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Mol Cell Biol, March 1998, p. 1388-1399, Vol. 18, No. 3
Department of Immunology, Mayo Clinic,
Guggenheim (3), Rochester, Minnesota 559051;
Institut de Cardiologie de Montreal, Montreal, Quebec, Canada
H1T 1C82; and
Cell Biology and
Metabolism Branch, National Institutes of Health, Bethesda, Maryland
20892-54303
Received 16 July 1997/Returned for modification 26 August
1997/Accepted 8 December 1997
T-cell antigen receptor (TCR) engagement activates multiple protein
tyrosine kinases (PTKs), including the Src family member, Lck, and the
Syk-related PTK, ZAP-70. Studies in ZAP-70-deficient humans have
demonstrated that ZAP-70 plays crucial roles in T-cell activation and
development. However, progress toward a detailed understanding of the
regulation and function of ZAP-70 during TCR signaling has been
hampered by the lack of a suitable T-cell model for biochemical and
genetic analyses. In this report, we describe the isolation and
phenotypic characterization of a Syk- and ZAP-70-negative somatic
mutant derived from the Jurkat T-cell line. The P116 cell line displays
severe defects in TCR-induced signaling functions, including protein
tyrosine phosphorylation, intracellular Ca2+ mobilization,
and interleukin-2 promoter-driven transcription. These signaling
defects were fully reversed by reintroduction of catalytically active
versions of either Syk or ZAP-70 into the P116 cells. However, in
contrast to ZAP-70 expression, Syk expression triggered a significant
degree of cellular activation in the absence of TCR ligation.
Transfection experiments with ZAP-70-Syk chimeric proteins indicated
that both the amino-terminal regulatory regions and the
carboxy-terminal catalytic domains of Syk and ZAP-70 contribute to the
distinctive functional properties of these PTKs. These studies
underscore the crucial role of ZAP-70 in TCR signaling and offer a
powerful genetic model for further analyses of ZAP-70 regulation and
function in T cells.
Ligation of the T-cell antigen
receptor (TCR) triggers a cascade of intracellular signals that
culminate in cytokine gene expression, proliferation, and the execution
of T-cell effector functions. Signal transmission from the TCR is
mediated by the sequential activation of two families of protein
tyrosine kinases (PTKs) (6, 30). Members of the Src family,
Lck and FynT, initiate this process by phosphorylating tyrosine
residues in the cytoplasmic domains of the CD3 and Genetic studies involving T-cell somatic mutants and gene-targeted mice
have dramatically underscored the critical roles of Lck and FynT in TCR
signaling. Targeted disruption of the Lck and FynT genes in mice causes
defects in T-cell development and TCR responsiveness to ligand
stimulation (1, 25, 39, 49, 54). Lck-negative somatic
mutants derived from the human leukemic T-cell line, Jurkat, or the
murine cytotoxic T-cell line, CTLL-2, also display severe defects in
TCR signaling (31, 50).
The importance of ZAP-70 in T-cell activation and development is
highlighted by the identification of a familial form of severe combined
immunodeficiency caused by loss of function mutations in both ZAP-70
alleles (2, 12, 19, 23). These patients lack mature
CD8+ T cells, and the low numbers of CD4+ T
cells that do exit the thymus are nonresponsive to stimulation by TCR
ligands. Mice rendered nullizygous at the ZAP-70 gene locus display an
even more severe phenotype characterized by the complete absence of
CD4+ and CD8+ T cells (40). Although
these studies indicate that ZAP-70 is critical for signaling through
the TCR, the actual contributions of this PTK to the tyrosine
phosphorylation of downstream substrates are poorly understood. Several
proteins, including SLP-76, The role of Syk in signal transduction through the TCR is less well
defined. Although Syk is expressed in developing Studies aimed toward understanding the regulation and function of
ZAP-70 and Syk in T cells would be greatly facilitated by the
availability of a genetically tractable T-cell model that is deficient
in expression of both PTKs. In this study, we provide the initial
description of a ZAP-70- and Syk-deficient somatic mutant derived from
the Jurkat E6 T-cell line. The P116 subclone displays severe defects in
TCR signaling functions that are corrected by reintroduction of
wild-type but not catalytically inactive ZAP-70. The TCR signaling
defects observed in P116 cells were also reversed by expression of Syk;
however, the activation requirements for Syk and ZAP-70 were notably
different in P116 cells. Transfection studies with ZAP-70-Syk chimeric
proteins revealed that both the amino-terminal regulatory and the
carboxy-terminal catalytic domains of these PTKs contributed to their
distinctive functional activities in P116 cells. The P116 cell line
therefore represents a useful model in which the roles of Syk family
PTKs in lymphocyte signaling can be dissected.
Reagents and cell lines.
The human CD3-
0270-7306/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Genetic Evidence for Differential Coupling of Syk
Family Kinases to the T-Cell Receptor: Reconstitution Studies in a
ZAP-70-Deficient Jurkat T-Cell Line
ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
subunits
(30, 53). These tyrosines are embedded within conserved
signaling modules termed immunoreceptor tyrosine-based activation
motifs (ITAMs) (28, 38, 44). The phosphorylated ITAMs serve
as docking sites for the recruitment of a Syk family PTK, most commonly
ZAP-70, to the activated receptor complex (10, 26, 53, 58).
The clustered Src and Syk family PTKs subsequently phosphorylate a series of cytoplasmic substrates, including PLC-
1, Vav, Slp-76, and
p36. However, the specific contribution of each receptor-associated PTK
to the phosphorylation of downstream substrates remains largely undefined.
-tubulin, and the human erythrocyte band
3 protein, have been identified as in vitro substrates for ZAP-70
(29, 59), and it is generally believed that ZAP-70 directly
phosphorylates specific target proteins in activated T cells
(8). In addition, ZAP-70 itself undergoes phosphorylation on
multiple tyrosine residues in response to TCR stimulation. These
phosphotyrosines may create binding sites for SH2 domain-containing
proteins, perhaps facilitating their phosphorylation by the colocalized
Src family PTKs, Lck and FynT (41).
T cells,
thymocyte maturation is not grossly impaired in Syk
/
mice, suggesting that the loss of this PTK is effectively compensated for by the coexpressed ZAP-70 (13, 52). The functional
overlap between Syk and ZAP-70 is further illustrated by the ability of either PTK to restore B-cell antigen receptor signaling in a
Syk/ B-cell line (36). However, accumulating
data suggest that Syk and ZAP-70 exhibit significant differences in
their mechanisms of activation by receptor-mediated stimuli
(34). Whereas binding of Syk to a phosphorylated ITAM is
sufficient to activate the Syk catalytic domain (15, 45,
48), ZAP-70 requires an additional stimulatory input from Lck,
which phosphorylates ZAP-70 at its regulatory Y493 residue
(9, 29, 35, 41, 57). These observations were recently
extended with the finding that Syk, but not ZAP-70, is capable of
reversing the TCR signaling deficits observed in both Lck-negative and
CD45-negative Jurkat T cells (14).
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
-specific
monoclonal antibody OKT3 (55) and the monoclonal antibody
(MAb) 9E10 (21), which recognizes a peptide from the Myc
polypeptide, were purified from murine ascites by chromatography over a
protein G-agarose affinity column. Monoclonal antiphosphotyrosine
antibody 4G10 was obtained from Upstate Biotechnology, Inc. (Lake
Placid, N.Y.). The rabbit polyclonal anti-PLC-1 and anti-
antisera have been described previously (42, 51). The sheep
polyclonal anti-SLP-76 antiserum was provided by Gary Koretzky (University of Iowa). The ZAP-70-specific antiserum was prepared by
immunization of rabbits with a keyhole limpet hemocyanin-coupled peptide corresponding to residues 326 to 341 of human ZAP-70.
-mercaptoethanol). Cells were
maintained at culture densities below 5 × 105 cells
per ml. J.CaM1 cells and K562 erythroleukemia cells were obtained from
American Type Culture Collection (Rockville, Md.). The B-cell lymphoma
Tf.wild (5) was kindly provided by David McKean.
Plasmids.
The wild-type ZAP-70 expression vector was
prepared by amplification of the full-length open reading frame of
ZAP-70 by reverse transcriptase PCR, with Jurkat cell-derived mRNA as
the template. The cDNA was sequenced to verify the fidelity of the PCR
and was cloned into the EcoRI-NotI restriction
sites in the mammalian expression vector, pcDNA3 (Invitrogen), to yield
the pcDNA3-ZAP plasmid. The 5' terminus of the ZAP-70 open reading
frame was modified to encode the Myc epitope tag recognized by MAb
9E10. The resulting expression vector was designated pcDNA3-mZAP. The cDNA encoding a catalytically inactive ZAP mutant (Lys369
Arg) was similarly tagged with the Myc epitope coding sequence and
was cloned into the pSX plasmid (57). The cDNA insert was excised from this plasmid with BamHI and subcloned into the
BamHI site of pBJneo to generate the pBJ-mZAPKD vector used
in the present study.
Derivation of ZAP-70-negative Jurkat somatic mutant. Jurkat cells were suspended at 5 × 105 cells per ml in standard growth medium containing 5 µg of ICR-191 (Sigma) per ml. After 6 h at 37°C, the mutagenized cells were washed three times with phosphate-buffered saline and resuspended at a cell density of 2 × 105 cells per ml in standard growth medium. The cells were cultured for 2 weeks to allow recovery from the mutagenesis procedure, and then selection for Ca2+ signaling mutants was initiated. The cells were loaded with the Ca2+ indicator dye, indo-1 (see below), and placed on-line in a FACStar Plus cell sorter (Becton-Dickinson). Intracellular Ca2+ mobilization was stimulated with 100 µM pervanadate. After approximately 90 s at 37°C, cells that failed to respond to the stimulus were collected by cell sorting. Preliminary experiments demonstrated that virtually 100% of unmutagenized Jurkat cells displayed a clear increase in their 405- to 495-nm fluorescence emission ratios (a direct reflection of the ratio of Ca2+-bound to -unbound indo-1) after exposure to pervanadate for 90 s. The sorted cells were diluted into standard growth medium, and cell numbers were expanded in culture, at which time the selection procedure was repeated. After four cycles, the sorted cell population contained greater than 50% pervanadate-nonresponsive cells. The cells were stained with fluorescein isothiocyanate-conjugated anti-CD3 (OKT3) MAb (Becton Dickinson), and TCR-positive cells were plated into 96-well plates at 1 cell per well with an automated cell depositing unit on a FACS Vantage cell sorter.
Intracellular free Ca2+ measurements. Jurkat cells were harvested from growth medium, and 15 × 106 cells were resuspended in 1.5 ml of solution no. 1 (Hanks balanced salt solution supplemented with 5 mM dextrose and buffered to pH 7.0 with 10 mM HEPES). Indo-1-acetoxymethylester (Calbiochem) was dissolved in dimethylsulfoxide and added to the cell suspension to a final concentration of 5 µM. After 30 min at 37°C, an equal volume of solution no. 2 (Hanks balanced salt solution containing 5 mM dextrose and buffered at pH 7.4 with 10 mM HEPES) was added and cells were incubated for an additional 30 min. The indo-1 loaded cells were washed with 10 volumes of solution no. 3 (solution no. 2 supplemented with 0.05% bovine serum albumin [BSA]) and resuspended in solution no. 3 at a density of 1 × 106 cells/ml. Samples were incubated for 5 min at 37°C, and pervanadate was added to a final concentration of 100 µM. The pervanadate stock solution was prepared by mixing 2.3 µl of 30% H2O2 (Sigma) with 1 ml of 20 mM Na3VO4 and allowing the mixture to react for 5 min at room temperature. Stimulus-induced changes in intracellular Ca2+ concentration ([Ca2+]i) in the cell population were determined by monitoring the fluorescence emission ratio at 405 to 495 nm as described above.
Northern analysis.
Total cellular RNA was isolated with
RNAzol B (Cinna/Biotecx Laboratories, Inc.). The RNA (30 µg) was
electrophoresed through a 1% agarose gel containing 6% formaldehyde
and transferred to a nitrocellulose membrane. The membrane was
sequentially probed with a XhoI-ScaI fragment
from the ZAP-70 cDNA and a PstI fragment from pIBI30-GADPH
(provided by Selina Shen-Kiang, Mt. Sinai School of Medicine, New York,
N.Y.) which hybridizes to the glyceraldehyde-3-phosphate dehydrogenase
(GAPDH) mRNA. The DNA probes were radiolabeled with [-32P]CTP with random oligonucleotide primers
(Oligolabelling kit, Pharmacia).
Immunoblot analysis. For ZAP-70 or Myc epitope tag immunoblots, 106 Jurkat cells were lysed in 30 µl of buffer A (20 mM Tris, 40 mM NaCl, 50 mM NaF, 30 mM sodium pyrophosphate, 5 mM EDTA [pH 7.4] supplemented with 1% Triton X-100, 10 µg of leupeptin per ml, 5 µg of pepstatin per ml, and 5 µg of aprotinin per ml). Postnuclear supernatants were mixed with 10 µl of 4× sodium dodecyl sulfate (SDS) sample buffer (32) and boiled for 5 min. Proteins were separated by SDS-polyacrylamide gel electrophoresis (PAGE) through a 10% gel and electrophoretically transferred to an Immobilon-P membrane (Millipore). The membrane was blocked overnight in Tris-buffered saline containing 0.2% Tween 20 (TBST) supplemented with 2% BSA. Membranes were probed for 1 h with ZAP-70 antiserum (1:500 in TBST) or 9E10 MAb (1 µg/ml in TBST containing 0.1% BSA). The 9E10 MAb immunoblots were incubated for 1 h with rabbit anti-mouse immunoglobulin G (IgG) (RaMIg) (Pierce). Immunoreactive proteins were detected with horseradish peroxidase-coupled protein A followed by the enhanced chemiluminescence reagent (Amersham).
For antiphosphotyrosine immunoblots, cells were harvested from standard growth medium and resuspended in ice-cold solution no. 2 at a density of 5 × 106 cells per ml. The solution contained either no antibody or 1 µg of OKT3 antibodies per ml. After 20 min on ice, the cells were pelleted and resuspended in either 50 µl of prewarmed solution no. 2 containing 10 µg of RaMIg per ml or 50 µl of 100 µM pervanadate. The cells were stimulated for 2 min at 37°C and then lysed with an equal volume of 2× lysis buffer (20 mM Tris [pH 7.4], 50 mM-glycerophosphate, 60 mM NaF, 40 mM sodium
pyrophosphate, 2 mM EDTA, 2 mM sodium orthovanadate, 2% Triton X-100,
and protease inhibitors). The cleared extracts were mixed with 20 µl
of 4× SDS sample buffer and heated at 100°C. After separation by
SDS-PAGE, the proteins were transferred to an Immobilon-P membrane. The membranes were blocked overnight in TBST containing 2% BSA and then
were probed for 1 h with 100 ng of antiphosphotyrosine antibody per ml. Membranes were washed in TBST and incubated for 1 h with 1 µg of RaMIg per ml. Washed membranes were then incubated with horseradish peroxidase-conjugated protein A, and proteins were detected
by enhanced chemiluminescence.
Tyrosine phosphorylation of PLC-1, SLP-76, and was examined with
a sequential immunoprecipitation-immunoblotting procedure. Cells were
harvested from growth medium, washed in phosphate-buffered saline, and
suspended in solution no. 2 at a density of 2.5 × 107
cells per ml. Aliquots (200 µl) were stimulated for various times with 100 µM pervanadate or 1 µg of OKT3 per ml at 37°C. Reactions were terminated with 800 µl of buffer A. After 5 min on ice,
insoluble material was cleared by centrifugation and the extracts were
mixed with 15 µl of packed protein A-Sepharose and 4 µl of
anti-PLC-1 or 10 µl of anti- antiserum for 1 h at 4°C.
SLP-76 immunoprecipitations were done with 2 µl of sheep polyclonal
anti-SLP-76 antiserum and 15 µl of protein G-Sepharose. The
immunoprecipitates were washed three times in lysis buffer A and boiled
in 30 µl of 2× SDS sample buffer. The solubilized proteins were
separated by SDS-PAGE, transferred to Immobilon-P, and probed with
antiphosphotyrosine antibodies. The blots were subsequently stripped
and reprobed with the corresponding PLC-1-, -, or SLP-76-specific
antibodies.
Transfections. Jurkat subclones were harvested from growth medium and resuspended at 4 × 107 cells per ml in fresh growth medium. For transient transfections, pcDNA3-based ZAP-70 or Syk expression plasmids were cotransfected along with the appropriate reporter plasmid. Aliquots of the cell suspension (107 cells) were placed in 0.4-cm-path-length cuvettes and, unless indicated otherwise, were mixed with 30 µg of total DNA in a final volume of 300 µl. The cells were electroporated with a BTX model T 820 square-wave electroporator (San Diego, Calif.) set at 300 V (pulse duration, 10 ms). For stable expression, P116 cells were electroporated with pcDNA3-ZAP or pBJ-mZAPKD cDNAs, and after 24 h, the cells were selected in standard growth medium supplemented with 2 mg of G418 per ml. The drug-resistant bulk population was cloned by limiting dilution, and individual subclones were screened for expression of wild-type or kinase-inactive ZAP-70 protein by immunoblot analysis of whole-cell lysates.
ZAP-70 kinase assays.
K562 cells (107/sample)
were electroporated with 40 µg of the indicated expression plasmids
as described above. The transfected cells were diluted to a density of
5 × 105 per ml in culture medium. After 24 h,
the cells were washed with phosphate-buffered saline and lysed in
ZAP-lysis buffer (25 mM Tris-HCl [pH 7.4], 150 mM NaCl, 1 mM sodium
orthovanadate, 5 mM EDTA, 1% Brij-96, and protease inhibitors). The
cleared extracts were mixed with 10 µg of 9E10 MAb and 15 µl of
protein A-Sepharose precoated with 5 µg of RaMIg. After 1 h at
4°C, the immunoprecipitates were washed once in ZAP-lysis buffer,
once in high-salt buffer (100 mM Tris-HCl [pH 7.4], 0.5 M LiCl, and
0.1% Brij-96), and twice in Z-kinase buffer (20 mM Tris [pH 7.4], 10 mM MgCl2, 10 mM MnCl2). Immune complex kinase
reactions were initiated with 25 µl of Z-kinase buffer containing 10 µM ATP, 20 µCi of [-32P]ATP, and 2 µg of
His6-cdb3. The samples were incubated for 5 min at 22°C,
and the reactions were terminated with 10 µl of 4× SDS sample
buffer. The samples were heated for 5 min at 100°C, and the
solubilized proteins were separated by SDS-PAGE and transferred to
Immobilon-P. Radiolabeled proteins were detected by autoradiography, and incorporation of 32P into His6-cdb3 was
quantitated with an AMBIS phosphorimager system.
Cellular infections with recombinant vaccinia viruses. Recombinant vaccinia viruses encoding ZAP-70 and Syk were kind gifts from A. M. Scharenberg and J. P. Kinet (Harvard Medical School, Boston, Mass.). Infections with wild-type and recombinant vaccinia viruses were performed as previously described (3). Jurkat subclones were infected with vaccinia viruses at a multiplicity of infection of 20:1 and were used in experiments at 3 h postinfection.
Luciferase assays. Cells were transfected with 10 µg of a pT81Luc-based reporter plasmid containing 2 kb of nucleotide sequence from the human interleukin-2 (IL-2) promoter (pIL2-Luc) or three tandem copies of the nuclear factor of activated T cells (NFAT) DNA binding site (pNFAT-Luc) (kind gifts of David McKean, Mayo Clinic). At 6 to 18 h posttransfection, the cells were stimulated for an additional 6 to 12 h with 20 ng of tetradecanoyl-o-phorbol-13-acetate (TPA) per ml plus 2 µM ionomycin (with 20 ng of TPA per ml and 1 µg of OKT3 antibody per ml) or with 1 µg of OKT3 antibody per ml alone. Cells were lysed, and luciferase activity was determined with a commercial kit (Promega) and a Berthold Lumat luminometer.
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RESULTS |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Isolation of a ZAP-70-negative Jurkat subclone. In an effort to generate new genetic model systems for studies of TCR signaling, we implemented a screening strategy designed to isolate Jurkat T-cell-derived somatic mutants bearing defects in downstream components of the pathway leading to the mobilization of intracellular Ca2+. Wild-type Jurkat E6 cells were mutagenized with the frameshift mutagen, ICR-191, and the resulting bulk population was repetitively selected for cells that failed to increase [Ca2+]i in response to pervanadate. The protocol was similar to that employed by Goldsmith and Weiss (24) except that the stimulus employed in the present study was pervanadate, a powerful inducer of PTK-dependent activation responses in T cells. The choice of pervanadate as the stimulus stemmed from the earlier observation that this agent triggered both protein tyrosine phosphorylation and [Ca2+]i increases in TCR-negative or CD45-negative Jurkat cells (46). Hence, the pervanadate-based selection procedure should facilitate the isolation of mutant cells bearing loss of function mutations in more distal elements of the Ca2+ response pathway in these cells.
The selected Jurkat subclones were rescreened for their abilities to mount a [Ca2+]i increase in response to anti-CD3 antibody (OKT3) or pervanadate stimulation. One of these clones, designated P116, was nonresponsive to antibody-mediated TCR cross-linkage and exhibited a delayed response to pervanadate in comparison to wild-type Jurkat cells (Fig. 1). FACS analysis of OKT3 antibody-stained P116 cells indicated that TCR expression on these cells was identical to that found on wild-type Jurkat cells (data not shown). In contrast to the P116 subclone, the Lck-negative Jurkat subclone, J.CaM1, failed to mobilize Ca2+ in response to either anti-CD3 antibodies or pervanadate. This phenotypic difference suggested that the signaling defects displayed by P116 cells were not explained by the loss of Lck. Indeed, immunoblot analysis of whole-cell lysates indicated that P116 cells contained wild-type levels of Lck as well as FynT and PLC-1 (data not shown).
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Arg) were derived in a similar
fashion. Reintroduction of ZAP-70 into P116 cells restored the
Ca2+ mobilization response to anti-CD3 antibody-mediated
TCR cross-linkage and eliminated the time lag for the
pervanadate-induced [Ca2+]i increase (Fig.
1). Neither response was reconstituted in the DK2 and DK33
transfectants, indicating that the PTK activity of ZAP-70 was essential
for coupling the TCR to the Ca2+-mobilizing machinery and
for the rapid [Ca2+]i increase triggered by
pervanadate.
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ZAP-70 reconstitutes TCR-dependent phosphorylation events in P116 cells. To determine the role of ZAP-70 in TCR-dependent protein tyrosine phosphorylation, ZAP-70-positive and -negative Jurkat subclones were stimulated with anti-CD3 antibodies or pervanadate and detergent-soluble proteins were immunoblotted with antiphosphotyrosine antibodies. TCR stimulation triggered rapid increases in the tyrosine phosphorylation of several proteins in wild-type Jurkat cells (Fig. 3). As reported previously (46), pervanadate stimulation provoked a more dramatic increase in overall protein tyrosine phosphorylation in these cells. In contrast, the ZAP-70-negative P116 cells showed no consistent increase in protein tyrosine phosphorylation in response to anti-CD3 antibodies. Interestingly, the P116 cells displayed a robust phosphorylation response to the pharmacologic stimulus, pervanadate. The latter characteristic clearly distinguished the phenotype of P116 cells from that of Lck-negative J.CaM1 cells, as the latter cells were refractory to either anti-CD3 antibody or pervanadate stimulation. The basal level of protein tyrosine phosphorylation in unstimulated J.CaM1 cells was also strikingly lower than that observed in either wild-type Jurkat or P116 cells. The protein tyrosine phosphorylation defects in P116 cells were reversed in the ZAP-70-transfected P116.c39 and P116.c40 cells but not in the DK.33 and DK.2 subclones (Fig. 3 and data not shown), which express a catalytically inactive form of ZAP-70. Thus, correction of the defective signaling phenotype exhibited by the P116 cells is dependent on the PTK activity of ZAP-70.
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1
(43, 47, 61). To determine the role of ZAP-70 in the
stimulus-dependent tyrosine phosphorylation of PLC-1, we stimulated
wild-type Jurkat, J.CaM1, or P116 cells with anti-CD3 antibodies or
pervanadate and immunoprecipitated PLC-1 from cell extracts prior to
antiphosphotyrosine immunoblotting (Fig.
4A). Both stimuli provoked clear
increases in the tyrosine phosphorylation of PLC-1 in wild-type
Jurkat cells. In contrast, J.CaM1 and P116 cells showed no increase in
PLC-1 tyrosine phosphorylation after a 2-min stimulation with either
anti-CD3 antibodies or pervanadate, indicating that both Lck and ZAP-70
are necessary for rapid modification of PLC-1 induced by these
agents. As expected, the PLC-1 phosphorylation defect was fully
reversed in the ZAP-70-transfected P116.c39 and P116.c40 cells,
confirming that the signaling abnormalities in P116 cells are explained
by the loss of ZAP-70.
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1
phosphorylation in P116 cells was inconsistent with the initial
observation (Fig. 1) that pervanadate exposure generated a delayed but
significant increase in [Ca2+]i in these
cells. This apparent discrepancy was resolved by a comparison of the
time courses of pervanadate-induced PLC-1 phosphorylation in
wild-type Jurkat and P116 cells (Fig. 4B). In wild-type Jurkat cells,
pervanadate stimulated a dramatic increase in the tyrosine phosphorylation of PLC-1 within 2 min, and a maximal phosphorylation response was observed after 10 min of stimulation. The time course of
PLC-1 tyrosine phosphorylation was clearly delayed in P116 cells.
The slower rate of PLC-1 phosphorylation in these cells correlated
with the abnormally slow rise in [Ca2+]i
induced by pervanadate. In contrast, J.CaM1 cells displayed only a
slight increase in PLC-1 phosphorylation after 30 min of exposure to
pervanadate, again paralleling the nonresponsiveness of these cells to
pervanadate in the Ca2+ mobilization assay. Taken together,
these results indicate that ZAP-70 is an essential component of the
pathway that links TCR ligation to PLC-1 tyrosine phosphorylation
and that the requirement for ZAP-70 can be at least partially
circumvented by the pharmacologic stimulus, pervanadate.
Effect of ZAP-70 deficiency on TCR-dependent transcriptional activation. The proximal signaling events initiated by TCR engagement, in conjunction with costimulatory signals supplied by CD28 ligation or phorbol esters, ultimately regulate the transcription of a complex set of target genes, including those encoding IL-2 and other cytokines (16, 17). To define the role of ZAP-70 in TCR-dependent IL-2 gene transcription, wild-type and mutant Jurkat cell lines were transiently transfected with the pIL2-Luc reporter plasmid, which contains the 5' enhancer region of the IL-2 gene linked to a luciferase reporter. The transfected cells were stimulated with anti-CD3 antibodies (OKT3) plus TPA, and luciferase activities were normalized to the activities measured in the same population of cells after exposure to ionomycin plus TPA (Fig. 5). Compared to wild-type Jurkat cells, ZAP-70-negative P116 cells exhibited a dramatic defect in TCR-dependent IL-2 promoter activation. As expected, the Lck-negative J.CaM1 cells were virtually nonresponsive to the stimulus provided by anti-CD3 antibodies in these transcriptional activation assays. The defective transcriptional response of P116 cells was corrected by stable expression of wild-type ZAP-70 (clones P116.c39 and P116.c40), but not by expression of a catalytically inactive ZAP-70 mutant (clones DK2 and DK33). Thus, the deficient expression of ZAP-70 in P116 cells effectively disrupts the coupling mechanism between TCR stimulation and IL-2 gene transcription in these cells.
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Reconstitution of early signaling events by Syk. In preliminary studies, we confirmed a previous report (22) that Jurkat E6 cells fail to express Syk and that the Jurkat E6-derived subclone, P116, is similarly negative for expression of this PTK (data not shown). The P116 cell line therefore offers a suitable host for comparative studies of the signaling functions of ZAP-70 and Syk in T cells. Unfortunately, repeated efforts to isolate stable Syk-transfected P116 subclones proved unsuccessful, suggesting that long-term expression of Syk might not be tolerated by these cells (see Discussion). To circumvent this problem, we adopted a vaccinia virus-based expression approach, which permitted an evaluation of the impact of Syk on early TCR signaling events in P116 cells (Fig. 6). Wild-type Jurkat, P116, and J.CaM1 cells were infected with recombinant vaccinia viruses encoding either ZAP-70 or Syk, and the cells were stimulated with anti-CD3 antibodies at 3 h postinfection. Introduction of either Syk or ZAP-70 increased both basal and anti-CD3 antibody-stimulated protein tyrosine phosphorylation in P116 cells. The increases in baseline tyrosine phosphorylation induced by Syk and ZAP-70 may be the result of the high level of overexpression achieved during vaccinia virus infection. In contrast, Syk, but not ZAP-70, augmented both basal and TCR-stimulated protein tyrosine phosphorylation in J.CaM1 cells. The latter finding is consistent with the recent report that only Syk is capable of reversing the TCR signaling defects in both J.CaM1 cells and CD45-deficient J45.01 cells (14).
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subunits of
the receptor itself. Previous studies suggested that the binding of the
ZAP-70 SH2 domains to the phospho-ITAMs of the subunit protected
the phosphotyrosines from dephosphorylation by CD45 and other protein
tyrosine phosphatases (8). This model can be tested directly
in the ZAP-70- and Syk-deficient P116 cell line. Stimulation of
wild-type Jurkat cells with anti-CD3 antibodies led to a dramatic
increase in the tyrosine phosphorylation of the TCR subunit (Fig.
7A). This response was markedly
attenuated in P116 cells. Interestingly, the defect in subunit
phosphorylation was reversed by expression of either wild-type or
catalytically inactive ZAP-70, indicating that the PTK activity of
ZAP-70 is not required for this response. On the other hand, expression of catalytically active Syk failed to enhance the tyrosine
phosphorylation of subunits in anti-CD3 antibody-stimulated P116
cells. A time course analysis confirmed that ZAP-70, but not Syk,
supported a rapid and transient increase in subunit phosphorylation
in response to TCR ligation (Fig. 7B). These results indicate that ZAP-70 and Syk differ significantly with respect to their abilities to
induce and/or maintain the phosphorylation of the ITAMs during TCR
stimulation.
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Syk reconstitution of TCR-dependent gene transcription. To further characterize the ability of Syk to reconstitute TCR signaling functions in P116 cells, transient transfection experiments were performed with pNFAT-Luc as a readout for transcriptional activation. In contrast to pIL2-Luc, the pNFAT-Luc reporter plasmid is transcriptionally activated by TCR cross-linkage alone, eliminating the potentially confounding effects of TPA from these assays (27, 63). P116 cells were cotransfected with the pNFAT-Luc reporter construct together with various amounts of plasmid DNA encoding epitope-tagged versions of ZAP-70 or Syk. In the absence of either PTK, anti-CD3 antibody stimulation failed to activate NFAT-dependent transcription in P116 cells, and this defect was effectively reversed by cotransfection of the cells with the ZAP-70 expression plasmid (Fig. 8A). Transient expression of Syk in P116 cells also reconstituted the transcriptional response to antibody-mediated TCR cross-linkage. However, unlike ZAP-70, Syk provoked a significant elevation in luciferase activity in unstimulated P116 cells, indicating that Syk-mediated NFAT activation was partially independent of TCR ligation. The difference in baseline luciferase activities observed in Syk- and ZAP-70-transfected P116 cells was not explained by the relative overexpression of Syk protein, as the two PTKs were expressed at comparable levels in these cells (Fig. 8A). Transfection of P116 cells with lower amounts (0.1 to 1 µg of plasmid DNA) of the Syk or ZAP-70 expression vectors resulted in a progressive reduction in anti-CD3 antibody-stimulated luciferase activity, indicating that lower amounts of either PTK could limit TCR signaling in P116 cells (62). Nonetheless, Syk expression, even at levels below those detectable with the tag-specific antibody, consistently elevated the basal level of reporter gene transcription in these cells.
|
Reconstitution of TCR signaling functions by ZAP-70-Syk chimeric proteins. The results described above suggested that the signaling functions of Syk and ZAP-70 may be differentially regulated in P116 cells. Several potential regulatory domains have been identified in Syk and ZAP-70, including two tandem SH2 domains, a linker region containing putative tyrosine phosphorylation sites, and a carboxy-terminal catalytic domain. To further understand the structural basis for the functional differences between Syk and ZAP-70, we transiently transfected P116 cells with expression vectors encoding Myc epitope-tagged ZAP-70-Syk chimeric proteins (Fig. 9A). These chimeric constructs contained the tandem SH2 domains and linker region of one Syk family member fused to the catalytic region and carboxy terminus of the other family member.
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DISCUSSION |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Using a Ca2+ mobilization-based selection strategy, we have isolated a ZAP-70-negative somatic mutant from the Jurkat E6 T-cell line. The P116 subclone expresses no detectable ZAP-70 mRNA or protein and, like the parental Jurkat E6 clone from which it was derived (22), also fails to express Syk. As predicted from studies of T cells from ZAP-70-deficient humans, P116 cells display global defects in TCR signaling functions, including the induction of protein tyrosine phosphorylation, Ca2+ release from intracellular stores, and IL-2 promoter-driven transcription. These signaling defects were corrected by reintroduction of either ZAP-70 or Syk into the mutant cell line. However, our results indicate that these two closely related PTKs differ significantly in terms of their mechanisms of activation in T cells.
The severe defects in TCR signaling in ZAP-70-negative P116 cells are
reminiscent of those found in the Lck-deficient J.CaM1 cell line
(50). However, comparative studies with pervanadate as the
stimulus suggest that the loss of Lck leads to a more fundamental impairment of the downstream signaling machinery than does the loss of
ZAP-70. Whereas J.CaM1 cells are virtually nonresponsive to
pervanadate, P116 cells mounted a significant but delayed activation response to this agent, which is capable of activating Jurkat cells in
a TCR-independent fashion (46). Notably, exposure of P116
cells to pervanadate led to the tyrosine phosphorylation and catalytic
activation of PLC-1, a pivotal event in the initiation of IL-2 gene
transcription (7, 16). The ability of the ZAP-70-deficient cells to respond to pervanadate indicates that these cells retain the
PTK activity needed to set in motion the TCR-linked signaling cascade;
however, this cascade cannot be engaged effectively by the TCR in the
absence of ZAP-70.
The absence of both ZAP-70 and Syk in P116 cells offered a unique opportunity to compare the regulation and functions of these PTKs in the context of both the native TCR complex and the Src family member, Lck. Introduction of either ZAP-70 or Syk reversed the TCR signaling defects in P116 cells; however, the Syk-expressing cells were partially activated even in the absence of TCR ligands. The relaxed ligand dependence of Syk-mediated signaling suggests that the activation threshold for Syk is significantly lower than that of ZAP-70 in P116 cells. This notion is consistent with previous observations that Syk aggregation or binding of Syk to phospho-ITAMs is sufficient to activate this PTK, whereas ZAP-70 requires an additional stimulatory input supplied by Lck, which phosphorylates ZAP-70 at its activating Y493 residue (4, 9, 41). The unique ability of Syk to restore TCR signaling functions in Lck-deficient J.CaM1 cells is fully consistent with the conclusion that Syk can be activated in an Lck-independent fashion (14) (Fig. 9C).
The most straightforward explanation for the above findings is that the Syk catalytic domain carries an intrinsically higher level of PTK activity than does that of ZAP-70. Indeed, the specific activity of Syk was approximately 100-fold higher than that of ZAP-70 when the two PTKs were expressed in COS-1 cells (37). These investigators used chimeric ZAP-70-Syk proteins to show that the higher catalytic activity was an intrinsic property of the Syk catalytic domain. We have drawn a similar conclusion from immune complex kinase assays done with ZAP-70-Syk chimeric proteins expressed in Jurkat-derived P116 cells (Fig. 10). However, our results further indicate that both the catalytic domains and N-terminal regulatory regions of ZAP-70 and Syk contribute to the distinctive behaviors of these PTKs in P116 cells.
In spite of its higher intrinsic catalytic activity, Syk does not activate Jurkat cells in a completely autonomous fashion. The constitutive signaling activity of Syk in Jurkat cells was dependent on the coexpression of Lck and a cell surface TCR complex. A reasonable scenario is that a baseline level of ITAM phosphorylation, mediated by Lck or FynT, triggers the recruitment of a few Syk molecules to the plasma membrane, where they undergo phosphorylation and activation by Lck. This process sets up a feed-forward amplification mechanism leading to further ITAM phosphorylation and Syk activation (via transphosphorylation by other activated Syk molecules) that eventually surpasses the threshold needed to trigger the signaling cascade normally controlled by TCR ligation. A similar Src family kinase-initiated activation loop has been proposed to explain the robust activation of Syk observed after relatively weak stimulation of Fc receptors on mast cells (20).
The constitutive activation of the TCR-linked signaling cascade by Syk may explain the apparent toxicity of long-term Syk expression in P116 cells. Repeated attempts to derive stable P116 transfectants expressing Syk met with failure, whereas P116 subclones that stably express ZAP-70 were generated readily under identical conditions of transfection and drug selection (2). The cytotoxic effect of Syk may be due to the stimulation of Fas-dependent apoptosis in the transfected cell population. P116 cells exhibit a profound defect in the induction of Fas ligand expression in response to anti-CD3 antibody stimulation (18). This abnormality is corrected by the reexpression of ZAP-70, indicating that this PTK is required for the delivery of TCR-mediated signals leading to the transcription of the Fas ligand gene. Ectopically expressed Syk should also restore TCR-dependent Fas ligand expression in P116 cells; however, the propensity of Syk to become active in the absence of TCR engagement may result in constitutive expression of Fas ligand and, in turn, increased susceptibility to Fas-mediated apoptosis. A survival advantage for Syk-negative Jurkat cells may explain the spontaneous outgrowth of Jurkat P116 cells bearing loss of function mutations in both Syk alleles (22). A physiologic correlate of this phenomenon may occur during normal T-cell development. Syk is expressed in double-positive and single-positive thymocytes; however, expression of this PTK declines precipitously in postthymic T cells (11). The loss of Syk as T cells enter the periphery may reflect the need to raise the threshold for initiation of signal output from the TCR in order to avoid the deleterious consequences (cell death or immune hyperreactivity) of inappropriate receptor activation.
To circumvent the problems associated with long-term Syk expression, we
used the vaccinia virus expression system to generate ZAP-70- or
Syk-expressing P116 cells for analyses of the early biochemical events
induced by TCR stimulation. Expression of either PTK corrected the
global defects in TCR-dependent protein tyrosine phosphorylation in
P116 cells. However, we found a striking difference in the tyrosine
phosphorylation of the TCR- subunit in ZAP-70- versus Syk-expressing
P116 cells. In nontransfected P116 cells, anti-CD3 antibodies
stimulated only a minor increase in tyrosine phosphorylation, and
this defect was fully reversed by expression of either catalytically
active or inactive ZAP-70. These results suggest that binding of the
SH2 domains of ZAP-70 to the phospho-ITAMs protects these motifs
from dephosphorylation by CD45 and other protein tyrosine phosphatases
(8). In contrast to the restorative effect of ZAP-70, Syk
failed to reconstitute anti-CD3 antibody-stimulated tyrosine
phosphorylation in P116 cells. This observation is not restricted to
the Jurkat model, as we have recently obtained identical results with a
Syk-expressing T-cell line derived from a ZAP-70-deficient human
patient (62).
The inability of Syk to support subunit phosphorylation suggests
that the interaction of Syk with the phospho-ITAMs differs fundamentally from that of ZAP-70. One possibility is that, unlike ZAP-70, Syk preferentially interacts with phosphorylated CD3-, -
,
or - ITAMs rather than with those residing in the cytoplasmic domain. Alternatively, Syk may bind to the phospho-ITAMs but fail
to protect them from dephosphorylation due to rapid dissociation of the
activated form of this PTK from the TCR complex. A recent study by
Keshvara et al. (33) identified a tyrosine residue (Tyr130 in murine Syk), located in the inter-SH2 domain
region, whose phosphorylation provokes both activation of the Syk
catalytic domain and dissociation of the activated PTK from the
B-cell antigen receptor complex. Interestingly, the inter-SH2 domain
region of ZAP-70 contains a tyrosine residue (Tyr126)
homologous to the regulatory Tyr130 of Syk.
Although Tyr126 has been identified as an in vitro
phosphorylation site in ZAP-70 (60), phosphorylation of this
residue has not been observed after isolation of ZAP-70 from activated
T cells. Perhaps a relatively low stoichiometry of phosphorylation at
Tyr126 promotes a persistent interaction between ZAP-70 and
the phospho-ITAMs and thereby more effectively protects these
phospho-ITAMs from attack by protein tyrosine phosphatases.
To further understand the structural basis for the functional
differences between Syk and ZAP-70, we expressed ZAP-70-Syk chimeric
proteins in either P116 cells or J.CaM1 cells. Unexpectedly, the
signaling characteristics of these chimeric PTKs were radically different in ZAP-70- versus Lck-deficient Jurkat cells. In P116 cells,
introduction of the Syk-ZAP chimera rendered NFAT activation superinducible by TCR ligation. The maximal level of reporter gene
expression induced by anti-CD3 antibody stimulation of the Syk-ZAP-expressing P116 cells was approximately 100-fold greater than
that conferred by reexpression of full-length ZAP-70 in these cells. On
the other hand, the converse ZAP-Syk chimera constitutively activated
NFAT, and, in stably-transfected P116 cells, rendered the tyrosine
phosphorylation of SLP-76 independent of TCR ligation. The results
obtained with the Syk-ZAP chimera suggest that the SH2 domains of Syk
couple more productively to either the CD3 or phospho-ITAMs than do
the SH2 domains of ZAP-70. In the case of the opposite ZAP-Syk chimera,
the more active Syk kinase domain may drive signaling in the presence
of a low-level ITAM phosphorylation in unstimulated P116 cells.
However, fusion of the ZAP-70 SH2 domains and hinge region to the Syk
catalytic domain apparently compromises the ability of this chimera to
respond further to the stimulus provided by antibody-mediated TCR
cross-linkage.
The strikingly disparate results obtained when the chimeric ZAP-70-Syk constructs were expressed in Lck-deficient J.CaM1 cells underscore the functional importance of the interplay between Syk family PTKs and Lck during T-cell activation. As recently reported with full-length Syk (14), the ZAP-Syk chimera reconstituted the coupling pathway between TCR stimulation and the NFAT activation in J.CaM1 cells. In contrast, transient expression of the converse Syk-ZAP chimera caused only a minor restoration of TCR signaling functions in these cells. Thus, the functional consequences of expressing the two chimeric proteins in J.CaM1 cells were essentially the mirror image of those obtained in P116 cells. The results obtained with J.CaM1 cells suggest that the capacity to function in an Lck-negative intracellular milieu maps to the catalytic domain of Syk. On the other hand, when Lck is not limiting (e.g., in P116 cells) the SH2 domains of Syk appear to mediate an increased level of signaling in response to TCR ligation. Thus, both the amino-terminal regulatory regions and the carboxy-terminal catalytic domains of Syk and ZAP-70 contribute to the distinctive functional characteristics of these PTKs in T cells.
In summary, the ZAP-70-deficient P116 cell line represents a genetically manipulatable model in which the structural and biochemical parameters that govern both the redundant and unique functions of Syk and ZAP-70 in T lymphocytes can be explored. This cell line is a powerful addition to the previously described battery of Jurkat T-cell somatic mutants for studies of the mechanisms of signal initiation and propagation through the TCR. From the therapeutic viewpoint, P116 cells should prove useful in efforts to develop ZAP-70- or Syk-targeted immunosuppressive agents for use in transplantation and in the treatment of autoimmune diseases.
| |
ACKNOWLEDGMENTS |
|---|
We thank David McKean for providing the luciferase reporter gene constructs, Andrew M. Scharenberg and Jean-Pierre Kinet for supplying recombinant vaccinia virus, and Gary Koretzky for the gift of SLP-76-specific antiserum. We also express our appreciation to Amnon Altman and Larry Karnitz for many helpful discussions pertaining to this study.
This work was supported by NIH grants GM47286 (R.T.A.) and CA47752 (P.J.L.) and by the Mayo Foundation. W.Z. is a Leukemia Society of America Fellow, and R.T.A. is a Leukemia Society of America Scholar.
| |
FOOTNOTES |
|---|
* Corresponding author. Mailing address: Department of Immunology, 301 Guggenheim Building, Mayo Clinic, Rochester, MN 55905.
Present address: Gerontology Research Center, National Institute on
Aging, Baltimore, MD 21224.
| |
REFERENCES |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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