From the Laboratory of Signal Transduction, La Jolla Cancer Research Center, The Burnham Institute, La Jolla, California 92037
Received for publication, July 20, 2000, and in revised form, November 14, 2000
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ABSTRACT |
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The 21-kDa dual specific protein phosphatase
VH1-related (VHR) is one of the smallest known phosphatases, and
its function has remained obscure. We report that this enzyme is
expressed in lymphoid cells and is not induced by T cell antigen
receptor like other dual specificity phosphatases. Introduction of
exogenous VHR into Jurkat T cells caused a marked decrease in the
transcriptional activation of a nuclear factor of activated T
cells and an activator protein-1-driven reporter gene in
response to ligation of T cell antigen receptors. The inhibition
was dose-dependent and was similar at different doses of
anti-receptor antibody. Catalytically inactive VHR mutants caused an
increase in gene activation, suggesting a role for endogenous VHR in
this response. In contrast, the activation of a nuclear factor
Members of the mitogen-activated protein kinase
(MAP1 kinase) family are
activated by a wide range of extracellular stimuli including growth and
differentiation factors and cytokines as well as ultraviolet radiation,
heat shock, and osmotic shock (reviewed in Refs. 1-3). Several
distinct MAP kinase cascades have been identified in mammalian cells
including the extracellular signal-regulated kinases Erk1 and Erk2,
which preferentially transmit signals that regulate cell growth and
differentiation, the c-Jun N-terminal kinases Jnk1 and Jnk2, the p38
kinases, which participate mainly in responses to stress, inflammation
and apoptosis, and the 80-kDa Erk5, which may regulate cell
proliferation (4). Activation of T lymphocytes by antigens is
accompanied by activation of all of these pathways (5-8) although
activation of the Jnks and p38s requires costimulation through CD28
(7), proinflammatory cytokines, or oxidative conditions that are often
present at the site of inflammation and lymphocyte activation.
At the molecular level, MAP kinases are activated by a dual threonine
and tyrosine phosphorylation within the motifs Thr-Glu-Tyr (Erk),
Thr-Pro-Tyr (Jnk), or Thr-Gly-Tyr (p38) in their activation loops by a
number of specific MAP kinase kinases (9). Conversely, inactivation of
MAP kinases is achieved by dephosphorylation of either or both residues
by protein Ser/Thr phosphatases, protein-tyrosine phosphatases, or dual
specific protein phosphatases (DSPs) (reviewed in Refs.10-12). In
fact, the number of identified phosphatases that inactivate MAP kinases
exceeds the number of known MAP kinase kinases, providing the first
example of an important process in which the regulatory emphasis is
more on the phosphatases than on the kinases (11).
Many members of the DSP group have assumed a role in MAP kinase
inactivation, and most are inducible nuclear proteins that play
important negative feedback roles in cellular signaling processes in
response to environmental signals such as mitogenesis, apoptosis, differentiation, and secretion of cytokines (10-12). The first cloned
member of this group was the VH1 protein from the vaccinia virus (13).
A closely related enzyme was subsequently found in mammalian cells and
termed VHR (VH1-related) (14). Although most DSPs identified to date
seem to be specific for Erk1 and Erk2, and a few prefer Jnk or p38, the
cellular target(s) for VHR have remained unclear. VHR also differs from
other DSPs in being much smaller, only 21 kDa.
We report that VHR is expressed in all examined lymphoid and
hematopoietic cell types and is constitutively expressed in T cells. We
also present evidence that VHR counteracts the Erk and Jnk MAP kinases,
but not p38, and all reporter genes that depend on Erk or Jnk. As a
catalytically inactive VHR behaved as a "dominant-negative" in many
experiments, we suggest that this may be the physiological function of
VHR in T lymphocytes.
Antibodies and Reagents--
The anti-VHR mAb was from
Transduction Laboratories (Los Angeles, CA). The 12CA5
anti-hemagglutinin mAb was from Roche Molecular Biochemicals.
The 9E10 hybridoma producing the mAb that recognizes the c-Myc epitope
tag and the OKT3 hybridoma that produces the anti-CD3 Polymerase Chain Reactions and Plasmids--
The cDNA for
VHR was amplified from a Jurkat cell cDNA library using the
polymerase chain-reaction (PCR) primer pair 5'-A ATT GCT AGC ATG TCG
GGC TCG TTC-3' and 5'-G CGC ATC GAT CTA GGG TTT CAA CTT-3' in a
polymerase chain reaction using PWO polymerase obtained from
Roche Molecular Biochemicals. The PCR product was sequenced and
subcloned into the pEF/HA vector (15), which adds a hemagglutinin (HA)
tag to the N terminus of the insert. The c-Myc-tagged Erk2 was in
pEF-neo, the HA-tagged Erk1 (from G. Baier, University of Innsbruck,
Austria) was in pEF-neo, HA-Jnk1 and HA-Jnk2 were in the pcDNA3
vector, and p38- Site-directed Mutagenesis--
To generate a catalytically
inactive mutant of VHR, the codon for Cys-124 was changed into a codon
for serine in the pEF/HA-VHR plasmid using the
TransformerTM site-directed mutagenesis kit as recommended
by the manufacturer (CLONTECH). The
substrate-trapping mutant of VHR (VHR-D92A) was generated using the
Quick Change (Stratagene, San Diego, CA) site-directed mutagenesis kit
as instructed by the manufacturer. Both mutations were verified by
nucleotide sequencing.
Cells and Transfections--
Jurkat T leukemia cells were kept
at logarithmic growth in RPMI 1640 medium supplemented with 10% fetal
calf serum, 2 mM L-glutamine, 1 mM sodium
pyruvate, nonessential amino acids, and 100 units/ml each of penicillin
G and streptomycin. 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 constant the
amount of DNA in each sample. Cells were used for experiments 24 h
after transfection. Human bone marrow was purchased from AllCells LLC
(Foster City, CA). Bone marrow leukocytes were obtained by hypotonic
lysis of the erythrocytes in water for 1 min followed by washing of the
cells in RPMI medium. Peripheral blood lymphocytes (~80% T cells)
were obtained from venous blood from healthy donors (Red Cross Blood
Bank, San Diego, CA) by Ficoll gradient centrifugation and by removal
of monocytes by adherence to plastic at 37 °C for 2 h. The
adherent cells were also recovered.
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.
Immunoblotting--
Proteins resolved by SDS-polyacrylamide gel
electrophoresis were transferred electrophoretically to nitrocellulose
filters, which were immunoblotted as before (15-19) with optimal
dilutions of mAbs followed by anti-mouse-Ig peroxidase. The blots were
developed by the enhanced chemiluminescence technique (ECL kit,
Amersham Pharmacia Biotech) according to the manufacturer's instructions.
Jnk Assays--
These assays were performed as before (16).
Briefly, 20 × 106 Jurkat T cells were transfected
with either empty vector or 5 µg of HA-tagged Jnk1 or Jnk2 plasmid
alone or plus 5 µg of VHR plasmid. Cells were harvested 2 days after
electroporation, either left untreated or stimulated with anti-CD3
Cells were lysed in 20 mM Tris/HCl, pH 7.4, 150 mM NaCl, 1% Nonidet P-40, 1 mM
Na3VO4, 25 mM MAP Kinase Assays--
These were done as before (17-19).
Briefly, 20 × 106 Jurkat T cells were transfected
with 5 µg of c-Myc-tagged Erk2 plasmid and 5 µg of VHR plasmid.
Empty vector was added to control samples to make constant the 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
was 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 [ Luciferase Assays--
Luciferase assays were performed as
described previously (16-19). Briefly, 20 × 106
cells were transfected with 2 µg of NFAT/AP-1-luc (or other
reporters) together with empty pEF/HA vector alone or VHR plasmids.
After stimulation for 6 h, the cells were lysed in 100 µl of 100 mM potassium phosphate, pH 7.8, 1 mM
dithiothreitol, and 0.2% Triton X-100. The final assay contained 50 µl of lysate, 100 µl of ATP solution (10 mM ATP, 35 mM glycylglycine, pH 7.8, and 20 mM
MgCl2), plus 100 µl of luciferin reagent (0.27 mM coenzyme A, 0.47 mM luciferin, 35 mM glycylglycine, pH 7.8, and 20 mM
MgCl2). The activity was measured in an automatic
luminometer (Monolight 2010, Analytical Luminescence Laboratory, Ann
Arbor, MI). The activity of a cotransfected Expression of VHR in Hematopoietic and Lymphoid Cells--
To
survey normal lymphoid cell types and organs for VHR expression, we
designed an oligonucleotide primer pair complementary to the ends of
the open reading frame to amplify VHR mRNA-derived sequences by the
PCR from a panel of cDNA libraries (CLONTECH). A fragment of the expected size (558 base pairs) was readily obtained from bone marrow, fetal liver, lymph node, peripheral blood
lymphocytes, spleen, thymus, tonsil, and the T leukemia cell line
Jurkat (Fig. 1A). The expression was
lowest in the thymus and highest in Jurkat. To verify that the
amplification products were derived from VHR, we cloned and sequenced
the fragment. The obtained sequence was 100% identical to the
published sequence of VHR (14) (data not shown).
To confirm the presence of VHR protein in hematopoietic cells, cell
lysate samples containing 70 µg of protein from isolated blood T
cells, monocytes, bone marrow leukocytes, or Jurkat T cells were
resolved by SDS-polyacrylamide gel electrophoresis, transferred to
nitrocellulose, and immunoblotted with the anti-VHR mAb. An ~21-kDa
band of very similar intensity was seen in these cells (Fig.
1A, right panels).
Generation of a HA-tagged VHR Expression Plasmid--
Next we
subcloned the sequenced open reading frame of VHR into the pEF/HA
vector, which adds an N-terminal HA tag to the insert. When the plasmid
was transfected by electroporation into Jurkat T cells, a 23-kDa
protein appeared as expected (the HA tag adds ~2 kDa). This protein
was immunoprecipitated by both an anti-HA mAb and an anti-VHR mAb and
was immunoblotted by both mAbs (Figs. 1 B and
2). In contrast, an endogenous protein of
21 kDa was immunoprecipitated and immunoblotted with only the anti-VHR
mAb (Fig. 1, B and C).
T Cell Activation Does Not Induce VHR Expression--
Because most
DSPs are inducible genes encoded by immediate early genes, we wanted to
determine whether this was also true for VHR. Jurkat T cells were
treated with 5 µg/ml of the activating anti-CD3 Active VHR Inhibits Anti-CD3 Plus Anti-CD28-induced Activation of a
Reporter Gene Taken from the Interleukin-2 Gene--
As a rapid
screening assay for a possible role of VHR in T cell activation, we
coexpressed VHR with a sensitive luciferase reporter gene, in which
luciferase transcription is under the control of a tandem NFAT/AP-1
element taken from the interleukin-2 gene promoter. This reporter
responds to T cell antigen receptor ligation with anti-CD3
In contrast to the effect of catalytically active VHR, expression of
the two catalytically inactive VHR mutants, C124S and D92A, had the
opposite effect, namely a substantial increase in gene activation in
response to anti-CD3 Active VHR Inhibits Anti-CD3 Plus Anti-CD28-induced Activation of
Jnk--
To further understand the effect of VHR on reporter gene
activation and to address the hypothesis that VHR acts on one or several members of the MAP kinase family, we measured the activation of
these kinases one by one. First, VHR was cotransfected with an
epitope-tagged Jnk2 in Jurkat T cells. Two days after transfection, the
cells were treated with anti-CD3 VHR Also Inhibits UV- and Heat Shock-induced Activation of
Jnk--
Next we tested whether VHR would also affect stress-induced
activation of Jnk2. Experiments were performed as above, but instead of
stimulating the cells with antibodies, we either irradiated them with
ultraviolet light or kept them at 45 °C for 20 min. Both treatments
activated Jnk2, and both were inhibited by active VHR (Fig. 3,
lanes 9-24). Activation of Jnk2 by heat shock was augmented
~2-fold by the catalytically inactive VHR mutant (39,203 cpm in
lane 24 versus 17,333 cpm in lane 20 incorporated
into the substrate), but the very strong activation of Jnk2 by
ultraviolet light was partly reduced by this mutant. This finding was
consistently seen in several independent experiments and may be due to
binding to Jnk2 resulting in some steric hindrance of the kinase. The amount of Jnk2 was found to be similar in all the samples (lower panels), and VHR and VHR-C124S were equally expressed in
lanes 13-16 and 21-24 (lower
panels). These results were obtained in two independent experiments.
Inhibition of Erk Activation by Active VHR--
Having established
that Jnk is sensitive to VHR, we tested Erk activation in similar
experiments. A Myc-tagged Erk2 was coexpressed with VHR or inactive
mutant VHR, and the cells were stimulated with the anti-CD3 VHR Has No Effects on Receptor- or UV-induced Activation of p38
Kinase--
p38 kinase is also activated in T cells in response to
receptor signaling and ultraviolet irradiation. However, coexpression of active or inactive VHR had no effect on these responses despite being well expressed (Fig. 4B). These results have been
obtained in several independent experiments and suggest that VHR has
specificity toward Jnk and Erk but not p38.
Effects of VHR on Downstream Targets for MAP Kinases--
To
further investigate the negative regulation of Jnk and Erk by VHR, we
decided to utilize a number of downstream targets of these kinases. In
intact cells, many of these targets are also affected by p38, but the
relative importance of the three principal MAP kinases varies
considerably from target to target. First we utilized a luciferase
reporter gene under the control of the Elk protein, which is
phosphorylated and activated mostly by Erk. To increase or decrease its
dependence on Erk, we coexpressed Erk, Jnk, or p38 with the reporter.
These experiments showed that the reporter was sensitive to VHR and
even more sensitive when the level of Erk was increased (Fig.
5A). Coexpression of p38 caused a smaller augmentation of reporter gene activation, and the
inhibition was less striking, as would be expected if p38 were more
involved relative to Erk in these cells. The effects of Jnk were
intermediate.
The activation of the c-Jun protein is principally mediated by Jnk and
can be measured using a c-Jun-GAL4 fusion protein, which controls the
expression of a cotransfected GAL4-sensitive luciferase (20). This
reporter was activated ~2-fold by stimulation of the T cells with
anti-CD3
Finally we used a luciferase reporter gene driven by a single AP-1 site
(without an adjacent NFAT element) that binds the dimeric Jun/Fos
complex referred to as AP-1. This reporter was strongly induced when
either Jnk or Erk was coexpressed, and in both cases VHR caused a
marked inhibition of the response. In contrast to the Elk reporter, the
AP-1 reporter was somewhat better responsive to Jnk and more inhibited
by VHR in the presence of Jnk. Nevertheless, all these results show
that both Erk and Jnk are similarly sensitive to inhibition by VHR,
whereas p38 is much less so, if at all.
The 185-amino acid VHR was the first human DSP to be cloned (14);
the enzyme has been crystallized, and its three-dimensional structure
has been solved (21). The structure revealed that the catalytic center
of VHR is composed of the same structural elements as in the classical
protein-tyrosine phosphatases like PTP1B (22) including the cysteine
(Cys-124) in the bottom of the catalytic cleft. Mutagenesis experiments
and kinetic analyses have confirmed that Cys-124 is essential for
catalysis (23, 24) and that Asp-92 participates in substrate
dephosphorylation by acting as proton donor for the reaction (25). The
dual specificity of VHR toward both phosphotyrosine and
phosphoserine/phosphothreonine in protein substrates is explained by
its catalytic pocket being less deep than in other protein-tyrosine
phosphatases. This allows all three phosphoamino acid residues to reach
the catalytic machinery (21). However, dephosphorylation of
phosphotyrosine is much preferred over phosphothreonine and occurs much
more rapidly (26).
Despite the detailed insights into its atomic structure and catalytic
mechanism, the physiological function of VHR has remained obscure. It
differs from the group of MAP kinase-specific DSPs in that it lacks
noncatalytic regulatory or targeting regions. For this reason, the
enzyme is also considerably smaller that other DSPs, only 21 kDa.
Nevertheless, a recent paper (27) reported that VHR can dephosphorylate
Erk1 and Erk2 and suggested that these two kinases are physiological
targets for VHR. Our findings agree with this report, but we also find
that Jnk is an equally preferred target for VHR in intact T cells. Like
Todd et al. (27), we find that p38 kinase is not affected.
It remains to be determined what gives VHR this specificity and how the
enzyme is targeted and regulated.
In T cells, the Erk kinases are activated very rapidly with peak
activities observed within minutes followed by a gradual decline
beginning within 5 min and a return to basal or near basal levels at
10-30 min. The inducible DSPs, primarily the Pac-1 protein in
lymphocytes (28), are not present at these time points but appear in
the nucleus some 30-60 min after cell stimulation. In this location,
they can dephosphorylate any remaining activated MAP kinases and
terminate the response. In fibroblasts lacking MKP-1 (29), also encoded
by an immediate early gene (30), the activation of MAP kinase also
proceeds normally. In contrast, when MKP-1 is expressed in cells under
a noninducible promoter, it strongly blocks MAP kinase activation by
active Ras (31) or by extracellular stimuli (30, 32, 33). At the early
time points, however, MAP kinase activation must be counteracted by phosphatases that are present in resting cells such as hematopoietic protein-tyrosine phosphatases (17, 18) and VHR. At present, no other
MAP kinase-specific phosphatases are known to be constitutively expressed in lymphocytes.
In its role as a negative regulator of Erk, VHR may be competing with a
number of other phosphatases (reviewed in Ref. 11). However, as a Jnk
regulator VHR is accompanied only by VH5 (M3/6 in the mouse (34)) and
perhaps by MKP-2, which dephosphorylates both Erk and Jnk with
comparable efficiency at least in vitro (35). Both of these
enzymes are inducible nuclear enzymes that may not be present in T
cells at least during the first 30-60 min after receptor stimulation.
Thus, VHR may be of particular importance in this cell type as a
negative regulator of Jnk1 and Jnk2, which are important signal
transducers in lymphocytes as well as other cell types (36, 37).
B-driven reporter was not affected. The inhibitory effects of VHR
were also seen at the level of the mitogen-activated kinases Erk1,
Erk2, Jnk1, Jnk2, and on reporter genes that directly depend on these
kinases, namely Elk, c-Jun, and activator protein-1. In contrast, p38
kinase activation was not affected by VHR, and p38-assisted gene
activation was less sensitive. Our results suggest that VHR is a
negative regulator of the Erk and Jnk pathways in T cells and,
therefore, may play a role in aspects of T lymphocyte physiology that
depend on these kinases.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
mAb were from
American Type Culture Collection (Manassas, VA). Both mAbs were used as
ascites. The mAb against CD28 was from PharMingen (San Diego, CA). The
polyclonal anti-extracellular signal-regulated kinase 2 (Erk2) was from
Santa Cruz Biotechnology Inc. A cDNA panel from hematopoietic and
lymphoid organs was from CLONTECH (Palo Alto, CA).
cDNA (from J. D. Lee, Scripps Clinic, La
Jolla, CA) was subcloned into the pEF-HA vector. GAL4-Elk was from J. Tian and G. Hauser (Burnham Institute, La Jolla, CA), and GAL4-c-Jun
and GAL4-luc were from T. Kawakami (La Jolla Institute for Allergy and
Immunology, San Diego, CA).
mAb plus anti-CD28 (9.3) mAb followed by the cross-linking with
anti-mouse Ig for 20 min at 37 °C, or UV-irradiated for 1 min at
room temperature using a transilluminator (FBTI-88,
=312 nm; Fisher
Scientific, Pittsburgh, PA). After irradiation, the cells were placed
in a 37 °C incubator for 1 h before lysis. Cells were
heat-shocked at 45 °C for 20 min.
-glycerol
phosphate, 1 mM dithiothreitol, 1 mM
phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, leupeptin, and soybean trypsin inhibitor, and the HA-tagged Jnk was
immunoprecipitated with 2 µg of anti-HA mAb (12CA5) as above. The
kinase reaction was performed for 30 min at 30 °C in 20 µl of
kinase buffer containing 25 mM HEPES, pH 7.5, 25 mM MgCl2, 25 mM
-glycerol
phosphate, 1 mM dithiothreitol, 0.1 mM
Na3VO4, 10 µM cold ATP, 5 µg of
GST-c-Jun, and 10 µCi of [
-32P]ATP. The reaction was
terminated by adding 20 µl of 2 × SDS sample buffer and heating
to 95 °C for 2 min. The samples were run on 10% SDS-polyacrylamide
gels and transferred onto nitrocellulose filters, and the labeled
proteins were visualized by autoradiography.
-32P]ATP. The
reactions were terminated by adding 20 µl of 2 × SDS sample
buffer and heating to 95 °C for 2 min. The samples were run on 12%
SDS-polyacrylamide gels were transferred onto nitrocellulose filters,
and the labeled proteins were visualized by autoradiography. The
presence of equal amounts of the immunoprecipitated Erk2 was verified
by immunoblotting using the anti-Erk2 antibody at 1:1000 dilution.
-galactosidase was
measured and used to normalize the luciferase activity for transfection efficiency.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Expression of VHR in lymphoid cells.
A, reverse transcriptase-polymerase chain reaction (RT-PCR)
assay for the expression of VHR mRNA in a panel of lymphoid tissues
and cell types (1 ng of DNA/sample). The band at 558 base pairs in the
upper panel is VHR, whereas the ~1-kilobase band in the lower
panel is the housekeeping enzyme glycerol-3-phosphate
dehydrogenase, a positive control. Right-hand panels
are anti-VHR mAb immunoblots of lysates of the indicated cells (70 µg
of protein/sample). B, anti-VHR immunoblot of
immunoprecipitates obtained with anti-HA or anti-VHR mAbs as indicated
from Jurkat T cells transfected with pEF-HA vector alone (lanes
1 and 2) or with HA-tagged VHR (lanes 3 and
4). C, anti-VHR immunoblot of Jurkat T cells incubated for
the indicated times at 37 °C with medium alone (upper
panel) or in the presence of 10 µg/ml anti-CD3 mAb and
anti-CD28 (lower panel). D, similar experiments
with normal blood T lymphocytes. Note that the amount of VHR remains
constant. PBL, peripheral blood lymphocytes, PBML, peripheral
blood mononuclear leukocytes.
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Fig. 2.
Inhibition of T cell antigen receptor-induced
NFAT/AP-1 activation by VHR. A, activation of a
luciferase reporter gene driven by NFAT/AP-1 (NFAT/AP-1 luc)
at different doses of VHR plasmid. The data represent the mean from
duplicate determinations in a representative of three independent
experiments. The inset is an anti-HA blot to show expression
of VHR in the transfectants (upper panel). B,
activation of the same reporter in cells stimulated with different
amounts of anti-CD3 mAb. The inset is an anti-HA blot of the
same cells (upper panel). C, activation of the
same reporter gene in cells cotransfected with 3 µg of the indicated
VHR plasmids. The inset is an anti-HA blot of the same cells
(upper panel). D, activation of the same reporter
gene by phorbol ester and/or ionomycin as indicated. The
inset is an anti-HA blot of the same cells (upper
panel). PMA, phorbol 12-myristate 13-acetate.
WT, wild type. E, activation of a nuclear factor
B-driven luciferase reporter gene (NF-
B
luc) in cells cotransfected with the indicated amount of VHR
plasmid. All luciferase activities are given as arbitrary light units
(arb. units), generally 1/1000 of the luminometer readout
values, and they were normalized for transfection efficiency using the
activity of a cotransfected
-galactosidase.
mAb OKT3 plus the
anti-CD28 mAb for 0-48 h and then immunoblotted with the anti-VHR mAb.
The amount of VHR immunoreactivity remained completely unchanged during
these experiments (Fig. 1C). The same experiment was also
performed with normal blood T cells with the same result (Fig.
1D). Thus, VHR is constitutively expressed in T cells prior
to receptor stimulation and is not induced.
mAbs
alone or together with coligation of the CD28 costimulatory molecule.
When these experiments were first performed with different amounts of
VHR plasmid, we observed that VHR inhibited the response in a
dose-dependent manner with a sharp drop in gene activation
between 2 and 5 µg of plasmid (Fig. 2A). The expression of
VHR protein correlated with the DNA dose (inset). A
titration of the amount of anti-CD3
showed that VHR did not shift
the anti-CD3
dose-response (e.g. by desensitizing the receptor) but
inhibited it to an equal degree at all doses (Fig. 2B).
or anti-CD3
plus anti-CD28 (Fig.
2C). This dominant-negative effect may result from a
displacement of endogenous, active VHR. If so, our results support the
notion that endogenous VHR suppresses reporter gene activation.
Catalytically active VHR also inhibited reporter gene activation
induced by phorbol ester and ionomycin, whereas the inactive C124S
mutant augmented it (Fig. 2D). In support of a role of VHR
in the signaling pathways that up-regulate NFAT/AP-1-driven
transcription, we found that coexpression of VHR with a luciferase
reporter under the control of a nuclear factor
B element had a small
stimulatory effect on its activation (Fig. 2E). Because
nuclear factor
B induction, in contrast to NFAT/AP-1, was not
inhibited in a dose-dependent manner, we conclude that VHR
does not inhibit this pathway even when expressed at higher levels. The
small stimulatory effect is of questionable significance but could be
secondary to inhibition of other pathways.
plus anti-CD28 on ice for 15 min,
washed twice with cold RPMI, and then incubated at 37 °C for 20 min
in the presence of a cross-linking sheep anti-mouse Ig antibody. After
cell lysis, the HA-tagged Jnk was immunoprecipitated with the 12CA5
anti-HA mAb and subjected to in vitro kinase assays using
GST-c-Jun as a substrate. These experiments revealed that expression of
active VHR inhibited the activation of Jnk, whereas the catalytically
inactive mutant had little effect (Fig.
3, lanes 1-8). The amount of
Jnk2 was found to be similar in all the samples (Fig. 3, lower
panel), and the VHR proteins were equally expressed in lanes
5-8 (lower panel). This result was obtained
in several independent experiments, and very similar results were
obtained with Jnk1.
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Fig. 3.
Inhibition of Jnk activation by VHR.
In vitro kinase assays (upper panels) with
GST-c-Jun N terminus as a substrate of HA-tagged Jnk2
immunoprecipitated from Jurkat T cells transfected with the indicated
plasmids and treated with medium (odd lane numbers),
anti-CD3e plus anti-CD28 (lanes 2, 4, 6, and 8),
UV irradiation (lanes 10, 12, 14, and 16), or
heat shock at 45 °C (lanes 18, 20, 22, and 24)
for 20 min. The expressions of Jnk2 and VHR are shown by anti-HA
immunoblots of lysates from the same transfectants (lower
panels).
mAb for
5 min (anti-CD28 is not required). After cell lysis, Erk2 was
immunoprecipitated with the 9E10 anti-Myc mAb and subjected to an
in vitro kinase reaction with myelin basic protein as a
substrate. These experiments revealed that Erk activation was reduced
by active VHR but not by the inactive mutant (Fig. 4A). However, in most
experiments, the inhibition was not as marked as the inhibition
observed for Jnk. The amount of Erk was equal in all samples (Fig.
4A, middle panel), and VHR was well
expressed (Fig. 4A, bottom panel). This result
was obtained in several additional experiments.
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Fig. 4.
VHR inhibits Erk activation but not p38
kinase activation. A, in vitro
kinase assay with myelin basic protein (MBP) as a substrate
of Myc-tagged Erk2 immunoprecipitated from Jurkat T cells transfected
with the indicated plasmids and treated with medium (odd lane
numbers) or anti-CD3 mAb (even lane numbers) for 5 min. Equal amounts of Erk in the assay were verified by anti-Erk
immunoblotting of the same filter (middle panel) and the
expression of VHR by anti-HA immunoblotting of lysates from the same
transfectants (lower panel). B, in
vitro kinase assay (upper panel) with GST-ATF2 as a
substrate of HA-tagged p38 immunoprecipitated from Jurkat T cells
transfected with the indicated plasmids and treated with medium alone
(odd lanes), UV irradiation (lanes 2, 4, 6, and
8), or anti-CD3
(lanes 10, 12, 14, and
16) for 5 min. Equal expression was verified by anti-HA
immunoblotting of lysates from the same transfectants (lower
panel).
View larger version (50K):
[in a new window]
Fig. 5.
Inhibition of downstream targets for MAP
kinases by VHR. A, activation of a luciferase reporter
gene driven by Elk (Elk-GAL4-luc). The data represent the
mean ± S.D. from triplicate determinations. The right-hand
panels represent an anti-Erk2 blot (upper panel), a
longer exposure of the anti-HA blot to show Jnk (middle
panel), and a shorter exposure of the anti-HA blot to show p38 and
VHR (lower panel) of the lysates of the transfectants. Note
that endogenous Erk with a slightly lower Mr is
visible in all lanes in the anti-Erk2 blot. B, activation of
a c-Jun-induced luciferase reporter gene (c-Jun-GAL4 luc).
The data represent the mean ± S.D. from triplicate
determinations. The right-hand panel is an anti-HA blot to
show Jnk and VHR. C, activation of a luciferase reporter
gene driven by AP-1 (AP-1 luc). The data represent the
mean ± S.D. from triplicate determinations. The right-hand
panel is an anti-HA blot to show Jnk1, Erk1, and VHR.
plus anti-CD28 but was much augmented by coexpression of
Jnk1 or Jnk2 (Fig. 5B). In both cases, a coexpressed VHR
caused a strong inhibition of the reporter.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
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We are grateful to Drs. Gottfried Baier, J. D. Lee, Jianmin Tian, Greg Hauser, and Toshiaki Kawakami for the kind gift of reagents.
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FOOTNOTES |
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* This work was supported by a fellowship from the Spanish Ministries of Education and Culture and by Grants AI48032, AI35603, AI41481, and AI40552 from the National Institutes of Health.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.
Current address: Gemini Science Inc., 10355 Science Center Dr.,
San Diego, CA 92121.
§ To whom correspondence should be addressed: Laboratory of Signal Transduction, La Jolla Cancer Research Center, The Burnham Institute, 10901 North Torrey Pines Road, La Jolla, CA 92037. Tel.: 858-713-6270; E-mail: tmustelin@burnhman-inst.org.
Published, JBC Papers in Press, November 20, 2000, DOI 10.1074/jbc.M006497200
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ABBREVIATIONS |
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The abbreviations used are: MAP kinase, mitogen-activated protein kinase; NFAT, nuclear factor of activated T cells; AP-1, activator protein-1; Erk, extracellular signal-regulated protein kinase; Jnk, c-Jun N-terminal kinase; AP-1, activator protein-1; DSP, dual specific protein phosphatase; VHR, VH1-related; mAb, monoclonal antibody; PCR, polymerase chain reaction; HA, hemagglutinin; GST, glutathione S-transferase.
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REFERENCES |
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