INTRODUCTION

Top Abstract Introduction Materials and Methods Results Discussion References

Stimulation of the T-cell antigen receptor (TCR) initiates a complex signaling cascade leading to clonal expansion, cytokine production, and differentiation. Activation of essential downstream signaling pathways, including the phosphatidylinositol-4,5-bisphosphate (PIP₂), Ras-mitogen-activated protein kinase (MAPK), and phosphatidylinositol-3-kinase pathways, is dependent upon the activity of protein tyrosine kinases (4, 42). Since the TCR lacks intrinsic tyrosine kinase activity, the expression of non-receptor tyrosine kinases is required for TCR signaling. In particular, members of the Src family of tyrosine kinases are thought to provide critical functions during the initial steps of TCR signaling.

Following receptor engagement, Src family kinases mediate the phosphorylation of the TCR on tyrosine residues within immunoreceptor-based tyrosine activation motifs (ITAMs) (6, 22, 53, 56). The ZAP-70 tyrosine kinase is then recruited to the TCR by binding to tyrosine phosphorylated ITAMs (6, 21, 22, 57), where it is activated by Src family kinases through tyrosine phosphorylation within the ZAP-70 catalytic domain (5, 27, 56). Once activated, ZAP-70 then phosphorylates key adapter proteins, such as LAT (62) and SLP-76 (58), which ultimately promote the activation of downstream signaling pathways. Loss of ZAP-70 (37, 60), LAT (13, 63), or SLP-76 (7, 39, 61) disrupts TCR signaling, impairs T-cell development, and blocks T-cell activation. In this model of TCR signaling, Src family kinases mediate both ITAM phosphorylation and ZAP-70 activation (4, 42, 55).

T cells express primarily two members of the Src family of tyrosine kinases, Lck and Fyn (9, 38), both of which have been implicated in TCR signaling. Both kinases have been shown to interact with the TCR (11, 14, 46, 51), and activated forms of the kinases enhance interleukin-2 (IL-2) production (1, 8, 29). However, studies of mice carrying null alleles of the Fyn and Lck genes indicate that at least during development the kinases have only partially overlapping functions. T-cell development is severely impaired in Lck^/ mice (35), while Fyn-deficient mice exhibit a much less drastic phenotype. Development of Fyn^/ thymocytes appears normal, although they have a diminished response to TCR stimulation, and mature T cells from Fyn^/ mice fail to produce IL-2 upon TCR stimulation in vitro (2, 49). The different phenotypes of Lck- and Fyn-deficient mice either reflect the relatively low expression of Fyn early in development (8, 38, 53) or indicate that these kinases provide predominantly distinct functions. Crossing the Lck- and Fyn-deficient mice exacerbates the developmental defects which are apparent in the Lck^/ mice (16, 54), suggesting that these Src family kinases possess some overlapping functions. In addition, expression of an activated form of Fyn as a transgene in Lck^/ mice can rescue certain aspects of T-cell development (16). In sum, these studies indicate that Fyn and Lck have only partially overlapping functions during T-cell development. However, since these kinases may participate in a variety of processes, it is not possible to attribute the observed developmental phenotypes exclusively to alterations in TCR signaling.

Investigations designed specifically to address the ability of Lck and Fyn to mediate TCR signal transduction have not revealed substantial differences in their function. Studies using heterologous cell systems indicate that both Lck and Fyn can mediate ITAM phosphorylation and ZAP-70 activation when coexpressed with the TCR  subunit and ZAP-70 (5, 6, 56). Similarly, activated forms of either Lck or Fyn are able to enhance TCR signaling and the production of IL-2 in T cells stimulated in vitro (1, 8, 29), although Fyn appears to be uniquely required for the activation of Pyk2 kinase following TCR stimulation (41). These results imply that although Lck and Fyn have distinct abilities to mediate T-cell development, they may function equivalently to mediate TCR signaling when expressed at similar levels, as is the case in mature T cells (38), or when activated. To address the capacity of Fyn to support TCR signal transduction, we have directly analyzed Fyn-mediated TCR signaling with a T-cell line which expresses Fyn but lacks functional Lck. Our results indicate that unlike Lck, which mediates TCR-signaling events necessary for full T-cell activation, Fyn mediates an alternative form of TCR signaling that results in a partial T-cell activation phenotype. TCR signaling mediated by Fyn is characterized by a distinct pattern of ITAM phosphorylation and the inability to activate ZAP-70 following its recruitment to the TCR. These findings indicate that the outcome of TCR signaling may be determined by differential usage of Src family tyrosine kinases.

	MATERIALS AND METHODS

Top Abstract Introduction Materials and Methods Results Discussion References

Cells and plasmids. The Jurkat leukemic T-cell line and its Lck-deficient derivative, JCaM1, were grown in RPMI 1640 medium supplemented with 10% fetal calf serum, glutamine, penicillin, and streptomycin. Stable transfectants of a JCaM1 derivative expressing a VP16-Tet repressor fusion protein (15) were obtained by electroporation and selection by growth in the presence of G418 and hygromycin. Fyn or Lck cDNA was subcloned into the pBP1 plasmid, in which expression is regulated by a cytomegalovirus promoter containing a triplication of the Tet operator site (10). Clones which expressed levels of TCR equivalent to that of the parental Jurkat cell line, as determined by fluorescence flow cytometry, were maintained for further analysis. Because mature T cells express nearly equivalent levels of Lck and Fyn kinases (38), we compared TCR signaling in clones expressing equivalent levels of these kinases. The levels of Fyn and Lck expression in the individual clones were determined by immunoblotting lysates from a defined number of cells for Lck and Fyn and then comparing the signal to known amounts of glutathione S-transferase (GST) fusion protein standards of Lck or Fyn (38). Using this approach, Lck expression in JCaM1/Lck clones and the parental Jurkat E6 cell line was estimated to be between 160,000 and 200,000 molecules per cell (data not shown). Similarly, the various JCaM1/Fyn clones used in this study express between 100,000 and 180,000 molecules of Fyn per cell, whereas vector plasmid-transfected control cells, JCaM1/Lck cells, and parental Jurkat cells each express approximately 5,000 molecules of Fyn per cell (not shown).

Immunoprecipitations and immunoblotting. Cells were stimulated at 37°C with TCR antibody C305 (59), harvested by brief centrifugation, and lysed in a mixture of 1% NP-40, 10 mM Tris (pH 7.8), 150 mM NaCl, 1 mM phenylmethylsulfonyl fluoride (PMSF), 0.4 mM sodium orthovanadate, 10 mM NaF, and 1 µg of leupeptin per ml. NP-40-soluble cell lysates were cleared by centrifugation at 14,000 × g for 10 min at 4°C. Lysates to be separated into soluble and particulate fractions were cleared at 750 × g for 10 min at 4°C, and the resulting supernatant was subjected to ultracentrifugation at 100,000 × g for 1 h. Proteins were immunoprecipitated from the particulate fraction after solubilizing the pellet with NP-40 lysis buffer containing 1% sodium dodecyl sulfate (SDS) and then adjusting the SDS concentration in both the soluble and particulate fractions to 0.1%. Lysates were further cleared by preincubation with fixed Staphylococcus aureus cells. The anti-TCR  (6B10.2; Zymed) and the anti-CD3 (Leu-4; Becton Dickinson) monoclonal antibodies were covalently coupled to protein A-Sepharose with dimethyl pimelimidate to eliminate interference of the immunoglobin during subsequent immunoblotting. SLP-76 was immunoprecipitated with a monoclonal antibody (Ab Solutions). ZAP-70, LAT, and PLC-1 were immunoprecipitated with rabbit antiserum (Upstate Biotechnology). Immunoprecipitates were collected on protein A-Sepharose beads and washed in lysis buffer containing 0.5 M NaCl. Immunoprecipitates or lysates were analyzed by immunoblotting with monoclonal antibodies to detect tyrosine phosphoproteins (4G10; Upstate Biotechnology), Lck (provided by Anne Burkhardt and Joe Bolen), Fyn (Transduction Laboratories), ZAP-70 (2F3.2), and PLC-1 (mixed monoclonal antibodies; Upstate Biotechnology). Phosphorylated extracellular signal-regulated kinases were detected with a phospho-ERK1/2 antibody (New England Biolabs). Primary antibodies were detected with a horseradish peroxidase-conjugated secondary antibody and enhanced chemiluminescence.

ZAP-70 in vitro kinase assay. ZAP-70 kinase activity was evaluated as described previously using ZAP-70 immunoprecipitates incubated with [-³²P]ATP and a substrate peptide of erythrocyte band III protein fused to GST (10). Samples were separated by SDS-polyacrylamide gel electrophoresis (PAGE) and visualized by autoradiography. Regions corresponding to GST-band III fusions were excised, and incorporation of ³²P was quantitated by measuring Cerenkov radiation levels.

Calcium measurement. Determination of intracellular Ca²⁺ ([Ca²⁺]_i) concentration was performed as described previously (10). Cells were loaded with the fluorescent calcium binding dye indo-1 (Molecular Probes) at 1 µM, washed extensively, and placed in a spectrofluorometer equipped with a water-jacketed cuvette holder at 37°C. Fluorescence intensity values were corrected for cell autofluorescence, and intracellular calcium concentration was determined using a K_d of calcium binding to indo-1 of 250 nM (17).

Ras activation assay. Cells (25 × 10⁶) were lysed in Ras assay lysis buffer (25 mM HEPES [pH 7.5], 150 mM NaCl, 10 mM MgCl₂, 1 mM EDTA, 1% NP-40, 0.25% sodium deoxycholate, and 10% glycerol) and cleared by centrifugation, and activated Ras was collected by incubation for 30 min at 4°C with glutathione-Sepharose beads precoated with 10 to 20 µg of Raf-1 Ras-binding domain (RBD) fused to GST (52). Following incubation, the beads were washed once with Ras assay lysis buffer, analyzed by SDS-PAGE, and immunoblotted with a pan-Ras monoclonal antibody (Transduction Laboratories).

Analysis of CD69 expression, IL-2 production, and growth inhibition. Cells were incubated at 37°C for 16 to 20 h with either medium alone, TCR antibody (C305; 1:500 dilution of ascites fluid, as either soluble antibody or antibody bound directly to the tissue culture plate), phytohemagglutinin (PHA) (0.3 µg/ml), phorbol myristate acetate (PMA) (50 ng/ml), or ionomycin (1 µM). CD69 cell surface expression was evaluated by fluorescence flow cytometry, following staining with a mouse anti-CD69 antibody (PharMingen) and a fluorescein isothiocyanate-conjugated goat anti-mouse secondary antibody. IL-2 secreted into the culture supernatant was measured by enzyme-linked immunosorbent assay using a monoclonal anti-human IL-2 antibody (BioSource) for capture, followed by detection with rabbit anti-IL-2 antiserum (Genzyme), and an alkaline phosphatase-coupled goat anti-rabbit antibody. Purified recombinant IL-2 (Genzyme) was used as a standard. For growth inhibition, cells were grown in medium alone or stimulated with PHA for 72 h. Viable cells were identified by the exclusion of trypan blue, and cell number was determined with a hemocytometer.

NFAT activation assay. Cells (10 × 10⁶) were transiently transfected with 25 µg of a 3× NFAT-luciferase reporter plasmid, rested for 48 h, and then incubated for 5 h at 37°C with medium alone, plate-bound C305 in combination with PMA, or PMA plus ionomycin. Luciferase activity in unstimulated and TCR-stimulated transfectants was expressed relative to that of the appropriate PMA plus ionomycin stimulated sample to account for differences in transfection efficiency between cell types.

	RESULTS

Top Abstract Introduction Materials and Methods Results Discussion References

Expression of Src kinases in the JCaM1 clones. To compare TCR signaling mediated by Lck and Fyn, we utilized the Jurkat-derived JCaM1 T-cell line, which is deficient in Lck activity (50) and has only a low level of endogenous Fyn (Fig. 1A). Quantitative analysis established that our JCaM1 and parental Jurkat cell lines express Fyn at approximately 1/30 the level of Lck found in Jurkat (data not shown; see Materials and Methods). Transfection of the JCaM1 cell line with a Fyn cDNA allowed us to examine Fyn-mediated TCR-signaling events which are independent of Lck and compare them to TCR signaling in JCaM1 cell lines expressing normal levels of Lck. Since mature T cells have similar levels of Lck and Fyn (38), we selected clones which expressed Fyn at levels which were essentially equivalent to the level of Lck expressed in Jurkat cells. TCR expression was also similar in all of the clones (data not shown). In the JCaM1 transfectants, the Lck and Fyn kinases were expressed under the control of a tetracycline-repressible TetR-VP16 fusion protein (15). Addition of tetracycline to the culture medium reduced kinase levels in a concentration-dependent manner, as shown in Fig. 1B for JCaM1/Lck cells.

View larger version (32K): [in this window] [in a new window]   FIG. 1.   Fyn and Lck expression in JCaM1 transfectants. (A) NP-40-soluble lysates from an equal number of JCaM1/Lck, JCaM1/Fyn, or plasmid vector-transfected cells were immunoblotted for the expression of Lck or Fyn. (B) JCaM1/Lck cells were grown in medium containing tetracycline (0 to 100 ng/ml) for at least 4 days, and NP-40-soluble cell lysates from equivalent cell numbers were immunoblotted for Lck to confirm tetracycline-regulated Lck expression. Tet, tetracycline.

Inducible tyrosine phosphorylation mediated by Lck or Fyn. We assessed the ability of Fyn to replace the function of Lck in JCaM1 by blotting NP-40-soluble cell lysates with an antiphosphotyrosine antibody (Fig. 2, top). Transfection of JCaM1 cells with Fyn restored both the basal level of tyrosine phosphorylation and the induction of tyrosine phosphorylation following TCR stimulation. However, the pattern of tyrosine phosphorylation induced by TCR stimulation of JCaM1/Fyn cells was distinct from that in JCaM1/Lck cells, particularly in the 50- to 75-kDa range (Fig. 2, top). A 70-kDa band is absent in JCaM1/Fyn lysates, and a 65-kDa protein is hyperphosphorylated relative to JCaM1/Lck. The distinct pattern of tyrosine phosphorylation did not appear to be due simply to a quantitative reduction in TCR signaling function in the JCaM1/Fyn cells since reducing signaling in the JCaM1/Lck cells by lowering Lck levels two- to threefold with the addition of tetracycline decreased the levels, but did not alter the pattern, of tyrosine phosphorylation in JCaM1/Lck cells.

View larger version (56K): [in this window] [in a new window]   FIG. 2.   Fyn expression in JCaM1 cells restores protein tyrosine phosphorylation. (Top) NP-40-soluble lysates were prepared from JCaM1/Lck cells grown in the absence or presence of tetracycline (Tet) (3 ng/ml), JCaM1/Fyn cells, or plasmid vector-transfected cells that were unstimulated or stimulated with a TCR antibody (C305) for 2 min at 37°C and immunoblotted with a phosphotyrosine antibody. The numbers at the right indicate the positions of molecular mass markers in kilodaltons. Arrowheads indicate differences in protein tyrosine phosphorylation between JCaM1/Lck and JCaM1/Fyn cells. Results are representative of more than 10 experiments. Similar results were observed with three additional JCaM1/Fyn clones. (Middle) CD3 was immunoprecipitated from lysates of cells which were unstimulated or anti-TCR stimulated for 2 min at 37°C and immunoblotted for tyrosine phosphoproteins. The positions of tyrosine-phosphorylated CD3 chains and ZAP-70 are indicated. Anti-CD3- immunoblotting demonstrated that equivalent amounts of CD3 were immunoprecipitated from all samples (results not shown). Results are representative of at least three experiments. Similar results were observed with two additional JCaM1/Fyn clones. (Bottom)  chain was immunoprecipitated from cell lysates and immunoblotted with phosphotyrosine antibody. Nonreducing conditions were used to accentuate the decrease in electrophoretic mobility caused by extensive phosphorylation of - dimers. Anti--chain immunoblotting showed that equivalent amounts of  were immunoprecipitated from each sample (results not shown). Results are representative of at least three experiments. Similar results were observed with two additional JCaM1/Fyn clones.

TCR tyrosine phosphorylation. One of the key targets of the Src family kinases which could be differentially phosphorylated following TCR stimulation are the ITAM sequences in the cytoplasmic domains of the TCR subunits. We analyzed the ability of Fyn to induce phosphorylation of the CD3 and  subunits. Each CD3 subunit (, , and ) possesses a single ITAM, while an individual  chain contains three ITAM sequences and is normally expressed as a disulfide-linked dimer. Immunoprecipitates of CD3 or  were prepared from unstimulated or TCR-stimulated cells and blotted with an antiphosphotyrosine antibody. We observed that TCR stimulation of JCaM1/Fyn induced substantially lower levels of CD3 phosphorylation than JCaM1/Lck cells (Fig. 2, middle). In addition, TCR stimulation of the JCaM1/Fyn cells resulted in a pattern of -chain phosphorylation which was distinct from the JCaM1/Lck cells (Fig. 2, bottom). In these experiments, -chain immunoprecipitates were analyzed under nonreducing conditions which accentuated the reduction in electrophoretic mobility caused by greater levels of ITAM phosphorylation. TCR stimulation of JCaM1/Fyn cells generated primarily a rapidly migrating form of phospho- compared to the electrophoretic mobility of the phosphorylated  species generated by TCR stimulation of JCaM1/Lck cells (Fig. 2, bottom). These differences in  phosphorylation are similar to those accompanying stimulation of human T-cell clones with agonist or altered peptide ligands (20). The differences in CD3 and -chain phosphorylation could not be explained by a quantitative reduction in TCR signaling in JCaM1/Fyn cells. Lowering Lck expression in JCaM1/Lck cells by including tetracycline in the growth medium decreased the overall level of tyrosine phosphorylation, including CD3 phosphorylation; however, even these reduced levels of phosphorylation exceeded those of JCaM1/Fyn cells (Fig. 2, middle). Similarly, overall -chain phosphorylation was reduced in JCaM1/Lck cells grown in tetracycline, but despite these lower levels of phosphorylation, the electrophoretic mobility of phospho- under nonreducing conditions resembled that of JCaM1/Lck cells grown in the absence of tetracycline (Fig. 2, bottom). These results indicate that TCR phosphorylation mediated by Fyn is qualitatively distinct from that of Lck.

Activation of ZAP-70. We determined if the apparent alterations in TCR phosphorylation which accompanied Fyn-mediated signaling would alter the recruitment of ZAP-70 to the TCR or its subsequent activation. To specifically test for the presence of tyrosine phosphoproteins associated with the  chain, immunoprecipitates of  chain from unstimulated or TCR-stimulated JCaM1/Lck or JCaM1/Fyn cells were resolved by SDS-PAGE under reducing conditions and immunoblotted for tyrosine phosphoproteins. Differences in the electrophoretic mobility of phospho- observed under nonreducing conditions (Fig. 2, bottom) are not observed under reducing conditions. While TCR stimulation of JCaM1/Fyn cells induced -chain phosphorylation, phosphorylated ZAP-70 was barely detectable in the  immunoprecipitates (Fig. 3, top). It was possible that the reduced level of tyrosine-phosphorylated ZAP-70 in the immunoprecipitates from the JCaM1/Fyn cells was due to lower levels of -chain phosphorylation. To address this possibility, we decreased the level of Lck expression in JCaM1/Lck cells by including tetracycline in the growth medium, thereby reducing the level of TCR-stimulated -chain phosphorylation in these cells. Our results indicate that there were still substantially greater levels of phospho-ZAP-70 in  immunoprecipitates from JCaM1 cells expressing lower levels of Lck than in JCaM1/Fyn cells, despite equivalent levels of -chain tyrosine phosphorylation.

View larger version (45K): [in this window] [in a new window]   FIG. 3.   Fyn-mediated TCR signaling fails to induce ZAP-70 phosphorylation despite the recruitment of ZAP-70 to the TCR. (Top)  chain was immunoprecipitated from cell lysates of JCaM1/Lck cells, JCaM1/Lck cells grown in the presence of tetracycline (Tet) (3 ng/ml), and JCaM1/Fyn cells which were either unstimulated or anti-TCR stimulated for 2 min at 37°C. The immunoprecipitates were analyzed under reducing conditions for tyrosine phosphoproteins. The positions of tyrosine-phosphorylated  chain and ZAP-70 are indicated. (Bottom) The  chain immunoprecipitates were analyzed for the presence of associated ZAP-70 by immunoblotting. Anti--chain immunoblotting demonstrated that equivalent amounts of  chain were immunoprecipitated from each sample (results not shown). Results are representative of at least three experiments. Similar results were obtained using two additional JCaM1/Fyn clones.

View larger version (26K): [in this window] [in a new window]   FIG. 4.   Activation of ZAP-70 is defective in JCaM1/Fyn cells. (A) ZAP-70 phosphorylation is defective in JCaM1/Fyn cells. Immunoprecipitates of ZAP-70 from JCaM1/Lck and JCaM1/Fyn cells which were unstimulated or anti-TCR stimulated for 2 min at 37°C were immunoblotted for phosphotyrosine (top) or ZAP-70 (bottom). Results are representative of at least five experiments. Similar results were obtained using two additional JCaM1/Fyn clones (not shown). (B) ZAP-70 kinase activation is defective in JCaM1/Fyn cells. In-vitro kinase activity was analyzed using ZAP-70 immunoprecipitates from unstimulated or TCR-stimulated JCaM1/Lck and JCaM1/Fyn cells, and an exogenous substrate consisting of a band III peptide was fused to GST. Incorporation of 32P into the GST-band III fusion protein was measured by counting Cerenkov radiation levels. ZAP-70 kinase activity is expressed relative to the activity from unstimulated JCaM1/Lck cells (mean ± standard error [SE]; n = 3). (C) Inhibition of tyrosine-phosphatase activity does not restore ZAP-70 phosphorylation in JCaM1/Fyn cells. JCaM1/Lck and JCaM1/Fyn cells were stimulated individually at 37°C either with bisperoxovanadium(phenanthroline) (20 µM) for 5 min or TCR antibody for 2 min or in combination by pretreatment with bisperoxovanadium(phenanthroline) for 3 min prior to inclusion of TCR antibody for an additional 2 min. ZAP-70 immunoprecipitates from each stimulation condition were analyzed for induction of tyrosine phosphorylation. Results are representative of two experiments.

Adapter molecule phosphorylation. Two key adapter molecules, LAT and SLP-76, have been identified as substrates of ZAP-70 and are essential for the activation of downstream signaling pathways (13, 58, 61, 62). Since the activation of ZAP-70 following TCR stimulation is defective in JCaM1/Fyn cells, we expected that the tyrosine phosphorylation of these adapters would be defective. Antiphosphotyrosine blots of LAT immunoprecipitates showed that TCR stimulation of JCaM1/Fyn cells failed to induce LAT tyrosine phosphorylation (Fig. 5A), whereas JCaM1/Lck cells induced LAT phosphorylation. TCR stimulation of plasmid vector-transfected JCaM1 cells also failed to induce LAT phosphorylation (data not shown). Additionally, phosphorylated LAT was not observed in immunoprecipitates from the particulate fraction of JCaM1/Fyn cell lysates, demonstrating that the failure to observe LAT phosphorylation in JCaM1/Fyn cells is not due to alterations in the intracellular distribution of LAT (data not shown). Consistent with these findings, no tyrosine-phosphorylated LAT was detected in immunoprecipitates of the Grb2 adapter protein from TCR-stimulated JCaM1/Fyn cells, whereas tyrosine-phosphorylated LAT associated with Grb2 in TCR-stimulated JCaM1/Lck cells (data not shown). Surprisingly, in contrast to LAT, antiphosphotyrosine blots of SLP-76 immunoprecipitates showed that TCR simulation of JCaM1/Fyn cells was able to induce SLP-76 tyrosine phosphorylation (Fig. 5B). The level of SLP-76 phosphorylation was similar to that found in immunoprecipitates from TCR-stimulated JCaM1/Lck cells. TCR stimulation of plasmid vector-transfected JCaM1 cells induced a level of SLP-76 phosphorylation which was detectable upon extended exposure, probably due to the expression of low levels of endogenous Fyn in these cells (not shown). These results suggest that despite the failure to activate ZAP-70 in JCaM1/Fyn cells following TCR stimulation, the SLP-76 adapter molecule is still inducibly phosphorylated and may be able to mediate the activation of downstream signaling pathways.

View larger version (41K): [in this window] [in a new window]   FIG. 5.   Fyn has differential effects on the phosphorylation of the T-cell adapter proteins LAT and SLP-76. Immunoprecipitates of LAT (A) and SLP-76 (B) from JCaM1/Lck and JCaM1/Fyn cells which were unstimulated or anti-TCR stimulated for 2 min at 37°C were immunoblotted with antiphosphotyrosine, anti-LAT, or anti-SLP-76. Results are representative of three experiments in each case. Similar results were obtained using three additional JCaM1/Fyn clones (results not shown).

Intermediate signaling events in JCaM1/Fyn cells. Since SLP-76 has been implicated in the activation of both the PIP₂ and Ras-MAPK pathways (58, 61), we examined these downstream signaling events in JCaM1/Fyn cells. Engagement of the TCR induces the phosphorylation and activation of phospholipase C-1 (PLC-1), resulting in the production of IP₃ and elevations in [Ca²⁺]_i. Activation of the PIP₂ pathway in JCaM1/Fyn cells was examined by evaluating the phosphorylation of PLC-1 and monitoring elevations in [Ca²⁺]_i. Antiphosphotyrosine immunoblots of PLC-1 immunoprecipitates from unstimulated and TCR-stimulated cells revealed that PLC-1 phosphorylation was induced to the same level in JCaM1/Fyn and JCaM1/Lck cells (Fig. 6A). TCR stimulation of vector plasmid-transfected JCaM1 cells did not induce detectable PLC-1 phosphorylation (not shown). Analysis of the elevation in [Ca²⁺]_i using cells loaded with the Ca²⁺-sensitive fluorescent dye indo-1 showed that TCR stimulation of JCaM1/Fyn and JCaM1/Lck cells elicited similar elevations in [Ca²⁺]_i (Fig. 6B). Because disturbances in activation of the PIP₂ pathway may be more apparent at submaximal levels of TCR stimulation, we also analyzed the elevations in [Ca²⁺]_i induced by lower concentrations of anti-TCR antibody. Even submaximal levels of TCR stimulation induced similar elevations in [Ca²⁺]_i in both JCaM1/Lck and JCaM1/Fyn cells (Fig. 6C). These results indicate that TCR stimulation is able to induce the activation of the PIP₂ pathway despite the lack of ZAP-70 activation.

View larger version (21K): [in this window] [in a new window]   FIG. 6.   Activation of the PIP2 pathway is intact in TCR-stimulated JCaM1/Fyn cells. (A) PLC-1 is tyrosine phosphorylated following TCR stimulation of JCaM1/Fyn cells. Immunoprecipitates of PLC-1 from JCaM1/Lck and JCaM1/Fyn cells which were unstimulated or TCR stimulated for 2 min at 37°C were immunoblotted with antiphosphotyrosine or anti-PLC-1. Results are representative of three experiments. (B) JCaM1/Lck and JCaM1/Fyn cells exhibit similar elevations in [Ca2+]i upon TCR stimulation. JCaM1/Lck, JCaM1/Fyn, or vector-transfected cells were loaded with the Ca2+-selective fluorescent indicator indo-1 and stimulated at 37°C with a saturating concentration of TCR antibody (C305). Results are representative of at least five experiments. (C) JCaM1/Lck and JCaM1/Fyn cells display similar sensitivities to TCR stimulation. Indo-1-loaded JCaM1/Lck or JCaM1/Fyn cells were stimulated with various dilutions of TCR antibody (C305), and the resulting elevations in [Ca2+]i were measured and expressed relative to the maximum for each cell type (mean ± SE; n = 3). Statistical analysis was performed using a one-way analysis of variance and Bonferroni's multiple comparison test. At each antibody dilution, JCaM1/Lck and JCaM1/Fyn cells induced elevations in [Ca2+]i which did not differ significantly (P > 0.05).

View larger version (22K): [in this window] [in a new window]   FIG. 7.   TCR stimulation of JCaM1/Fyn cells activates the Ras-MAPK signaling pathway. (A) Ras activation is similar in JCaM1/Lck and JCaM1/Fyn cells. Activated Ras was affinity purified from lysates of unstimulated or TCR-stimulated JCaM1/Lck, JCaM1/Fyn, and vector-transfected cells (2 min at 37°C) using an RBD-GST fusion protein. Ras content was determined by immunoblotting. Results are representative of three experiments. (B) The kinetics of ERK activation in JCaM1/Fyn cells following TCR stimulation is distinct from that in JCaM1/Lck cells. ERK phosphorylation was evaluated by immunoblotting lysates with a phospho-ERK1/2 antibody. JCaM1/Lck or JCaM1/Fyn cells were stimulated with a TCR antibody for 0 to 10 min at 37°C as indicated. TCR stimulation of vector plasmid-transfected cells did not induce ERK phosphorylation (results not shown). Results are representative of three experiments.

Downstream signaling in JCaM1/Fyn cells. Stimulation through the TCR is able to elicit changes in gene expression and cell growth as a result of downstream signaling events. To confirm that TCR stimulation of JCaM1/Fyn cells was indeed capable of inducing downstream signaling, we examined the expression of the CD69 activation marker, growth arrest following stimulation, and production of the IL-2 cytokine. Stimulation with PHA induced the expression of CD69 in JCaM1 cells expressing either Lck or Fyn, although the levels attained were greater in the JCaM1/Lck cells (Fig. 8A). Stimulation with a TCR antibody also induced CD69 expression in both JCaM1/Lck and JCaM1/Fyn cells, but neither PHA nor anti-TCR antibody was able to induce CD69 expression in plasmid vector-transfected JCaM1 cells (data not shown). In addition, treatment with PHA blocked cell growth to similar extents in both JCaM1/Lck and JCaM1/Fyn cells, while PHA had no effect on the growth of vector-transfected JCaM1 cells (Fig. 8B). In contrast to these results, stimulation of JCaM1/Fyn cells with either PHA or plate-bound TCR antibody, in combination with PMA, induced 5- to 10-fold less IL-2 than similar treatment of JCaM1/Lck cells (Fig. 8C). TCR-independent stimulation with PMA and the Ca²⁺ ionophore ionomycin elicited similar levels of IL-2 from both cell types. To determine if the deficits in IL-2 production in JCaM1/Fyn cells were due to impaired NFAT activity, we transfected cells with an NFAT-regulated luciferase reporter gene construct and measured luciferase expression induced by TCR stimulation. While TCR stimulation of JCaM1/Fyn cells was able to induce some NFAT activity, it was substantially less than that of JCaM1/Lck cells (Fig. 8D). These findings suggest that TCR signaling mediated by Fyn is able to activate some downstream signaling pathways, but not others, resulting in a partial activation phenotype.

View larger version (26K): [in this window] [in a new window]   FIG. 8.   TCR stimulation of JCaM1/Fyn cells elicits partial activation of downstream signaling pathways. (A) TCR stimulation of JCaM1/Fyn cells induces expression of the CD69 activation antigen. JCaM1/Lck or JCaM1/Fyn cells were incubated overnight with medium alone (unstim), PHA (0.3 µg/ml), or PMA (50 ng/ml). Following stimulation, the cells were analyzed for expression of the CD69 activation antigen by fluorescence flow cytometry. TCR antibody also induced CD69 expression in JCaM1/Lck and JCaM1/Fyn cells but not in vector plasmid-transfected JCaM1 cells (results not shown). (B) TCR stimulation of JCaM1/Fyn cells induces inhibition of cell growth. Cells were plated in culture medium in the absence or presence of PHA (1 µg/ml) for 72 h at 37°C. The number of viable cells in each sample was determined by counting cells which excluded trypan blue (mean ± SE; n = 3). (C) IL-2 production is impaired in JCaM1/Fyn cells following TCR stimulation. Cells were incubated overnight at 37°C in medium with PMA (50 ng/ml) in combination with PHA (0.3 µg/ml) or with immobilized TCR antibody (1:1,000 dilution of C305 ascites fluid). IL-2 content in the culture medium was determined by enzyme-linked immunosorbent assay. IL-2 production by JCaM1/Fyn cells is expressed relative to that of JCaM1/Lck cells (mean ± SE; n = 3). (D) NFAT activation is markedly reduced in JCaM1/Fyn cells. Activation of the NFAT transcription factor was measured by transiently transfecting JCaM1/Lck, JCaM1/Fyn, or vector control cells with a 3× NFAT-luciferase reporter construct. Transfected cells were incubated at 37°C for 5 h with medium alone or with PMA in combination with immobilized C305 or ionomycin. To control for differences in transfection efficiency, luciferase activity induced by TCR stimulation is expressed relative to that induced by treatment with PMA and ionomycin (mean ± SE; n = 5).

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

The Lck and Fyn tyrosine kinases have been implicated in the initiation of TCR signal transduction, but it is unclear whether they utilize the same signaling mechanism or activate the same downstream signaling pathways. Analyses of Lck- and Fyn-deficient mice suggest that these kinases provide unique and overlapping functions in thymocytes (2, 16, 35, 49, 54), although these studies do not address whether these differences reflect distinctions in the initiation of the TCR signal or in other processes. The relatively low level of Fyn expression early in thymocyte development (8, 38, 53) may also contribute to the distinct phenotypes exhibited by Lck- and Fyn-deficient mice. Studies in heterologous cell systems indicate that Lck and Fyn exhibit a very similar ability to execute the initial steps in TCR signaling, including the phosphorylation of the TCR  subunit and activation of ZAP-70 (5, 6, 56). However, these systems may lack T-cell-specific regulatory proteins and may not express the transfected TCR signaling proteins at the levels and proportions observed in T cells. In this study, we have directly examined the ability of Fyn to mediate TCR signaling in T cells, independent of the contribution of Lck. Our results demonstrate that Fyn is capable of mediating TCR signaling but uses a mechanism which is distinct from that of Lck and only partially activates downstream signaling events. Moreover, Fyn-mediated TCR signaling resembles the initial biochemical signaling events that have been previously described for TCR stimulation by altered peptide ligands (31, 48).

Stimulation of the TCR in cells which express Fyn, but lack Lck, induced tyrosine phosphorylation, but phosphorylation of the TCR was altered compared to cells expressing Lck. Fyn mediated only weak phosphorylation of CD3, and the pattern of  chain phosphorylation was distinct from the pattern observed with Lck. The qualitative differences in TCR phosphorylation persisted even when the level of Lck-mediated signaling was reduced to match that of Fyn. Despite the altered TCR phosphorylation, Fyn was still capable of mediating recruitment of ZAP-70 to the TCR complex, although the phosphorylation and activation of ZAP-70 were defective. These results indicate that while both Fyn and Lck can initiate TCR signaling, the mechanisms they use are distinct.

The inability of cells expressing Fyn to induce the phosphorylation of the LAT protein is consistent with the loss of ZAP-70 activation, since LAT is a likely substrate of ZAP-70 (62). In contrast, SLP-76, also a substrate of ZAP-70 (44, 58), was tyrosine phosphorylated following TCR stimulation, suggesting that an alternative means of TCR signaling is utilized by Fyn. Previous studies indicate that TCR signaling mediated by Lck induces SLP-76 phosphorylation as a result of ZAP-70 activation and is largely dependent upon LAT expression (13, 44, 58). Our results show that Fyn induces SLP-76 phosphorylation through a distinct mechanism, although further experiments are required to determine if Fyn and ZAP-70 mediate the same pattern of SLP-76 phosphorylation (12, 58). Since both Fyn (28) and SLP-76 (36) interact with the adapter protein SLAP-130/FYB, it is possible that SLP-76 is recruited to Fyn by SLAP-130/FYB, either directly or indirectly via the SKAP55 adapter protein (28, 32, 33). Assembly of this complex could allow SLP-76 to become phosphorylated by Fyn independent of ZAP-70 activation. A recent study showed that cotransfection of Fyn, SLAP-130/FYB, and SLP-76 was able to potentiate downstream signaling (45). Once phosphorylated, SLP-76 may participate in activation of both the PIP₂ and the Ras-MAPK pathways (61).

TCR stimulation of JCaM1/Fyn cells induced phosphorylation of PLC-1 and elevations in [Ca²⁺]_i, as well as activation of Ras and ERK1/2. Consistent with the activation of these intermediate signaling pathways, TCR stimulation induced the expression of the CD69 activation antigen and caused inhibition of cell growth. However, Fyn-mediated TCR signaling elicited only modest amounts of IL-2 production, due at least in part to substantially reduced activation of the NFAT transcription factor. This reduction in NFAT activation may reflect the transient nature of ERK activation in JCaM1/Fyn cells (Fig. 7). Consistent with this, we have observed that the induction of AP-1 DNA binding activity in JCaM1/Fyn cells is deficient compared to that of JCaM1/Lck cells (data not shown). These results indicate that Fyn is able to mediate the activation of TCR signaling pathways by a distinct mechanism which bypasses the normal functioning of the ZAP-70 tyrosine kinase.

The differences in the initiation of TCR signaling mediated by Lck and Fyn may be due to distinct substrate preferences of the catalytic domains of the kinases or the result of distinct interactions of the kinases with the TCR or other molecules. Inherent catalytic differences between the kinases appear unlikely, given the similar ability of Fyn and Lck to mediate -chain phosphorylation and ZAP-70 activation when expressed in Cos cells (6) or activation of a CD16-CD7-ZAP-70 chimera following co-cross-linking with either a Fyn or a Lck transmembrane chimera (26). Consistent with this, we do not observe substantial differences in the ability of Fyn and Lck to phosphorylate the cytoplasmic domains of CD3 or  chain in vitro (data not shown). However, these systems may lack key regulatory elements which are present in the complete TCR complex or expressed exclusively in T cells. As such, the distinct interaction of the kinases with the intact TCR or other molecules may influence their signaling properties. For example, Fyn has been demonstrated to associate with ITAMs present in the cytoplasmic domains of the TCR (14, 46). It is possible that this interaction specifies a binding orientation of Fyn which determines the pattern of ITAM phosphorylation and results in inefficient ZAP-70 phosphorylation by Fyn. The high levels of expression which are attainable in heterologous cell systems may permit ZAP-70 and Src family kinases to interact independently of the expression of TCR subunits, which could mask differences based upon distinct TCR binding. Distinct signaling function of the Src family kinases could also reflect differences in the SH2 and SH3 domains, since these regions could influence substrate preferences and subcellular localization. Despite the high degree of homology between the Lck and Fyn kinases, the precise nature of their interactions with substrates may determine the specific signaling pathways which are elicited by TCR stimulation.

The inability of JCaM1/Fyn cells to produce substantial amounts of IL-2 following TCR stimulation, although other downstream signaling events are induced, resembles the phenotype of T cells stimulated under conditions which lead to nonresponsiveness (anergy) (23, 47). In particular, our results are consistent with the hypothesis that Fyn may mediate TCR-signaling events leading to anergy following stimulation with altered peptide ligand (APL) or under conditions in which the strength of TCR signal is decreased (20, 30). The biochemical events which accompany TCR stimulation with APL closely parallel our findings with Fyn-mediated TCR signaling. In both cases, the phosphorylation of CD3 is reduced and the pattern of -chain phosphorylation is altered. In mouse or human T cells, APL preferentially induces a less completely phosphorylated form of  than agonist peptides (20, 25, 31, 48). The pattern of agonist- and APL-mediated  phosphorylation in human cells closely resembles that observed in JCaM1/Lck and JCaM1/Fyn cells, respectively (20). In addition, stimulation of JCaM1/Fyn cells, like stimulation of T-cell clones with APL, leads to the recruitment of ZAP-70 to the TCR but does not lead to ZAP-70 activation. The similarity between signaling events which accompany partial activation by APL and the TCR signaling events in JCaM1/Fyn cells suggests a model whereby TCR signaling mediated by Fyn in the absence of Lck leads to partial activation and the induction of anergy.

Our results imply that T cells may regulate TCR signaling by altering the expression, activity, or localization of Lck and Fyn. Changes in the relative expression of Lck and Fyn have been described during T-cell development (38), following TCR stimulation (34, 43), and in T cells from lpr mice (24). Under these conditions, TCR-signaling events may be mediated selectively by either Lck or Fyn. In addition, sequestering Lck away from the TCR by virtue of its association with CD4 or other molecules could limit Lck-mediated TCR signaling (18, 19, 30). In fact, stimulation of the TCR in the absence of CD4 corecruitment can lead to the induction of T-cell nonresponsiveness (30). Furthermore, exclusive activation of Lck or Fyn catalytic function could also lead to differential TCR signaling (3). Further studies are required to determine whether differential TCR signaling results from the utilization of these mechanisms.

In summary, we present biochemical and cellular evidence that Fyn is able to mediate TCR signaling transduction but that it utilizes a mechanism which is distinct from that utilized by Lck. This alternative signaling mechanism leads to only a partial induction of downstream signaling events, resulting in deficient production of IL-2. These findings suggest that the outcome of TCR stimulation is likely to be dependent upon whether Lck or Fyn plays a predominate role in mediating signaling events.