The Zap70 protein tyrosine kinase controls TCR-linked signal transduction pathways and is critical for T cell development and responsiveness. Following engagement of TCR, the Zap70 undergoes phosphorylation on multiple tyrosine residues that are implicated in the regulation of its catalytic activity and interaction with signaling effector molecules downstream of the TCR. We have shown previously that the CT10 regulator of kinase II (CrkII) adapter protein interacts with tyrosine-phosphorylated Zap70 in TCR-engaged T cells, and now extend these studies to show that Tyr315 in the Zap70 interdomain B region is the site of interaction with CrkII. A point mutation of Tyr315 (Y315F) eliminated the CrkII-Zap70 interaction capacity. Phosphorylation of Tyr315 and Zap70 association with CrkII were both dependent upon the Lck protein tyrosine kinase. Previous studies demonstrated the Tyr315 is the Vav-Src homology 2 (SH2) binding site, and that replacement of Tyr315 by Phe impaired the function of Zap70 in TCR signaling. However, fluorescence polarization-based binding studies revealed that the CrkII-SH2 and the Vav-SH2 bind a phosphorylated Tyr315-Zap70-derived peptide with affinities of a similar order of magnitude (Kd of 2.5 and 1.02 μM, respectively). The results suggest therefore that the biological functions attributed to the association of Zap70 with Vav following T cell activation may equally reflect the association of Zap70 with CrkII, and further support a regulatory role for CrkII in the TCR-linked signal transduction pathway.

Tlymphocyte activation is dependent upon a sustained physical interaction of the TCR, with a peptide-bound MHC molecule on the surface of APCs. This interaction results in a temporal and spatial organization of multiple cellular elements at the T cell-APC-contact region, referred to as the immunological synapse (1, 2). The transient assembly of functional molecular complexes in activated T cells enables the recruitment of enzymes and other effector molecules to specific compartments at the immunological synapse, thus activating a tightly regulated gene transcription program, which leads to cell differentiation and acquisition of effector function.

Among the earliest detectable effects in engaged T cells are the phosphorylation of multiple cellular substrates on tyrosine residues, including the cytoplasmic tails of the TCR subunits (3, 4, 5). This step is essential for the recruitment of signaling effector molecules to the receptor site and their assembly into functional activation complexes. Whereas the TCR subunits are devoid of enzymatic activity, several nonreceptor protein tyrosine kinases (PTKs),3 which are constitutively associated with, or recruited to the activated TCR, mediate the phosphorylation of the receptor subunits, as well as other substrates at the immunological synapse. These include the Src family members, Lck and Fyn, which constitutively associate with the TCR/CD3 and the CD4/CD8 coreceptors, respectively, and Zap70, a cytosolic PTK that is recruited to the activated TCR and interacts with its tyrosine-phosphorylated receptor subunits (6, 7). Activation of these PTKs at the receptor site induces the phosphorylation and/or up-regulation of critical enzymes, such as phospholipase Cγ1 and PI3K, which produce second messengers that serve as signal amplifiers (8, 9). In addition, they phosphorylate scaffold proteins that have no enzymatic activity, such as linker for activation of T cells, SH2 domain-containing leukocyte protein of 76 kDa, and Cbl, which function as docking sites for Src homology 2 (SH2)/SH3-containing effector molecules (10).

A third type of proteins, which play important roles in the assembly of multimolecular complexes at the receptor site, includes cytoplasmic adapter proteins, such as growth factor receptor-bound protein 2 (Grb2), Shc, and CT10 regulator of kinase (Crk). These proteins are devoid of enzymatic activity, but possess different protein-protein or protein-phospholipid interaction domains (11, 12). They potentially regulate cell growth and differentiation by coupling proximal biochemical events, initiated by cell surface receptor engagement, with distal signal-transducing pathways.

The Crk adaptor proteins are implicated in signaling pathways that regulate diverse cellular functions, and lead to cell growth (13), differentiation (14, 15), transformation (16, 17), and death (18). They are involved in coordinating signaling pathways linked to surface receptors for distinct extracellular ligands, such as integrins (19), ILs (20), and growth factors (20, 21, 22). In addition, Crk proteins appear to be involved in signal transduction from Ag receptors on B and T lymphocytes (23, 24, 25, 26, 27). However, in contrast to adapter proteins, such as Grb2 and Shc, which function as putative positive regulators of TCR-coupled signaling pathways, Crk is implicated in the negative regulation of TCR-proximal signaling events, which may regulate the termination of TCR-induced activation signals, or promote T cell suppression and induction of immune anergy (23). T cell anergy can be induced by different activation conditions, including the engagement of the TCR in the absence of costimulation (24, 25, 28, 29). It is possible therefore that the Crk adapter proteins are involved in the regulation of the inductive phase (13, 30). A single study suggested that Crk associates with negative regulatory proteins, such as Cbl and C3G, in anergic, but not in responsive T cells (30), but is questionable in view of more recent findings demonstrating a positive role for C3G in multiple cell signaling pathways (31).

We have previously shown that CrkII associates with tyrosine-phosphorylated and enzymatically active Zap70 PTK in TCR-stimulated T cells (27). In addition, it forms a cell activation-dependent trimolecular complex with the PI3K regulatory subunit, p85, and the Cbl protein (26). We now demonstrate that the Zap70 Tyr315 is critical for the interaction with CrkII. A point mutation of Tyr315 (Y315F) eliminated the CrkII-Zap70 interaction. Tyrosine phosphorylation of Zap70 is dependent on the Lck PTK, which either directly phosphorylates Zap70, or serves as an upstream regulator of a Zap70-specific PTK (32). Tyr315 was found to serve as a potential binding site for the Vav-SH2 domain and to be critical for several Zap70-dependent T cell functions (33). We found that the CrkII-SH2 and the Vav-SH2 interact with the phosphorylated Tyr315 with affinities of a similar order of magnitude, suggesting that this site is equally important for Zap70 regulation by both molecules. In addition, it is possible that binding competition between Vav and CrkII, and their relative abundance at the vicinity of Zap70 may determine selected Zap70-induced biological functions.

Aprotinin, leupeptin, and Triton X-100 were from Sigma-Aldrich. AEBSF was from ICN Biomedicals; nitrocellulose membranes were from Schleicher & Schuell Microscience; ECL, glutathione-Sepharose 4B, and protein A-Sepharose were from Amersham Biosciences.

Anti-phosphotyrosine (pY) (4G10) was from Upstate Biotechnology; a mouse mAb specific for CrkI/CrkII was from BD Transduction Laboratories; and anti-Cbl and anti-GST mAb were from Santa Cruz Biotechnology. The hybridoma, 9E10, produces IgG1 mAb against the human Myc-derived epitopic tag SMEQKLISEEDLN (34), was obtained from the American Type Culture Collection, and used for the preparation of ascites in BALB/c mice. The hybridoma, HA.11 (clone 16B12), produces IgG1 mAb that was raised against a 12-aa peptide (CYPYDVPDYASL) and specifically recognizes the influenza hemagglutinin (HA) epitope. This mAb was obtained from Covance Research Products. Rabbit anti-Zap70 polyclonal antiserum was raised against GST fusion protein containing aa 255–345 of human Zap70, a gift from J. Bolen (DNAX, Palo Alto, CA) (35). HRP-conjugated sheep anti-mouse, or donkey anti-rabbit Ig Abs, and HRP-conjugated protein A were from Amersham Biosciences.

Myc-tagged full-length Zap70 in pSXSRα expression vector was prepared and its sequence was verified, as described (36). HA-tagged wild-type (WT) Zap70 and Tyr292→Phe mutant in pSXSRα were a gift of A. Altman (La Jolla Institute of Allergy and Immunology, San Diego, CA). Single point mutations (Tyr292→Phe and Tyr315→Phe) and a deletion mutant (Δ265–331) of Myc-tagged Zap70 in pSXSRα (37) were a gift of A. Weiss (University of California, San Francisco, CA).

pGEX-5X vectors were from Amersham Pharmacia Biotech, and pGEX plasmids encoding different GST-CrkII fusion proteins were gifts of M. Matsuda (National Institute of Health, Tokyo, Japan). All pGEX plasmids were used for transformation of Escherichia coli DH5α cells (Invitrogen Life Technologies). Recombinant fusion proteins were prepared by bacterial growth in Luria-Bertani and protein induction with 0.1 mM isopropyl-1-thio-β-d-galactopyranoside (Promega). After ∼3 h of incubation, the bacteria were resuspended in a lysis buffer containing 50 mM Tris-HCl (pH 8.0), 100 mM NaCl, 1 mM PMSF, and 1% Triton X-100, and further disrupted by sonication. Following centrifugation at 10,000 × g for 20 min, the induced proteins were adsorbed to bead-immobilized glutathione.

In vitro binding studies for pull-down assays were performed by incubation of bead-adsorbed GST or GST fusion proteins (5 μg/sample) with cell lysates at 4°C on a rotator for 3 h. The beads were then washed three times in a lysis buffer, and bound proteins were eluted by boiling in sample buffer and subjected to SDS-PAGE under reducing conditions, followed by immunoblotting.

Human leukemic Jurkat T cells, Jurkat cell-derived Zap70-deficient P116 cells, and African green monkey SV40-transformed kidney-derived Cos-7 cells were maintained at a logarithmic growth phase in complete RPMI (RPMI 1640 supplemented with 5% heat-inactivated FCS, 2 mM l-glutamine, 50 U/ml penicillin, 50 μg/ml streptomycin (all from Biological Industries), and 5 × 10−6 M 2-ME (Sigma-Aldrich)) in 75-cm2 growth-area tissue culture flasks (Cell-Cult; Sterilin) in an atmosphere of 7.5% CO2 at 37°C.

Preparation and characterization of the P116 subline, a Zap70-deficient somatic mutant, which is derived from the Jurkat E6 T cell line, were previously described (38). P116 cells, and P116 that stably express Myc-tagged WT Zap70, or a Myc-tagged single Tyr→Phe mutation at position 315 or 319, were gifts of R. Abraham (Burnham Cancer Institute, La Jolla, CA). Jurkat or P116 cells (10 × 106/100 μl) were stimulated with freshly prepared 1% pervanadate (perVO4) (10 mM Na3VO4 containing 1% H2O2) for 30 min at 37°C.

Cell lysates were prepared by resuspension of cells in a lysis buffer containing 25 mM Tris-HCl (pH 7.5), 150 mM NaCl, 5 mM EDTA, 1 mM Na3VO4, 50 mM NaF, 10 μg/ml each of leupeptin and aprotinin, 2 mM AEBSF, and 1% Triton X-100, followed by a 20-min incubation on ice. Lysates were centrifuged at 13,000 × g for 30 min at 4°C, and the nuclear-free supernatants were used for immunoprecipitation studies, or mixed with equal volumes of 2× SDS sample buffer, vortexed, boiled for 5 min, and analyzed by SDS-PAGE.

Immunoprecipitation was performed by using an optimal dilution of polyclonal antisera or mAbs that were preabsorbed on protein A-Sepharose beads for 1 h at 4°C. Excess Abs were removed by three washes in cold PBS, and Ab-coated beads were incubated with cell lysates for 2–3 h at 4°C. Immune complexes were precipitated by centrifugation, followed by extensive washing in a lysis buffer. Immunoprecipitated proteins were then fractionated by SDS-PAGE and immunoblotted with specific Abs.

Samples of cell lysates, GST fusion proteins, GST fusion protein-bound molecules, or Ab immunoprecipitates were resolved by electrophoresis on 10% acrylamide gels using Bio-Rad MiniPROTEAN II Cell. Proteins in the gels were either stained with Coomassie brilliant blue (Sigma-Aldrich) or blotted onto nitrocellulose membranes (Schleicher & Schuell Microscience) at 100 V for 45 min in a Bio-Rad Mini Trans-Blot transfer cell. After 1 h blocking at 37°C with 3% BSA in PBS, nitrocellulose membranes were incubated with the indicated primary Abs, followed by incubation with HRP-conjugated sheep anti-mouse, or donkey anti-rabbit, Ig, or with HRP-conjugated protein A. Immunoreactive proteins were visualized using an ECL reagent and autoradiography.

Peptide arrays were constructed according to the Spot-synthesis methods (39, 40). Acid-hardened cellulose membranes prederivatized with polyethylene glycol (AbiMed-Langfield) were spotted with a grid of 9-fluorenylmethyloxycarbonyl (Fmoc) β-alanin (Bachem) before peptide synthesis. Standard Fmoc chemistry was used throughout. Fmoc-protected and -activated amino acids were spotted in high density 24 × 18 Spot arrays on 130 × 90-mm membranes using an AbiMed ASP422 robot. All washing, Fmoc, and side chain deprotection steps were done manually in polypropylene containers. The amino acids were at a concentration of 0.25 M and were spotted at a volume of 0.2 μl, twice for each coupling reaction. After overnight blocking of the membrane in TBS containing 10% skim milk and 0.05% Tween 20 (T-TBS), the membranes were probed with GST or GST-CrkII-SH2 fusion proteins (10 μg/ml for 3 h at room temperature). This incubation was followed by three washes with T-TBS and incubation of the membranes with anti-GST Abs (1:1,000 in T-TBS). After additional three washes with T-TBS, the membranes were incubated for 1 h with HRP-conjugated anti-mouse IgG (1:10,000 in T-TBS), and bound proteins were visualized using an ECL reagent and autoradiography.

Fifteen-mer peptides corresponding to Zap70 residues 311–325 (Table I) were synthesized on an Applied Biosystems 431A automated peptide synthesizer using a standard Fmoc solid-phase chemistry. Phosphotyrosine was directly incorporated as its Nα-fluorenylmethyloxycarbonyl-O-phosphoro-l-tyrosine derivative. Amino acid activation, peptide elongation, and cleavage of the peptide from the resin were performed as described (41, 42). Crude peptides were then precipitated and purified by HPLC (41, 42). Peptide identity was confirmed by electrospray mass spectroscopy and amino acid analysis. Fluorescein labeling of the peptides at their amino terminus was conducted through reaction with 5 (and 6)-carboxyfluorescein succinimidyl ester (Molecular Probes) and HPLC purification. The concentrations of the peptide stock solutions were determined according to their absorbance at 270 nm using extinction coefficients of 1752 M−1 cm−1 for pTyr and 840 M−1 cm−1 for Tyr (43).

Table I.

Binding affinity values of CrkII-SH2 and Vav-SH2 domains to fluorescein-labeled Zap70 peptidesa

PeptideSequenceDissociation Constant (Kd, M) (SE)
GST-CrkII-SH2GST-Vav-SH2
F-YY Fluorescein-DTSVY315ESPY319SDPELK >100 >100 
F-pYY Fluorescein-DTSVpY315ESPY319SDPELK 2.5 ± 0.24 1.02 ± 0.20 
F-pYpY Fluorescein-DTSVpY315ESPpY319SDPELK 1.07 ± 0.25 0.53 ± 0.08 
PeptideSequenceDissociation Constant (Kd, M) (SE)
GST-CrkII-SH2GST-Vav-SH2
F-YY Fluorescein-DTSVY315ESPY319SDPELK >100 >100 
F-pYY Fluorescein-DTSVpY315ESPY319SDPELK 2.5 ± 0.24 1.02 ± 0.20 
F-pYpY Fluorescein-DTSVpY315ESPpY319SDPELK 1.07 ± 0.25 0.53 ± 0.08 
a

Binding affinity values of CrkII-SH2 and Vav-SH2 domains to fluorescein-labeled Zap70 peptides. Fluoresceinated probes are short synthetic peptides corresponding to Zap70 residues 311–325 in which both Tyr315 and Tyr319 are phosphorylated (F-pYpY), only Tyr315 is phosphorylated (F-pYY), or none of the tyrosine residues is phosphorylated (F-YY). Tyrosine and phosphotyrosines within the sequence of the peptides are indicated by bold letters. Kd values were determined by nonlinear regression analysis using the GraphPad software, and represent average values ± SE of three independent measurements.

The equilibrium dissociation constants (Kd) were determined by using fluorescence polarization measurements on a Beacon Fluorescence Polarization System (Invitrogen Life Technologies). Binding studies were conducted with 5 nM fluorescein-labeled probe dissolved in PBS containing 1 mM DTT and mixed with 0.02–15 μM GST-CrkII-SH2 or GST-Vav-SH2 protein (final volume 100 μl). The reaction mixtures were allowed to stand for 5 min at room temperature before each measurement. All binding studies were conducted at 22°C. The polarization value represents a ratio of light intensities and is expressed in millipolarization units. Nonlinear regression analysis and dissociation constant values were obtained using the GraphPad prism, version 3.0 for Windows (GraphPad).

In a previous work, we demonstrated the association of CrkII with tyrosine-phosphorylated Zap70 from activated T cells (27). To further analyze the mechanism of interaction between the two molecules, we have now used Zap70 mutants that are transiently expressed in Cos-7 cells, or constitutively expressed in P116 cells, a Jurkat T cell-derived subline that is deficient in Zap70. To confirm the association between CrkII and Zap70 in activated Jurkat T cells, and the lack of Zap70 in the P116 cells, we treated both cell types with pervanadate and tested the ability of bead-immobilized CrkII to pull down Zap70. Results (Fig. 1) confirm the presence of Zap70 in the whole cell lysate of Jurkat, but not P116 cells. In addition, they demonstrate that GST-CrkII pulls down Zap70 from pervanadate-treated, but not resting Jurkat T cells. The findings that GST-CrkII pulls down a 70-kDa protein band from Jurkat, but not P116 cells reconfirm that the 70-kDa protein is identical with Zap70. An internal control showing usage of equal amounts of GST-CrkII in all four GST-CrkII pull-down samples is provided by the cross-reactive response of the anti-GST-Zap70 antiserum with the GST moiety of the GST-CrkII fusion protein.

FIGURE 1.

Bead-immobilized GST-CrkII pulls down a Zap70-immunoreactive 70-kDa protein from activated Jurkat, but not P116 T cells. Jurkat T cells or Zap70-deficient Jurkat-derived P116 cells (4 × 107/group) were treated with 1% pervanadate (perVO4) at 37°C for 30 min. Cell lysates were then incubated with GST or GST-CrkII fusion proteins immobilized to glutathione agarose beads on a rotator at 4°C. After 3 h of incubation followed by extensive washings in lysis buffer, the beads were boiled for 5 min in sample buffer, and eluted proteins were subjected to SDS-PAGE under reducing conditions. The proteins were electroblotted onto a nitrocellulose membrane that was incubated with anti-Zap70 Abs, and immunoreactive proteins were visualized by reaction with an HRP-conjugated secondary Ab, development with immunoperoxidase ECL detection system, and autoradiography. Control whole cell lysate (CT-WCL) was run in parallel to show the presence or absence, and the position of the Zap70 protein band. Arrowheads indicate the position of the anti-Zap70-immunoreactive protein band and the GST-CrkII fusion protein (which is recognized by anti-GST Abs produced against the GST-Zap70 immunogen). Results are representative of three independent experiments.

FIGURE 1.

Bead-immobilized GST-CrkII pulls down a Zap70-immunoreactive 70-kDa protein from activated Jurkat, but not P116 T cells. Jurkat T cells or Zap70-deficient Jurkat-derived P116 cells (4 × 107/group) were treated with 1% pervanadate (perVO4) at 37°C for 30 min. Cell lysates were then incubated with GST or GST-CrkII fusion proteins immobilized to glutathione agarose beads on a rotator at 4°C. After 3 h of incubation followed by extensive washings in lysis buffer, the beads were boiled for 5 min in sample buffer, and eluted proteins were subjected to SDS-PAGE under reducing conditions. The proteins were electroblotted onto a nitrocellulose membrane that was incubated with anti-Zap70 Abs, and immunoreactive proteins were visualized by reaction with an HRP-conjugated secondary Ab, development with immunoperoxidase ECL detection system, and autoradiography. Control whole cell lysate (CT-WCL) was run in parallel to show the presence or absence, and the position of the Zap70 protein band. Arrowheads indicate the position of the anti-Zap70-immunoreactive protein band and the GST-CrkII fusion protein (which is recognized by anti-GST Abs produced against the GST-Zap70 immunogen). Results are representative of three independent experiments.

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Several studies have demonstrated that T cell activation is followed by phosphorylation of Zap70 on multiple tyrosine residues (36, 44, 45). Some of the phosphotyrosine residues were implicated in the regulation of Zap70 catalytic activity (36, 45, 46, 47), while others, predominantly those located at the interdomain B region (see Fig. 2), were found to mediate interaction with distinct SH2-containing effector molecules (33, 37, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56). To test whether interdomain B-residing phosphotyrosyl residues are required for the interaction with CrkII, we used Cos-7 cells that transiently overexpress Myc-tagged WT Zap70 or Myc-tagged Zap70Δ265–331 proteins. The cells were then treated with 1% pervanadate, and the ability of the Zap70 proteins to interact with CrkII was tested. Pull-down assays with bead-immobilized fusion proteins and immunoblot with anti-Zap70 antiserum demonstrated that Zap70WT, but not Zap70Δ265–331, interacted with CrkII (Fig. 3,A). Anti-Myc immunoblot of the above cell lysates confirmed the expression of equal levels of Zap70 proteins and demonstrated their position on the gel, which coincided with the position of the bands that reacted with anti-Zap70 Abs (Fig. 3 B).

FIGURE 2.

A schematic representation of the Zap70 protein structure demonstrating the two tandem SH2 domains, the C-terminal kinase domain, an intervening region, termed interdomain B. A partial amino acid sequence of interdomain B and the position of its three regulatory tyrosine residues are also indicated.

FIGURE 2.

A schematic representation of the Zap70 protein structure demonstrating the two tandem SH2 domains, the C-terminal kinase domain, an intervening region, termed interdomain B. A partial amino acid sequence of interdomain B and the position of its three regulatory tyrosine residues are also indicated.

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FIGURE 3.

Truncation of the Zap70 interdomain B region abrogates its ability to interact with CrkII. Cos-7 cells (10 × 107/quvette) were transfected with 5 μg of pEF-Lck and 5 μg of either Myc-tagged WT Zap70 or Myc-tagged Zap70Δ265–331 cDNA, cultured for 48 h at 37°C, and then stimulated with 1% pervanadate (perVO4) for additional 30 min. Cell lysates were then incubated with 5 μg of bead-immobilized GST or GST-CrkII fusion proteins for 3 h on a rotator at 4°C. After extensive washings, the beads were boiled for 5 min in sample buffer, and eluted proteins were subjected to SDS-PAGE under reducing conditions. Proteins were electroblotted onto a nitrocellulose membrane that was incubated with anti-Zap70 Abs, and treated with an immunoperoxidase ECL detection system and autoradiography (A). To control for protein expression, Ab specificity, and electrophoretic mobility of the expressed proteins, a parallel gel was run with total cell lysates and anti-Myc immunoprecipitates, followed by immunoblotting with anti-Myc mAbs (B). Control whole cell lysate was run in parallel to show the presence and position of the WT and mutated Zap70 protein bands. Molecular size markers (in kDa) are indicated on the left and arrowheads mark the position of the indicated protein bands. Results are representative of two independent experiments.

FIGURE 3.

Truncation of the Zap70 interdomain B region abrogates its ability to interact with CrkII. Cos-7 cells (10 × 107/quvette) were transfected with 5 μg of pEF-Lck and 5 μg of either Myc-tagged WT Zap70 or Myc-tagged Zap70Δ265–331 cDNA, cultured for 48 h at 37°C, and then stimulated with 1% pervanadate (perVO4) for additional 30 min. Cell lysates were then incubated with 5 μg of bead-immobilized GST or GST-CrkII fusion proteins for 3 h on a rotator at 4°C. After extensive washings, the beads were boiled for 5 min in sample buffer, and eluted proteins were subjected to SDS-PAGE under reducing conditions. Proteins were electroblotted onto a nitrocellulose membrane that was incubated with anti-Zap70 Abs, and treated with an immunoperoxidase ECL detection system and autoradiography (A). To control for protein expression, Ab specificity, and electrophoretic mobility of the expressed proteins, a parallel gel was run with total cell lysates and anti-Myc immunoprecipitates, followed by immunoblotting with anti-Myc mAbs (B). Control whole cell lysate was run in parallel to show the presence and position of the WT and mutated Zap70 protein bands. Molecular size markers (in kDa) are indicated on the left and arrowheads mark the position of the indicated protein bands. Results are representative of two independent experiments.

Close modal

To narrow down the region within interdomain B, which is relevant for the interaction with CrkII, we first used Jurkat TAg cells transfected with HA-tagged WT Zap70, and HA- or Myc-tagged expression vectors of Zap70 with Tyr→Phe mutations at position 292 or 315, respectively. Cell treatment with pervanadate, pull-down of cell lysate proteins using bead-immobilized GST or GST-CrkII fusion proteins, and immunoblot with anti-Zap70 mAbs demonstrated the presence of Zap70 in all cell lysates and in all groups treated with GST-CrkII (Fig. 4,A). These anti-Zap70-immunoreactive protein bands can include both the endogenous and the overexpressed Zap70 molecules. However, reblotting of the same membranes with anti-tag mAbs, which selectively detect the overexpressed proteins, revealed that Zap70WT and Zap70Y292F, but not Zap70Y315F, were pulled down by GST-CrkII (Fig. 4 B). Binding of Zap70 was specific for CrkII and was not observed with bead-immobilized GST.

FIGURE 4.

Binding of a transiently overexpressed Zap70 from activated Jurkat T cells to GST-CrkII requires Tyr315. Jurkat TAg cells (10 × 107/quvette) were transfected with 5 μg of HA-tagged WT Zap70, HA-tagged Zap70Y292F, or Myc-tagged Zap70Y315F cDNA. The cells were cultured for 48 h at 37°C, followed by treatment with 1% pervanadate (perVO4) for additional 30 min. Cell lysates were then incubated with 5 μg of bead-immobilized GST or GST-CrkII fusion proteins for 3 h on a rotator at 4°C. After extensive washings, the beads were boiled for 5 min in sample buffer, and eluted proteins were subjected to SDS-PAGE under reducing conditions. Proteins from the gel were electroblotted onto a nitrocellulose membrane that was immunoblotted with anti-Zap70 Abs (A), followed by stripping of the membranes and reblotting with either anti-HA or anti-Myc Abs, as indicated (B). Control whole cell lysate was run in parallel, to show the presence and position of the WT and mutated Zap70 protein bands. Molecular size markers (in kDa) are indicated on the left and arrowheads mark the position of the indicated protein bands. Results are representative of two independent experiments.

FIGURE 4.

Binding of a transiently overexpressed Zap70 from activated Jurkat T cells to GST-CrkII requires Tyr315. Jurkat TAg cells (10 × 107/quvette) were transfected with 5 μg of HA-tagged WT Zap70, HA-tagged Zap70Y292F, or Myc-tagged Zap70Y315F cDNA. The cells were cultured for 48 h at 37°C, followed by treatment with 1% pervanadate (perVO4) for additional 30 min. Cell lysates were then incubated with 5 μg of bead-immobilized GST or GST-CrkII fusion proteins for 3 h on a rotator at 4°C. After extensive washings, the beads were boiled for 5 min in sample buffer, and eluted proteins were subjected to SDS-PAGE under reducing conditions. Proteins from the gel were electroblotted onto a nitrocellulose membrane that was immunoblotted with anti-Zap70 Abs (A), followed by stripping of the membranes and reblotting with either anti-HA or anti-Myc Abs, as indicated (B). Control whole cell lysate was run in parallel, to show the presence and position of the WT and mutated Zap70 protein bands. Molecular size markers (in kDa) are indicated on the left and arrowheads mark the position of the indicated protein bands. Results are representative of two independent experiments.

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To further substantiate these findings, we took advantage of the Jurkat-derived P116 cell lines, which stably express different Zap70 mutants. We found that GST-CrkII pulled down Zap70WT that was transfected into P116 cells, as well as the Zap70Y319F mutant (Fig. 5,A). However, Zap70 was not detected in the GST-CrkII pull-down from lysates of Zap70Y315F P116 cells (Fig. 5, B and C). The faint band of a ∼70-kDa protein band in the last lane of Fig. 5,B is distinct from the Zap70Y315F protein, because it does not react with the tag-specific anti-Myc mAb. The Zap70Y315F mutant protein was expressed in the P116 cells and could be detected in the whole cell lysate by the Zap70-specific Abs (Fig. 5,B). Nevertheless, there was a slight possibility that the pulled down Zap70Y315F was less well detected by anti-Zap70 Abs, if the Y315F mutation impaired an immunodominant epitope, resulting in a lower affinity of this mutated protein to the Zap70 Abs. To eliminate this possibility, we reblotted the same membrane with a mAb specific for the Myc tag. The results in Fig. 5,C confirmed the presence of the Zap70Y315F in the whole cell lysate, but not in the GST-CrkII pull down (Fig. 5 C).

FIGURE 5.

Zap70 Tyr315 mediates the T cell activation-induced interaction of Zap70 with CrkII in stable transfected P116 cells. P116 cells that constitutively express WT Zap70, Myc-Zap70Y319F, or Myc-Zap70Y319F cDNA were treated with 1% pervanadate (perVO4) for 30 min. Cell lysates were then incubated with 5 μg of bead-immobilized GST or GST-CrkII fusion proteins for 3 h on a rotator at 4°C. After extensive washings, the beads were boiled for 5 min in sample buffer and eluted proteins were subjected to SDS-PAGE under reducing conditions. Proteins from the gel were electroblotted onto nitrocellulose membranes that were immunoblotted with anti-Zap70 Abs (A and B), followed by stripping and reblotting with anti-Myc mAbs (C). Control whole cell lysate was run in parallel to show the presence and position of the WT and mutated Zap70 protein bands. Molecular size markers (in kDa) are indicated on the left and arrowheads mark the position of the indicated protein bands. Results are representative of two independent experiments.

FIGURE 5.

Zap70 Tyr315 mediates the T cell activation-induced interaction of Zap70 with CrkII in stable transfected P116 cells. P116 cells that constitutively express WT Zap70, Myc-Zap70Y319F, or Myc-Zap70Y319F cDNA were treated with 1% pervanadate (perVO4) for 30 min. Cell lysates were then incubated with 5 μg of bead-immobilized GST or GST-CrkII fusion proteins for 3 h on a rotator at 4°C. After extensive washings, the beads were boiled for 5 min in sample buffer and eluted proteins were subjected to SDS-PAGE under reducing conditions. Proteins from the gel were electroblotted onto nitrocellulose membranes that were immunoblotted with anti-Zap70 Abs (A and B), followed by stripping and reblotting with anti-Myc mAbs (C). Control whole cell lysate was run in parallel to show the presence and position of the WT and mutated Zap70 protein bands. Molecular size markers (in kDa) are indicated on the left and arrowheads mark the position of the indicated protein bands. Results are representative of two independent experiments.

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The results to date indicate that the phosphorylated Zap70 Tyr315 is critical for CrkII association with Zap70. Although the obvious interpretation is that pTyr315 represents a dominant binding site for the CrkII-SH2, one cannot rule out the possibility that other tyrosine residues mediate the interaction with CrkII. In such a case, the Tyr315→Phe mutation may alter the topology of the mutated Zap70 and thereby prevent it from undergoing phosphorylation on other functional tyrosine residues. We therefore tested the ability of Zap70(Y315F) to undergo tyrosine phosphorylation in pervanadate-treated P116 T cells and compared it with that of WT Zap70 and the Zap70(Y319F) mutant. The results (data not shown) demonstrated that the three forms of Zap70 were expressed in P116 cells at roughly similar levels, and further indicated that the Zap70(Y315F) mutant was not impaired in its ability to undergo tyrosine phosphorylation.

The Lck PTK is linked to the CD4 and CD8 cytoplasmic tails and is involved in the early phase of the T cell activation response (57). It is considered as the major PTK, which phosphorylates the TCRζ and CD3 subunits, and is therefore critical for the early activation events in TCR-engaged T cells. Phosphorylation of the TCRζ promotes the association of Zap70 with the receptor, followed by Lck-mediated phosphorylation of Zap70, and up-regulation of its catalytic activity (32). Nevertheless, the tyrosine residues that are phosphorylated by Lck remain to be fully established. To test whether Tyr315 is a downstream target for phosphorylation by Lck (or a Lck-dependent PTK), we overexpressed the Myc-tagged CrkII protein in the Jurkat-derived Lck-deficient cells, JCaM.1, and Jurkat cells as a control, treated the cells with pervanadate, and tested whether CrkII Abs can coimmunoprecipitate Zap70. The results (Fig. 6) demonstrated that Zap70 coimmunoprecipitated with CrkII from a lysate of Jurkat, but not JCaM.1 cells. This is despite the fact that JCaM.1 and Jurkat express similar levels of Zap70 protein. Anti-pY immunoblotting of the same membrane demonstrated that the level of tyrosine-phosphorylated proteins in JCaM.1 cells is much lower than that of Jurkat cells, suggesting that Lck is a critical upstream PTK for a wide range of substrate proteins in activated T cells.

FIGURE 6.

T cell activation-induced CrkII binding to Zap70 is dependent upon Lck. Jurkat and JCaM1 (Lck-deficient Jurkat cell-derived mutant subline) cells were transfected with 5 μg of Myc-tagged CrkII and cultured for 48 h at 37°C. Cells were then left untreated or incubated with 1% pervanadate (perVO4) for additional 30 min at 37°C. Cell lysates were prepared and incubated with anti-CrkII Ab preadsorbed to bead-immobilized protein A for 3 h on a rotator at 4°C, followed by extensive washings. Bound proteins were eluted from the beads and subjected to SDS-PAGE under reducing conditions. Proteins were then electroblotted onto nitrocellulose membranes and immunoblotted sequentially with anti-Zap70 (A), anti-pY (B), or anti-CrkII (C) Abs. Immunoreactive protein bands were visualized using an immunoperoxidase ECL detection system, followed by autoradiography. Control whole cell lysate (CT-WCL) was run in parallel to show the presence and position of the Zap70 protein band. Molecular size markers (in kDa) are indicated on the left. Arrowheads indicate the position of the Zap70 and CrkII protein bands.

FIGURE 6.

T cell activation-induced CrkII binding to Zap70 is dependent upon Lck. Jurkat and JCaM1 (Lck-deficient Jurkat cell-derived mutant subline) cells were transfected with 5 μg of Myc-tagged CrkII and cultured for 48 h at 37°C. Cells were then left untreated or incubated with 1% pervanadate (perVO4) for additional 30 min at 37°C. Cell lysates were prepared and incubated with anti-CrkII Ab preadsorbed to bead-immobilized protein A for 3 h on a rotator at 4°C, followed by extensive washings. Bound proteins were eluted from the beads and subjected to SDS-PAGE under reducing conditions. Proteins were then electroblotted onto nitrocellulose membranes and immunoblotted sequentially with anti-Zap70 (A), anti-pY (B), or anti-CrkII (C) Abs. Immunoreactive protein bands were visualized using an immunoperoxidase ECL detection system, followed by autoradiography. Control whole cell lysate (CT-WCL) was run in parallel to show the presence and position of the Zap70 protein band. Molecular size markers (in kDa) are indicated on the left. Arrowheads indicate the position of the Zap70 and CrkII protein bands.

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To confirm that Crk-SH2 preferentially interacts with the Tyr315-containing Zap70 epitope at the molecular level, we used a Spot-membrane analysis.

Twelve-mer synthetic peptides representing three overlapping frames of a sequence derived from aa 311–324 of Zap70 were synthesized and spotted on cellulose membranes, as previously described (39, 40).

Each frame included three peptides: a nonphosphorylated peptide and phosphorylated peptides either at position Tyr315 or Tyr319. The filters were blocked with 10% skim milk in TBS-T and probed with 10 μg of GST or GST-CrkII-SH2 fusion proteins for 3 h at room temperature. Protein binding was evaluated by membrane washings, followed by immunoblotting with GST-specific Abs. The results (Fig. 7) demonstrate binding of GST-CrkII-SH2 to pTyr315-containing sequences, but not to nonphosphorylated or pTyr319-containing sequences. Almost identical results were obtained with the three sets of peptides representing aa 311–322, 312–323, or 313–324 of Zap70. No binding was observed with the GST alone.

FIGURE 7.

GST-CrkII-SH2 binding to membrane-coupled Zap70-derived short peptides demonstrates a selective binding to pTyr315-containing peptides. Twelve-mer peptides derived from aa 311–324 of Zap70, and representing three overlapping frames, were synthesized and spotted on a cellulose membrane. The peptides in each frame (A–C) were either nonphosphorylated, or phosphorylated at positions corresponding to the Zap70-Tyr315 or -Tyr319. The membranes were probed with soluble GST or GST-CrkII-SH2 fusion proteins, followed by immunoblotting with anti-GST mAbs. Sequences of synthetic peptides are indicated above each lane. Results are representative of two independent experiments.

FIGURE 7.

GST-CrkII-SH2 binding to membrane-coupled Zap70-derived short peptides demonstrates a selective binding to pTyr315-containing peptides. Twelve-mer peptides derived from aa 311–324 of Zap70, and representing three overlapping frames, were synthesized and spotted on a cellulose membrane. The peptides in each frame (A–C) were either nonphosphorylated, or phosphorylated at positions corresponding to the Zap70-Tyr315 or -Tyr319. The membranes were probed with soluble GST or GST-CrkII-SH2 fusion proteins, followed by immunoblotting with anti-GST mAbs. Sequences of synthetic peptides are indicated above each lane. Results are representative of two independent experiments.

Close modal

To determine the extent of interaction of the CrkII-SH2 and the phosphorylated Tyr315 of Zap70, we used a fluorescence polarization assay. In this assay, polarization measurements reflect the average angular displacement of the fluorophore, which occurs between the absorption and subsequent emission of a photon, and is dependent upon the rate and extent of rotational diffusion during the lifetime of the excited state. Because probes with low molecular mass are very flexible, they have relatively low polarization values when free in solution. However, upon binding to a high molecular mass target protein, the polarization of the probe increases and the differences in fluorescence polarization values can be translated into actual binding affinity values.

Binding studies were performed on a Beacon Fluorescence Polarization system using purified rGST-CrkII-SH2 polypeptide and N-terminal fluorescein-labeled 15-mer synthetic peptides corresponding to Zap70 residues 311–325. The fluoresceinated probe was either nonphosphorylated or phosphorylated on Tyr315. In addition, we also included a double-phosphorylated peptide in which both Tyr315 and Tyr319 were phosphorylated, to test whether a phosphotyrosyl residue at position 319 can affect the CrkII-SH2 binding to pTyr315. The assay was performed in the presence of 5 nM fluorescein-labeled synthetic peptide and addition of different concentrations of purified GST-CrkII-SH2, until binding saturation has been reached. Binding of GST-CrkII-SH2 to the fluoresceinated doubly phosphorylated (Tyr315 + pTyr319; F-pYpY) or singly phosphorylated (pTyr315; F-pYY) peptides reached saturation at micromolar concentration (see Table I), suggesting a single-site biomolecular interaction (Fig. 8, A and B). In contrast, the nonphosphorylated Zap70-derived peptide (F-YY; Fig. 8 C) did not exhibit binding to GST-CrkII-SH2. Control studies with GST alone did not show appreciable binding to any of the phosphopeptides, even at a mM concentration range (data not shown), thus reconfirming previously published control studies with GST (58).

FIGURE 8.

Fluorescence polarization analysis of GST-CrkII-SH2 and GST-Vav-SH2 binding to fluorescein-labeled peptides corresponding to Zap70 residues 311–325. Solutions with the indicated concentrations of GST-CrkII-SH2 (A–C) or GST-Vav-SH2 (D–F) fusion proteins in mixtures with 5 nM fluorescein-labeled peptide probes were monitored for fluorescence polarization at 22°C. The synthetic fluoresceinated probes correspond to Zap70 residues 311–325 in which both Tyr315 and Tyr319 are phosphorylated (F-pYpY; A and D), only Tyr315 is phosphorylated (F-pYY; B and E), or none of the tyrosine residues is phosphorylated (F-YY; C and F). Results of the saturation curves are presented in millipolarization (mP) units and Michaelis-Menten plots, and dissociation constant values were obtained using the GraphPad prism program.

FIGURE 8.

Fluorescence polarization analysis of GST-CrkII-SH2 and GST-Vav-SH2 binding to fluorescein-labeled peptides corresponding to Zap70 residues 311–325. Solutions with the indicated concentrations of GST-CrkII-SH2 (A–C) or GST-Vav-SH2 (D–F) fusion proteins in mixtures with 5 nM fluorescein-labeled peptide probes were monitored for fluorescence polarization at 22°C. The synthetic fluoresceinated probes correspond to Zap70 residues 311–325 in which both Tyr315 and Tyr319 are phosphorylated (F-pYpY; A and D), only Tyr315 is phosphorylated (F-pYY; B and E), or none of the tyrosine residues is phosphorylated (F-YY; C and F). Results of the saturation curves are presented in millipolarization (mP) units and Michaelis-Menten plots, and dissociation constant values were obtained using the GraphPad prism program.

Close modal

The dissociation constants of the CrkII-SH2-peptide complexes were 2.5 μM for the F-pYY and 1.07 μM for the F-pYpY (Table I), suggesting that phosphorylation of Tyr319 increases the binding affinity of the CrkII-SH2 to pTyr315 by ∼2-fold. These binding affinity values were at the range that is similar to that reported earlier for other SH2 or phosphotyrosine binding (PTB) domains (58, 59, 60, 61, 62), suggesting that CrkII-SH2 binding to pTyr315 Zap70 potentially represents a physiological meaningful biochemical event.

Because Zap70 Tyr315 has already been reported to serve as a preferred binding site for the Vav-SH2 domain (33), we used the same assay to determine the Vav-SH2-binding affinity to the pTyr315 Zap70, and compare it with that of the CrkII-SH2 domain. The results demonstrated that the Vav-SH2 dissociation constants for the singly and doubly tyrosine-phosphorylated Zap70-derived peptides corresponded to 1.05 and 0.5 μM, respectively (Fig. 8, D–F, and Table I). These values indicate that the Vav-SH2 binds pTyr315 Zap70 with ∼2-fold higher affinity than that of the CrkII-SH2. Nevertheless, because tyrosine phosphorylation of Zap70 in mature peripheral blood T cells is a rapid and transient event, it is highly likely that both CrkII and Vav can interact with activated Zap70, depending on their abundance and proximity to the phosphorylated Tyr315 Zap70.

A previously published work reported an interaction between Crk and Vav in activated B cells (63), and because Vav, but not Crk, undergoes strong phosphorylation by T cell PTKs, it is possible that the two molecules interact by binding of the Crk-SH2 to phosphotyrosyl-containing Vav epitopes. However, if CrkII interaction with tyrosine-phosphorylated Vav occurs at an affinity that is higher than that of the CrkII-SH2 binding to tyrosine-phosphorylated Zap70, it would be possible to assume that Vav competes with Zap70 for interaction with CrkII by two different mechanisms. The first is demonstrated in this study and involves the binding of the two SH2-containing molecules to a single epitope comprising pTyr315 on Zap70. A second possibility is that a phosphotyrosyl-containing Vav epitope occupies the SH2 domain in a fraction of CrkII proteins, and thereby decreases the cellular pool of unbound CrkII, which is available for interaction with Zap70.

To test whether CrkII may indeed associate with Vav in activated T cells, we treated Jurkat cells with pervanadate and compared the ability of Zap70 and Vav to associate with an immobilized GST-CrkII-SH2 fusion protein. For positive and negative controls, we included in the assay GST fusion proteins from SH2-containing molecules that represent Vav-binding (Grb2) and nonbinding noncatalytic region of tyrosine kinase (Nck) proteins. In this experiment (Fig. 9), we did not detect any association between Vav and the GST-CrkII-SH2 fusion protein. This is despite the fact that the pervanadate treatment induced the phosphorylation of Vav on tyrosine residues (data not shown) and the ability of this tyrosine-phosphorylated Vav to interact with the GST-GRB2 fusion protein (Fig. 9 B, lane 4). Specificity of the pull-down assay is further substantiated by the lack of association of both Zap70 and Vav with the GST and GST-Nck fusion proteins. Additionally, reciprocal coimmunoprecipitation studies failed to show physical interactions between CrkII and Vav in activated T cells (data not shown).

FIGURE 9.

CrkII does not bind tyrosine-phosphorylated Vav in activated T cells. Jurkat cells were treated with pervanadate (perVO4; 1% at 37°C for 30 min), and cell lysates were incubated with the indicated bead-immobilized GST fusion proteins (5 μg/group) for 3 h at 4°C. Bound proteins were eluted from the gel, subjected to SDS-PAGE under reducing conditions, electroblotted onto a nitrocellulose membrane, and sequentially immunoblotted with anti-Zap70 (A) or anti-Vav (B) Abs. Lysates of resting and perVO4-treated cells, as well as anti-Zap70 (A) and anti-Vav (B) immunoprecipitation were included for reference. Control whole cell lysate (CT-WCL) was run in parallel to show the presence and position of the Zap70 and Vav protein bands. Molecular size markers (in kDa) are indicated on the left and arrowheads mark the position of Zap70 (A) and Vav (B) proteins. Results are representative of three independent experiments.

FIGURE 9.

CrkII does not bind tyrosine-phosphorylated Vav in activated T cells. Jurkat cells were treated with pervanadate (perVO4; 1% at 37°C for 30 min), and cell lysates were incubated with the indicated bead-immobilized GST fusion proteins (5 μg/group) for 3 h at 4°C. Bound proteins were eluted from the gel, subjected to SDS-PAGE under reducing conditions, electroblotted onto a nitrocellulose membrane, and sequentially immunoblotted with anti-Zap70 (A) or anti-Vav (B) Abs. Lysates of resting and perVO4-treated cells, as well as anti-Zap70 (A) and anti-Vav (B) immunoprecipitation were included for reference. Control whole cell lysate (CT-WCL) was run in parallel to show the presence and position of the Zap70 and Vav protein bands. Molecular size markers (in kDa) are indicated on the left and arrowheads mark the position of Zap70 (A) and Vav (B) proteins. Results are representative of three independent experiments.

Close modal

We have previously shown a cell activation-dependent interaction between Zap70 and the CrkII adapter protein in human and mouse T cells (27), and in this study extended these findings to demonstrate that the interaction is mediated by binding of the CrkII-SH2 domain to the phosphorylated Tyr315 on Zap70, a putative regulatory site in the interdomain B region.

The Zap70 PTK was shown to be essential for both positive and negative selection processes during T cell development in the thymus, and indispensable for the efficient activation of TCR-linked signaling pathways in mature T cells (64, 65). The function of Zap70 is regulated by a TCR-induced Lck-mediated transphosphorylation as well as autophosphorylation of multiple tyrosine residues (45). These tyrosines play a role in the regulation of the Zap70 kinase activity, its interaction with specific effectors and regulators, and its potential recruitment to distinct subcellular locations (36, 44, 45).

Site-directed mutagenesis of Zap70 and analysis of the mutated gene products revealed that Tyr493 and Tyr492 in the activation loop of the kinase domain serve as positive and negative regulators, respectively, of the Zap70 catalytic activity (33, 37, 38, 39). In contrast, three tyrosine residues within interdomain B (at positions 292, 315, and 319) regulate Zap70 function by serving as temporal binding sites for the recruitment of other SH2- or PTB-domain-containing proteins (34, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50).

Phosphorylation of Tyr292 was found to promote the association of Zap70 with Cbl (41, 47), a negative regulator of T cell function (41, 47, 55). The Cbl protein possesses an E2-dependent ubiquitin-protein ligase activity (56), and its binding to pTyr292 may serve as a signal for the ubiquitination and degradation of Zap70, and thereby to down-regulation of the T cell activation response. In accordance, expression of the Zap70(Y292F) mutant induced hyperactive phenotype in response of T cells to TCR cross-linking, and enhanced the transcriptional activity of the NF-AT and IL-2 promoters (39, 40, 47). In addition, phosphorylation of Tyr315 was found to promote the association of Zap70 with Vav (42, 57), while phosphorylation of Tyr319 promoted Zap70 interaction with Lck and phospholipase Cγ1 (50, 51).

Studies on Zap70(Y315F) in cells and mice demonstrated that Tyr315 is important for optimal activity of Zap70 during the development of T cells in the thymus and activation of mature T cells in the lymph nodes and peripheral blood (33, 54, 56). Expression of Zap70(Y315F) in Syk-deficient DT40 cells impaired the function of Zap70 in BCR signaling by reducing the BCR-induced tyrosine phosphorylation of Zap70, Vav, SH2 domain-containing leukocyte protein of 76 kDa, and Shc, and attenuating the BCR-induced NF-AT-regulated responses (33). Nevertheless, mutation of Tyr315 did not affect Zap70 kinase activity nor its binding affinity to phosphorylated TCRζ ITAMs (33). The regulatory effects of Zap70(Y315F), together with the ability of pTyr315 to bind Vav, have led Wu et al. (33) to suggest that the effects of Zap70(Y315F) on T cell activation are due to its lack of ability to associate with Vav.

Analysis of TCR transgenic mice, carrying a Zap70(Y315F)-encoding gene, revealed a significant reduction in the rate of positive selection of T cells and delayed occurrence of negative selection in the thymus (54). In addition, the mutation resulted in decreased constitutive levels of tyrosine-phosphorylated TCRζ chains in thymocytes, and decrease in the inducible levels of tyrosine-phosphorylated TCRζ chains in both thymocytes and lymph node T cells (54). These studies confirmed that the intrinsic catalytic activity of Zap70(Y315F) was intact, but in disagreement with the findings of Wu et al. (33), demonstrated an impaired binding of the Zap70(Y315F) to tyrosine-phosphorylated TCRζ ITAMs (54, 56). Additional studies by di Bartolo et al. (56) suggested that the defective ITAM-binding capacity of Zap70(Y315F) is independent of the putative role of Tyr315 as a binding site for Vav. This assumption was based on the phenotype of Vav-deficient T cells, which exhibited normal levels of tyrosine phosphorylation of TCRζ and Zap70 (66, 67). If Vav binding to Zap70 would have been essential for Zap70 interaction with the TCR, as observed in Zap70(Y315F) T cells, a reduction in the tyrosine phosphorylation levels of TCRζ and Zap70 would have also been anticipated in Vav-deficient T cells.

Recruitment of signaling proteins to the site of the engaged TCR and formation of the immunological synapse are assumed to be essential for the sequential phosphorylation and activation of multiple effector molecules (68). Vav association with Zap70 may represent one important step along the TCR downstream signaling pathway (69). For example, it may serve as a mean for Zap70 to induce T cell activation-dependent rearrangement of the cytoskeleton through the activation of the Vav guanine nucleotide exchange factor on members of the ρ family of small G proteins (70, 71).

The subcellular distribution of Zap70 in activated T cells is still a controversial issue, and is largely dependent on the method and/or cell type used for the analysis. Some studies show minimal quantities of Zap70 at the glycolipid-enriched membrane microdomains (72), while others demonstrate significant levels of Zap70 at the detergent-insoluble membrane domain (73). Both glycolipid-enriched membrane microdomains and detergent-insoluble membrane domains are assumed to represent membrane regions that concentrate at the T cell-APC contact area. It is possible therefore that Vav and CrkII interact with two (or more) different populations of Zap70 molecules located at different subcellular compartments and mediate distinct signaling events. Further analysis of the potential role of CrkII in T cell signaling awaits the preparation of appropriate knockout models.

The Vav protein, similar to Zap70, is required for T cell development, predominantly at the stage of transition of double-positive thymocytes into mature single-positive T cells (74). Nevertheless, the effects of Vav deficiency, or expression of Zap70(Y315F), on TCR-coupled signaling events are not identical. For example, the TCR-induced Ca2+ response is impaired in Vav-deficient thymocytes, but not in Zap70(Y315F)-expressing thymocytes (54). In contrast, CD5 expression, or the constitutive tyrosine phosphorylation levels of TCRζ were decreased in Zap70(Y315F)-expressing thymocytes, but not in Vav-deficient thymocytes (54). The opposite effects were induced by the absence of Vav or expression of the Zap70(Y315F) mutant, indicating that Tyr315 in Zap70 is not among the major phosphotyrosine-based motifs that connect Vav to the TCR signaling machinery. Magnan et al. (54) have suggested that the effects noted in Zap70(Y315F) T cells were not necessarily related to the lack of interaction between Vav and Zap70. Because many of the tyrosine residues that undergo phosphorylation in activated cells serve as docking sites for SH2- or PTB-containing effector molecules, it is possible that pTyr315 functions as a binding site for signaling molecules other than Vav. The present study suggests that some of the effects attributed to the Zap70(Y315F) mutant in the murine or human T cells may reflect its inability to associate with CrkII and promote CrkII-specific signaling events.

To obtain more information about the relative binding affinity of CrkII-SH2 to Zap70 pTyr315, we synthesized fluoresceinated 15-mer peptides with sequences flanking both sides of Tyr315 of Zap70, and using a fluorescence polarization method, determined their extent of interaction with the CrkII-SH2 protein. Our studies revealed that the equilibrium dissociation constants (Kd) of GST-CrkII-SH2 to the pTyr315 peptide corresponded to 2.5 μM. Fluorescence polarization-based SH2 domain-binding studies for other proteins revealed Kd values ranging from 10−5 to 10−7 M. Thus, the Src-SH2 interacted with its tyrosine-phosphorylated ligand with a Kd value of 0.24 μM (59), and STAT1-SH2 and STAT6-SH2 domains interacted with a tyrosine-phosphorylated IFN-γ-derived peptide, or IL-4R-derived peptide with Kd values of 0.12 and 2 μM, respectively (60). Binding studies with PTB domains yielded affinity values within the same range of dissociation constants. The dNumb PTB domain interacted with its corresponding ligands with Kd values of 0.5–1.8 μM, and the same peptides interacted with the mNumb PTB with somewhat lower affinities, ranging from 1.3 to 3.9 μM (58). In addition, the Shc PTB domain interacted with Trk-derived phosphopeptide with a Kd of 0.04 μM (75), while the Kd for the IRS-1 PTB domain interacting with an IL-4R-derived phosphopeptide was 6 μM (61).

In vivo and in vitro binding studies demonstrate that CrkII-SH2 interacts preferentially with the phosphorylated Tyr315, and comparative analysis of the binding affinities of the singly (Tyr315) and doubly (Tyr315 and Tyr319) phosphorylated peptides demonstrated that phosphorylation of Tyr319 does not significantly affect the binding of CrkII-SH2 to the peptide. This is despite the fact that both Tyr315 and Tyr319 are within a YXXP sequence motif (YESP vs YSDP), which was predicted to be a highly selective binding site for CrkII-SH2 (76). Furthermore, both sequences possess a similar interim charge, which includes a single negatively charged residue. Nevertheless, screening of the phosphopeptide library with a CrkII-SH2 probe revealed a preference for a negatively charged residue at +1 position, as found adjacent to Tyr 315, but not Tyr319 (76). In addition, a positively charged (or nonpolar) amino acid is predicted to be the preferred residue at +2 position, while the residue at +2 position relative to Tyr319 possesses a negative charge (76). Thus, the preferred binding of pTyr315 over the pTyr319 is also justified according to the predicted binding model obtained with the phosphopeptide library.

Another possible explanation for the difference between the two positively charged YXXP motifs relates to the observation that Tyr315XXP includes a glutamic acid at the +1 position, while Tyr319XXP includes an aspartic acid at the +2 position. As a result, these positively charged residues might face two opposite directions within the helix backbone and impose the formation of different hydrogen bonds with the CrkII-SH2 domain.

We suggest that the CrkII interaction with the Zap70 PTK is a potentially important signaling event downstream of the TCR (27). The ability of Crk to interact with Tyr315 in activated T cells, with an affinity that is comparable to that of Vav, suggests that the two proteins may compete for binding to the same site, and perhaps can link the activated TCR to two different signaling pathways. Alternatively, it is possible that Vav binding to tyrosine-phosphorylated Zap70 represents an early activation event that promotes T cell activation, while replacement of Vav by CrkII may occur at a later time point and initiate biochemical events leading to termination of the activation response. Replacement of Vav by CrkII may result from posttranslational modification of the Zap70-bound Vav protein (i.e., by tyrosine phosphorylation), which results in conformational changes in the molecule that decreases its affinity to pTyr315. A recent study by Sasahara et al. (77) confirmed our findings that Crk proteins interact with Zap70 of activated T cells and provided additional data that support a role for the Zap70-associated CrkL in Wiskott-Aldrich syndrome protein-regulated formation of F-actin and rearrangement of cytoskeletal elements at the immunological synapse.

Finally, we would like to emphasize that a productive engagement of a T cell does not result in recruitment of the entire pool of Zap70 molecules into the immunological synapse (78). It is possible therefore that Vav and CrkII interact with two different populations of Zap70 molecules that are located at different subcellular compartments and mediate distinct signaling events. Further analysis of the potential role of CrkII in T cell signaling awaits the preparation of appropriate knockout models.

Although the present work concentrates on the CrkII-Zap70 interaction mechanism, it is worthwhile mentioning that the CrkII alternative spliced form, CrkI, shares identical SH2 and SH3C regions, and is therefore likely to possess the ability to associate with tyrosine-phosphorylated Zap70 with a similar binding affinity. The biological importance of expression of two alternative spliced forms of c-Crk is not known yet, but may provide in the future additional information as to the potential selective regulation of signaling pathways by the two Crk isoforms, which may involve the SH2-mediated interaction of CrkII and CrkI with Zap70, and their differential interaction with other effector molecules via the distinct C termini of the two proteins.

We thank Dr. Piers Nash for his help with the Spot-membrane synthesis, and Drs. R. T. Abraham, A. Altman, J. Bolen, M. Matsuda, and A. Weiss for their gifts of reagents.

The authors have no financial conflict of interest.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1

This work was supported in part by the Yael Research Fund, the Israel Science Foundation, the Israel Cancer Research Fund, the Chief Scientist’s office, Israel Ministry of Health, the Israel Cancer Association, the German-Israeli Foundation for Scientific Research and Development, and the USA-Israel Binational Science Foundation.

3

Abbreviations used in this paper: PTK, protein tyrosine kinase; Fmoc, 9-fluorenylmethyloxycarbonyl; HA, hemagglutinin; PTB, phosphotyrosine binding domain; pY, phosphotyrosine; SH, Src homology; WT, wild type; Crk, CT10 regulator of kinase; Grb2, growth factor receptor-bound protein 2; Nck, noncatalytic region of tyrosine kinase.

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