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. 1998 May;18(5):2855–2866. doi: 10.1128/mcb.18.5.2855

Genetic Evidence of a Role for Lck in T-Cell Receptor Function Independent or Downstream of ZAP-70/Syk Protein Tyrosine Kinases

Jane Wong 1,2,3, David Straus 4, Andrew C Chan 1,3,5,6,7,*
PMCID: PMC110664  PMID: 9566904

Abstract

T-cell antigen receptor (TCR) engagement results in sequential activation of the Src protein tyrosine kinases (PTKs) Lck and Fyn and the Syk PTKs, ZAP-70 and Syk. While the Src PTKs mediate the phosphorylation of TCR-associated signaling subunits and the phosphorylation and activation of the Syk PTKs, the lack of a constitutively active Syk PTK has prohibited the analysis of Lck function downstream of these initiating signaling events. We describe here the generation of an activated Syk family PTK by substituting the kinase domain of Syk for the homologous region in ZAP-70 (designated as KS for kinase swap). Expression of the KS chimera resulted in its autophosphorylation, the phosphorylation of cellular proteins, the upregulation of T-cell activation markers, and the induction of interleukin-2 gene synthesis in a TCR-independent fashion. The KS chimera and downstream ZAP-70 or Syk substrates, such as SLP-76, were still phosphorylated when expressed in Lck-deficient JCaM1.6 T cells. However, expression of the KS chimera in JCaM1.6 cells failed to rescue downstream signaling events, demonstrating a functional role for Lck beyond the activation of the ZAP-70 and Syk PTKs. These results indicate that downstream TCR signaling pathways may be differentially regulated by ZAP-70 and Lck PTKs and provide a mechanism by which effector functions may be selectively activated in response to TCR stimulation.

Signaling through the T-cell antigen receptor (TCR) requires the sequential activation of the Src and Syk families of protein tyrosine kinases (PTKs) (for reviews, see references 8, 11, and 70). The Src PTKs Lck and Fyn phosphorylate the two conserved tyrosine residues in the immunoreceptor tyrosine-based activation motif (designated as ITAM) (7), which is present in each of the TCR signaling subunits. In turn, phosphorylation of the ITAMs mediates the interaction of the Syk family PTKs ZAP-70 and Syk with the receptor. Both ZAP-70 and Syk have tandemly arranged amino (N)-terminal Src homology 2 (SH2) domains that are separated from their carboxy (C)-terminal catalytic domain by a hinge domain. These two PTKs have ∼55% amino acid identity and share overlapping functions in a variety of cell lines. Moreover, the coexpression of ZAP-70 and Syk in the thymus enables these two PTKs to play overlapping roles in T-cell development and TCR activation (2, 9, 12, 19, 24, 28).

Lck, a member of the Src family of PTKs, is also required for both T-cell development and TCR function (34, 47, 61). The catalytic activity of Lck is required for phosphorylation of the TCR ITAM sequences and ZAP-70 (32). In addition, the SH2 domain of Lck plays an important role in stabilization of the CD4/CD8-TCR-major histocompatibility complex and is required for efficient ζ-chain phosphorylation, TCR-mediated intracellular Ca2+ concentration ([Ca2+]i) mobilization, and interleukin-2 (IL-2) synthesis (18, 44, 60, 65, 76). The ability of isolated Lck SH2 and SH3 domains to interact with tyrosine-phosphorylated or proline-rich effector molecules, respectively, suggests a potential role for Lck either downstream or independent of ZAP-70 (14, 22, 53, 63, 77). However, one of the effector molecules bound to the Lck SH2 domain is ZAP-70, and expression of an Lck molecule with a nonfunctional SH2 domain fails to induce ZAP-70 phosphorylation (44, 60). Hence, analysis of the functional roles of Lck downstream of ZAP-70 activation has been hampered by the lack of a constitutively activated form of a Syk family kinase.

We report here the generation of such a constitutively active form of a Syk family kinase PTK, accomplished by substituting the kinase domain of Syk for the kinase domain of ZAP-70 (designated as the KS [for kinase swap] chimera). Expression of the KS chimera results in constitutive T-cell activation through signaling pathways normally mediated by activation of ZAP-70 and Syk following TCR cross-linking. By utilizing this constitutively active chimera, we obtained genetic evidence of a role for Lck, subsequent to or independent of the activation of ZAP-70, in TCR function.

MATERIALS AND METHODS

Cells, antibodies, and FACS analysis.

HeLa cells, Jurkat cells, and Jurkat cell derivatives JCaM1.6, J.RT3-T3.5, and J449.3 were maintained as previously described (27, 38, 73). Antibodies used included 12CA5, an antihemagglutinin (anti-HA) epitope monoclonal antibody (MAb; Boehringer Mannheim); 2F3.2, an anti-ZAP-70 MAb (Upstate Biotechnology, Inc. [UBI]); 4G10, an antiphosphotyrosine MAb (pY; UBI); PY20, an anti-pY MAb (Santa Cruz Biotechnology), an anti-PLCγ1 antiserum (UBI); an anti-Vav antiserum (Santa Cruz Biotechnology); anti-human CD69 MAb (Becton Dickinson); an anti-SLP-76 antiserum (6); an anti-ZAP-70 antiserum, raised against a peptide encoding amino acids 327 to 343; and an anti-Syk antiserum, raised against a peptide encoding amino acids 308 to 335. Analysis of cell surface markers was performed by fluorescence-activated cell sorter (FACS) analysis with a FACSCALIBUR (Becton Dickinson).

Construction of cDNAs.

Chimeric and truncated PTKs were produced by PCR-directed mutagenesis and confirmed by dideoxynucleotide sequencing. Epitope-tagged versions of the PTKs were produced by appending the HA epitope (YPYDVPDYA) to their N termini (21). IL-2– and nuclear factor of activated T cells (NFAT)-luciferase constructs and cytomegalovirus-chloramphenicol acetyltransferase (CMV-CAT) cDNAs were gifts from K. Murphy and T. Chatila (Washington University, St. Louis, Mo.).

Expression and analysis of proteins.

Infection of HeLa cells with recombinant vaccinia virus was performed as previously described (49). Following an 8-h infection, cells (5 × 105) were lysed in 0.5 ml of 1% Nonidet P-40–150 mM NaCl–10 mM Tris-HCl (pH 8.0) containing protease and phosphatase inhibitors. Cellular debris was removed by centrifugation at 10,000 × g for 10 min at 4°C. Clarified supernatants were then used for protein analysis.

Protocols for transfection of Jurkat cells and derivatives thereof have been previously reported (6, 38). For stable transfections, 107 cells were electroporated with 25 μg of DNA at 270 V and 1,060 μF at a resistance setting of 6 (R6) (BTX ElectroCell Manipulator 600). Forty-eight hours after electroporation, J449.3 cells were selected in medium containing 1 μg of hygromycin, 10 μg of tetracycline, and 2 mg of neomycin per ml. Selection of Jurkat cells with pApuro vectors was accomplished with medium containing 0.5 μg of puromycin per ml. Transient transfections were performed under the electroporation conditions of 250 V, 960 μF, and R6 (BTX). In brief, 2 × 107 cells were electroporated with 60 μg of IL-2–luciferase or NF-AT–luciferase and 80 μg of empty vector or the PTK cDNA. Cells were harvested at 48 h and replated at a concentration of 106/ml in medium alone, in medium containing phorbol myristate acetate (PMA) and either an anti-TCR MAb (C305 or 235) or phytohemagglutinin (PHA), or in medium containing PMA and ionomycin. Following 6 h of stimulation, luciferase assays were performed as previously described (62). Transfection efficiency was determined by cotransfection of 5 μg of a CMV-CAT reporter as previously described (38).

T-cell activation, immunoprecipitation, kinase assays, and Western blot analysis.

T-cell clones were analyzed under resting and TCR-stimulated conditions as previously described (6). Concentrations of anti-TCR MAbs used for stimulation were 1:250 for both 235 (anti-CD3 MAb, courtesy of Shu Man Fu) and C305 (anti-Tiαβ MAb, courtesy of Arthur Weiss). For biochemical analysis, cells were stimulated for 2 min at 37°C prior to lysis. Lysates were clarified by centrifugation and subsequently incubated with the appropriate antiserum for 90 min at 4°C and then with protein A-Sepharose (Pharmacia) for 60 min at 4°C. Samples were washed three times with lysis buffer and boiled in 3× Laemmli sample buffer.

For analysis of enzymatic activity, washed immunoprecipitates were incubated with 10 μCi of [γ-32P]ATP and 1 μg of a glutathione S-transferase (GST)–band III exogenous substrate in kinase buffer for 10 min at room temperature as previously described (10, 56). Proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis transferred to Immobilon (Millipore, Bedford, Mass.) or nitrocellulose membranes, blotted with the appropriate antibodies, and developed by enhanced chemiluminescence (Amersham) in accordance with the manufacturer’s recommendations.

RESULTS

Generation and expression of chimeric PTKs.

To investigate the functional and structural homologies between ZAP-70 and Syk, we generated three chimeric PTKs in which various domains of human ZAP-70 were replaced with the homologous regions of human Syk (Fig. 1A). These included the following: (i) a chimera in which the ZAP-70 kinase domain, amino acids (aa) 331 to 619, was replaced with the Syk kinase domain, aa 358 to 630 (designated as the KS chimera); (ii) a chimera in which the hinge region located between the C-terminal SH2 and catalytic domains of ZAP-70, aa 254 to 326, was replaced with the homologous region within Syk, aa 255 to 354 (designated as H); and (iii) a chimera in which a region encompassing the ZAP-70 transactivation domain, aa 476 to 523, was replaced with a homologous region encompassing the Syk transactivation domain, aa 505 to 552 (designated as TAD).

FIG. 1.

FIG. 1

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Expression of PTKs in HeLa cells. (A) Schematic representation of chimeric PTKs. Chimeras of ZAP-70 and Syk PTKs were generated by PCR-directed mutagenesis. Three chimeric PTKs, KS, H, and TAD, were generated by exchange of the kinase domains, the hinge regions, and the transactivation domains, respectively. (B) Autophosphorylation of the KS chimera. HeLa cells were infected with recombinant vaccinia virus encoding the chimeric and wild-type PTKs individually as described in Materials and Methods. Cells (2 × 105) were lysed, and lysates were used for immunoprecipitation or immunoblotting studies with an anti-pY MAb (top panel) and anti-ZAP-70 or anti-Syk antiserum (bottom panel). Lanes: 1, wild-type (WT) ZAP-70; 2, TAD chimera; 3, KS chimera; 4, wild-type Syk; 5, H chimera. To ensure that equimolar amounts of each of the PTKs were analyzed, expression of each of the wild-type and chimeric PTKs was determined by blotting with anti-ZAP-70 or anti-Syk antibodies and normalized to standardized amounts of baculovirus-encoded GST–ZAP-70 or GST-Syk protein (data not shown) (6, 10). No differences between tagged and untagged versions of Syk were observed (data not shown). The experiment shown here is representative of five independent experiments. Molecular weight standards (in thousands) are depicted at the left margin. (C) Autoactivation and phosphorylation of HeLa cell proteins by the KS chimera. HeLa cell lysates infected with chimeric or wild-type PTK as described in for panel B were analyzed for tyrosine phosphorylation of cellular proteins. MW, molecular weight standards. (D) The tyrosine residues within the transactivation loop contribute to, but are not absolutely required for, autoactivation and autophosphorylation. HeLa cells (2 × 105) were infected with wild-type Syk (lanes 1), the KS chimera (lanes 2), or the KS chimera in which Tyr 518 and Tyr 519 in Syk were mutated to phenylalanine [KS(YYFF)] (lanes 3). Anti-HA immunoprecipitates of each PTK were analyzed in an in vitro kinase assay using an exogenous substrate (top panel) and immunoblotting with an anti-pY MAb (third panel from top). Coomassie blue staining demonstrates comparable levels of the GST-band III exogenous substrate (second panel from top), and immunoblotting analysis with an anti-HA MAb demonstrates comparable levels of the expressed PTK (bottom panel).

ZAP-70, Syk, and the three chimeric PTKs were expressed in HeLa cells. Each PTK was immunoprecipitated with either anti-ZAP-70 or anti-Syk antiserum and analyzed by immunoblotting with an anti-pY MAb. Only the KS chimera was phosphorylated on tyrosine residues (Fig. 1B, top, lane 3). In contrast, no tyrosine phosphorylation was observed with wild-type ZAP-70, wild-type Syk, the TAD chimera, or the H chimera (Fig. 1B, top, lanes 1, 2, 4, and 5). All chimeric PTKs retained enzymatic activity, which indicated that the exchanging of homologous regions within ZAP-70 and Syk had not disrupted their overall structures (data not shown).

Since the KS chimera could undergo autophosphorylation, we also analyzed the ability of the KS chimera to undergo autoactivation to phosphorylate cellular proteins. Again, only the expression of the KS chimera resulted in significant tyrosine phosphorylation of HeLa cellular proteins (Fig. 1C, lane 3); tyrosine phosphorylation of cellular proteins was substantially diminished (>50-fold difference) in HeLa cells expressing wild-type ZAP-70, Syk, the TAD chimera, or the H chimera (Fig. 1C, lanes 1, 2, 4, and 5). In addition, analysis of HeLa cells expressing the KS chimera at 1/10 the level of the wild-type or other chimeric PTKs still demonstrated significantly augmented phosphorylation of cellular proteins (data not shown). Together, these data demonstrate that expression of the KS chimera in HeLa cells induces greater than a 10-fold increase in tyrosine phosphorylation of cellular proteins compared to wild-type ZAP-70 or Syk.

Since the tyrosine residues within the transactivation loop of Syk are required for Syk activation (40), we analyzed the enzymatic activity and phosphorylation status of wild-type Syk, the KS chimera, and the KS chimera in which both tyrosine residues (i.e., Tyr 518 and 519) were mutated to phenylalanine [designated as KS(YYFF) in Fig. 1D]. Both the KS and KS(YYFF) chimeras were tyrosine phosphorylated and demonstrated increased enzymatic activity compared to wild-type Syk (Fig. 1D). Enzymatic activation of the KS(YYFF) mutant was slightly decreased compared to that of the KS chimera, consistent with the ability of Syk to contribute to autoactivation (20). However, the enzymatic activity of the KS(YYFF) chimera was still substantially greater than that of the Syk holoenzyme, suggesting that the increased phosphorylation of cellular proteins observed in HeLa cells expressing the KS chimera (Fig. 1B to D) is, in large part, independent of the tyrosine residues within the transactivation loop that are phosphorylated by Src PTKs or by autophosphorylation (20, 40).

Characterization of the KS chimeric PTK in T cells.

To analyze the functional effects of the KS chimera, stable clones were established following transfection of the Jurkat leukemic T-cell line (J6) with plasmids which express wild-type ZAP-70, wild-type Syk, or the KS chimera. Each PTK was appended with a common HA epitope tag to permit direct comparison of their levels of expression. Since constitutive activation of T cells may result in activation-induced cell death, the KS chimeric and Syk PTKs were expressed via a tetracycline-regulated promoter. Multiple clones of the KS chimera, wild-type ZAP-70, or wild-type Syk PTKs were isolated, and their levels of expression were analyzed (Fig. 2A and data not shown). Data for two representative clones (2E4 and 3G6) which differed in their basal levels of expression of the KS chimera are shown in Fig. 2A (lanes 2 to 5). While the two clones demonstrated different levels of expression of the KS chimera under nonpermissive conditions (i.e., in the presence of tetracycline [lanes 2 and 4]), withdrawal of tetracycline resulted in increased expression of the KS chimera (lanes 3 and 5). The KS chimera was consistently expressed at less than one-third the level of overexpressed wild-type ZAP-70 or Syk PTK (lanes 6 to 8).

FIG. 2.

FIG. 2

FIG. 2

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Biochemical analysis of cells expressing the KS chimera. (A) Expression of KS, Syk, and ZAP-70. Stable transfectants of the KS chimera (2E4 and 3G6) and Syk (5G4) were expressed under the control of a tetracycline-regulated promoter in Jurkat cells (lanes 2 to 5, 7, and 8) (37). In addition, ZAP-70 was overexpressed under the control of an actin promoter (lane 6) (38). Parental Jurkat 449 cells are included as a control (C) (lane 1). Expression of the HA-epitope-tagged PTKs was analyzed by Western blotting. Since the KS chimera was expressed at approximately one-third the level of wild-type ZAP-70 or Syk, immunoprecipitates from 4 × 107 cells of the KS-expressing clones (lanes 2 to 5) were analyzed, compared to 2 × 107 cells expressing HA-Syk or HA–ZAP-70 (lanes 6 to 8). Expression of the KS chimera was maximal at 24 h following tetracycline withdrawal and did not change for the 72-h period of analysis (data not shown). Lanes: 1, parental cells (449); 2; KS clone 2E4 with tetracycline (nonpermissive conditions); 3, KS clone 2E4 without tetracycline (permissive conditions); 4, KS clone 3G6, nonpermissive conditions; 5, KS clone 3G6, permissive conditions; 6, ZAP-70 (wt24); 7, Syk clone 5G4, nonpermissive conditions; 8, Syk clone 5G4, permissive conditions. (B) Phosphorylation of KS in T cells. The epitope-tagged KS (clone 2E4; 4 × 107 cells), wild-type ZAP-70 (clone wt24; 107 cells), and wild-type Syk (clone 5G4; 107 cells) were immunoprecipitated in resting or TCR-activated cells and analyzed by Western blotting with an anti-pY MAb (top) or an anti-HA MAb (bottom). Both KS- and Syk-expressing clones were analyzed under permissive conditions. These data are representative of three independent experiments. (C) Tyrosine phosphorylation of cellular proteins by the KS chimera. Lysates (5 × 106 cells/lane) from stable transfectants were analyzed by immunoblotting with an anti-pY MAb. Lanes: 1, control (C) Jurkat cells (449); 2, KS cells (clone 2E4) under nonpermissive conditions; 3, KS cells (clone 2E4) under nonpermissive conditions; 4, control parental Jurkat cells (449); 5, KS cells (clone 2E4) under permissive conditions; 6, KS cells (clone 2E4) under permissive conditions. Lanes 1 to 3 represent resting cells, while lanes 4 to 6 represent cells stimulated with an anti-CD3 MAb (235) for 2 min at 37°C. All lanes were derived from the same gel and exposure, although the molecular weight markers originally placed between lanes 3 and 4 were cropped from the final photograph. These data are representative of three independent experiments and of three independent clones expressing the KS chimera. (D) Tyrosine phosphorylation of SLP-76, an in vivo downstream substrate of ZAP-70. SLP-76 was immunoprecipitated from control (C) parental cells (clone 449; 2 × 107 cells/lane) (lanes 1 and 2) or KS cells (clone 2E4; 2 × 107 cells/lane) (lanes 3 and 4) and analyzed by Western blotting with an anti-pY MAb (top panel) or an anti-SLP-76 MAb (H3 MAb) (bottom panel). Unstimulated cells are represented in lanes 1 and 3, while TCR-activated cells are represented in lanes 2 and 4. These data are representative of a minimum of three independent experiments and of two independent clones expressing the KS chimera. (E) Tyrosine phosphorylation of Vav. Vav was immunoprecipitated from control (C) parental 449 cells (2 × 107 cells/lane) (lanes 1 and 2) or KS cells (clone 2E4; 2 × 107 cells/lane) (lanes 3 and 4) and analyzed by Western blotting with an anti-pY MAb (top panel) or an anti-Vav MAb (bottom panel). Unstimulated cells are represented in lanes 1 and 3, while TCR-activated cells are represented in lanes 2 and 4. These data are representative of a minimum of three independent experiments and of two independent clones expressing the KS chimera.

To analyze the biochemical characteristics of these transfected PTKs, we immunoprecipitated the KS chimera, wild-type ZAP-70, or wild-type Syk PTK from resting or TCR-activated cells. Both wild-type ZAP-70 and wild-type Syk were phosphorylated on tyrosine residues following TCR cross-linking, although a very low level of phosphorylation of ZAP-70 and Syk was observed in unstimulated cells after longer exposures (Fig. 2B, lanes 5 to 8). In contrast, the KS chimera immunoprecipitated from cells (2E4) under permissive conditions was phosphorylated on tyrosine residues in unstimulated cells, and its level of phosphorylation was only slightly augmented following TCR cross-linking (Fig. 2B, compare lanes 3 and 4).

Analysis of the biochemical pathways activated by the KS chimera.

To determine if the KS chimera would activate the biochemical pathways mediated by wild-type ZAP-70 and Syk, we first analyzed the pattern of tyrosine phosphorylation of cellular proteins in cells expressing the KS chimera (Fig. 2C). Minimal phosphorylation of cellular proteins was observed in parental (449) cells or in cells (2E4) under the nonpermissive conditions (Fig. 2C, lanes 1 and 2). Upon tetracycline withdrawal, expression of the KS chimera resulted in the induction of tyrosine phosphorylation of a number of cellular proteins (Fig. 2C, lane 3). Moreover, the pattern of phosphorylation of these proteins was qualitatively similar to, though quantitatively less than, the pattern induced following TCR cross-linking of parental or KS-expressing cells (Fig. 2C, lanes 4 to 6).

Since the pattern of phosphorylation of cellular proteins by the KS chimera appeared similar to that of proteins phosphorylated following TCR cross-linking, we assessed the phosphorylation status of specific cellular proteins implicated as effector proteins downstream of ZAP-70. We and others have previously demonstrated that SLP-76 represents a downstream in vivo substrate of both ZAP-70 and Syk (6, 55). Whereas SLP-76 was inducibly tyrosine phosphorylated following TCR cross-linking in parental cells (Fig. 2D, lanes 1 and 2), SLP-76 was constitutively tyrosine phosphorylated in cells expressing the KS chimera (Fig. 2D, lane 3). A 72,000-molecular-weight tyrosine phosphoprotein representing the KS chimera coimmunoprecipitated with SLP-76 in the basal state, consistent with our previous demonstration of the association of SLP-76 with activated ZAP-70 following TCR cross-linking (6). Consistent with the further increase in tyrosine phosphorylation of the KS chimera following receptor activation, SLP-76 also demonstrated a small increase in tyrosine phosphorylation following TCR cross-linking (Fig. 2D, lane 4). Interestingly, the association of the KS chimera with SLP-76 decreased following TCR engagement, although the decrease was not a consistent result; this may reflect activation of protein tyrosine phosphatases by TCR activation.

In addition to SLP-76, Vav was also phosphorylated on tyrosine residues in unstimulated cells expressing the KS chimera (Fig. 2E, lane 3). Consistent with the ability of SLP-76 to interact with Vav following TCR stimulation of parental cells (51, 66, 75) (Fig. 2E, lanes 1 and 2), cells expressing the KS chimera demonstrated constitutive association of tyrosine-phosphorylated SLP-76 with Vav. Additional cellular proteins, including phospholipase Cγ1 (PLCγ1) and, to a lesser extent, cbl (Casitas B-lineage lymphoma), were also found to be phosphorylated on tyrosine residues in unstimulated cells expressing the KS chimera and were all further phosphorylated following TCR cross-linking (data not shown).

Analysis of the functional parameters activated by the KS chimera.

In addition to analyzing the phosphorylation status of cellular proteins, we also assessed the ability of the KS chimera to upregulate the CD69 T-cell activation marker. Resting parental T cells (clone 449) demonstrated minimal (3.6%) CD69 expression (Fig. 3A, top left panel). Unstimulated cells examined under nonpermissive conditions also demonstrated a low level of CD69 expression which was comparable to that of resting parental 449 cells (data not shown). In contrast, when examined under permissive conditions, two representative clones which express the KS chimera demonstrated increased expression of CD69 (70.3% for clone 2E4.1 and 53.8% for clone 3G6) in the absence of TCR stimulation (Fig. 3A, top middle and right panels). Activation of all cells with PMA or following TCR cross-linking resulted in further upregulation of CD69 expression (Fig. 3A, bottom panels, and data not shown).

FIG. 3.

FIG. 3

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Functional analysis of cells expressing the KS chimera. (A) Upregulation of CD69, an early TCR activation marker, by the KS chimera. Cells expressing the KS chimera (clones 2E4.1 and 3G6) were analyzed for CD69 expression by FACS analysis. Parental 449 T cells are also shown (left panels). A total of 106 cells were analyzed under each set of experimental conditions. The top panels represent resting cells examined under permissive conditions. The bottom panels represent cells examined following stimulation with PMA. These data are representative of five independent experiments. (B) Receptor-independent IL-2 gene synthesis in cells expressing the KS chimera. Jurkat T cells were transiently transfected with wild-type or chimeric PTKs and a reporter plasmid encoding the IL-2 promoter. Cells were harvested after 36 h and replated in medium alone, medium containing PMA and an anti-TCR MAb (235), or medium containing PMA and PHA. Luciferase activity was assessed following 6 h of stimulation and normalized for CAT activity as previously described (62). The experiment shown here is representative of at least five independent experiments. Similar results were obtained for stable clones.

Finally, we analyzed the ability of cells overexpressing ZAP-70, Syk, or the KS chimera to regulate IL-2 synthesis. A reporter plasmid consisting of the IL-2 promoter fused to luciferase was transiently transfected with each PTK into Jurkat cells. The activity of the IL-2 promoter was analyzed in resting cells, in cells stimulated with a combination of PMA and an anti-CD3 MAb (235), or in cells stimulated with a combination of PMA and PHA. While expression of wild-type ZAP-70 or wild-type Syk resulted in activation of the IL-2 promoter only following TCR stimulation, activation of the IL-2 promoter in cells expressing the KS chimera was observed without receptor engagement (Fig. 3B). In fact, the degree of basal activity observed with expression of the KS chimera was comparable to the level of IL-2 promoter activity in receptor-activated cells overexpressing wild-type ZAP-70 or wild-type Syk. IL-2 gene transcription was augmented following TCR engagement in cells expressing the KS chimera, indicating that the TCR signaling pathway remained intact in these cells. Expression of the TAD or H chimeric PTKs had no effect on basal IL-2 promoter activity (data not shown). Similar data were observed when a reporter construct consisting of the NF-AT promoter element was used (data not shown). Together, these data demonstrate that expression of the KS chimera results in activation of biochemical pathways utilized by wild-type ZAP-70 and Syk in TCR signaling that culminate in CD69 expression and IL-2 gene synthesis.

Signaling by the KS chimera is independent of the TCR.

Since expression of other PTKs in T cells, such as the epidermal growth factor receptor, can induce TCR signaling through cross-talk mechanisms between the epidermal growth factor and T-cell receptors (35), we undertook studies to determine whether the transcriptional activation of the IL-2 promoter observed with the KS chimera was mediated through a receptor or via a receptor-independent mechanism. Hence, we transiently cotransfected the KS chimera with an IL-2 reporter gene into a Jurkat cell derivative devoid of a surface TCR (J.RT3-T3.5 [73]). Consistent with the ability of the KS chimeric PTK to activate the IL-2 promoter in Jurkat T cells without engagement of the TCR, transcriptional activation of the IL-2 promoter was observed in the TCR-negative Jurkat derivative at a level comparable to that in TCR-positive Jurkat T cells (Fig. 3B and 4A).

FIG. 4.

FIG. 4

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Constitutive activation of T cells by the KS chimera does not require a surface TCR complex. (A) IL-2 gene synthesis induced by the KS chimera does not require a surface TCR. The KS chimera was transiently transfected into a TCR-negative variant of the Jurkat cell line with an IL-2–luciferase construct. Cells were analyzed as described in the legend to Fig. 3B. Luciferase activity induced by PMA and ionomycin was comparable to the level induced in TCR-positive T cells (Fig. 3B). These data are representative of two independent experiments. (B) The TCR ζ chain is not tyrosine phosphorylated by the KS chimera. The TCR ζ chain was immunoprecipitated from 4 × 107 parental Jurkat cells (C) (lanes 1 and 2), from KS-expressing cells under nonpermissive conditions (clone 2E4) (lanes 3 and 4), and from KS-expressing cells under permissive conditions (clone 2E4) (lanes 5 and 6). Immunoprecipitates from 4 × 107 resting (lanes 1, 3, and 5) or TCR-stimulated (lanes 2, 4, and 6) cells were analyzed by immunoblotting with an anti-pY MAb (top panel) or an anti-ζ antiserum (bottom panel). These data are representative of two independent experiments.

To further confirm the ability of the KS chimera to activate T cells in a receptor-independent fashion, we assessed the tyrosine phosphorylation status of the TCR ζ chain in TCR-positive cells (Fig. 4B). Similar to the TCR-induced phosphorylation of the TCR ζ chains in parental cells or cells analyzed under nonpermissive conditions (Fig. 4B, lanes 1 to 4), cells expressing the KS chimera also demonstrated TCR-inducible ζ phosphorylation (Fig. 4B, upper panel, compare lanes 5 and 6). No phosphorylation of the ζ chain was observed in unstimulated cells under the permissive conditions (Fig. 4B, upper panel, lane 5). Together, these data demonstrate that the ability of the KS chimera to activate signaling pathways downstream of ZAP-70 and Syk, culminating in IL-2 gene transcription, is independent of the signaling events proximal to ZAP-70, including the requirement of a functional TCR and ITAM phosphorylation.

Functional role for Lck downstream of ZAP-70 and Syk PTKs in TCR activation.

Since our studies in HeLa cells indicated that the KS chimera could function in an Lck-independent fashion, we addressed whether the KS chimera could bypass the signaling defects observed in Lck-deficient T cells. Stable clones expressing wild-type Syk or the KS chimera were established in the Lck-deficient JCaM1.6 leukemic cell line (27, 61). To ensure that the KS chimera could undergo autoactivation and autophosphorylation in an Lck-independent fashion, we first analyzed the phosphorylation status of wild-type Syk or the KS chimera expressed in JCaM1.6 cells. Consistent with our observations in HeLa cells, the KS chimera, when expressed in JCaM1.6 cells, was tyrosine phosphorylated and occurred independently of receptor engagement (Fig. 5A, lanes 3 to 6). In contrast, wild-type Syk was not tyrosine phosphorylated in resting or TCR-activated JCaM1.6 cells (clone 3H3), despite being expressed at higher levels than the KS chimera in clone 5F7 (Fig. 5A, lanes 5 to 8). Hence, consistent with our results in HeLa cells, the KS chimera can undergo autoactivation independently of Lck.

FIG. 5.

FIG. 5

FIG. 5

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Functional evidence for Lck acting downstream or independently of ZAP-70 activation. (A) Tyrosine phosphorylation of the KS chimera is receptor and Lck independent. Immunoprecipitates of KS (lanes 1 to 6) or wild-type Syk (lanes 7 and 8) from Jurkat cells (clone 2E4) (lanes 1 and 2) or JCaM1.6 cells (clones 4D11, 5F7, and 3H3) (lanes 3 to 8) were analyzed by immunoblotting with an antiphosphotyrosine MAb (top) or an anti-HA MAb (bottom). A total of 4 × 107 cells were analyzed per immunoprecipitate. The JCaM1.6 clones were expressed under the control of the actin promoter. The 2E4 clone was examined under permissive conditions. (B) Tyrosine phosphorylation of SLP-76 by the KS chimera is receptor and Lck independent. Immunoprecipitates of SLP-76 from Jurkat cells (lanes 1 to 3 and 6 to 8) or JCaM1.6 cells (lanes 4, 5, 9, and 10) expressing the KS chimera (clone 2E4 [lanes 3 and 8] and clone 4D11 [lanes 4 and 9]), wild-type Syk (clone 5G4 [lanes 2 and 7]) and clone 3H3, [lanes 5 and 10]), or wild-type ZAP-70 (clone wt24 [lanes 1 and 6]) were analyzed by immunoblotting with an anti-pY MAb (top panel) or an anti-SLP-76 (H3) MAb (bottom panel). A total of 2 × 107 cells were analyzed per immunoprecipitate. Clones 5F4 and 2E4 were analyzed under permissive conditions. These data are representative of four independent experiments. (C) Tyrosine phosphorylation of Vav by the KS chimera. Immunoprecipitates of Vav from JCaM1.6 cells (lanes 1 and 2), JCaM1.6 cells expressing Syk (clone 5G4) (lanes 3 and 4), or JCaM1.6 cells expressing the KS chimera (clone 4D11) (lanes 5 and 6) were analyzed by immunoblotting with an anti-pY MAb (top panel) or an anti-Vav MAb (bottom panel). A total of 2 × 107 cells were analyzed per immunoprecipitate. These data are representative of two independent experiments. On substantially longer exposures, two minor nonspecific bands migrating above and below Vav were observed in lanes 4 and 5. (D) Lck is required downstream of ZAP-70 activation for CD69 expression. JCaM1.6 cells, Jurkat cells expressing the KS chimera (clone 2E4), or JCaM1.6 cells expressing the KS chimera (clones 4D11 and 7E7) were analyzed for CD69 expression as described in the legend to Fig. 3A. A total of 106 cells were analyzed under each set of experimental conditions. The top panels represent resting cells, while the bottom panels represent cells examined following treatment with PMA as described in Materials and Methods. The percentage of CD69+ cells is quantitated above each bracket. These data are representative of three independent experiments. (E) Lck is required downstream of ZAP-70 activation for IL-2 gene synthesis. Wild-type and chimeric PTKs were individually transiently transfected with an IL-2 promoter into the Lck-deficient variant Jurkat T-cell line JCaM1.6. Luciferase activity was detected in unstimulated and TCR-activated cells as described in Materials and Methods. A parallel experiment was performed in parental Jurkat cells for comparison purposes. These data are representative of at least three independent experiments.

Analysis of the phosphorylation status of SLP-76 demonstrated similar results. While SLP-76 was phosphorylated in a receptor-dependent fashion in Jurkat cells expressing wild-type ZAP-70 or wild-type Syk (Fig. 5B, lanes 1, 2, 6, and 7), it was constitutively phosphorylated in Jurkat (clone 2E4 [lanes 3 and 8]) and JCaM1.6 (clone 4D11 [lanes 4 and 9]) cells expressing the KS chimera. Hence, phosphorylation of SLP-76 occurs independently of receptor stimulation and Lck. In contrast, SLP-76 is not phosphorylated in resting or TCR-activated JCaM1.6 cells expressing Syk or ZAP-70 (Fig. 5B, lanes 5 and 10, and data not shown). Hence, the KS chimera still retains its ability to phosphorylate, in vivo, downstream substrates of ZAP-70 and Syk in the absence of Lck.

Vav was also phosphorylated in resting JCaM1.6 cells expressing the KS chimera (clone 4D11 [Fig. 5C, top panel, lanes 5 and 6]), but it was not phosphorylated in resting JCaM1.6 parental cells (lanes 1 and 2), although its level of phosphorylation in the 4D11 cells was lower than that of Jurkat cells expressing the KS chimera (Fig. 2E, lane 3, and data not shown). Intriguingly, Vav phosphorylation was further increased following TCR cross-linking in JCaM1.6 cells expressing the KS chimera (Fig. 5C, top panel, compare lanes 5 and 6), suggesting that both the Src and Syk families of PTKs likely contribute to Vav phosphorylation. In contrast, phosphorylation of Vav was not detected in JCaM1.6 parental cells or in JCaM1.6 cells expressing Syk, under resting conditions or following TCR cross-linking (Fig. 5C, top panel, lanes 1 to 4).

To determine if expression of the KS chimera could restore downstream signaling defects in Lck-deficient cells, we analyzed the ability of the KS chimera to upregulate CD69 in JCaM1.6 cells. While Lck-sufficient cells expressing the KS chimera (2E4.1) demonstrated increased expression of CD69 in unstimulated cells (70%), CD69 expression in two representative JCaM1.6 clones expressing the KS chimera was indistinguishable from that of parental JCaM1.6 cells (Fig. 5D, top panel). Hence, while expression of the KS chimera in Lck-deficient cells is sufficient for tyrosine phosphorylation of KS, SLP-76, and, to a lesser degree, Vav, expression is not sufficient to bypass the absence of Lck in upregulating CD69.

Finally, we analyzed the ability of the KS chimera to bypass the absence of Lck to upregulate IL-2 synthesis. Consistent with the inability of the KS chimera to upregulate CD69, expression of the KS chimera in JCaM1.6 cells resulted in a sevenfold-lower level of basal IL-2 gene transcription than that in Jurkat cells (Fig. 5E). The residual basal function observed may be due to the ability of Fyn to partially compensate for the absence of Lck (30, 67). In addition, overexpression of neither wild-type ZAP-70 nor wild-type Syk in JCaM1.6 cells resulted in basal or receptor-dependent IL-2 gene transcription. Together, these data suggest that in addition to the proximal signaling functions established for Lck, which include phosphorylation of the receptor signaling subunit-encoded ITAMs and ZAP-70, Lck plays roles in TCR function downstream of the ZAP-70 and Syk PTKs.

DISCUSSION

We have described the generation of a constitutively active form of the Syk family of PTKs by exchange of the catalytic domains of ZAP-70 and Syk. Expression of this chimeric PTK in HeLa and Jurkat T cells resulted in tyrosine phosphorylation of the KS chimera that was independent of the Src PTKs and TCR activation, respectively. In addition, expression of the KS chimera in stably transfected Jurkat T-cell clones resulted in constitutive tyrosine phosphorylation of a number of cellular proteins normally phosphorylated following TCR cross-linking that reside downstream (e.g., SLP-76, Vav, PLCγ1, and cbl), but not upstream (e.g., the TCR ζ-chains), of ZAP-70. Phosphorylation of these substrates, in turn, resulted in transcriptional activation of both the NF-AT and IL-2 promoter elements and expression of T-cell activation markers in the absence of receptor engagement. Finally, the KS chimera did not exert any aberrant effects on the endogenous TCR signaling machinery, since TCR cross-linking still resulted in tyrosine phosphorylation of ζ and endogenous ZAP-70. While we cannot exclude the possibility that the KS chimera may activate biochemical pathways not normally activated by wild-type ZAP-70 and Syk PTKs, these data together support the hypothesis that the KS chimera is mediating its effects through the normal TCR-mediated signaling pathways downstream of ZAP-70.

The ability of the KS chimera to autoactivate implies that ZAP-70 and/or Syk PTKs may possess some basal intrinsic inhibitory conformation that is disrupted with the generation of the KS chimera. Multiple lines of evidence suggest that such inhibitory constraints may exist. First, the p42 tryptic fragment of Syk that encodes solely its catalytic domain has an approximately twofold-greater enzymatic activity than the holoenzyme (79). Second, an antibody directed to the C terminus of Syk inhibits the ability of doubly phosphorylated ITAMs to activate Syk and recognizes only a subpopulation of activated Syk (36, 56). Third, the activated and nonactivated forms of Syk exhibit different trypsin sensitivities (36). Conformational changes, as determined by biophysical and biostructural methods, have also been described for ZAP-70 (41). Alternatively, but not exclusively, the KS chimera may have lost inhibitory elements that normally regulate ZAP-70 and Syk in their basal states. Consistent with this latter possibility is the inability of the SHP-1 protein tyrosine phosphatase to efficiently dephosphorylate and downregulate the catalytic activity of the KS chimera in insect Sf9 cells (data not shown). In contrast, coinfection of SHP-1 with activated ZAP-70 or Syk resulted in the downregulation of PTK activity (52) (data not shown). Hence, the resultant substitution of the Syk domain for the ZAP-70 kinase domain may alleviate intrinsic inhibitory effects, resulting in its dysregulation. Similar inhibitory mechanisms have been demonstrated for the Src PTK Hck. The resolution of the Hck holoenzyme structure has revealed inhibitory constraints, mediated by both its SH2 and SH3 domains, that also affect Hck enzymatic activity (46, 57). Additional molecular dissections of potential inhibitory domains within ZAP-70 and Syk are presently under way.

Functional role for Lck downstream of ZAP-70.

The generation of constitutively active mutants permits one to complement biochemical studies with genetic analyses. While expression of the KS chimera in Jurkat T cells resulted in constitutive activation of a number of signaling pathways, expression of the KS chimera in JCaM1.6 T cells failed to bypass the deficiency in Lck in mediating IL-2 gene expression or upregulation of activation markers. We and others have demonstrated that membrane localization and transphosphorylation of ZAP-70 at Tyr 493 by Lck are required for efficient T-cell function (10, 29, 37, 38, 69, 71, 78). Studies in a variety of cellular systems have demonstrated that ZAP-70 is required for both receptor-mediated calcium mobilization and Ras activation (38, 39, 50, 54). The data presented in this paper provide genetic evidence for a functional role of Lck downstream of ZAP-70 or independent of the phosphorylation and activation of ZAP-70. The constitutive phosphorylation of SLP-76 by the KS chimera in Lck-deficient cells favors a model in which the TCR signaling pathway diverges, with ZAP-70 and Lck mediating distinct subsets of signaling pathways downstream of ZAP-70. However, the convergence of these distinct signaling pathways is required for efficient T-cell function. This additional level of regulation provides mechanisms by which the interaction of Lck with other signaling molecules may affect Lck activity through SH2 and/or SH3 interactions or recompartmentalizes Lck into distinct subcellular localizations and modulates the biological response of a given TCR-activating event. Studies by Mustelin and colleagues have suggested that Syk may phosphorylate the SH2 domain of Lck to mediate downstream signaling functions (16). Both the SH2 and SH3 domains of Lck have been described to interact with a variety of signaling molecules, including the TCR ζ chain, ZAP-70, phosphatidylinositol 3-kinase, the HS1 protein, the Lck-binding protein 1 (LckBP1), Nef, and GTPase-activating protein (5, 14, 18, 22, 53, 60, 63, 65, 77). Recent studies suggest that the SH3 domain of Lck plays a role in T-cell function independent of ZAP-70 activation (16a). Mutation of the SH3 domain of Lck inhibited TCR-mediated Erk activation but did not affect ITAM or ZAP-70 phosphorylation. Studies have also indicated the involvement of the Lck SH2 domain in stabilization of the interaction of CD4 with the class II major histocompatibility complex (76), colocalization of CD4 with the activated TCR complex (18), and regulation of Lck enzymatic activity through interactions mediated via its C-terminal tyrosyl residue (1, 58, 68, 72). In addition, Lck may mediate TCR-independent signaling pathways and molecules such as CD28 and Itk to effect full T-cell function (3, 4, 25, 26, 31). The ability of the KS chimera to bypass the proximal signaling functions of Lck (i.e., ζ and ZAP-70 phosphorylation) will permit us to determine if these additional functions attributable to the SH2 domain of Lck are upstream or downstream of ZAP-70 activation.

Src dependence of the Syk PTK.

Our inability to bypass the deficiency in Lck by overexpression of Syk, as measured by SLP-76, Vav, or Syk tyrosine phosphorylation, CD69 induction, or IL-2 gene synthesis (Fig. 5 and data not shown), is in contrast with recent studies showing that transient expression of Syk in JCaM1.6- or CD45-deficient J45.01 T cells was sufficient to reconstitute TCR-mediated NF-AT transcriptional activation (13, 74). In addition, expression of Syk in COS cells under certain conditions has been demonstrated to result in ITAM phosphorylation independently of Src PTKs (43, 80). Our stable clones derived from JCaM1.6 T cells that express Syk or ZAP-70 demonstrate no basal or TCR-induced phosphorylation of these kinases. However, higher levels of Syk expression in fibroblasts and insect Sf9 cells have been demonstrated to result in Syk autoactivation (6, 15, 42). A recent study of Syk activation demonstrated that while Syk could be activated through autophosphorylation, Src-dependent transphosphorylation was still required for the initiation of activation while autophosphorylation appeared to function in signal amplification (20). Moreover, expression of a Syk transgene which is sufficient to reconstitute thymocyte development in zap-70−/− mice does not restore the developmental defects observed in CD45−/− mice (28). Finally, while syk−/− mice do not exhibit any T-cell developmental abnormalities, recent studies indicate that ZAP-70 and Syk play overlapping roles in mediating pre-TCR function. Whereas zap-70−/− mice accumulate CD4+ CD8+ thymocytes, mice deficient in both zap-70 and syk are blocked at an earlier developmental stage and accumulate CD4− CD8− thymocytes (12, 48). These data indicate that either ZAP-70 or Syk is sufficient to mediate the transition of CD4− CD8− to CD4+ CD8+ thymocytes. Moreover, mice deficient in both zap-70 and syk demonstrate a developmental block similar to that of mice deficient in both lck and fyn. Hence, Syk alone is unable to regulate the pre-TCR transition in the absence of Src PTKs. Together, these observations also support a requirement of Src PTKs in Syk function.

Once activated, however, Syk, but not ZAP-70, appears to be able to more efficiently amplify its own autophosphorylation. Hence, while ZAP-70 and Syk may have similar upstream signaling requirements (i.e., CD45 and Lck) for their initiation of activation, the biochemical differences in the modulation of downstream effector functions by activated ZAP-70 and Syk may alter the thresholds for signaling mediated by these two PTKs. Latour et al. have recently analyzed a similar panel of chimeric PTKs in COS cells and described a 100-fold difference in the enzymatic activities of the ZAP-70 and Syk kinase domains (42). While these regulatory differences between ZAP-70 and Syk are reflected in differences in the efficiencies of proximal signaling events, such as calcium responses and cytolysis, they do not appear to cause significant differences in ITAM-based αβ-T-cell development or in antibody- or mitogen-induced proliferative responses (12, 28, 37). However, it is intriguing to speculate that differential expression of ZAP-70 and Syk may be able to alter the strength of the proximal signaling events and convert a low-affinity ligand or an altered peptide ligand response (reviewed in references 33 and 59) into a high-affinity or wild-type peptide response. In addition, the selection of a TCR repertoire may be altered depending on whether the ZAP-70 or Syk PTK is activated in a given developmental stage.

Finally, in contrast to αβ-T-cell function, ZAP-70 and Syk play distinct roles in the development and function of γδ T cells, in the activation of the high-affinity immunoglobulin G and E receptors, in natural killer cell function, and in integrin-mediated signaling (17, 23, 28, 45, 64). Hence, it is likely that in these and other cellular systems, less-constrained signaling motifs may result in selective activation of Syk but not ZAP-70. Additional in vivo studies to translate these observed potential mechanistic differences between ZAP-70 and Syk into functional differences in biological outcomes are ongoing.

ACKNOWLEDGMENTS

We thank Eric Brown, Matt Thomas, Andrey Shaw, and Julie Bubeck Wardenburg for critical reading of the manuscript.

This work was supported in part by grants from the National Institutes of Health (RO1CA71516 and 5T32DK07126). A.C.C. is a Pew Scholar in the Biomedical Sciences.

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