INTRODUCTION

Top Abstract Introduction Procedures Results Discussion References

T cell activation is thought to be initiated by the interaction of the T cell antigen receptor (TCR)¹ with two distinct classes of nonreceptor protein-tyrosine kinases (recently reviewed in Refs. 1-3). Biochemical as well as genetic evidence suggests that members of the Src family of nonreceptor protein-tyrosine kinases, represented in T cells by the Fyn and Lck proteins, are involved in this process. Reconstitution experiments of antigen receptor signaling in fibroblasts (4), as well as the biochemical and functional characterization of a Jurkat cell line (JCaM1.6) that lacks Lck kinase activity (5, 6), strengthened the view that Src kinases are responsible for a very important initial step in the antigen receptor signaling cascade, namely the phosphorylation of the intracellular domains of TCR-associated proteins. This includes the  homodimer, as well as the CD3 complex composed of the , , and  chains. With the help of chimeric receptors (7, 8), a distinct motif, now termed ITAM, which is present in all of the above mentioned TCR-associated polypeptides, was found to be sufficient for the induction of T cell activation in various experimental systems (9-11). The ITAM motif is phosphorylated on tyrosine residues in vivo following antigen receptor stimulation. This phosphorylation event was shown to be responsible for the recruitment of the cytoplasmic ZAP-70 kinase to the phosphorylated receptor via phosphotyrosine-SH2 domain interactions (6, 12, 13).

Surprisingly, significant activation of the ZAP-70 kinase mediated by its binding to ITAMs could not be demonstrated (14), but it was suggested that the recruitment of ZAP-70 into the antigen receptor complex provided a platform for "cross-talk" between the Src and ZAP-70 kinases, which leads to phosphorylation and activation of ZAP-70 (15, 16). Co-precipitation analyses demonstrated that ZAP-70 interacts directly with Lck (17-19); furthermore, the protein-tyrosine kinase activity of Lck is required for the phosphorylation of ZAP-70 Tyr 493 in vivo (16). This phosphorylation event correlated with the functional activity of ZAP-70. Multiple phosphorylation of ZAP-70, mediated by autophosphorylation, by Src kinases, or by other cellular kinases, is thought to result in the association of downstream signaling components by SH2 domain-phosphotyrosine interactions (14, 20-23).

In this study, we describe gain-of-function mutants of the ZAP-70 and Syk tyrosine kinases (ZAP YY597/598FF, Syk YYY624-626FFF), which activate Jurkat cell lines. Our results implicate novel regulatory mechanisms that control the cellular activity of these kinases, possibly involving inhibitory proteins.

	EXPERIMENTAL PROCEDURES

Top Abstract Introduction Procedures Results Discussion References

Cell Lines and Antibodies-- TAg-Jurkat T cells (24), Jurkat E6 cells, Lck kinase-deficient JCaM1.6 (5), and Syk-deficient DT40 chicken B cells (25) were maintained in RPMI 1640, 10% fetal calf serum, 10 µg/ml gentamicin. COS-7 cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum and 10 µg/ml gentamicin.

Plasmids and Mutagenesis-- P5C7, a derivative of pRK5 (26) containing a modified polylinker region, was used as the mammalian expression vector. Cytoplasmic Ig fusion proteins (cIg) and transmembrane CD16/CD7 chimeras have been described previously (15, 27). CD16/7/control is a construct that lacks the cytoplasmic domain. The transmembrane Ig fusion proteins (sIg) comprise the leader sequence from CD5, extracellular CH2 and CH3 heavy chain domains from human IgG₁, the CD7 transmembrane domain, and the intracellular portions of TCR- (sIg-) or a control polypeptide (sIg-control).

Transient Transfections and Luciferase Assay-- DEAE-dextran transfections of COS-7 cells were performed as described previously (30). Jurkat T cells were transiently transfected using the EasyjecT Plus electroporation system (Eurogentec). Cells (1.3 × 10⁷ in complete medium) were mixed for 5 min at room temperature with 10 µg of luciferase reporter plasmid plus the indicated amount of cytomegalovirus promoter expression plasmids. TAg-Jurkat T cells were pulsed at 310 V and 1200 microfarads, JCaM1.6 cells at 240 V and 1200 microfarads and immediately transferred to 10 ml of complete medium, routinely resulting in transfection efficiencies of 60-70% for TAg and 30-40% for JCaM1.6. Twelve to twenty hours after transfection, cells were aliquoted and stimulated for 8 h in 1 ml of complete medium using 0.5 µg/ml ionophore A23187 (Sigma), 50 ng/ml phorbol 12-myristate 13-acetate (PMA, Sigma), 2 µg/ml OKT3, or 2 µg/ml anti-human IgG antibody, or left untreated. Syk-deficient DT40 cells were transfected as described (31) and stimulated with ionophore plus PMA as above or with 3 µg/ml M4 anti-BCR antibody for 8 h. Cells were subsequently washed in Tris-buffered saline and lysed in 50 µl of reporter lysis buffer (Promega). Luciferase activity was quantified in a microplate scintillation counter (Packard) after mixing 20 µl of cell lysate with 100 µl of luciferase reagent (Promega). Luciferase activities are expressed relative to the ionophore/PMA-stimulated sample and are average values of triplicate experiments with standard deviations less than 20%.

Cell Lysis and Immunoprecipitations-- For immunoprecipitations, cells were transfected as described above. T cells were lysed 12-20 h after transfection, COS-7 cells after 48 h. Stimulation of Jurkat cells was performed in 50-100 µl of RPMI by adding 2 µg of OKT3 or anti-human IgG for the indicated time period at 37 °C. For SDS total cell lysates, SDS was added to a concentration of 1% following stimulation. Detergent lysis buffer contained 1% Brij 97, 20 mM Tris, pH 7.5, 150 mM NaCl, 1 mM EDTA, 50 mM NaF, 10 mM Na₄P₂O₇, 1 mM Na₃VO₄, 10 µg/ml leupeptin, 5 µg/ml aprotinin, and 1 mM phenylmethylsulfonyl fluoride (all from Sigma). Alternatively, 1% digitonin was used as detergent for OKT3 immunoprecipitations. After 20 min at 4 °C, lysates were centrifuged at 20,000 × g to remove insoluble material. Anti-ZAP-70 antibody was added at 1 µg/ml and OKT3 at 5 µg/ml, respectively, whereas Ig fusion proteins were directly collected on Protein A-6MB-Sepharose beads (Amersham Pharmacia Biotech). Immunoprecipitates were washed three times in lysis buffer prior to dissociation in SDS sample buffer. Proteins were separated on SDS-polyacrylamide gels and transferred onto nitrocellulose membranes. Immunodetections were performed using horseradish peroxidase-conjugated secondary antibodies (Dianova) and chemiluminescence (Amersham Pharmacia Biotech).

In Vitro Kinase Assays-- ZAP-70 and its mutant variants were transiently expressed in TAg-Jurkat T cells and purified by immunoprecipitation as described above. Additionally, precipitates were washed once in 20 mM Tris, pH 7.5, 0.3 M LiCl and twice in kinase buffer (10 mM Tris, pH 7.5, 10 mM MnCl₂). Autophosphorylation experiments were performed for 10 min at 25 °C in 25 µl of kinase buffer in the presence of 10 µCi of [-³²P]ATP (~3000 Ci/mmol). For substrate phosphorylation assays, 5 µg of purified glutathione S-transferase-SAM-C and 10 µM unlabeled ATP were included in each reaction. Samples were boiled in SDS loading buffer, separated by SDS-polyacrylamide gel electrophoresis, transferred to nitrocellulose, and analyzed by autoradiography, Ponceau S staining, and immunodetection. Quantification of radioactive signals was done using a microchannel array detector (InstantImager, Packard).

Intracellular Calcium Measurement-- Jurkat E6 and JCaM1.6 cells were transfected with chimeric transmembrane CD16/7/Syk mutants by infection with recombinant vaccinia virus as described (15). After 6 h, cells were washed and loaded with the fluorescent calcium probe Fluo-3 (Molecular Probes) for 1 h in Hanks' buffered saline solution, washed, and analyzed by flow cytometry (Coulter Epics XL). Expression levels were monitored by anti-CD16 immunofluorescence.

	RESULTS

Top Abstract Introduction Procedures Results Discussion References

Expression of ZAP YF-C Stimulates the IL-2 Promoter in Jurkat Cells-- The ZAP-70 and Syk tyrosine kinases are highly homologous. One exception is ZAP-70, which is bears a unique extended C terminus. However, a cluster of tyrosines (YY597/598 in ZAP-70, YYY624-626 in Syk) located close to the C terminus, is conserved between the two kinases (Fig. 1A). In order to test whether these tyrosines are important for cellular activation events mediated by ZAP-70, we altered the residues by site-directed mutagenesis (ZAP YY597/598FF, ZAP YF-C). ZAP YF-C as well as wild type or mutant control constructs of ZAP-70 were transiently expressed in several cell lines at similar levels (Fig. 1B and data not shown).

View larger version (29K): [in this window] [in a new window]   Fig. 1.   Overexpression of ZAP-70 mutants. A, schematic representations of ZAP-70 mutants. ZAP YF-C is a novel substitution mutant affecting two conserved tyrosine residues (597/598) located immediately C-terminally to the kinase domain. B, transient overexpression of ZAP-70 or mutant derivatives in TAg-Jurkat T cells. Cell lysates were resolved by 12% SDS-polyacrylamide gel electrophoresis and immunodetected with an anti-ZAP-70 mAb.

View larger version (35K): [in this window] [in a new window]   Fig. 2.   Increased transcriptional activation of the IL-2 promoter and NF-AT-elements in YF-C-transfected TAg-Jurkat cells. A, TAg-Jurkat cells were transfected with 10 µg of IL-2 promoter luciferase reporter plasmid and 15 µg of control vector or different ZAP-70 mutant expression vectors. Comparable expression of ZAP-70 constructs was verified by Western blotting (data not shown). Cells were left unstimulated, or were stimulated with OKT3 alone, PMA alone, PMA and the OKT3 anti-CD3-epsilon mAb, or PMA and ionophore. Luciferase activities are expressed as percent of the ionophore/PMA-stimulated sample of each transfection and are average values of triplicate experiments with standard deviations less than 20%. B, TAg-Jurkat cells were transfected with 10 µg of NF-AT luciferase reporter plasmid and were otherwise treated and analyzed as above. C, TAg-Jurkat cells were co-transfected with 10 µg of IL-2 promoter reporter construct and the indicated amounts of wild-type ZAP-70, ZAP Y292F, or ZAP YF-C and analyzed as described above. Luciferase activity of PMA (left panel) or PMA/OKT3-stimulated cells is expressed as -fold induction compared with the control transfection with the cloning vector.

Biochemical Characterization of the ZAP YF-C Mutant in Vivo-- To analyze whether the gain-of-function mediated by ZAP YF-C correlated with increased substrate phosphorylation in vivo, we investigated the cellular tyrosine phosphorylation pattern of TAg-Jurkat cells that had been transfected with ZAP YF-C or control constructs (Fig. 3). Vector-transfected Jurkat cells that had been stimulated with OKT3 were found to be substantially enriched in a number of tyrosine-phosphorylated proteins as compared with unstimulated control cells (Fig. 3A, first and last lanes, respectively). Overexpression of either ZAP-70, ZAP Y492F, or kinase-inactive ZAP-70 in unstimulated cells does not result in increased tyrosine phosphorylation of cellular proteins except for a band at 68-70 kDa. This band corresponds at least in part to the overexpressed ZAP-70 variants (data not shown). However, this background phosphorylation, induced by overexpression and seen for all ZAP-70 versions, does not correlate with substantial IL-2 promoter activation (Fig. 2). In contrast, ZAP YF-C overexpression resulted in a markedly different pattern (Fig. 3A). First, the 70-kDa band was found to be considerably more highly phosphorylated as compared with the controls. Second, other cellular proteins showed enhanced phosphorylation following ZAP YF-C overexpression, namely at 21-23 kDa, at 55-56 kDa, and at about 150 kDa. The 21-23-kDa phosphorylated bands appeared particularly striking because they corresponded precisely to the described sizes of the phosphorylated TCR  chain. Thus, ZAP YF-C overexpression induces the phosphorylation of a characteristic subset of cellular proteins, as compared with OKT3-stimulated tyrosine phosphorylation (Fig. 3).

View larger version (42K): [in this window] [in a new window]   Fig. 3.   Overexpression of ZAP-YF-C in Jurkat cells is sufficient to induce tyrosine phosphorylation of a distinct subset of cellular proteins, including the p22/23 isoform of TCR-. A, anti-phosphotyrosine immunoblot of total detergent lysates of TAg-Jurkat cells transfected with 30 µg of the indicated ZAP-70 mutant expression vectors. One sample of cells transfected with control vector was stimulated for 2 min with OKT3 as a control. Proteins were resolved on 12% polyacrylamide gels. Arrows indicate proteins specifically hyperphosphorylated on tyrosines after expression of ZAP YF-C. Comparable expression levels of ZAP-70 mutants were verified by stripping the blot and reprobing with an anti-ZAP-70 mAb (shown in Fig. 1). B, TCR immunoprecipitates with anti- mAb OKT3 from transfected TAg-Jurkat cells were immunodetected with an anti-phosphotyrosine mAb. Cells were either left unstimulated or stimulated for 2 min with OKT3 prior to lysis. Immunoprecipitates were separated on 12% polyacrylamide gels under reducing conditions. Bands at 52 and 33 kDa correspond to OKT3 heavy and light chains.

ZAP YF-C Displays Moderately Enhanced Catalytic Activity in Vitro-- We subsequently analyzed whether the catalytic activity of ZAP YF-C was activated in respect to exogenous substrates. ZAP YF-C as well as positive or negative controls were expressed in TAg-Jurkat cells, immunoprecipitated via a monoclonal antibody, and used in in vitro kinase assays. The in vitro substrate used to determine their respective kinase activities was a fragment of the SAM68 protein (14) that had been expressed in, and purified from, E. coli. The positive control mutant, ZAP Y492F, which had been shown to have a substantially enhanced catalytic activity in vitro (16, 32), was consistently found to be a more potent kinase in this assay than the wild type ZAP-70 protein (4-fold enhancement, Fig. 4A). ZAP YF-C displayed about 1.6-fold enhanced kinase activity (Fig. 4A).

View larger version (35K): [in this window] [in a new window]   Fig. 4.   In vitro kinase activities of ZAP-70 mutants and association with phospho-. A, in vitro catalytic activities of ZAP-70 mutants using 5 µg of purified glutathione S-transferase-SAM68 C-terminal fragment (amino acids 331-443) as a substrate. ZAP-70 mutants were purified by anti-ZAP-70 mAb-mediated immunoprecipitation from lysates of transfected TAg-Jurkat cells (1.3 × 107) and allowed to phosphorylate the substrate for 10 min at 25 °C in the presence of radioactively labeled ATP. Proteins were resolved on 8% SDS-polyacrylamide gels, transferred to nitrocellulose, and visualized by autoradiography. Membranes were stained with Ponceau S and immunoblotted with anti-ZAP-70 mAb to ensure comparable amounts of substrate and ZAP-70 variants, respectively. Quantifications of radioactive signals were obtained using the InstantImager (Packard). B, in vitro autophosphorylation. ZAP-70 mutants were expressed as cytoplasmic Ig fusion proteins (comprising the CH2 and CH3 domains of human IgG) in the cytoplasm of TAg-Jurkat cells and purified by precipitation on Protein A-Sepharose. Proteins were subjected to autophosphorylation in the presence of radioactively labeled ATP and analyzed as described above. Membranes were stained with Ponceau S to ensure comparable amounts of purified cytoplasmic Ig-ZAP-70 chimeras. C, in vitro association of ZAP-70 mutants to phosphorylated sIg-. A chimeric TCR- chain comprising an extracellular IgG CH2/CH3 fusion part, the transmembrane domain of CD7 and the intracellular domain of  (sIg-) was co-expressed with p56-Lck in COS-7 cells to achieve strong tyrosine-phosphorylation of ITAMs. EDTA-containing lysates were mixed with lysates of COS cells transfected with different ZAP-70 mutants. Complexes of sIg- and ZAP-70 mutants were precipitated on Protein A, resolved on 8% SDS-polyacrylamide gels, and transferred to nitrocellulose. Amounts of co-purified ZAP-70 variants were analyzed by anti-ZAP-70 immunodetection.

ZAP YF-C-induced T Cell Activation Is Dependent on the Presence of Lck-- Since it has been suggested that ZAP-70 requires the Lck tyrosine kinase for its in vivo activity (16), we analyzed whether ZAP YF-C was uncoupled from known upstream signaling events. Jurkat JCaM1.6 cells that do not express kinase-active Lck (5) were used for this part of the study. Fig. 5A shows that JCaM1.6 cells were completely unresponsive to ZAP YF-C expression or other stimuli, unless the TCR signaling pathway of these cells was reconstituted by Lck. Co-transfection experiments revealed that ZAP YF-C had a similar gain-of-function activity in JCaM1.6 cells when Lck was present. Thus, Lck appears to be a necessary component of the pathway utilized by the ZAP YF-C mutant.

View larger version (23K): [in this window] [in a new window]   Fig. 5.   ZAP YF-C-induced hyperactivation is dependent on p56-Lck and is not observed in Syk-deficient B cells. A, Lck kinase-deficient JCaM1.6 Jurkat cells were transfected and analyzed as described in Fig. 2A using the IL-2 luciferase reporter. For reconstitution with p56-Lck (left panel), cells were co-transfected with 5 µg of Lck expression vector. B, Syk-deficient DT40 Chicken B cells were co-transfected with 10 µg of NF-AT luciferase reporter plasmid and 20 µg of ZAP-70 mutant expression vectors. Cells were stimulated with ionophore plus PMA or 3 µg/ml anti-BCR M4 monoclonal antibody or left untreated and analyzed as described.

ZAP YF-C Reconstitutes the B Cell Antigen Receptor Signal Transduction Pathway in Syk-negative DT40 cells, but Does Not Hyperactivate Them-- In order to test whether the observed effects were specific for T cell activation, we transfected ZAP YF-C, along with relevant controls, into the Syk-negative DT40 cell line that had previously been used to study the cellular function of ZAP-70 mutants (16, 29, 31, 39, 40).

Syk YF-C Hyperactivates TAg-Jurkat Cells and Compensates for the Signaling Lesion of JCaM1.6 Cells-- It was recently reported that the Syk tyrosine kinase can be phosphorylated in vitro at positions 625-626 (41, 42), which are homologous to the tyrosine residues 597/598 in ZAP-70. Furthermore, previous studies showed that Syk is not strictly dependent on the activity of Lck (15, 43-46). We therefore made the corresponding Syk YF-C mutant and tested its ability to activate the IL-2 promoter in TAg-Jurkat cells. In this part of the study, intracellular Ig fusion proteins (27) of Syk or Syk YF-C were used to ensure comparable expression levels (Fig. 6A). Fig. 6B (left panel) shows that overexpression of both wild type Syk or Syk YF-C enhanced the responsiveness of cells that were stimulated by OKT3 and PMA. However, in cells that were treated with PMA only, Syk YF-C stimulated the IL-2 promoter 10-fold, whereas wild type Syk had a moderate (2.5-fold) effect. We then expressed Syk YF-C in JCaM1.6 cells (Fig. 6B, right panel). Syk YF-C but not wild type Syk activated the IL-2 promoter in PMA-treated JCaM1.6 cells dramatically, essentially overcoming the need for TCR occupancy. It should be noted that wild type Syk is capable of activating TAg-Jurkats or JCaM1.6 cells at substantially higher expression levels (data not shown), consistent with previous reports (44, 45). However, our data show that Syk YF-C has a markedly increased activity when compared with the wild type kinase. Thus, the activating potential of the Syk YF-C mutation is independent of the presence of Lck.

View larger version (23K): [in this window] [in a new window]   Fig. 6.   Syk YF-C is a gain-of-function mutant in TAg-Jurkat and JCaM1.6 cells. A, TAg-Jurkat cells (left panel) or JCaM1.6 cells (right panel) were co-transfected with 10 µg of IL-2 promoter reporter construct and 3.5 µg of cytoplasmic Ig-Syk fusion protein expression vector. Lysates were resolved on SDS-polyacrylamide gels, blotted, and immunodetected with anti-Ig antibodies. B, cells were transfected as above and analyzed as described under Fig. 2A.

Intracellular Calcium Mobilization Mediated by a Syk YF-C Chimera-- Finally, we tested the ability of the Syk YF-C mutant to induce intracellular calcium mobilization, an important receptor proximal event known to be inducible by Syk/ZAP-70 family tyrosine kinases. To this end, tripartite chimeras were constructed that comprised the extracellular domain of the CD16 antigen, the CD7 transmembrane domain, and either the wild type Syk or Syk YF-C molecules as intracellular portions (15). The fusion proteins were expressed by using recombinant vaccinia viruses in Jurkat E6 or JCaM1.6 cells (Fig. 7A), two cell types in which virus dependent overexpression of the Syk chimeras was comparable. Fig. 7B (left panel) shows that expression of the 16/7/Syk chimera induces a constitutive high level of intracellular calcium in E6 cells, as was described previously (15). In the case of Syk YF-C, however, calcium levels were superinduced by aggregation of the chimera.

View larger version (24K): [in this window] [in a new window]   Fig. 7.   Membrane-targeted and cross-linked Syk YF-C displays enhanced capacity to mobilize intracellular calcium. A, chimeric Syk constructs comprising the extracellular domain of CD16 were expressed in Jurkat E6 (left panel) or JCaM1.6 cells (right panel) by infection with recombinant vaccinia virus. Expression levels were analyzed by anti-CD16 immunofluorescence and flow cytometry. B, transfected cells were loaded with the calcium-sensitive fluorescent indicator Fluo-3 and analyzed by flow cytometry for 360 s. After 80 s, OKT3 or anti-CD16 antibody were added to the cells, followed by cross-linking with anti-mouse IgG after 130 s (arrows). Mean Fluo-3 fluorescence is indicated as -fold intensity compared with CD16/7/control.

	DISCUSSION

Top Abstract Introduction Procedures Results Discussion References

Gain-of-function mutations have been successfully used in the past to study signal transduction pathways, because they provide a genetic tool to analyze sequential events within signaling cascades. In this study we describe novel gain-of-function mutants of ZAP-70 (ZAP YF-C) and Syk, in which tyrosines 597/598 or 624-626, respectively, have been substituted by phenylalanines. Tyrosines 625-626 of Syk had previously been shown to be autophosphorylated in vitro (41, 42) and therefore appeared to be good targets for a functional analysis. Previously published data were furthermore compatible with functional tyrosine phosphorylation of ZAP-70 at multiple residues (14, 20). We found that expression of ZAP YF-C resulted in an activation of the IL-2 promoter or the NF-AT element in TAg-Jurkat cells. Furthermore, it led to hyperactivation of JCaM1.6 cells when these were reconstituted with Lck. Biochemical analyses revealed that ZAP YF-C induces phosphorylation of the TCR  chain and other cellular substrates. The YF-C mutation of ZAP-70 also appears to induce cellular responses in a cell specific fashion. Consistently, expression of a Syk YF-C chimera in JCaM1.6 cells activated the IL-2 promoter and strongly induced intracellular calcium mobilization in response to antibody-mediated clustering.

ZAP YF-C expression correlates well with phosphorylation of cellular substrates, but in vitro data show that a direct modulation of the kinase activity is rather unlikely. The other described gain-of-function mutant of ZAP-70, Y292F, has also not been found to be associated with enhanced kinase activity in vitro (31). Since Syk has been reported to be phosphorylated at the YF-C site in vitro, the most likely explanations for the observed phenotypes are (a) enhanced recruitment of a second kinase or (b) the sequestration of an inhibitory protein (e.g. a phosphatase).

The first possibility seems less likely, since enhanced recruitment of Lck or activation of Lck was not found (data not shown), and the in vitro kinase activity of immunoprecipitated ZAP YF-C protein complexes, which might contain associated kinases, was not significantly enhanced. However, this does not rule out an as yet unknown kinase.

It was recently suggested that the activating potential of the Y292F mutation is based on the loss of interaction of phosphorylated Y292 with c-Cbl (47). If an analogous inhibitory mechanism is the basis for the YF-C phenotype, then it is unlikely to be mediated by c-Cbl as well, because the phenotypes of the mutants are different. In our hands, YF-C has a stronger activating potential as compared with Y292F in Jurkat cells and, more significantly, ZAP YF-C-mediated cellular activation appears to be restricted to the T cell lineage, whereas Y292F is hyperactive in Syk-negative DT40 cells (29, 31). Our data confirmed previous results, which had shown that the Y492F mutation of ZAP-70 activates B cells only. It therefore appears that common as well as cell-specific effector mechanisms of Syk/ZAP-70 type kinases exist, regulating downstream signaling events in hematopoietic cells.

We have found that cellular substrates are phosphorylated upon ZAP YF-C expression. Among these was the 23-kDa isoform of the TCR  chain. Direct phosphorylation by ZAP YF-C can most likely be ruled out because  is a poor substrate for ZAP-70 (48). Consistent with this finding,  phosphorylation by ZAP YF-C was not found to be enhanced in vitro (data not shown). However, the observed phosphorylation of p23  is compatible with either the activation of a second kinase or the sequestration of a phosphatase.

In summary, we have identified tyrosines in the C-terminal regions of ZAP-70 and Syk that regulate their own functional activity in T cells.