From the Division of Cell Biology, La Jolla Institute for Allergy and Immunology, San Diego, California 92121
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ABSTRACT |
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The hematopoietic tyrosine phosphatase (HePTP) is predominantly expressed in thymocytes and T lymphocytes and at lower levels in other hematopoietic cells. Expression of the gene is enhanced by the T cell growth factor interleukin-2, suggesting a role for HePTP in T cell proliferation or differentiation. We report that HePTP blocks T cell antigen receptor (TCR)-induced transcriptional activation of a reporter gene driven by a nuclear factor of activated T cells(NFAT)/AP-1 element taken from the interleukin-2 gene promoter. This effect was specific to HePTP and was abolished by a mutation (C270S) that impaired its phosphatase activity. Co-expression of HePTP also reduced TCR-induced activation of the mitogen-activated protein kinase Erk2 and the TCR-induced appearance of phosphorylated Erk. In contrast, HePTP did not affect the activation of the N-terminal c-Jun kinase, Jnk. Together these findings suggest that HePTP plays an active negative role in TCR signaling by dephosphorylating one or several signaling molecules between the receptor and the mitogen-activated protein kinase pathway.
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INTRODUCTION |
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One of the earliest biochemical events seen in T lymphocytes triggered through the T cell antigen-receptor (TCR)1is an enhanced, but transient, phosphorylation of a number of cellular proteins on tyrosine residues. Inhibition of this event by pharmacological agents prevents T cell activation as measured by both functional read-outs and biochemical assays (1, 2). It has become evident that several protein tyrosine kinases (PTKs) and the CD45 protein tyrosine phosphatase (PTPase) play crucial roles (reviewed in Ref. 3) and that the TCR-induced cascade of transient tyrosine phosphorylation events depends on a dynamic interplay between these and, presumably, many additional PTKs and PTPases. In addition to CD45 (4-7), only three other PTPases have been implicated in T cell activation, namely SHP1 (8, 9), SHP2 (10), and the low molecular weight PTPase, LMPTP-B (11).
The hematopoietic protein tyrosine phosphatase (HePTP) was originally
cloned from human T lymphocytes (12-14). The gene is expressed in
thymus and at lower levels in spleen, but not in nonhematopoietic
tissues. The exon/intron structure of the HePTP gene (15) is quite
similar to that of the phosphatase domains of human CD45. In contrast
to CD45, however, the 38-kDa HePTP consists of only a single PTPase
domain, which occupies the C-terminal three-fourths of the enzyme and
is preceded by a ~70-amino acid noncatalytic N terminus. Presently,
very little is known about the physiological function of HePTP, but
some indications may be derived from the findings that its gene maps to
chromosome 1q32.1 (13), which is a site for frequent chromosomal
abnormalities in preleukemic myeloproliferative disease (16, 17), and
that gene amplification and overexpression of HePTP have been reported for leukemic cells (13). In addition, expression of HePTP in NIH3T3
cells resulted in altered cell morphology and decreased contact
inhibition (13). Together, these findings suggest a role for HePTP in
the regulation of cell proliferation, survival, or differentiation.
This possibility is supported by the up-regulation of HePTP mRNA in
T cells in response to interleukin-2 (IL-2) (18), and by the rapid
phosphorylation of HePTP on tyrosine in RBL-2H3 mast cells stimulated
through their FcRI (19).
Since HePTP is preferentially expressed in T cells and may be involved in the regulation of cell proliferation, we decided to investigate the involvement of this enzyme in TCR-induced T cell activation. We report that HePTP had a strong inhibitory effect on the transcriptional activation of a reporter gene driven by three tandem nuclear factor of activated T cells (NFAT)/AP-1 elements derived from the 5' IL-2 gene promotor, while a catalytically inactive C270S mutant HePTP did not. HePTP also reduced TCR-induced activation of the mitogen-activated protein kinase (MAPK) Erk2, but not of the N-terminal c-Jun kinase (Jnk). Based on these findings we suggest that HePTP plays a negative role in TCR signaling by acting on signaling molecules upstream of MAPK and transcriptional activation of the IL-2 gene.
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MATERIALS AND METHODS |
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Antibodies and Reagents--
The anti-Tyr(P) mAb 4G10 was from
Upstate Biotechnology Inc. (Lake Placid, NY). The 12CA5
anti-hemagglutinin mAb was from Boehringer Mannheim and was used for
immunoprecipitation, and the 16B12 anti-hemagglutinin was from Babco
(Richmond, CA) was used for immunoblotting. The 9E10 hybridoma
producing the mAb that recognizes the c-Myc epitope tag and the OKT3
hybridoma that produces the anti-CD3 mAb were from American Type
Culture Collection (Bethesda, MD). Both mAbs were used as ascites. The
polyclonal anti-extracellular signal-regulated kinase 2 (Erk2) was from
Santa Cruz Biotechnology Inc., and the polyclonal anti-phospho-Erk was from New England Biolabs.
Plasmids-- The cDNA for HePTP was obtained from Dr. Brent Zanke, and the coding region was subcloned into the pEF/HA vector (20), which adds a hemagglutinin (HA) tag to the N terminus of the insert. HePTP was also subcloned into the pGEX-2T prokaryotic expression vector with a glycine kinker. The cDNAs for LMPTP (11) and VHR were generated using the polymerase chain reaction and a Jurkat cell cDNA library and were cloned into pEF/HA and verified by sequencing. The cDNAs for SHP1 (from M. Thomas), TCPTP (from Deborah Cool), and Syk (21) were also cloned into pEF/HA, while PTP36 (from M. Ogata) and Lck (22) were in pEF-neo (driven by the same promotor, but without N-terminal tag) and c-Cbl (from A. Veillette) in pMX139. The c-Myc-tagged Erk2 (from C. Marshall) was in pEF-neo. The NFAT/AP-1-luc construct (from G. Crabtree) contains three tandem NFAT/AP-1 sites driving the expression of a luciferase gene.
Site-directed Mutagenesis-- To generate a catalytically inactive mutant of HePTP, the codon for Cys-270 was changed into a codon for serine in the pEF/HA-HePTP plasmid using the transformerTM site-directed mutagenesis kit as recommended by the manufacturer (CLONTECH). The resulting C270S mutation was verified by sequencing. The C216S mutant of TCPTP was generated and verified by the same procedure.
Cells and Transfections-- Jurkat T leukemia cells and two variants of this cell line: J-TAg (from Dr. M. Karin), which is stably transfected with simian virus 40 large T antigen, and JCaM1.6 (from Dr. A. Weiss), which lacks Lck (23), were kept at logarithmic growth in RPMI 1640 medium with 5% fetal calf serum, L-glutamine, and antibiotics. These cells were transiently transfected with a total of 5-10 µg of DNA by electroporation at 950 microfarads and 240 V. Empty vector was added to control samples to make a constant amount of DNA in each sample. Cells were used for experiments 48 h after transfection. COS-1 cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum. These cells were transfected by lipofection with a total of 5-10 µg of DNA and grown for 48 h prior to the experiments.
NFAT/Luciferase Assays--
These assays were performed as
described previously (24). Briefly, cells were transiently transfected
with 2 µg of NFAT/AP-1-luc together with empty vector or PTK and/or
PTPase plasmids. Two days later, the cells were either stimulated with
the anti-CD3 mAb OKT3 (5 or 10 µg/ml) or left untreated. After
8 h, the cells were washed with phosphate-buffered saline and
lysed in 100 µl of lysis buffer (100 mM potassium
phosphate buffer, pH 7.8, 1 mM dithiothreitol, 0.2% Triton
X-100). Lysates were clarified by centrifugation at 15,000 × g for 5 min. The final assay contained 50 µl of lysate
plus 100 µl of ATP solution (10 mM ATP in 35 mM glycylglycine, pH 7.8, 20 mM
MgCl2) plus 100 µl of luciferin reagent (0.27 mM coenzyme A, 0.47 mM luciferin, 35 mM glycylglycine, pH 7.8, 20 mM
MgCl2). The activity was measured in an automatic
luminometer (Monolight 2010, Analytical Luminescence Laboratory, Ann
Arbor, MI). The total protein concentration in each cell lysate was
determined by the Bradford protein assay and was used to normalize the
luciferase activity. Typically, the variation in protein concentration
between samples was less than 20%.
Immunoprecipitation-- Cells were lysed in 20 mM Tris/HCl, pH 7.5, 150 mM NaCl, 5 mM EDTA containing 1% Nonidet P-40, 1 mM Na3VO4, 10 µg/ml aprotinin and leupeptin, 100 µg/ml soybean trypsin inhibitor, and 1 mM phenylmethylsulfonyl fluoride and clarified by centrifugation at 15,000 rpm for 20 min. The clarified lysates were preabsorbed on protein G-Sepharose and then incubated with antibody for 2 h, followed by protein G-Sepharose beads. Immune complexes were washed three times in lysis buffer, once in lysis buffer with 0.5 M NaCl, again in lysis buffer, and either suspended in SDS sample buffer or used for in vitro kinase assays.
MAPK Assays--
These were performed as described previously
(20, 22). Briefly, 20 × 106 JCaM1 cells were
transfected with 5 µg of c-Myc-tagged Erk2 plasmid, 1 µg of HePTP
plasmid, and 5 µg of Lck plasmid. Empty vector was added to control
samples to make constant amount of DNA in each sample. Cells were
harvested 2 days after electroporation, divided into two
samples/transfection, and either stimulated with OKT3 (5 µg/ml) for 5 min at 37 °C or left untreated. Cells were lysed as described above
and the Myc-tagged Erk2 immunoprecipitated with 2 µg of the 9E10
anti-Myc mAb followed by 25 µl of protein G-Sepharose beads. The
kinase reaction was performed for 30 min at 30 °C in 20 µl of
kinase buffer containing 20 mM Tris/HCl, pH 7.5, 150 mM NaCl, 20 mM MgCl2, 10 µg of
myelin basic protein, 1 µM ATP,and 10 µCi of
[-32P]ATP. The reactions were terminated by adding 20 µl 2 × SDS sample buffer and heating to 95 °C for 2 min. The
samples were run on 12% SDS-polyacrylamide gels, transferred onto
nitrocellulose filters, and the labeled proteins visualized by
autoradiography. The presence of equal amounts of the
immunoprecipitated Erk2 was verified by Western blotting using the
anti-Erk2 antibody at 1:1000 dilution, anti-mouse-Ig-peroxidase, and
the blots developed by the enhanced chemiluminescence technique (ECL
kit, Amersham Pharmacia Biotech) according to the manufacturer's
instructions.
PTPase Assays-- The catalytic activity of immunoprecipitated HePTP from Jurkat T cells was measured as described earlier (11). The reaction was for 30 min at 37 °C in 100 µl of 50 mM sodium citrate buffer, pH 5.5, with 5 mM p-nitrophenyl phosphate as a substrate. The production of p-nitrophenol was measured as absorbance at 410 nm.
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RESULTS |
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Cloning of HePTP and Characterization of the HePTP Antisera-- The coding region of HePTP was amplified by the polymerase chain reaction technique using oligonucleotide primers corresponding to both ends of the open reading frame and with human HePTP (clone HEPTP2761 PBSSKM from B. Zanke) as the template. The 1019-base pair amplification product was cloned into the pEF-HA vector and sequenced. The obtained nucleotide sequence was 100% identical to the published sequence (12). Transient expression of the pEF/HA-HePTP construct in COS or J-TAg cells resulted in the appearance of a ~40-kDa (the tag adds ~2 kDa) protein that was both immunoblotted and immunoprecipitated with the anti-HA tag mAbs 12CA5 and 16B12. The expression plasmid was also well expressed in Jurkat, JCaM1, and particularly well in J-TAg cells. Very similar expression levels were seen with the catalytically inactive C270S mutant of HePTP. Fig. 1d shows a PTPase assay of anti-tag immunoprecipitates obtained from 20 × 106 J-TAg cells transfected with empty pEF/HA vector or the HePTP constructs or another PTPase TCPTP and its inactive (C216S) mutant. As expected, the wild-type enzymes had easily measurable catalytic activity, while the two mutants did not. The amounts of wild-type and mutated proteins were similar (Fig. 1d, inset).
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HePTP Suppresses TCR-induced Activation of an NFAT/AP-1 Element from the IL-2 Promoter-- Activation of T cells through the TCR plus accessory molecules leads to the transcriptional activation of the IL-2 gene, followed by production and secretion of IL-2. Subsequently, this lymphokine binds to specific high-affinity receptors present mainly on activated T cells and drives the cells through the cell cycle, resulting in the clonal proliferation of T cells. TCR-induced activation of the IL-2 gene is conveniently measured by transfecting T cells with a luciferase reporter construct driven by elements from the IL-2 gene 5' promoter.
First, we transfected J-TAg cells with the NFAT/AP-1-luc reporter together with HePTP or empty pEF/HA vector. Two days after transfection the cells were treated with OKT3 for 6 h, lysed, and the activity of the induced luciferase measured. As can be seen in the upper panel of Fig. 2., co-expression of HePTP strongly reduced the capacity of the TCR/CD3 to activate NFAT/AP-1. This result was obtained in three independent experiments (duplicate determinations in each). However, due to the SV40 large T antigen, these cells express high levels of transiently transfected constructs, making the system potentially prone to artifacts. Therefore, we next used JCaM1 cells, in which pEF-based constructs are expressed at levels close to the endogenous amounts of signaling proteins (25). These cells also lack endogenous Lck (23) and have very low levels of Syk (26) and do not respond to anti-CD3 mAbs unless either kinase is re-expressed. First, we transfected these cells with Lck alone or in combination with wild-type HePTP or catalytically inactive HePTP-C270S together with the NFAT/AP-1-luc reporter. Two days after transfection the cells were treated with OKT3 for 8 h, lysed, and the luciferase activity measured. As shown in the lower panel of Fig. 2., expression of Lck restored the capacity of the TCR/CD3 to activate NFAT/AP-1. In comparison, co-transfection of active HePTP reduced the activation of the reporter construct to less than 10%, while the C270S mutant did not have any significant effect. This result was obtained in four independent experiments (duplicate determinations in each), and control blots showed that HePTP and Lck were similarly expressed in all cases (Fig. 2, lower panel inset).
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Effect of HePTP on TCR-induced Erk2 Activation-- As the IL-2 promoter activation experiments above suggest that HePTP may play a role in TCR-induced T cell activation somewhere between the TCR and the IL-2 gene, we next tested a more receptor-proximal read-out for TCR signaling, namely the activation of the MAPK pathway. JCaM1 cells were transiently co-transfected with a Myc-tagged Erk2 MAPK together with HePTP or HePTP-C270S. Two days after transfection, each sample was divided in half; one-half was left unstimulated while the other half was treated with anti-CD3 mAbs for 5 min at 37 °C. After cell lysis, the tagged Erk2 was immunoprecipitated with the 9E10 anti-c-Myc mAb, and after washing these precipitates were subjected to in vitro kinase assays with myelin basic protein as a substrate and the phosphorylation of the substrates was visualized by autoradiography. As can be seen in Fig. 4, the tagged Erk2 was not significantly activated in cells co-transfected with empty pEF/HA vector (lanes 1 and 2). Co-expression of Lck (lanes 3 and 4) enabled the anti-CD3 mAb to cause severalfold activation of Erk2. Furthermore, HePTP had a significant inhibitory effect on MAPK activation whereas HePTP-C270S augmented the effect. The amount of Erk2 was found to be similar in all the samples (Fig. 4, middle panel), and HePTP and HePTP-C270S were equally expressed in lanes 5-8 (lower panel). The expression of Lck was also similar in lanes 3-8 (not shown). This result was obtained in several independent experiments.
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HePTP Also Blocks TCR-induced Phosphorylation of Endogenous Erk-- To further verify that the effect of HePTP on Erk2 truly reflects an inhibitory effect on the intracellular mechanism of MAPK activation rather than a direct effect in the kinase assays or some other artifactual event connected to the transfected Erk, we analyzed lysates from JCaM1 cells transfected with Lck with or without HePTP, HePTP-C270S, TCPTP, or TCPTP-C216S by immunoblotting with antibodies specific for the phosphorylated and activated form of Erk. These blots (Fig. 5, upper panel) showed that phospho-Erk was only induced by OKT3 treatment in cells expressing Lck and that catalytically active HePTP reduced this induction. In contrast, the C270S mutant of HePTP, as well as the TCPTP constructs, did not. Control blots showed that all samples contained similar amounts of endogenous MAPK (second panel) and that HePTP and TCPTP were well and equally expressed (third panel), as was Lck (bottom panel). This result was obtained in three independent experiments.
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HePTP Does Not Affect the Jnk Pathway-- To further understand the negative effect of HePTP on NFAT/AP-1 activation, we decided to also study the effect of this PTPase on the Jnk kinase pathway, which is involved in the activation of c-Jun, a component of the AP-1 complex. Jurkat cells were transfected with empty vector, Jnk1 (in pEF/HA), alone or together with HePTP. Two days after transfection, samples were treated with OKT3 and anti-CD28 (mAb 9.3) plus sheep anti-mouse Ig antibody or with the secondary antibody alone. 20 min later, the cells were lysed and the HA-tagged Jnk1 immunoprecipitated with the 12CA5 anti-HA mAb and subjected to in vitro kinase assays using glutathione S-transferase-c-Jun as a substrate. These experiments consistently failed to reveal any effect of HePTP despite good activation of Jnk1 and good expression of both Jnk1 and the PTPase (not shown). We conclude that the substrate(s) for HePTP are involved in MAPK regulation, but not in Jnk activation.
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DISCUSSION |
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Reversible tyrosine phosphorylation is a critical mechanism for
the regulation of signal transduction, cell growth, differentiation, and development. These processes involve the balanced action of both
PTKs and PTPases, which also regulate each other in a complex manner.
In T cells, the repertoire of PTKs involved in the earliest signaling
events initiated by ligation of the TCR and its co-receptors is
relatively well characterized. In contrast, the PTPases that regulate
these PTKs and that counteract their phosphorylation of key signaling
molecules are largely unknown. The transmembrane receptor-like PTPase
CD45 was the first PTPase implied in T cell activation (3-7). This
enzyme dephosphorylates the C-terminal negative regulatory tyrosine in
the Src family PTKs Lck (6, 7) and Fyn (28). This function of CD45 is
not induced by TCR triggering, but occurs in a constitutive manner and
is critical for T cell for activation, presumably by maintaining a
large fraction of Lck and Fyn in a potentially active state (3) and by
promoting a relatively high level of basal tyrosine phosphorylation of
the activation motifs in the TCR- and CD3 chains. Another
PTPase, the SH2 domain-containing PTPase SHP1, has been
implied as a negative regulator of signaling from the TCR as well as
many other hematopoietic receptors. The evidence for its direct
participation in TCR signaling, however, is not compelling, and it may
be that SHP1 is mainly involved in signaling by inhibitory receptors,
such as p58KIR, p70KIR, Fc
RIIB, and CD22 (29). We have previously
added the second SH2 domain-containing PTPase, SHP2 (10), and the low molecular weight PTPase, LMPTP-B (11), to the list of PTPases that may
participate in TCR signaling. SHP2 is tyrosine-phosphorylated, while
LMPTP-B is dephosphorylated upon TCR triggering, but there is currently
no evidence for any functional role of these PTPases in T cell
activation. Thus, the PTPases that dephosphorylate TCR-
, the
activation sites in Lck (Y394) or Fyn (Y417), or the multiple phosphorylation sites in Zap and Syk, or the multitude of tyrosine phosphorylation sites in all the substrates for these PTKs, remain unknown.
In this paper we show that another T cell-expressed PTPase, HePTP, has a strong negative effect in TCR-induced T cell activation. We show that expression of HePTP blocks the induction of NFAT/AP-1-driven transcription and the phosphorylation and activation of Erk. This effect of HePTP did not require gross overexpression of the enzyme and was unique to HePTP in comparison with several other PTPases (TCPTP, SHP1, PTP36, LMPTP, and VHR). The effect also required the catalytic activity of HePTP. In some experiments (e.g. Fig. 4), the catalytically inactive C270S mutant of HePTP even had a stimulatory effect, perhaps due to competition with endogenous HePTP. It is also worth noting that expression of HePTP did not detectably affect the overall profile of cellular tyrosine phosphorylation (not shown). Together, these experiments indicate that HePTP dephosphorylates relatively few components of the TCR signaling machinery, at least one of which is located upstream of the MAPK Erk2. In contrast, our experiments with TCPTP do not reveal any effects of this PTPase in TCR signaling.
It is worth noting that HePTP blocked NFAT/AP-1 induction to a greater extent (80-100%) than it reduced MAPK activation (~50%). This difference could be due to the dephosphorylation of additional signaling molecules by HePTP leading to inhibition of other parallel pathways required for NFAT/AP-1 activation. Alternatively, the reduction of MAPK activation seen at 5 min is sufficient to keep the machinery responsible for NFAT/AP-1 activation at 8 h after receptor triggering below a critical threshold. It is conceivable that HePTP also shortens the duration of the MAPK response.
In conclusion, our results introduce HePTP as a potentially important negative regulator of T cell activation and indicate that at least one site of HePTP action is located between the TCR and the phosphorylation and activation of MAPKs. Further experiments will be needed to determine more precisely where in the signaling cascade that HePTP acts and to establish its exact role and importance in T cell activation.
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ACKNOWLEDGEMENT |
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We are grateful to Dr. Brent Zanke for the kind gift of the HePTP cDNA.
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FOOTNOTES |
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* This work was supported by Grants GM48960, AI35603, AI41481, and AI40552 from the National Institutes of Health. This is publication number 221 from the La Jolla Institute for Allergy and Immunology.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: Division of Cell
Biology, La Jolla Institute for Allergy and Immunology, 10355 Science
Center Dr., San Diego, CA 92121. Tel.: 619-558-3547; Fax: 619-558-3526;
E-mail: tomas_mustelin{at}liai.org.
1 The abbreviations used are: TCR, T cell antigen receptor; Erk2, extracellular signal-regulated protein kinase 2; HePTP, hematopoietic cell PTPase; MAPK, mitogen-activated protein kinase; NFAT, nuclear factor of activated T cells; PTK, protein tyrosine kinase; PTPase, protein tyrosine phosphatase; LMPTP, low molecular weight PTPase; VHR, vaccinia H1-related; TCPTP, T cell PTPase.
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REFERENCES |
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