From the § Laboratory of Biological Chemistry, Gerontology Research Center, NIA, National Institutes of Health, Baltimore, Maryland, 21224 and § Cell Biology and Metabolism Branch, NICHD, National Institutes of Health, Bethesda, Maryland, 20892
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ABSTRACT |
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Oxidative stress in T cells induces signaling events similar to those initiated by T cell antigen receptor engagement, including tyrosine phosphorylation and activation of the critical protein-tyrosine kinase ZAP-70. Distal signaling events such as the activation of mitogen-activated protein kinases and downstream transcription factors are also initiated by oxidative stimuli. In this study P116, a ZAP-70-negative Jurkat T cell line, was used to investigate the role of ZAP-70 in mediating activation of Erk in response to H2O2. Consistent with the hypothesis that ZAP-70 is required for activation of Erk in response to an oxidative stimulus, Erk1 and Erk2 could be rapidly activated in Jurkat cells but not in P116 cells upon addition of H2O2. P116 cells became competent for H2O2-induced Erk activation upon stable transfection with wild-type ZAP-70. An in vivo ZAP-70 substrate, SLP-76, implicated in Erk activation, also became rapidly tyrosine-phosphorylated in Jurkat cells, but not in P116 cells, upon treatment with H2O2. Surprisingly, although ZAP-70 was required for H2O2-mediated Erk activation, Erk activation in response to T cell antigen receptor engagement did not require ZAP-70. In addition to demonstrating a requirement for ZAP-70 in H2O2-stimulated Erk activation, these results provide the first evidence for the existence of a ZAP-70-independent pathway for Erk activation in T cells.
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INTRODUCTION |
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Naïve, mature T lymphocytes undergo proliferation and acquisition of effector function when they encounter antigen on the surface of specialized antigen-presenting cells that also express required co-stimulatory surface proteins. Our understanding of the molecular signaling pathways that are initiated by these stimulatory events has advanced considerably over the past decade, largely as a result of studies in transformed T cell lines in which T cell antigen receptor (TCR)1 engagement has been mimicked by agents such as lectins and anti-TCR antibodies, which cause aggregation of the TCR (1-3). Oxidants such as H2O2, pervanadate, and ultraviolet light have been found to mimic the intracellular signals initiated by TCR aggregation and have also been used to study this signaling pathway (4).
The earliest detectable event upon TCR engagement is the tyrosine
phosphorylation of a number of substrates involved in downstream signaling events. These include TCR and the
,
, and
chains of CD3, ZAP-70, Itk, phospholipase C
1, p95Vav, SLP-76,
c-Cbl, p36Lat as well as others (1, 2). The accumulation of
these tyrosine-phosphorylated proteins is the result of a shift in the
net balance of competing protein-tyrosine kinases (PTK) and protein
tyrosine phosphatase activities and results in part from the activation
of several PTKs, including members of the Src, Syk/ZAP-70, and Btk/Itk
families of PTKs (1, 3). Three PTKs in particular, Fyn, Lck, and ZAP-70, have been extensively studied, and their involvement in TCR
signaling has been demonstrated by a number of different approaches (3).
The cascade of early TCR-proximal signaling events is initiated by the
phosphorylation of conserved tyrosine residues within immunoreceptor
tyrosine-based activation motifs (ITAMs) present within the CD3 and
TCR chains. This event requires the activity of a src-family PTK (5,
6). Phosphorylated ITAMs act as high affinity binding sites for certain
SH2 domain-containing proteins, including ZAP-70, which binds to ITAMs
via its two SH2 domains (5, 7, 8). Recruitment of ZAP-70 to the TCR is required for the subsequent tyrosine phosphorylation and activation of
ZAP-70 (9, 10), which requires the activity of a heterologous kinase,
presumably Lck (11-13). ZAP-70 appears to function both as a kinase,
phosphorylating downstream signaling proteins such as SLP-76 (14, 15),
p36LAT,2 and
tubulin (16) and as a scaffolding protein by binding particular SH2
domain-containing proteins by virtue of key sites of tyrosine phosphorylation within ZAP-70 (17). To date, biochemical analyses have
identified a number of proteins that can bind to
tyrosine-phosphorylated ZAP-70, including c-cbl, p95Vav,
p59fyn, p56lck, SHP-1, c-abl, and Ras-GAP
(1-3).
These early TCR-proximal signaling events lead to the activation of
several other signaling molecules, including phospholipase C1,
protein kinase C, and the low molecular weight G protein Ras (2).
Together, these signals combine to activate multiple transcription
factors that contribute toward the production of the essential
autocrine growth factor interleukin 2. Activated, GTP-bound Ras
contributes to this process by activating the Raf-1/mitogen-activated protein kinase kinase/Erk signaling pathway, which is required for the
production and activation of the critical transcription factor AP-1
(2). The importance of the signaling pathway between the TCR and Ras is
underscored by the discovery that anergy is the result of a block in
this pathway (18, 19).
How the activation of the TCR-proximal protein-tyrosine kinases causes activation of the Ras/Raf-1/mitogen-activated protein kinase kinase/Erk pathway remains an open question, even though their importance in this pathway was established by studies demonstrating that PTK inhibitors could block TCR-initiated activation of this pathway (20). A role for ZAP-70 has also been suggested on the basis of studies in which overexpression of dominant-negative ZAP-70 in Jurkat T cells could block Erk activation and activation of the interleukin 2 promoter in response to TCR cross-linking (10). SLP-76, which is an in vivo substrate of ZAP-70 (14, 15, 21, 22), can also cause Erk activation when overexpressed in Jurkat T cells (23).
In the present study we have reexamined the importance of ZAP-70 in regulating Erk activity in T cells in response to two different stimuli: 1) TCR cross-linking with the anti-CD3 monoclonal antibody, OKT3 and 2) an oxidative stimulus, hydrogen peroxide. Using the ZAP-70-negative Jurkat T cell line, P116, we find that ZAP-70 is required for the activation of Erk1 and 2 in response to H2O2 stimulation. Interestingly, we also find a ZAP-70-independent pathway for the activation of Erk 1 and 2 in P116 cells in response to TCR cross-linking, which is also independent of SLP-76 tyrosine phosphorylation. The implications of the presence of two pathways for the activation of Erk1 and 2 with differing requirements for ZAP-70 is discussed.
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EXPERIMENTAL PROCEDURES |
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Cells and Antibodies--
E6 Jurkat T cells, the ZAP-70-negative
Jurkat cell mutant, P116, and all stably transfected cell lines derived
thereof were maintained as described (9, 24). The characteristics of
the P116 cells have been described (24). In brief, they were derived from E6 Jurkat by mutagenesis and selection for inability to flux calcium in response to pervanadate. They lack ZAP-70 protein, and
normal signaling capacity is restored by transfection with wild-type
ZAP-70. P116 cells express normal levels of other signaling proteins,
such as TCR/CD3 chains, Lck, Fyn-T, phospholipase C1, SLP-76, and
Erk 1 and 2 (24). The OKT3 monoclonal antibody to human CD3
and the
polyclonal rabbit antiserum specific for ZAP-70 have been described
(9). The anti-phosphotyrosine monoclonal antibody, 4G10, and a sheep
antiserum against human SLP-76 were from Upstate Biotechnology, Inc.
(Lake Placid, NY). The anti-Erk2 polyclonal antiserum used for
immunoprecipitation was from Santa Cruz Biotechnology, Inc. (Santa
Cruz, CA). A Transduction Laboratories (Lexington, KY) monoclonal
antibody to Erk2 was used to blot for total Erk2 protein. A rabbit
polyclonal antiserum specific only for Erk phosphorylated at both
Thr-183 and the Tyr-185 and therefore specific for activated Erk was
obtained from Promega (Madison, WI).
Plasmids and Transfection--
The cDNA encoding
full-length, wild-type ZAP-70 fused with a C-terminal 9-amino acid
myc epitopic tag was subcloned as a BamHI fragment from the previously described pSXSR-ZAPmyc
plasmid (12) into the BamHI site of pBJneo to generate
pBJ-ZAPmyc. The pBJ-ZAPKDmyc vector, which
expresses a kinase-dead, dominant-negative form of ZAP-70, was
similarly constructed from ZAP-70 cDNA carrying a K369R mutation,
which was generated by site-directed mutagenesis using the Transformer
kit (CLONTECH Laboratories, Inc. (Palo Alto, CA).
P116 transfectants stably expressing wild-type or kinase-dead ZAP-70
were prepared as described previously (25).
Stimulation of Cells and Erk Kinase Assay--
The cells were
serum-starved for 16 h before being harvested and washed in
4 °C RPMI using minimal manipulation of the cells, since it had been
noted that extensive manipulation of the cells resulted in high basal
Erk activity. The cells were resuspended to 5 × 107
cells/ml in 4 °C RPMI and maintained on ice until stimulated. After
equilibration to 37 °C for 5-10 min, the cells were stimulated by
the addition of either OKT3 (1:100 of ascites),
H2O2 (10 mM, unless otherwise
indicated), or 50 ng/ml PMA. The duration of stimulation was 3 min
unless otherwise indicated. Stimulation was terminated by the addition
of 5 volumes of 4 °C lysis buffer (20 mM HEPES, pH 7.4, 1% Triton X-100, 50 mM -glycerophosphate, 2 mM EGTA, 10 mM sodium fluoride, 1 mM sodium orthovanadate, 10% glycerol, 10 µg/ml
leupeptin, 10 µg/ml aprotinin, 100 µg/ml
4-(2-aminoethyl)-benzenesulfonyl fluoride). After a 30-min
incubation on ice, post-nuclear lysates were prepared by a 10-min
centrifugation at 21,000 × g at 4 °C. The lysates
were then either directly analyzed by immunoblotting or subjected to
immunoprecipitation followed by immunoblotting or kinase assay.
Immunoprecipitates that were to be analyzed by immunoblotting were
washed three times with lysis buffer supplemented with 150 mM NaCl. Those immunoprecipitates that were to be subjected to immune complex kinase assays were washed two times each sequentially with lysis buffer, LiCl wash buffer (100 mM Tris-HCl, pH
7.5, 0.5 M LiCl, 0.1% Triton X-100, 1 mM
dithiothreitol), and kinase buffer (20 mM MOPS, pH 7.2, 2 mM EGTA, 10 mM MgCl2, 1 mM dithiothreitol, 0.1% Triton X-100). Immunoprecipitated
Erk was subjected to an immune complex kinase assay essentially as
described previously (26).
Electrophoresis, Immunoblotting and Autoradiography-- Samples to be analyzed by electrophoresis were prepared in NuPAGE sample buffer by heating to 100 °C for 5 min. Samples were analyzed on NuPAGE 4-12% gradient gels either in MOPS or MES sample buffers and then transferred to nitrocellulose membranes according to the manufacturer's instructions (NOVEX, San Diego, CA). Immunoblots were developed by the ECL system of Amersham Pharmacia Biotech according to manufacturer's instructions. Autoradiograms were obtained by exposure of BMR film (Kodak, Rochester, NY).
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RESULTS |
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ZAP-70 Is Required for TCR and H2O2-induced Accumulation of Tyrosine-phosphorylated Proteins-- H2O2 has been shown previously to cause accumulation of tyrosine-phosphorylated proteins in Jurkat T cells (27, 28). To test the role of ZAP-70 in this process, we examined tyrosine phosphorylation of proteins in cell lysates from ZAP-70-negative and ZAP-70-replete Jurkat T cells (Fig. 1). Hydrogen peroxide within the concentration range of 1-10 mM did not stimulate tyrosine phosphorylation of cellular substrates in the P116 cells. Normal Jurkat cells, on the other hand, showed a rapid accumulation of tyrosine-phosphorylated proteins in response to incubation with H2O2. The effect of H2O2 was concentration-dependent, and within the concentration range tested (1-10 mM), had a greater effect on tyrosine phosphorylation than that seen with anti-CD3 cross-linking, causing robust phosphorylation of proteins that were only minimally phosphorylated by anti-CD3. OKT3 stimulation of the P116 cells caused only weak tyrosine phosphorylation, with only proteins of 42, 85, and 150 kDa responding. Of note is the absence in both OKT3 and H2O2-stimulated P116 cells of the tyrosine-phosphorylated 36- and 76-kDa proteins that are predominant substrates in normal Jurkat cells and which are likely to correspond to the Grb2-associated p36 protein and SLP-76. These results are consistent with the severe early signaling defect that has been reported for P116 cells (24).
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ZAP-70 Is Required for Erk Activation in Response to H2O2 but Not TCR Cross-linking-- In the same experiment, the requirement for ZAP-70 in mediating Erk activation in response to TCR cross-linking or H2O2 was also assessed, using both an immune complex kinase assay and immunoblotting for doubly phosphorylated, active Erk in whole-cell lysates. The presence of equal amounts or Erk2 in the immunoprecipitations and of Erk 1 and 2 in the Jurkat and P116 whole-cell lysates was confirmed by immunoblotting for these proteins (not shown). Erk2 was immunoprecipitated from the cell lysates and was used in an in vitro kinase assay using myelin basic protein as an exogenous substrate. Stimulation of normal Jurkat cells with either anti-CD3 or H2O2 (1-10 mM) gave an apparent maximal activation of Erk2 (Fig. 2A). H2O2 was effective in activating Erk2 down to a concentration of 10 µM (the lowest concentration tested) and demonstrated a graded concentration-response relationship between 10 µM and 1 mM (not shown). Activated Erk1 and Erk2 could also be detected in immunoblots of lysates from Jurkat T cells stimulated with H2O2 or OKT3, confirming the results of the kinase assay (Fig. 2B). However, in the P116 cells, the effect of H2O2 on Erk activation was undetectable by anti-active Erk immunoblotting of whole-cell lysates and only weakly detectable in the immune complex kinase assay. Unexpectedly, although the P116 cells were unable to activate Erk in response to H2O2, they remained capable of activating Erk in response to CD3 cross-linking. Please note that the apparent slower migration of Erk1 and 2 isolated from Jurkat cells as compared with P116 was not a consistent observation of these studies (see Fig. 4). The presence of ZAP-70 in Jurkat cells and its absence in P116 cells was demonstrated by immunoprecipitation of ZAP-70 followed by ZAP-70 immunoblotting (Fig. 2C). A replicate membrane was blotted for phosphotyrosine and showed that both anti-CD3 and H2O2 caused increased tyrosine phosphorylation of ZAP-70 (Fig. 2D).
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SLP-76 Tyrosine Phosphorylation Occurs in Jurkat but Not in P116 Cells in Response to Erk-activating Stimuli-- Having found a dependence upon ZAP-70 for stimulating Erk activation in response to H2O2, we examined the tyrosine phosphorylation status of SLP-76, an in vivo substrate for ZAP-70 that has previously been implicated in Erk activation in response to TCR engagement. In Jurkat cells, anti-CD3 and H2O2 caused a striking increase in tyrosine phosphorylation of SLP-76 (Fig. 3). The effect of H2O2 was concentration-dependent and robust. In addition to increased tyrosine phosphorylation, there was also an increase in the association of tyrosine-phosphorylated proteins with SLP-76. However, in the ZAP-70-negative P116 cells, neither anti-CD3 nor H2O2 had any effect on SLP-76 tyrosine phosphorylation or its association with other tyrosine-phosphorylated proteins.
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Expression of Wild-type ZAP-70 but Not Kinase-dead ZAP-70 in P116 Cells Restores Ability to Activate Erk in Response to H2O2-- If the failure of P116 cells to activate Erk in response to H2O2 is actually due to the absence of ZAP-70, then reconstitution of these cells with wild-type ZAP-70 should correct this loss of signaling capacity. To test this, we compared Erk activation in P116 cells to P116 cells that had been stably transfected with either myc-tagged, wild-type ZAP-70 or myc-tagged, kinase-dead ZAP-70 carrying a K369R mutation. Three independent clones expressing wild-type ZAP-70 (P.WT2, P.WT5, and P.WT18) and two independent clones expressing kinase-dead ZAP-70 (P.DK2 and P.DK33) were analyzed. The clones expressed varying amounts of ZAP-70, as detected by an anti-ZAP-70 immunoblot, and only the results from the clones expressing the highest levels of ZAP-70 are shown (Fig. 4A). The P.WT18 clone had a similar level of ZAP-70 expression as that seen in Jurkat cells. Wild-type ZAP-70 restored the capacity of P116 cells to activate Erk1 and 2 in response to stimulation by H2O2 while having no apparent effect on OKT3-stimulated Erk activation in these cells. The relative amount of activated Erk detected in the different clones was directly correlated with the relative level of wild-type ZAP-70 expressed (not shown). It should be noted that the slower electrophoretic migration of ZAP-70 in the P.WT18 and P.DK2 clones was due to the myc tag (12). As shown previously, stimulation of normal Jurkat cells with anti-CD3 or H2O2 caused rapid tyrosine phosphorylation of ZAP-70 (Fig. 4B), which was correlated with the increased activation of Erk. Kinase-dead ZAP-70 did not support Erk activation in response to H2O2 in the stably transfected P116 cells, indicating that the kinase activity of ZAP-70 is required for Erk activation in response to H2O2. Surprisingly, the expression of kinase-dead ZAP-70 in P116 cells blocked the ZAP-70-independent activation of Erk1 and 2 that was observed in response to CD3 cross-linking. The ability to block OKT3-mediated Erk activation in K369R-ZAP-70-transfected P116 cells was correlated with the relative expression level of the mutant ZAP-70 (not shown). The ZAP-70 expressed in the P.DK2 cells became strongly tyrosine-phosphorylated upon CD3 cross-linking despite the absence of its own kinase activity and was only weakly phosphorylated in response to H2O2.
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DISCUSSION |
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The studies reported here were initiated to gain a better
understanding of the mechanism whereby H2O2 can
mimic TCR signaling. It had previously been demonstrated that oxidative
stressors such as H2O2 and UV can cause the
accumulation of tyrosine-phosphorylated proteins, raise the
intracellular concentration of Ca2+, and activate NF-B
and AP-1 in Jurkat T cells (27, 33, 34). ZAP-70 was found to be among
the proteins that became tyrosine-phosphorylated in response to UV or
H2O2 treatment of Jurkat T cells, resulting in
activation of its autocatalytic activity (27). These former studies did
not, however, examine the role of ZAP-70 in signaling the presence of
these agents to downstream signaling events. In this study we
demonstrate that ZAP-70 is a key mediator of the signals generated in T
cells by oxidative stress. ZAP-70-negative Jurkat T cells showed no
increase in tyrosine phosphorylation of cellular proteins nor Erk
activation in response to H2O2 but could be
made competent for H2O2-induced Erk activation
by stable transfection with wild-type ZAP-70 cDNA. A cDNA
encoding a kinase-dead mutant of ZAP-70 could not restore competence,
indicating a requirement for ZAP-70 kinase activity in mediating Erk
activation.
How ZAP-70 leads to Erk activation in response to H2O2 (or CD3 cross-linking) remains unclear. The principal pathway leading to Erk activation involves activation of Ras, which can be activated either by inhibiting the activity of Ras-specific GTPase activating proteins (GAPs) or by promoting the binding of GTP to Ras by the action of a guanine nucleotide exchange factor. Although there is evidence that TCR cross-linking results in decreased Ras-GAP activity, the mechanism of this response remains unknown (20). Other studies have suggested that TCR engagement can stimulate the recruitment of Ras-specific guanine nucleotide exchange factors such as SOS to the plasma membrane as a consequence of tyrosine phosphorylation of plasma membrane-associated proteins. As in other cell types, this process appears to involve the adapter protein Grb2, which uses its N-terminal SH3 domain to bind to a proline-rich region of SOS and its SH2 domain to bind tyrosine-phosphorylated membrane proteins. Grb2 can directly bind to tyrosine-phosphorylated TCR ITAMs in vitro (35, 36) and binds in vivo to a membrane-associated 36-kDa protein that is rapidly tyrosine-phosphorylated after TCR stimulation (reviewed in Ref. 37). This 36-kDa protein has recently been shown to be a specific substrate for ZAP-70 in vitro,2 and it may be that the requirement for ZAP-70 in H2O2-stimulated Erk activation may be for tyrosine phosphorylation of this protein.
Alternatively, a second substrate for ZAP-70, SLP-76, has also been suggested to be involved in TCR-stimulated Erk activation (14, 15, 21-23), and ZAP-70 may be required to phosphorylate SLP-76 in order to get Erk activation in response to H2O2. That SLP-76 may have to become tyrosine-phosphorylated in order to activate Erk is suggested by the fact that overexpression of a truncation mutant of SLP-76, which lacks the prominent sites of tyrosine phosphorylation, fails to augment Erk activation in response to TCR stimulation. This is in contrast to full-length SLP-76, which activates Erk (23). We did observe SLP-76 tyrosine phosphorylation in Jurkat cells, but not in P116 cells, in response to either TCR cross-linking or incubation with H2O2 despite the presence of equivalent levels of SLP-76 in the two cell lines (not shown). This is consistent with SLP-76 being downstream of ZAP-70 in the pathway leading to Erk activation. However, SLP-76 tyrosine phosphorylation cannot be an absolute requirement for Erk activation in T cells, since Erk activation could occur in OKT3-stimulated P116 cells in the absence of detectable SLP-76 tyrosine phosphorylation.
How SLP-76 then affects Erk activation remains to be established; however, like p36, it too is able to bind to Grb2. Unlike p36 though, this association is mediated by the C-terminal SH3 domain of Grb2. Whether or not Grb2 can simultaneously engage SOS and SLP-76 remains controversial. In addition to the proline-rich SH3 domain acceptor site, the hematopoietic-specific SLP-76 has a C-terminal SH2 domain and a cluster of N-terminal tyrosines that become phosphorylated upon TCR engagement. SLP-76 thereby forms complexes with many signaling molecules, and activates Erk, NF-AT, and AP-1 when overexpressed in T cells (22, 38). It seems likely that some of these SLP-76-associated proteins may modulate the activity of proteins involved in Ras activation and that ZAP-70-dependent tyrosine phosphorylation may regulate some of these events.
The ability of exogenous oxidants such as H2O2
to usurp myriad signaling pathways in many different cell types
indicates the existence of multiple regulatory molecules that can be
controlled at least in part by oxidation-reduction mechanisms,
suggesting that endogenous reactive oxygen species (ROS) may play a
role in normal signaling pathways. In fact, it has recently been shown that a number of cytokine and growth factor receptors, including the
receptors for interleukin 1, tumor necrosis factor ,
platelet-derived growth factor, and epidermal growth factor, generate
reactive oxygen species as second messengers, which are required for
the activation of key downstream signaling molecules such as NF-
B and phospholipase C
1 (39-44). One mechanism of action of ROS is through the inactivation of protein tyrosine phosphatases by oxidizing a critical conserved cysteine residue within the active site (reviewed in Ref. 45). It has been proposed that a transient inhibition of
protein tyrosine phosphatases is required in addition to activation of
PTKs in order to increase the basal tyrosine phosphorylation of key
signaling proteins above a threshold capable of propagating a
stimulatory signal. Interestingly, it has been suggested for many years
that T cell activation may also require the production of ROS on the
basis that treatment of T cells with antioxidants renders them
unresponsive to mitogenic stimuli (46-48). In addition, acute exposure
of T cells to H2O2 results in interleukin 2 production and increased proliferation (Ref. 49 and references
therein).
Taken together, the above observations lead us to speculate that ZAP-70 may normally be regulated, in part, by an upstream enzymatic activity that produces an ROS. To speculate further, it may be that it is the presence or strength of such an ROS signal that determines whether a given antigen-MHC TCR ligand gives rise to an agonist, partial agonist, or antagonist biochemical response (50). Perhaps activation of the TCR-proximal PTKs in the absence of ROS production and consequent protein tyrosine phosphatase inhibition results in the partial phosphorylation pattern seen with partial agonists and antagonists. The ability of ZAP-70 to activate Erk in response to ROS may also be important at sites of inflammation, where activated neutrophils and monocytes generate large quantities of ROS (51). This may provide a mechanism for these cells to regulate the proliferative capacity and activity of T cells present at inflammatory sites. The testing of these hypotheses and the identification of the enzyme(s) involved in ROS production is the subject of continuing investigation.
The most unexpected finding of these studies was the discovery that TCR cross-linking in P116 cells can initiate a ZAP-70-independent activation of Erk1 and 2. Most current models for TCR-initiated Erk activation invoke ZAP-70 activation as part of this process, and indeed, overexpression of dominant-negative ZAP-70 in Jurkat T cells results in a block in their ability to activate Erk in response to TCR ligation (10). However, these earlier studies may need to be reinterpreted in light of our finding that stable expression of kinase-dead ZAP-70 blocked the ZAP-70-independent activation of Erk in P116 cells. The ability of kinase-dead ZAP-70 to block OKT3-stimulated Erk activation in P116 cells is not due to an inhibitory effect on Syk activation, as there is no Syk present in P116 cells. The P116 cells were derived from the E6.1 Jurkat subclone and are not only ZAP-70-negative but are also negative for Syk protein and mRNA (24, 52). We are currently working to understand how Erk is activated in the absence of ZAP-70 in these cells and how it is that dominant-negative ZAP-70 is able to block a signaling pathway that does not involve ZAP-70 or Syk.
In conclusion, we have found that two stimuli that are considered to mimic the intracellular signaling events initiated by TCR engagement give completely different responses in a ZAP-70-negative Jurkat T cell line. The oxidative stressor H2O2 demonstrates a requirement for ZAP-70 in mediating Erk activation, whereas OKT3 cross-linking of CD3 can activate Erk in the absence of ZAP-70. This is the first demonstration of a ZAP-70-independent pathway for Erk activation in T cells and adds to the complexity of the signaling pathways that can lead to Erk activation in response to TCR engagement. Further study will be required to determine whether or not this pathway is functionally relevant in normal TCR signaling.
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ACKNOWLEDGEMENTS |
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We thank Dr. R. Abraham for sharing the P116 cells with us and Drs. N-P. Weng, L. E. Samelson, N. Holbrook, and Y. Liu for critical review of the manuscript and helpful discussions.
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FOOTNOTES |
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* 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: Gerontology Research Center, MSC-12, 5600 Nathan Shock Dr., Baltimore, MD 21224-6825. Tel.: 410-558-8054; Fax: 410-558-8107; E-mail: wanger{at}grc.nia.nih.gov.
1 The abbreviations used are: TCR, T cell antigen receptor; PTK, protein-tyrosine kinase; ITAM, immune receptor tyrosine-based activation motif; GAP, GTPase activating protein; ROS, reactive oxygen species; MOPS, 4-morpholinepropanesulfonic acid; MES, 4-morpholineethanesulfonic acid; PMA, phorbol 12-myristate 13-acetate.
2 W. Zhang and L. E. Samelson, submitted for publication.
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REFERENCES |
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