From the Laboratorium für Molekulare Biologie,
Genzentrum der Universität München, Feodor Lynen Strasse
25, D-81377 München, Germany and the § NIEHS, National
Institutes of Health, Research Triangle Park,
North Carolina 27709
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ABSTRACT |
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The Syk family tyrosine kinases play a crucial role in antigen receptor-mediated signal transduction, but their regulation and cellular targets remain incompletely defined. Following receptor engagement, phosphorylation of tyrosine residues within ZAP-70 and Syk is thought to control both kinase activity and recruitment of modulatory factors. We report here the characterization of novel mutants of ZAP-70 and Syk, in which conserved C-terminal tyrosine residues have been replaced by phenylalanines (ZAP YF-C, Syk YF-C). Both mutant kinases display a prominent gain-of-function phenotype in Jurkat T cells, as demonstrated by lymphokine promoter activation, tyrosine phosphorylation of potential targets in vivo, and elevated intracellular calcium mobilization. While the presence of p56-Lck was required for ZAP YF-C-induced signaling, Syk YF-C showed enhanced functional activity in Lck-deficient JCaM1 Jurkat cells. Our results implicate the C terminus of Syk family kinases as an important regulatory region modulating T cell activation.
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INTRODUCTION |
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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.
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EXPERIMENTAL PROCEDURES |
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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.
Antibodies used were as follows: 2F3.2 mAb directed against ZAP-70 (UBI), 4G10 mAb recognizing phosphotyrosine (UBI), OKT3 anti-CD3-epsilon mAb (purified from hybridoma supernatant on Protein A), antigen affinity-purified goat anti-human IgG Fc-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.
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RESULTS |
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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).
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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).
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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).
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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.
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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).
Co-transfection of either wild type ZAP-70 or ZAP YF-C resulted in reconstitution of the B cell antigen receptor (BCR) signaling pathway (Fig. 5B), but we did not find an enhanced response of ZAP YF-C, as compared with wild type ZAP-70. However, the cells were sensitive to hyperactivation in principle, because the ZAP Y292F and ZAP Y492F mutants augmented the anti-BCR response dramatically, as was described previously (29, 31). We conclude that ZAP YF-C exerts its function in a cell-specific manner.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.
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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.
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DISCUSSION |
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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.
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ACKNOWLEDGEMENTS |
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We thank E.-L. Winnacker for support and encouragement, Brian Seed for the surface immunoglobulin vector, Tomohiro Kurosaki and Michael Reth for the Syk-negative DT40 cells, Edgar Serfling for the NF-AT reporter construct, and members of the laboratory for discussion and advice.
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FOOTNOTES |
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* This work was supported by grants from the Deutsche Forschungsgemeinschaft (SFB 190) and the Bundesministerium für Bildung und Forschung.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed. E-mail: kolanus{at}lmb.uni-muenchen.de.
1 The abbreviations used are: TCR, T cell receptor; BCR, B cell receptor; mAb, monoclonal antibody; IL, interleukin; PMA, phorbol 12-myristate 13-acetate; sIg, surface immunoglobulin; cIg, cytoplasmic immunoglobulin; ITAM, immunoreceptor tyrosine activation motif.
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REFERENCES |
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