Tyrosine 319 in the Interdomain B of ZAP-70 Is a Binding Site for the Src Homology 2 Domain of Lck*

Michele PelosiDagger §, Vincenzo Di BartoloDagger §parallel , Virginie MounierDagger , Dominique MègeDagger , Jean-Marc PascussiDagger , Evelyne DufourDagger , Arnaud Blondel**, and Oreste AcutoDagger Dagger Dagger

From the Dagger  Molecular Immunology Unit and the ** Cellular Biochemistry Unit, Institut Pasteur, 25-28 Rue du Docteur Roux, 75724 Paris Cedex 15, France

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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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.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 (zeta , and CD3 gamma , delta , and epsilon ) 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).

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.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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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-zeta 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.

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 beta  chain transcript (22), was maintained in complete RPMI 1640 medium. beta WT160 was obtained from 31-13 cells by stable transfection of the TCR beta -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.

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 beta -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 pSRalpha -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.

The GST-(Delta 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).

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 beta -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.

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 pSRalpha -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 pSRalpha -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). beta -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).

Phosphopeptide Analysis-- The GST-(Delta 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 [gamma -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.

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 [gamma -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
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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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 Calpha 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 Cbeta and Cgamma 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 Rbeta 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.

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).


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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-(Delta SH2)ZAP-70 proteins were autophosphorylated in vitro in the presence of [gamma -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.

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-(Delta SH2)ZAP). Three different fusion proteins were used: the wild-type construct (GST-(Delta SH2)ZAP-WT) and the two mutants where Tyr315 or Tyr319 were replaced by phenylalanine (GST-(Delta SH2)ZAP-Y315F and GST-(Delta SH2)ZAP-Y319F, respectively). When GST-(Delta SH2)ZAP-WT was autophosphorylated in vitro in the presence of [gamma -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-(Delta SH2)ZAP-WT construct, essentially Tyr315 and Tyr319 are phosphorylated (16).

Comparison of the CNBr phosphopeptide pattern of autophosphorylated GST-(Delta SH2)ZAP-Y315F and GST-(Delta SH2)ZAP-Y319F to the GST-(Delta 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-(Delta SH2)ZAP-WT and GST-(Delta SH2)ZAP-Y315F constructs, whereas virtually no peptide from the CNBr-cleaved GST-(Delta 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.

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 beta -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 pSRalpha -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 pSRalpha -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 beta 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 pSRalpha -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.

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 beta WT160, a 31-13 derivative in which TCR expression was rescued by stably transfecting the TCR-beta 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 beta 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.

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.


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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 pSRalpha -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 pSRalpha -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 [gamma -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.

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-zeta (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 pSRalpha -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.

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.


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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 pSRalpha -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

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-(Delta 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.

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 Rbeta D'1 (44) of the LckSH2, providing further stabilization of the complex.

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.

    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.

    FOOTNOTES

* 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).

parallel Recipient of an Association pour la Recherche sur le Cancer fellowship. Supported by the Human Frontier Science Program.

Dagger Dagger 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.

    ABBREVIATIONS

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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ABSTRACT
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EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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