From the Molecular Immunology Unit and the ** Cellular
Biochemistry Unit, Institut Pasteur, 25-28 Rue du Docteur Roux, 75724 Paris Cedex 15, France
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ABSTRACT |
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T-cell antigen receptor-induced signaling
requires both ZAP-70 and Lck protein-tyrosine kinases. One essential
function of Lck in this process is to phosphorylate ZAP-70 and
up-regulate its catalytic activity. We have previously shown that after
T-cell antigen receptor stimulation, Lck binds to ZAP-70 via its Src homology 2 (SH2) domain (LckSH2) and, more recently, that
Tyr319 of ZAP-70 is phosphorylated in
vivo and plays a positive regulatory role. Here, we investigated
the possibility that Tyr319 mediates the
SH2-dependent interaction between Lck and ZAP-70. We show
that a phosphopeptide encompassing the motif harboring Tyr319, YSDP, interacted with LckSH2, although with a lower
affinity compared with a phosphopeptide containing the optimal binding motif, YEEI. Moreover, mutation of Tyr319 to phenylalanine
prevented the interaction of ZAP-70 with LckSH2. Based on these
results, a gain-of-function mutant of ZAP-70 was generated by changing
the sequence Y319SDP into Y319EEI. As a result
of its increased ability to bind LckSH2, this mutant induced a dramatic
increase in NFAT activity in Jurkat T-cells, was hyperphosphorylated,
and displayed a higher catalytic activity compared with wild-type
ZAP-70. Collectively, our findings indicate that
Tyr319-mediated binding of the SH2 domain of Lck is crucial
for ZAP-70 activation and consequently for the propagation of the
signaling cascade leading to T-cell activation.
Lck and ZAP-70, members of the Src and Syk families of nonreceptor
PTKs,1 respectively, control
in a sequential manner T-cell antigen receptor (TCR)-proximal signaling
(reviewed in Ref. 1). TCR triggering stimulates phosphorylation by Lck
of the immunoreceptor tyrosine-based activation motifs (ITAMs) present
in the signal transducing subunits ( Recent studies in our laboratory have shown that Tyr319, in
the linker region between the two tandem SH2 domains and the kinase domain (interdomain B; see Fig. 1) of ZAP-70, is a TCR-induced phosphorylation site and has an essential positive role in the regulation of the kinase (16). Indeed, mutation of Tyr319
to phenylalanine, although not affecting the basal kinase activity, strongly reduced the activation-induced tyrosine phosphorylation of
ZAP-70 and the up-regulation of its catalytic activity following TCR
stimulation. Consistently, overexpression of the mutant Y319F in
T-cells severely reduced the phosphorylation of ZAP-70 substrates (SLP-76 and LAT) and the TCR-dependent activation of NFAT
and interleukin-2 production (16).
We and others have previously demonstrated that after TCR stimulation,
Lck binds to ZAP-70 via its SH2 domain (LckSH2), raising the
possibility that this association is required for ZAP-70 activation by
Lck (11, 17, 18). Consistently, expression in an Lck-deficient cell
line of an SH2 point mutant of Lck was unable to restore the
TCR-mediated activation, a defect that correlated with the lack of
Lck-ZAP-70 interaction and the dramatic reduction of ZAP-70 tyrosine
phosphorylation (18).
The facts that mutation of either Tyr319 or
Tyr493 leads to a dramatic decrease in ZAP-70 function (7,
16) and that ZAP-70-Lck interaction plays a critical role in TCR
signaling suggested that tyrosine Tyr319 may represent a
binding site for LckSH2. In this work we provide evidence supporting
this notion.
The minimal sequence critical for interaction with the SH2 domains of
Src PTKs comprises the three amino acids immediately C-terminal to a
phosphorylatable tyrosine residue (19). By using a degenerated
phosphopeptide library, it has been shown that the motif pYEEI is
preferentially selected by the SH2 domain of members of the Src family
of PTKs (19). We built a molecular model of the motif encompassing
Tyr319 of ZAP-70 (YSDP) complexed to LckSH2 and found that
despite the divergence between the Y319SDP and the optimal
motif YEEI, the former possesses structural features compatible with
its binding to LckSH2. Competition experiments of the binding between
in vivo phosphorylated ZAP-70 and LckSH2 using a synthetic
peptide comprising Y319SDP confirmed that this motif
efficiently interacts with LckSH2, although less well than a
YEEI-containing peptide. Moreover, we demonstrate that mutation of
Tyr319 to phenylalanine abolishes the interaction of ZAP-70
with the LckSH2, whereas mutation of the neighbor Tyr315,
another residue phosphorylated in vivo following TCR
triggering (16), was ineffective. Based on these data, we generated a
mutant of ZAP-70 in which the natural sequence encompassing
Tyr319 was changed to Y319EEI (ZAP-YEEI). This
mutant showed a strong gain-of-function phenotype. Indeed, although its
basal kinase activity and its capacity to bind to phosphorylated ITAMs
were unaffected, ZAP-YEEI, expressed in Jurkat cells: (i) was
hyperphosphorylated, an event that correlated with an augmented
catalytic activity, and ii) induced a strong increase in NFAT activity.
The gain-of-function phenotype of ZAP-YEEI was dependent on the cell
surface expression of the TCR. These data are discussed in the context
of a model in which the SH2-mediated binding of Lck to
Tyr319 provides an essential step in the activation of
ZAP-70.
Antibodies--
Polyclonal anti VSV-G rabbit antiserum
(anti-tag) directed against an 11-amino acid synthetic peptide
determinant (an additional cysteine was added to the C terminus for
coupling to the carrier) derived from vesicular stomatitis virus
glycoprotein (VSV-G) was generated in our laboratory using keyhole
limpet hemocyanin as a carrier; anti ZAP-70 antiserum (4.06) was
produced in our laboratory as already described (20). The following
mouse monoclonal Abs (mAbs) were used: 101.5.2 (anti-human TCR Vb8, IgM
kindly provided by E. L. Reinherz, Dana-Farber Cancer Institute,
Boston, MA); 4G10 (anti-phosphotyrosine IgG2b, purchased from Upstate
Biotechnology, Lake Placid, NY); clone 29 (anti ZAP-70 C terminus
IgG2a, purchased from Transduction Laboratories, Lexington, KY); and
P5D4 (anti VSV-G-tag IgG1; hybridoma kindly provided by T. E. Kreis, Department of Cell Biology, University of Geneva, Switzerland
(21)).
Synthetic Peptides--
The following synthetic peptides were
purchased from Chiron Technologies, Clayton Victoria, Australia: YEEI
(sequence EPQYEEIPI) and pYEEI (sequence EPQpYEEIPI) (where pY
indicates a phosphotyrosine residue), derived from the hamster polyoma
MT sequence (19); pYSDP (sequence ESPpYSDPEE), pYESP (sequence
TSVpYESPYS), and pYTPE (sequence SDGpYTPEPA) encompassing residue
Tyr319, Tyr315, and Tyr292 of human
ZAP-70, respectively. A peptide (phospho-ITAM) corresponding to the
first ITAM motif of the human TCR- Cell Lines--
The human leukemia Jurkat T-cell line was
maintained in RPMI 1640 supplemented with 10% fetal calf serum, 10 mM Hepes, 2 mM L-glutamine, 100 units/ml penicillin, and 100 µg/ml streptomycin (Life Technologies,
Inc.) (complete RPMI 1640 medium). 31-13 T-cell line, a derivative of
the Jurkat T-cell line, which does not express the TCR due to the lack
of the TCR Constructions and Plasmids--
NFAT-luciferase (NFAT-luc)
reporter construct (10) was kindly provided by C. Baldari (University
of Siena, Italy). pSV-bgal vector (Promega) contained the
The GST-( Model Building--
The structure of the SH2 domain of Lck with
the phosphotyrosine in its binding pocket was derived from the
crystallographic atomic coordinates of the LckSH2-pYEEI peptide complex
(25) (Protein Data Bank code 1LCJ). The CHARMM academic program was
used to built sequentially the amino acids (SDP) ab initio. For each structure of the previous step (the crystallographic one for
the first step, i.e. the pY residue) the next amino acid was
appended either in its standard Cell Transfection, Activation, and NFAT Luciferase Activity
Assays--
Transient transfections of Jurkat cells were performed by
electroporation (260 V, 960 microfarads) as already described (10) with
the indicated doses of pSR Phosphopeptide Analysis--
The GST-( Immunoprecipitation and SH2 Binding Assays--
Jurkat T-cells
transfectants were stimulated with pervanadate 5 min at 37 °C or
left unstimulated and lysed in 1% Nonidet P-40-containing buffer (10).
Immunoprecipitation, SH2 binding assays, immunoblotting, and detection
of proteins by enhanced chemiluminescence (Amersham Pharmacia Biotech)
were performed as described previously (10, 11).
Phosphopeptide Binding Assays--
For phosphopeptide
competition binding assays, 5 × 107 Jurkat cells were
activated 5 min at 37 °C with pervanadate and lysed in 1% Nonidet
P-40 containing buffer (10). One-tenth of the lysate was incubated on
10 ml of MBP-LckSH2 beads 1 h at 4 °C in 45 µl of 20 mM Tris, pH 7.5, 150 mM NaCl, 1% Nonidet P-40, containing increasing concentrations of competitor phosphopeptide (11).
LckSH2-bound protein were separated on SDS-PAGE and subjected to
immunoblotting with an anti-ZAP-70 mAb (clone 29). Phospho-ITAM peptide
binding was performed as described previously (20).
In Vitro Kinase Assays--
Transfected cells were lysed in 1%
Nonidet P-40-containing buffer and expressed ZAP-70-WT or mutant was
immunoprecipitated by anti VSV-G tag antiserum. Anti VSV-G tag
immunoprecipitates were washed four times in Nonidet P-40 containing
buffer and twice with kinase buffer (25 mM MES buffer, pH
6.5, 10 mM MnCl2) and then incubated at room
temperature for 5 min in 25 µl of kinase buffer containing 0.5 µM of [ YSDP Motif from the Interdomain B of ZAP-70 Is Able to Bind
LckSH2--
The similar phenotype displayed by Y319F and Y493F ZAP-70
mutants (7, 16) together with the critical role of the SH2-mediated interaction between ZAP-70 and Lck in TCR signaling (11, 18), suggested
that Tyr319 could be a binding site for the LckSH2.
However, this residue is located in the motif YSDP (Fig.
1), which considerably diverges from
YEEI, an optimal binding sequence for the SH2 domain of Src PTKs (19).
Thus, we used molecular modeling to address the question of whether the
sequence YSDP could fit the LckSH2-binding site. The molecular modeling
was based on the crystallographic atomic coordinates of the
LckSH2-pYEEI peptide complex (25). To avoid any bias, this model was
built with no other information than the structure of the LckSH2
domain, with the pY residue lodged in its binding pocket (for further
details see "Experimental Procedures"). As shown in Fig.
2, the structural resemblance between the
built pYSDP peptide (yellow in the figure) and the pYEEI
(gray in the figure) was striking. The backbone of the two
peptides almost superimposed, with a root mean square deviation for the
C
To confirm the prediction arising from the molecular modeling,
i.e. that Y319SDP could bind to LckSH2, we
initially carried out a series of competition experiments, employing a
previously described binding assay (11). Lysates from activated Jurkat
cells were incubated with LckSH2 beads in the presence of different
concentrations of a pYSDP-containing nonapeptide. The binding capacity
of this peptide was compared relatively to three nonamer
phosphopeptides containing the optimal motif (pYEEI) or encompassing
Tyr292 (pYTPE) or Tyr315 (pYESP) of ZAP-70, two
residues that lie in the interdomain B of the kinase and are
phosphorylated in vivo (8, 16). An unphosphorylated version
of the nonamer peptide containing the YEEI motif was used as a control.
LckSH2-bound proteins were separated by SDS-PAGE and analyzed by
anti-ZAP-70 immunoblot. Only ZAP-70 from activated cells specifically
bound to LckSH2 beads (Fig. 3C
and data not shown).
Fig. 3A shows that the phosphopeptide pYEEI was able to
effectively compete for the binding of ZAP-70 to LckSH2, even at the lowest concentration tested (compare 11 µM of pYEEI
peptide with 300 µM of the YEEI control peptide). The
signal had almost disappeared at 300 µM pYEEI.
Phosphopeptide pY319SDP was also able to compete for ZAP-70
binding, although less effectively than pYEEI. Indeed, repeated
competition experiments allowed us to estimate that
pY319SDP bound to LckSH2 with about ~10-fold lower
affinity than the optimal phosphopeptide pYEEI, even though this
difference in affinity appears to be less pronounced in the experiment
of Fig. 3A. In analogous experiments, the
pY315ESP-containing phosphopeptide was a much less
effective competitor than Y319SDP, whereas
pY292TPE was totally ineffective in competing for the
binding of ZAP-70 to LckSH2 (Fig. 3A). These findings
demonstrate that although less efficient than the optimal sequence
pYEEI, the Tyr(P)319-containing motif of ZAP-70 can bind LckSH2.
Tyr319 of ZAP-70 Is Required for Interaction with the
SH2 Domain of Lck--
To provide further evidences that
Tyr319 of ZAP-70 functions as an LckSH2-binding site, we
performed binding experiments by using different Jurkat-T-cell lines
stably overexpressing ZAP-70 (ZAP-WT) or ZAP-70 mutants in which
Tyr315 or Tyr319 had been replaced by
phenylalanine (ZAP-Y315F and ZAP-Y319F, respectively) (16). All the
constructs were tagged at the C terminus with a VSV-G-derived peptide
(21). The 15.8 cell line expressing ZAP-WT was used as a control. The
cell lines 1.40 and 1.60 expressed ZAP-Y319F, whereas the cell lines
2.21/14 and 2.21/2 expressed the ZAP-Y315F mutant. The relative
expression levels of ZAP-70 tagged constructs in these cell lines,
which have been previously characterized (16), are shown in Fig.
3B. Because previous studies have indicated that the
ZAP-Y319F mutant is considerably less phosphorylated than ZAP-WT (16),
Jurkat cell transfectants were activated by pervanadate, a stratagem
that enables to bypass these differences. It is reasonable to assume
that under these experimental conditions, homogeneous tyrosine
phosphorylation of all ZAP-70 constructs was attained, with the obvious
exception of the mutated tyrosines in ZAP-Y315F and ZAP-Y319F. Lysates
were incubated with LckSH2 coupled to beads, and bound proteins were analyzed by SDS-PAGE and anti-tag immunoblotting. Fig. 3C
shows that despite comparable levels of phosphorylated ZAP-70
constructs present in the lysates from stimulated cells (upper
panel), only ZAP-WT and ZAP-Y315F were able to bind to LckSH2
(lower panel), whereas binding was completely abolished in
Y319F-expressing clones. The slightly higher amount of ZAP-Y315F bound
to LckSH2 compared with ZAP-WT is explained by the higher expression
level of the mutant in the 2.21/14 and 2.21/2 cell lines (Fig.
3B). These results demonstrate that Tyr319 is
required for the binding of ZAP-70 to LckSH2 and confirmed previous
findings by Wu et al. (28) showing that Tyr315
of ZAP-70 is not involved in this interaction.
3-Phosphorylated Tyr319 Binds Specifically to
LckSH2--
The results shown in Fig. 3C did not allow
exclusion of the possibility that the inability of the mutant ZAP-Y319F
to bind the Lck SH2 was due to an indirect effect, e.g.
inhibition of the phosphorylation of another tyrosine residue (16). To
address the direct involvement of Tyr319 in the binding of
the LckSH2, we made use of a GST-ZAP-70 fusion protein, which lacks the
SH2 domains but contains the interdomain B and the catalytic domain
(residues 255-619) of human ZAP-70 sequence (GST-(
Comparison of the CNBr phosphopeptide pattern of autophosphorylated
GST-( Substitution of the Sequence Y319SDP for
Y319EEI in ZAP-70 Results in a Gain-of-function
Phenotype--
The above data indicated that the sequence
Y319SDP of ZAP-70 functions as a docking site for LckSH2,
even though this motif does not closely match an optimal binding
sequence YEEI (19). Consistent with this, the affinity of the pYEEI for
LckSH2 is higher than that of pYSDP (Fig. 3A). Therefore, we
speculated that the introduction of a YEEI sequence in place of
Y319SDP in ZAP-70 would lead to a stronger interaction of
Lck with ZAP-70 and consequently to a gain-of-function. Thus, a ZAP-70 mutant (ZAP-YEEI) was generated in which the sequence
Y319SDP was changed to Y319EEI (Fig. 1).
C-terminally tagged ZAP-YEEI or ZAP-WT constructs were transiently
expressed in Jurkat cells together with an NFAT-luc reporter plasmid
that was used as a read-out for cellular activation (10, 29). As
previously demonstrated (10), overexpression of ZAP-WT in
unstimulated Jurkat cells induced a moderate increase in NFAT
activity (Fig. 4A). The
activating effect of ZAP-WT overexpression in the absence of the TCR
cross-linking is likely to depend on ZAP-70 being recruited to the
phosphorylated ITAMs (see also below). Indeed, we and others have
previously shown that overexpression of tandem SH2 domain of ZAP-70
leads per se to an increase of ITAM tyrosine
phosphorylation, as a consequence of protecting them from
dephosphorylation by protein-tyrosine phosphatases (10, 30, 31). Thus,
when overexpressed, ZAP-70 generates its own binding sites on the
ITAMs. In agreement with the initial hypothesis, expression of ZAP-YEEI
at levels similar to ZAP-WT induced a much higher NFAT activity in
unstimulated Jurkat cells (Fig. 4A). When the cells were
stimulated by TCR cross-linking, we found that transfection of both
ZAP-WT and ZAP-YEEI induced an increase in NFAT transcriptional
activity compared with cells transfected with the empty vector, with
ZAP-YEEI being more efficient than ZAP-WT (Fig. 4A).
Generalized effects on gene expression by ZAP-YEEI can be excluded
because no enhancement of the expression levels of a
These data demonstrate that changing the sequence Y319SDP
into Y319EEI results in a gain-of-function of ZAP-70 and
further support the notion that Y319SDP sequence in the
interdomain B of ZAP-70 harbors the binding site for LckSH2.
The gain-of-function phenotype of ZAP-YEEI was not observed in T-cells
lacking surface expression TCR-CD3 complex. Indeed, when ZAP-WT or
ZAP-YEEI was expressed in 31-13 cells, a TCR-negative cell line derived
from Jurkat (22), no NFAT activation was observed (Fig. 4C).
On the other hand, when expressed in ZAP-YEEI Shows an Increased Tyrosine Phosphorylation and Kinase
Activity--
As the Y319SDP to Y319EEI
mutation is expected to result in an increased affinity of the
SH2-mediated interaction between Lck and ZAP-70, the gain-of-function
phenotype of ZAP-YEEI should be mediated by a more efficient
phosphorylation/activation of the mutant by Lck. Indeed, when ZAP-WT
and ZAP-YEEI were expressed in unstimulated Jurkat cells,
immunoprecipitation experiments followed by anti-phosphotyrosine immunoblotting revealed that the phosphorylation level of ZAP-YEEI was
considerably higher than that of ZAP-WT (Fig.
5A). Such an increased
phosphorylation level of ZAP-YEEI did correlate with an augmented
kinase activity. As shown in Fig. 5B, when anti-tag immunoprecipitates from transiently transfected Jurkat cells were subjected to an in vitro kinase assay, using a GST-Band III
fusion protein as an exogenous substrate, ZAP-YEEI displayed a kinase activity significantly higher compared with ZAP-WT. Indeed, after normalization for the amount of immunoprecipitated protein, the catalytic activity of ZAP-YEEI was found to be 3.8 ± 1.0-fold (mean ± S.D., n = 4) higher than that of ZAP-WT.
Under the conditions employed, we did not see phosphorylated bands at
55-60 kDa that might correspond to autophosphorylated Lck
coprecipitating with ZAP-70 or ZAP-YEEI. This suggests that the amount
of Lck and/or its kinase activity was negligible and did not contribute
significantly to GST-band III phosphorylation.
Consistent with the lack of gain-of-function of ZAP-YEEI in
TCR-negative 31-13 cells (Fig. 4C), neither ZAP-WT nor
ZAP-YEEI were detectably phosphorylated when expressed in these cells
(Fig. 5A). Moreover, the kinase activity of ZAP-YEEI in
TCR-negative 31-13 cells was found to be comparable with that of ZAP-WT
(Fig. 5B). Indeed, the ratio of ZAP-YEEI to ZAP-WT kinase
activity in these cells was 0.82 ± 0.28 (mean ± S.D.,
n = 4). These results formally demonstrate that the
Y319EEI mutation did not affect the basal kinase activity
of ZAP-70, thus ruling out the possibility that increased catalytic
activity of ZAP-YEEI was due to a structural alteration of the
molecule. These findings also indicate that the hyperphosphorylation
and the increased kinase activity of ZAP-YEEI were dependent on the presence of the ITAMs, as previously shown for the increase in NFAT
activity induced by this mutant (see Fig. 4C).
Mutation of Y319SDP to Y319EEI in ZAP-70
Does Not Affect Its Binding to Receptor ITAMs--
One possible
explanation for the gain-of-function phenotype of ZAP-YEEI was that the
Y319EEI mutation leads to an increased binding to
phosphorylated ITAMs in the TCR-CD3 complex. To address this question,
TCR-negative 31-13 cells were transiently transfected with ZAP-WT or
ZAP-YEEI, and cellular lysates were mixed with a biotinylated, doubly
phosphorylated peptide encompassing the N-terminal ITAM of TCR- Increased Binding of ZAP-YEEI to LckSH2--
We finally verified
that ZAP-YEEI was able to bind LckSH2 with higher efficiency compared
with ZAP-WT. ZAP-YEEI and ZAP-WT constructs were transiently expressed
in Jurkat cells, and cell lysates were incubated with LckSH2 beads. The
precipitates were subjected to SDS-PAGE and immunoblotted with an
anti-tag monoclonal antibody. Fig. 7
shows that despite similar levels of expression of ZAP-WT and ZAP-YEEI,
binding to LckSH2 was detected only for the latter, although some
binding could be also observed for ZAP-WT after longer exposure of the
immunoblot (not shown). This finding demonstrates that ZAP-YEEI does
interact with higher efficiency with LckSH2 compared with ZAP-WT. It
should be noted, however, that this result cannot be uniquely
attributed to the increased affinity of binding to LckSH2 of ZAP-YEEI
compared with ZAP-WT. Indeed, as shown in Fig. 5A, ZAP-YEEI
has an increased tyrosine phosphorylation compared with ZAP-WT and, in
principle, accumulation of phosphorylated Tyr319 could in
part account for this result.
In a previous work we have demonstrated that Tyr319,
located in the interdomain B of ZAP-70, is a TCR-inducible
phosphorylation site and that it plays a critical role in ZAP-70
function (16). We have proposed that the dominant negative phenotype
observed for ZAP-Y319F could be explained if Tyr319 is the
binding site for a molecule involved in the positive regulation of
ZAP-70 activity, such as Lck (16). We now provide much evidence indicating that Tyr319 of ZAP-70 is a binding site for
LckSH2. First, a phosphorylated peptide encompassing Tyr319
was able to compete for the binding of ZAP-70 to LckSH2 (Fig. 3A). Moreover, mutation of Tyr319 to
phenylalanine impaired the binding of ZAP-70 to LckSH2 (Fig. 3C). Finally, CNBr fragments of ZAP-70 containing
phosphorylated Tyr319 specifically bound LckSH2 (Fig.
3D). In agreement with the notion that Lck interacts with
ZAP-70 by binding to phosphorylated Tyr319, a ZAP-70 mutant
(ZAP-YEEI) in which the Y319SDP sequence has been changed
to the optimal binding site for the SH2 domain of Lck, YEEI, displays a
gain-of-function phenotype. Indeed, when expressed in Jurkat cells,
ZAP-YEEI showed an increased tyrosine phosphorylation and catalytic
activity (Fig. 5), resulting in an augmented ability to induce
NFAT-dependent transcription (Fig. 4). These data also
reveal that an increase in the affinity of the SH2-mediated interaction
between Lck and ZAP-70 has dramatic functional consequences on TCR signaling.
Our previous work and data from other laboratories have suggested a
model in which, once associated to the ITAMs, ZAP-70 autophosphorylates on several tyrosines including Tyr319 (6, 16), thus
generating a binding site for LckSH2. The subsequent SH2-mediated
interaction between Lck and ZAP-70 would allow phosphorylation of
Tyr493 in the activation loop, thus ensuring an effective
phosphorylation/activation of ZAP-70 by Lck (7, 9). However,
contribution of other PTKs (e.g. Lck) in the phosphorylation
of Tyr319 cannot be ruled out (7).
The property of Src PTKs as regulators of PTKs belonging to other
families is illustrated by several examples, including the focal
adhesion kinase, Itk, and Btk (32-35). In particular, the mechanism we
suggest for the activation of ZAP-70 by Lck closely resembles that
proposed for focal adhesion kinase (32, 33). In that system, Src (or
Fyn) binds through its SH2 domain to an autophosphorylated site
(Tyr397) and positively regulates focal adhesion kinase
function by phosphorylating in turn Tyr576 and
Tyr577 in the kinase activation loop.
Besides Tyr319, other tyrosine residues of ZAP-70 that have
been shown to be phosphorylated in vivo following TCR
engagement are Tyr292, Tyr492,
Tyr493, and Tyr315 (7, 8, 16). However,
mutational analysis has suggested that phosphorylation of
Tyr292 and Tyr492 has a negative regulatory
role (9, 36, 37). Moreover, we have previously shown that
Tyr492 and Tyr493 are not required for the
association of ZAP-70 with Lck (10). Finally, this work and previous
data by Wu et al. (28) demonstrate that Tyr315
is not involved in the binding of LckSH2 to ZAP-70. These
considerations together with our data indicate that among the ZAP-70
tyrosines found to be phosphorylated in vivo,
Tyr319 is the most likely docking site for LckSH2.
Other tyrosine residues of ZAP-70 have been shown to be phosphorylated
in vitro, namely Tyr69, Tyr126, and
Tyr178 in the N-terminal region of the protein containing
the tandem SH2 domains (8). So far, in vivo phosphorylation
of these residues has not been demonstrated. Moreover, our binding
experiments using CNBr-digested GST-( It has been reported that the tyrosines in the activation loop are the
docking site for LckSH2 on Syk. We have shown that this is not the case
for ZAP-70 (10). Moreover, Syk was shown to function independently of
Lck, because it can rescue the signaling defect of Lck-negative JCaM1
cells (39, 40). Thus, the SH2-mediated interaction of Lck with Syk may
have a different functional significance than Lck binding to ZAP-70
(41).
Although the pY319SDP sequence diverges from the optimal
binding motif (pYEEI) for SH2 of Src-PTKs (19), it should be noted that
other previously identified binding sites for Src-PTKs SH2 (e.g. on platelet-derived growth factor and CSF-1 receptors;
Refs. 42 and 43) were also found to differ considerably from the optimal sequence. Molecular modeling predicts that pYSDP could be
accommodated within the LckSH2-binding site (Fig. 2) with a main chain
conformation similar to that shown for a pYEEI peptide complexed to
LckSH2 (25). According to our model, proline +3 of the pYSDP motif
would interact with the top of the Y+3 binding pocket, which in the
crystal structure is occupied by the isoleucine of the pYEEI peptide.
Consistent with the notion that a proline residue can substitute for
isoleucine in position +3, a P to I mutation in the sequence
Y319SDP of ZAP-70 generated a mutant whose expression in
Jurkat cells induced NFAT at levels similar to
ZAP-WT.3
Interestingly, a similar interaction of a proline residue with the
second binding pocket was previously shown for the Lck C-terminal
pY505QPQP regulatory sequence (44) and for the homologous
residue of Hck (45) in the sequence pY527QQQP. In the case
of the pYSDP peptide, our model suggests that the backbone would fit
with an extended conformation on the surface of the LckSH2 and that the
aspartate +2 side chain would be in a favorable position for
interacting with the arginine R We show that a pYEEI containing peptide binds LckSH2 with higher
affinity than a pYSDP peptide (Fig. 3A). Moreover,
conversion of the motif Y319SDP into Y319EEI
results in a ZAP-70 mutant having increased kinase activity and
tyrosine phosphorylation when expressed in Jurkat cells (Fig. 5,
A and B). Consistently, this mutant displays a
gain-of-function phenotype (Fig. 4), in agreement with the hypothesis
that an increased affinity of the SH2-mediated interaction between Lck
and ZAP-70 favors phosphorylation/activation of the latter. Indeed, we
observed that when expressed in Jurkat cells, ZAP-YEEI bound to LckSH2 with higher efficiency compared with ZAP-WT (Fig. 7).
A higher binding affinity of LckSH2 to ZAP-70 may also result in
increased Lck activity, by stabilizing an "open" (active) conformation of the enzyme and/or favoring the recruitment of Lck in
the proximity of specific substrates other than ZAP-70. We cannot
exclude the possibility that this mechanism contributes in part to the
enhanced NFAT activity induced by ZAP-YEEI.
Because ZAP-YEEI binds to phosphorylated ITAMs with affinity comparable
with that of ZAP-WT (Fig. 6) and because the mutation does not increase
the basal catalytic activity of the PTK (Fig. 5B), we can
exclude the possibility that the effects of Y319SDP to
Y319EEI mutation are due to a structural alteration of the
molecule, confirming that the most likely explanation for the phenotype observed for ZAP-YEEI is its increased affinity for LckSH2. Moreover, ZAP-YEEI displays its gain-of-function phenotype only in cells expressing a functional TCR (Fig. 4C), suggesting that the
molecule is not constitutively active and needs to be recruited to the plasma membrane to interact with Lck and activate the signaling pathways usually stimulated by ZAP-70.
The phenotype of ZAP-YEEI suggests that the SH2-mediated Lck-ZAP-70
interaction is critical for setting the threshold of TCR-induced responses. Indeed, an interaction with a suboptimal affinity between ZAP-70 and Lck, mediated by LckSH2 binding to Y319SDP
motif, may be better suited for keeping T-cell activation under tight
control. Our results also point at the SH2-mediated interaction of Lck
to ZAP-70 as a new and highly specific pharmacological target for the
manipulation of T-cell responses.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, and CD3
,
, and
) of
the TCR complex (2, 3). Phosphorylated ITAMs become then competent to
bind the cytoplasmic ZAP-70 kinase via its two tandemly arranged SH2
domains (2, 4, 5). By this mechanism, ZAP-70 is recruited to the
stimulated TCRs and becomes itself phosphorylated on tyrosine, as a
result of autophosphorylation (6) and transphosphorylation catalyzed by
Lck (2, 7, 8). The latter event, which implicates phosphorylation of
Tyr493 in the activation loop of the catalytic domain, is
required for augmenting ZAP-70 catalytic activity (7, 9) with
consequent phosphorylation of its substrates and propagation of the
signaling cascade (7, 10). Besides the direct regulation of its kinase activity, tyrosine phosphorylation of ZAP-70 appears to have additional roles such as providing binding sites for SH2 or
phosphotyrosine-binding domain-containing enzymes or adapter proteins
that may act as downstream effectors and/or regulators of ZAP-70
activity (6, 11-15).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
chain (residues 48-66) plus a
4-amino acid linker at the N terminus (sequence:
SGSGNQLYNELNLGRREEYDVLD) was synthesized as diphosphorylated on
Tyr51 and Tyr62 form (by F. Baleux, Department
of Organic Chemistry, Institut Pasteur, Paris, France).
Phospho-ITAM was purified by reverse-phase high pressure liquid
chromatography and biotinylated at the N terminus by Biotin sulfo-NHS
(Pierce). The purity and the molecular mass of the peptides were
confirmed by ion electrospray ionization mass spectrometry.
chain transcript (22), was maintained in complete RPMI
1640 medium.
WT160 was obtained from 31-13 cells by stable
transfection of the TCR
-chain (22) and maintained in complete RPMI
1640 medium also containing 2 g/liter of G-418 (Life Technologies,
Inc.). Jurkat stable transfectants expressing ZAP-70-WT (clone 15.8),
ZAP-70-Y319F (clones 1.40 and 1.60), and ZAP-70Y315F (clones 2.21/14
and 2.21/2) were previously described in Ref. 16.
-galactosidase gene driven by the SV40 promoter/enhancer. The ZAP-WT
construct, bearing a C-terminal 11-amino acid epitope VSV-G-tag was
described previously (10). The ZAP-Y319EEI mutant was
derived from this construct by polymerase chain reaction: the 5' primer
(nucleotides 713-734 of ZAP-70 sequence) encompassed a MluI
unique site; the 3' primer encoded for the Y319EEI mutation
(CCC GAG CTC CTC TAT CTC TTC GTA GGG GCT C) and contained a
SacI site. The MluI-SacI-digested
(nucleotides 713-1179) polymerase chain reaction product was ligated
with both a SacI-NsiI fragment (nucleotides
1179-1736) and a 3.8-kilobase ZAP-70WT-VSV-G pBs fragment restricted
with MluI and NsiI (10). Finally, the 1951-base pair ZAP-70YEEI-VSV-G construct (ZAP-70-Y319EEI) was
excised with EcoRI and XbaI and subcloned into
pSR
-puro vector (a gift of R. Sekaly, Institut de Recherche
Cliniques de Montreal, Canada). The complete sequences of ZAP-70-WT and
ZAP-70-Y319EEI were verified by standard dideoxy DNA sequencing.
SH2)ZAP-70WT fusion protein (containing residues 255-619
of human ZAP-70) and derived Tyr315 and Tyr319
mutants (obtained by polymerase chain reaction using
oligonucleotide-directed mutagenesis and confirmed by nucleotide
sequencing) were expressed in COS-1 cells and purified by glutathione
affinity chromatography.2
GSTBandIII fusion protein, containing 26 residues corresponding to
the N terminus of cytoplasmic fragment of the erythrocyte band III
(MEELQDDYEDMMEENLEDEEYEDPDI) (23, 24) was kindly provided by Dr.
A. M. Brunati (Department of Biological Chemistry, University of
Padova, Italy).
-sheet conformation or rotated every
30 degrees along its phi angle. Then the system was subjected to 10 ps
of molecular dynamics at 3000 K for each rotamer. The conformations
were saved every 0.1 ps, minimized, and grouped into clusters. The
lowest energy structure of each cluster (about a dozen) was kept to
initiate the next step of the building. The resulting structure was
both the one with the lowest energy and the one belonging to the
largest cluster. We used a sigmoidal dielectric constant to mimic the
effect of the solvent while enabling fast evaluations of the energy
minimizations or molecular dynamics and therefore a wide sampling.
-puro vector without insert (empty vector)
or containing ZAP-WT or ZAP-Y319EEI, 10 µg of NFAT-luc
plasmid, and 30 µg of pSV-bgal plasmid. The total amount of plasmidic
DNA was equalized with the empty pSR
-puro vector. Transfected cells
were cultured for 24 h, and then 105 cells were seeded
in 100 ml of growth medium into U-bottomed 96-well plates. 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) (Sigma) and calcium
ionophore A23187 (2 µg/ml) (Sigma).
-Galactosidase and luciferase
assays were performed by using the specific assay systems (Promega).
Luciferase activity was determined in triplicate samples using an
automated luminometer (Lumat LB 9501, Berthold, Wildbad, Germany) and
expressed as a percentage of maximal response (percentage of phorbol
12-myristate 13-acetate/ionophore).
SH2)ZAP-70 fusion
proteins (WT and mutant) were autophosphorylated for 30 min at room
temperature in 1 mM Tris, pH 7.4, 7.5 mM NaCl,
25 mM Hepes, 10 mM MnCl2, 0.05%
Nonidet P-40 and in the presence of 10 µCi of
[
-32P]ATP. Reaction products were separated by
SDS-PAGE and 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 (26). Cleavage products were separated on Tris-Tricine
SDS-PAGE as described (16, 27). Gels were dried and exposed for
autoradiography. For LckSH2 binding, CNBr cleavage products were
resuspended in 1% SDS, 20 mM Tris, pH 7.4, 150 mM NaCl and then diluted 10-fold in 1% Nonidet P-40, 20 mM Tris, pH 7.4, 150 mM NaCl and incubated for
1 h at 4 °C in the presence of MBP-LckSH2 Sepharose beads (11).
Beads were washed, and bound peptides were analyzed in Tris-Tricine
SDS-PAGE, as described above.
-32P]ATP (specific radioactivity,
20 mCi/mmol) and 20 ng of GST-Band III. The reaction was stopped by
adding an equal volume of 2× Laemmli sample buffer containing 200 mM dithiothreitol, 10 mM EDTA and boiling for 5 min. Samples were resolved by SDS-PAGE and transferred onto
polyvinylidene difluoride membranes (Millipore). Membranes were treated
with KOH 1 M for 1 h at 55 °C and dried before
autoradiography. An aliquot of each sample was used to quantify the
amount of immunoprecipitated proteins, by immunoblotting with an anti
ZAP-70 antiserum (4.06) and probing the Western blots with
125I-labeled protein A. In both the kinase assay and the
protein quantitation, radiolabeled bands corresponding to ZAP-70
(ZAP-70 WT and ZAP-70-Y319EEI) or the phosphorylated
GST-Band III were visualized by autoradiography and quantitated by
using Image Quant software after scanning in a PhosphorImager (Amersham
Pharmacia Biotech).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
of residues Y+1, Y+2, and Y+3 of 0.48 Å. This was also the case
for the side chains of the proline residue +3 of the YSDP peptide and
the side chain of the isoleucine +3 of the YEEI peptide, fitting on the
Y+3 binding pocket (root mean square deviation for the C
and C
of
the side chains of the Y+3 proline and isoleucine residues of 0.47 Å).
Moreover, as shown in Fig. 2B, the aspartate side chain of
the residue Y+2 of the YSDP peptide would be in a favorable position
for interacting with the arginine R
D'1 of the LckSH2. The data from
this molecular modeling show that the binding pocket of LckSH2 can
accommodate the YSDP motif, suggesting that no structural constraints
would impede this intermolecular interaction.
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Fig. 1.
Schematic representation of the overall
structure of ZAP-70. The sequence of the portion of interdomain B
encompassing Tyr292, Tyr315, and
Tyr319 is also shown, both in ZAP-70 WT and in the mutant
ZAP-YEEI.
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Fig. 2.
Model for the pYSDP peptide in the binding
pocket of LckSH2 compared with the consensus peptide pYEEI.
A, lateral view, showing the structural resemblance between
the built peptide and the consensus one and showing how proline is
fitting in the Y+3 binding pocket. B, global view of the
binding site, showing the accommodation of each residue of the built
peptide in their respective binding pockets. Nitrogen, oxygen, and
carbon atoms are displayed in blue, red, and
cyan, respectively, unless otherwise mentioned. The solvent
accessible surface of LckSH2 is shown for atoms within 7.5 Å from the
peptide. Carbon atoms of the model peptide YSDP and of the consensus
peptide YEEI are colored in yellow and gray,
respectively.
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Fig. 3.
Tyr319 of ZAP-70 mediates the
binding of LckSH2. A, specific phosphopeptide
competition of ZAP-70 binding to LckSH2. Lysates were prepared from
5 × 107 Jurkat cells activated with pervanadate, and
aliquots corresponding to 5 × 106 cells incubated
with MBP-LckSH2 Sepharose beads in the presence of competitor peptides
at the indicated concentrations. The peptides were EPQYEEIPI (YEEI) and
EPQpYEEIPI (pYEEI) from hamster polyoma MT sequence and
ESPpY319SDPEE (pYSDP), TSVpY315ESPYS (pYESP),
and SDGpY292TPEPA (pYTPE) from ZAP-70 sequence. The beads
were washed, and bound proteins were analyzed by SDS-PAGE and
immunoblotting with an anti-ZAP-70 mAb. B, 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 lines were subjected to SDS-PAGE and
immunoblotted with the anti-tag antiserum. WB, Western blot.
C, Y319F mutation abolishes SH2-mediated ZAP-70-Lck
association. 4 × 106 cells from the indicated cell
lines were left unstimulated ( ) or treated with pervanadate
(PV) and subsequently lysed in 1% Nonidet P-40 containing
buffer. For each sample one aliquot (equivalent to 106
cells) was immunoprecipitated by anti-tag antisera and immunoblotted
with an anti-phosphotyrosine mAb (upper panels). The rest of
the lysate (equivalent to 4 × 106 cells) was
incubated 2 h with MBP-LckSH2 Sepharose beads. The beads were
washed, and the associated proteins were analyzed by SDS-PAGE and
immunoblotting with an anti-tag mAb (lower panels).
D, ZAP-70-derived peptides containing Tyr319,
but not Tyr315, specifically bind to LckSH2.
GST-(
SH2)ZAP-70 proteins were autophosphorylated in vitro
in the presence of [
-32P]ATP and subjected to CNBr
cleavage as indicated under "Experimental Procedures." An aliquot
of the cleavage products was directly separated on a Tris-Tricine
SDS-PAGE (left panel). Equal amounts of radioactivity were
loaded in each lane. Another aliquot was subjected to LckSH2 binding,
and bound peptides were subsequently analyzed by Tris-Tricine SDS-PAGE
(right panel). Gels were dried and exposed for
autoradiography.
SH2)ZAP). Three
different fusion proteins were used: the wild-type construct
(GST-(
SH2)ZAP-WT) and the two mutants where Tyr315 or
Tyr319 were replaced by phenylalanine
(GST-(
SH2)ZAP-Y315F and GST-(
SH2)ZAP-Y319F, respectively). When
GST-(
SH2)ZAP-WT was autophosphorylated in vitro in the
presence of [
-32P]ATP and chemically cleaved with
CNBr, two major tyrosine phosphorylated peptides could be detected in
Tris-Tricine SDS-PAGE slabs (indicated as I and
II in Fig. 3D). We have previously demonstrated
that peptides I and II are phosphorylated at both Tyr315
and Tyr319, with peptide I likely extending from
Asp311 to Met359 and peptide II being a partial
cleavage product including peptide I (16). The other minor
phosphopeptides visible in the Tris-Tricine gel autoradiography are
likely to be partial cleavage products also containing phosphorylated
Tyr315 and Tyr319 (16). Indeed, as we have
previously shown, in the GST-(
SH2)ZAP-WT construct, essentially
Tyr315 and Tyr319 are phosphorylated (16).
SH2)ZAP-Y315F and GST-(
SH2)ZAP-Y319F to the
GST-(
SH2)ZAP-WT showed no qualitative differences (Fig.
3D, left panel), thus indicating that neither
mutation grossly altered protein autophosphorylation in
vitro (note that an equal amount of radioactivity was loaded in
each lane). However, when CNBr-cleaved proteins were incubated with
LckSH2, binding could be detected for peptides from the
GST-(
SH2)ZAP-WT and GST-(
SH2)ZAP-Y315F constructs, whereas
virtually no peptide from the CNBr-cleaved GST-(
SH2)ZAP-Y319F was
able to bind to LckSH2 (Fig. 3D, right panel).
These results formally demonstrate the implication of phosphorylated
Tyr319 in the binding to LckSH2.
-galactosidase
gene driven by a constitutive promoter was noticed in transfected cells
(not shown). The activating effect of ZAP-YEEI in unstimulated cells
was dose-dependent, reaching a plateau at 10 µg of
transfected DNA (Fig. 4B), and was 5-20-fold higher than
that observed with ZAP-WT.
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Fig. 4.
Substitution of the sequence
Y319SDP to Y319EEI in ZAP-70 results in a
gain-of-function phenotype. A, induction of NFAT
activity by ZAP-YEEI. Jurkat cells were transiently transfected with 10 µg of the empty pSR -puro vector or with 10 µg of the vector
encoding for ZAP-YEEI or ZAP-WT and co-transfected with an NFAT-luc
reporter plasmid (10 µg). 24 h after transfection, cells were
left unstimulated for 8 h (open bars) or stimulated
with anti-TCR mAb (filled bars) for 8 h and
subsequently assayed for NFAT-driven luciferase activity. B,
dose dependence of NFAT induction by ZAP-YEEI. Jurkat cells were
transiently transfected with increasing amounts of ZAP-YEEI
(squares) or ZAP-WT (circles) constructs and
co-transfected with a NFAT-luciferase reporter plasmid. Transfection of
10 µg of empty pSR
-puro plasmid gave no increase in NFAT activity
over the basal level (triangles). 32 h after
transfection unstimulated Jurkat cells were assayed for NFAT-driven
luciferase activity. C, the expression of either ZAP-WT or
ZAP-YEEI in 31-13 cells does not induce NFAT activity. TCR-negative
31-13 and TCR-positive
WT160-derivative cell lines were transiently
transfected with the indicated amounts of ZAP-YEEI (filled
bars) or ZAP-WT constructs (open bars) or with 10 µg
of the empty pSR
-puro vector (hatched bars) and
co-transfected with a NFAT-luciferase reporter plasmid. 32 h after
transfection, unstimulated cells were assayed for NFAT-driven
luciferase activity. In all the experiments, luciferase activities are
expressed as a percentage of the maximal activity, as measured after
stimulation with phorbol 12-myristate 13-acetate + Ca2+
ionophore A23187. One representative experiment is shown of three that
gave similar results. Error bars, S.D. (n = 3). The inset in each panel shows an anti-tag immunoblot
confirming that comparable amounts of ZAP-WT and ZAP-YEEI were
expressed in the different transfectants.
WT160, a 31-13 derivative in
which TCR expression was rescued by stably transfecting the TCR-
chain (22), ZAP-YEEI led to NFAT activation in absence of TCR
stimulation (Fig. 4C), as previously shown for Jurkat cells (Fig. 4, A and B). A moderate effect of NFAT
activation was also observed when ZAP-WT was expressed in
WT160
(Fig. 4C). These experiments demonstrated that to fulfill
its increased signal transducing capacity, ZAP-YEEI required the
expression of the ITAMs at the cell membrane. Thus, as observed in
TCR-negative 31-13 cells, overexpression of ZAP-YEEI was not sufficient
to exert its effect but required recruitment of the mutant kinase to
the TCR, where it was able to activate downstream signaling pathways.
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Fig. 5.
ZAP-YEEI shows an increased tyrosine
phosphorylation and kinase activity. A, ZAP-YEEI mutant
is hyperphosphorylated in unstimulated Jurkat T-cells but not in
TCR-negative 31-13 T-cells. Jurkat and TCR-negative 31-13 cell lines
were transiently transfected with the indicated amounts of ZAP-WT or
ZAP-YEEI constructs or with 5 µg of the empty pSR -puro vector.
24 h after transfection, lysates from 2 × 106
transfected cells where immunoprecipitated (IP) with an
anti-tag antiserum. The protein complexes were then separated on
SDS-PAGE and immunoblotted with an anti-tag mAb (upper
panel). The same filter was stripped and reprobed with an
anti-phosphotyrosine mAb (lower panel). B,
tyrosine kinase activity of ZAP-WT and ZAP-YEEI in Jurkat and
TCR-negative T-cells. Jurkat or TCR-negative 31-13 T-cells were
transfected with 10 µg of an empty vector pSR
-puro or of a ZAP-WT-
or ZAP-YEEI-encoding plasmid. 24 h after transfection cells were
lysed and immunoprecipitated with the anti-tag antiserum. The
immunoprecipitated ZAP-70 was subjected to an in vitro
kinase assay in the presence of [
-32P]ATP and the
exogenous substrate GST-Band III. The reaction products were separated
on an SDS-PAGE and transferred on polyvinylidene difluoride membrane,
and the phosphorylated proteins were detected by autoradiograpy
(lower panel). The amount of protein kinase in each sample
was quantitated by immunoblotting an aliquot of the same
immunoprecipitate with an anti-ZAP-70 antiserum followed by detection
with 125I-labeled protein A (upper panel). Both
32P-labeled GST-band III and 125I radioactivity
associated to ZAP-70 were quantitated by PhosphorImager scanning. The
relative catalytic activities of ZAP-WT and ZAP-YEEI were obtained by
normalizing P32-GST-band III band volumes for the
respective 125I-labeled band volume. The experiment shown
is representative of four independent determinations. WB,
Western blot.
(phospho-ITAM). Phospho-ITAM-bound proteins were precipitated by the
addition of avidin beads and subjected to SDS-PAGE and immunoblotting
with an anti-tag mAb. Fig. 6 shows that
similar amounts of ZAP-WT and ZAP-YEEI bound to the phospho-ITAM,
demonstrating that the mutation does not increase the affinity of
ZAP-70 for the phosphorylated ITAMs.
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Fig. 6.
Mutation of Y319SDP to
Y319EEI in ZAP-70 does not affect its binding to
phosphorylated ITAMs. TCR-negative 31-13 T-cells were transfected
with 10 µg of either an empty pSR -puro vector or a ZAP-WT or a
ZAP-YEEI construct. After transfection (24 h) cells were lysed, and the
lysates were mixed with 6 µM of biotinylated, doubly
phosphorylated ITAM peptide (phospho-ITAM), followed by the addition of
avidin beads to collect complexes. Band protein were analyzed by
SDS-PAGE and immunoblotted with an anti-tag mAb. The amount of
expressed ZAP-70 tag proteins in each lysate was determined by anti-tag
immunoblot. The experiment shown is representative of three independent
determinations. WB, Western blot.
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Fig. 7.
Binding of the mutant ZAP-YEEI to
LckSH2. Jurkat T-cells were transfected with 10 µg of either an
empty pSR -puro vector or a ZAP-WT or a ZAP-YEEI construct. After
transfection (24 h) cells were lysed, and the lysates were incubated
for 2 h with MBP-LckSH2 Sepharose beads. The beads were washed,
and the associated proteins were analyzed by SDS-PAGE and
immunoblotting with an anti-tag mAb. The amount of expressed ZAP-70 tag
proteins in each lysate was determined by anti-tag immunoblot.
WB, Western blot.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
SH2)ZAP demonstrate that the
presence of residues Tyr69, Tyr126, and
Tyr178 is dispensable for the binding of ZAP-70-derived
phosphopeptides to LckSH2 (Fig. 3D). It has been recently
demonstrated that mutation of C-terminal Tyr597 and
Tyr598 results in a gain-of-function mutant of ZAP-70 (38),
suggesting a negative role for the phosphorylation of these residues.
Moreover, mutation of Tyr474 has been shown to generate a
dominant negative mutant of ZAP-70, possibly as a result of its
defective interaction with the adapter protein Shc (15). However,
contrary to Tyr319, mutation of Tyr474 does not
affect the activation-induced kinase activity of ZAP-70. Based on the
phenotype of these mutants, it seems extremely unlikely that residues
Tyr474 or Tyr597/Tyr598 are
involved in the binding of LckSH2.
D'1 (44) of the LckSH2, providing
further stabilization of the complex.
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ACKNOWLEDGEMENTS |
---|
We thank Drs. C. Baldari, F. Baleux, A. M. Brunati, T. E. Kreis, A. Isacchi, G. Magistrelli, E. L. Reinherz, and R. Sekaly and for kind help in providing materials; Drs. S. Pellegrini and R. Weil for critical reading the manuscript and for suggestions; Dr. L. A. Pinna for encouragement in this work; 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 Centre National de la Recherche Scientifique, 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.
§ These authors contributed equally to this work.
¶ Recipient of a Fondation pour la Recherche Médicale fellowship. Supported by a post-doctoral fellowship of the University of Padova (Department of Biological Chemistry).
Recipient of an Association pour la Recherche sur le Cancer
fellowship. Supported by the Human Frontier Science Program.
To whom correspondence should be addressed: Molecular
Immunology Unit, Dept. of Immunology, Inst. Pasteur, 25, Rue du Docteur Roux, 75724 Paris Cedex 15, France. Tel.: 33-1-4568-8637; Fax: 33-1-4061-3204; E-mail: oacuto{at}pasteur.fr.
2 G. Magistrelli, R. Bosotti, B. Valsasina, C. Visco, R. Perego, S. Toma, O. Acuto, and A. Isacchi, submitted for publication.
3 M. Pelosi, D. Mège, and O. Acuto, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are: PTK, protein-tyrosine kinase; TCR, T-cell antigen receptor; SH2, Src homology 2; ITAM, immunoreceptor tyrosine-based activation motif; NFAT, nuclear factor of activated T-cells; mAb, monoclonal antibody; VSV-G, vescicular stomatitis virus-glycoprotein G; GST, glutathione S-transferase; MBP, maltose-binding protein; NFAT-luc, NFAT-luciferase; WT, wild type; PAGE, polyacrylamide gel electrophoresis; MES, 4-morpholineethanesulfonic acid; Tricine, N-[2- hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine.
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