotyrosine mAb of total cell lysates from nonactivated
and activated cells expressing Phe474ZAP-70 with lysates of
similarly treated parental cells did not show any obvious difference in
either the basal or the induced pattern of
tyrosine-phosphorylated proteins (Fig. 3B).
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Mutation of ZAP-70 at Tyr474 Prevents Shc Binding to
the TCR Chain--
Both ZAP-70 and Shc bind the phosphorylated
chain ITAMs (7, 8, 22). We have previously shown that Shc association with ZAP-70 is required for Shc binding to phospho-
(16). We thus
expected a reduction in the levels of Shc-associated phospho-
in
cells expressing Phe474ZAP-70. The presence of
tyrosine-phosphorylated
chain in ZAP-70-specific and Shc-specific
immunoprecipitates was tested by immunoblotting with
anti-phosphotyrosine mAb. As previously reported (16), and as shown in
Fig. 4, two
chain isoforms differing in the levels of tyrosine
phosphorylation, pp21 and pp23, were found in association with ZAP-70,
whereas only pp23 was found in association with Shc. Although no
difference in ZAP-70 bound phospho-
could be detected in cells
expressing Phe474ZAP-70, a significant reduction in the
levels of Shc-bound phospho-
was observed in the presence of the
ZAP-70 mutant (Fig. 4). Immunoblotting the stripped filters with
anti-ZAP-70 (Fig. 4A) or anti-Shc (Fig. 4B) mAb
showed that similar amounts of specific protein were present in the
immunoprecipitates from the two cell lines (data not shown). This
result is consistent with a priming role of ZAP-70 in the interaction
between Shc and tyrosine-phosphorylated
.
Phosphorylation of the Shc CH Domain by ZAP-70--
The
association between ZAP-70 and Shc induced by TCR engagement, as well
as the reduction in Shc phosphorylation in cells expressing
Phe474ZAP-70, suggests that Shc phosphorylation might be
the direct outcome of this interaction. To address this issue, Shc was
immunoprecipitated from lysates of nonactivated Jurkat cells and
subjected to an in vitro kinase assay with
[-32P]ATP in the presence or absence of recombinant
GST-ZAP-70. The reaction products were resolved by SDS-polyacrylamide
gel electrophoresis and visualized on the fixed and dried gel by
PhosphorImager analysis. The results are presented in Fig.
5A. When Shc
immunoprecipitates were incubated with recombinant ZAP-70, a
phosphorylated band migrating at approximately 52 kDa, corresponding to
the Shc isoform, which becomes phosphorylated in response to TCR
triggering (16, 23), was observed. In addition, a strong band of about
96 kDa, corresponding to autophosphorylated GST-ZAP-70, was also
detected. To confirm that ZAP-70 can phosphorylate Shc and to identify
the Shc domain where this phosphorylation occurs, an in
vitro kinase assay with recombinant ZAP-70 was carried out on each
of the three domains of Shc, and the reaction products were analyzed as
described above. As shown in Fig. 5B (upper
panel), only the Shc CH domain was effectively phosphorylated by
ZAP-70. Phosphorylated GST-ZAP-70 could also be visualized in these
experiments after longer exposures (data not shown). Similar amounts of
the GST fusion proteins, subjected to SDS-polyacrylamide gel
electrophoresis and stained with Coomassie Blue, show that, in contrast
to the PTB and the SH2 fusion proteins, the GST-CH protein is unstable
as a full-length protein and is found as a set of major proteolytic
products (Fig. 5B, lower panel), which correspond to the
bands phosphorylated by recombinant ZAP-70 in the in vitro
kinase assay. The same pattern of degradation products was consistently
found in different batches of GST-CH from independent cultures of the
E. coli clone transformed with the GST-CH
plasmid.3 In vitro
kinase assays using Lck showed that Lck could weakly phosphorylate the
Shc CH domain; however, 32P incorporation was about 85-fold
less than in parallel samples treated with ZAP-70, as determined by
PhosphorImager analysis. Thus the Shc CH domain can be effectively and
specifically phosphorylated by ZAP-70.
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DISCUSSION |
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The data presented provide evidence for a role of Tyr474 of ZAP-70 in Shc recruitment and phosphorylation and in TCR signaling. A tyrosine to phenylalanine substitution at position 474 results, in fact, in a ZAP-70 mutant able to transdominantly inhibit endogenous ZAP-70 signaling to downstream targets leading to activation of the transcription factor NF-AT, which specifically drives the response of the interleukin-2 gene promoter to TCR generated signals (reviewed in Ref. 33). To date, two other tyrosine residues involved in positive regulation of ZAP-70 have been identified. The first is Tyr493, which is phosphorylated by Lck and which is required for catalytic activation of ZAP-70 (13, 37). The second is Tyr315, which is the binding site for Vav, a putative guanine nucleotide exchange factor for the Rac/Rho/cdc42 family of small GTP-binding proteins, which plays a key role in lymphocyte development and activation (38-42). Two other tyrosine residues, Tyr292 and Tyr492, are implicated in negative regulation of ZAP-70, potentially by promoting interactions with inhibitory proteins (43, 44). Furthermore, two-dimensional phosphopeptide mapping has revealed the existence of additional phosphorylation sites, which might also include Tyr69, Tyr126, and Tyr178, shown by Watts et al. (34) to be phosphorylated in vitro by Lck. These phosphotyrosine residues might represent the docking sites for important signaling proteins, such as Lck itself, Fyn, Ras-GAP, abl, and Cbl, which have been reported to associate with ZAP-70 either in vitro or in vivo (14, 15, 17). Although Tyr474 of ZAP-70 might interact with additional molecular partners, our data suggest that at least one of the functions of Tyr474 of ZAP-70 is to recruit Shc and that this interaction might be required for productive antigen receptor signaling.
The obvious consequence of Shc recruitment to the activated TCR complex
is activation of the Ras pathway (reviewed in Refs. 20 and 21). Shc has
been reported to directly bind tyrosine-phosphorylated chain in
response to TCR triggering (22). However, the low affinity of this
interaction, as measured in in vitro binding experiments
with the
1 ITAM, as well as the capacity of Grb2 to directly bind
phospho-
, would suggest a secondary role for Shc in Grb2/Sos
recruitment to Ras (45). We have previously reported that binding of
Shc to phospho-
is dependent on Shc association with ZAP-70,
suggesting that ZAP-70 interaction with the Shc PTB domain is
responsible for Shc recruitment in the proximity of the
chain, to
which it can subsequently bind through its SH2 domain (16). This is
strongly supported by the data presented in this paper showing a
significant reduction in Shc-bound phospho-
in the presence of
Phe474ZAP-70. The combined interaction of Shc with both
ZAP-70 and phospho-
, achieved through its dual phosphotyrosine
binding specificity, might stabilize Shc on the activated TCR and
therefore compensate for the low affinity of the interactions between
Shc and individual phosphorylated
chain ITAMs, thus reproposing Shc
as an important player in TCR-induced Ras activation. In this respect,
the capacity of Shc to simultaneously bind two Grb2 molecules has been
proposed as an important means to stabilize the Ras exchange factor Sos in the Shc/Grb2/Sos complexes (46).
Shc is composed of three distinct domains, the amino-terminal PTB
domain and the carboxyl-terminal SH2 domain, both of which are involved
in interactions with tyrosine-phosphorylated proteins, and the CH
domain, which contains both Tyr239/Tyr240 and
Tyr317 (reviewed in Ref. 21). The dual specificity of the
tyrosine binding domains, as well as the potential of each domain to
bind multiple phosphoproteins in activated
T-cells,4 suggests that,
besides Ras activation, Shc might play a complex role in TCR signaling
by contributing to the assembly around the activated TCR of a large
multimolecular signaling complex, for which we have proposed the term
"transduceosome" (16). The chain is found in association with
the TCR/CD3 complex predominantly as a disulfide bond-linked homodimer.
Each
chain has three ITAMs, implying that, when fully
phosphorylated, each
homodimer could recruit up to six molecules of
ZAP-70. An important outcome of ZAP-70 recruitment to phospho-
is
tyrosine phosphorylation of ZAP-70 both by Lck and by
autophosphorylation, which results in the generation on ZAP-70 of
multiple binding sites for signaling proteins, most of which have the
potential to recruit additional molecular partners. Hence, ZAP-70
interaction with the activated TCR appears to be a key step in the
assembly of a functional transducing complex. In this context, it is
interesting to notice that overexpression of ZAP-70 does not result in
significant NF-AT activation (13, 44, 47), in agreement with our
finding that no NF-AT activity could be detected in the absence of
stimulation in cells expressing Phe474ZAP-70, where the
basal level of total ZAP-70 activity is higher than in parental cells.
Conversely, targeting ZAP-70 to the plasma membrane, either by
engineering a heterologous membrane localization site (47) or by using
synthetic ligands and their binding proteins (9) is sufficient for
ZAP-70 function, underlining the importance of ZAP-70 localization to
the cytosolic side of the plasma membrane for productive signaling. The
contribution of ZAP-70 to transduceosome assembly may be further
amplified by association with Shc, with its multivalent capacity to
recruit other components of the transducing complex.
A puzzling issue is the failure of ZAP-70 to become activated in
response to TCR engagement by altered peptide ligands, which, despite
their similarity with peptide antigens, fail to activate T-cells. When
TCR is engaged by a specific altered peptide ligand, ZAP-70 is
recruited to phospho-, but this event is followed neither by
activation nor by phosphorylation of ZAP-70 (48, 49). A clue to this
question has been recently provided. Inducing alternative configurations of membrane targeted ZAP-70 using chemical inducers of
dimerization has shown that orientation is an important parameter in
ZAP-70 function (9). Because, of the two major phosphorylated species
of
, pp21 and pp23, only pp21
can be found in T-cells presented
with an altered peptide ligand (48, 49), a possibility is that only
when bound to pp23
can ZAP-70 acquire the correct orientation. In
this respect, the differential binding of Shc to pp23
is
particularly interesting. Because Shc only binds to pp23
and this
binding depends on a previous interaction of Shc with ZAP-70 (Ref. 16
and present results), our data suggest that the dual interaction of Shc
with ZAP-70 and pp23
might contribute to conferring the correct
orientation to ZAP-70, resulting in productive TCR signaling
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ACKNOWLEDGEMENTS |
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We thank S. Grassini for skilled technical assistance, S. Censini for automated DNA sequencing, L. Gamberucci for photographic artwork, and G. Benocci for secretarial assistance. We also gratefully acknowledge A. Weiss for the kind gift of ZAP-70 cDNA, R. Kurrle for the gift of the mAb BMA, and S. Plyte for useful suggestions and critical reading of the manuscript.
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FOOTNOTES |
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* This work was generously supported by the Italian Association for Cancer Research. The financial support of the MURST (quota 60%) is also acknowledged.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 Italian Association for Cancer Research. Present address: Dept. of Cellular and Developmental Biology, University "La Sapienza," Via degli Apuli 1, 00185 Rome, Italy.
To whom correspondence should be addressed. Tel.:
39-577-298873; Fax; 39-577-298898; E-mail: baldari{at}unisi.it.
The abbreviations used are: TCR, T-cell antigen receptor; PTK, protein tyrosine kinase; mAb, monoclonal antibody; ITAM, immunoreceptor tyrosine-based activation motif; CAT, chloramphenicol acetyltransferase; GST, glutathione S-transferase.
2 G. Magistrelli, R. Bosotti, B. Valsasina, C. Visco, R. Perego, S. Toma, O. Acuto, and A. Isacchi, submitted for publication.
3 S. Pacini, L. Lanfrancone, and C. T. Baldari, unpublished observations.
4 S. Pacini and C. T. Baldari, unpublished data.
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REFERENCES |
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