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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 91 No. 11 (June 1), 1998:
pp. 4232-4241
T-Cell Receptor Signaling Pathway Exerts a Negative Control on
Thrombin-Mediated Increase in [Ca2+]i and
p38 MAPK Activation in Jurkat T Cells: Implication of the Tyrosine
Kinase p56Lck
By
Laurence Maulon,
Sandrine Guérin,
Jean-Ehrland Ricci,
Dariush FarahiFar, Jean-Philippe Breittmayer, and
Patrick Auberger
From CJF INSERM 96.05, Activation des Cellules
Hématopoiétiques, Faculté de Médecine, Nice
Cédex, France; INSERM U343, Hôpital de l'Archet, Nice,
France; and INSERM U364, Faculté de Médecine, Nice
Cédex, France.
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ABSTRACT |
Activation of the mitogen-activated protein kinase (Erk) and c-Jun
terminal kinase is a well-documented mechanism for the seven
transmembrane spanning receptors. We have previously shown that
thrombin stimulation of the T-leukemic cell line Jurkat induced a
transient increase in [Ca2+]i and tyrosine
phosphorylation of several cellular proteins. Here, we
have analyzed p42-44 MAPK, JNK and p38 MAPK activation using Jurkat
T-cell lines deficient in either the tyrosine kinase p56Lck (JCaM1) or
the tyrosine phosphatase CD45 (J45.01). Our results demonstrate that
p56Lck and CD45 exert a negative control on thrombin-induced p38 MAPK
activation and [Ca2+]i release in Jurkat
cells. Thrombin receptor expression was identical on the different cell
lines as assessed by FACS analysis. Tyrosine phosphorylation of p38
MAPK was drastically increased after thrombin stimulation of JCaM1 or
J45.01 cells, as compared with parental cells (JE6.1). P42-44 MAPK and
JNK activity also enhanced after thrombin treatment of JE6.1 and JCaM1
cell lines, whereas basal kinase activity was higher in J45.01 cells
and was not further stimulated by thrombin. Thrombin and thrombin
receptor agonist peptide-induced [Ca2+]i
mobilization paralleled p38 MAPK activation in JCaM1 and J45.01 cells.
Moreover, reconstitution of J45.01 and JCaM1 cell lines with either
CD45 or Lck is accompanied by restoration of a normal thrombin-induced
[Ca2+]i response and p38MAPK
phosphorylation. These data show that a component of the T-cell
receptor signaling pathway exerts a negative control on
thrombin-induced responses in Jurkat T cells. Accordingly, we found
that thrombin enhanced tyrosine phosphorylation of p56Lck and decreased
p56Lck kinase activity in J45.01 cells. Our results are consistent with
a negative role for p56Lck on thrombin-induced
[Ca2+]i release and p38 MAPK activation in
Jurkat T-cell lines.
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INTRODUCTION |
THE THROMBIN RECEPTOR is a member of the
seven transmembrane receptor family that can transduce mitogenic
stimulation by coupling to heterotrimeric G proteins, Gi and/or
Gq,1-4 leading to activation of adenylate cyclase and
phospholipase C respectively. Receptor activation occurs through
proteolysis by thrombin at a specific site in the N-terminal portion of
the receptor unmasking a sequence that functions as a ligand for the
receptor.5,6 Synthetic peptides corresponding to this
sequence mimic the action of thrombin.6-9 T-leukemic cell
lines stimulated with thrombin or the thrombin receptor agonist peptide
show increased cytoplasmic free calcium, phospholipase C stimulation,
protein kinase C activation and as a consequence NF B
activation.1,10 We have previously shown that thrombin and
thrombin receptor agonist peptide-stimulated tyrosine phosphorylation
of several cellular proteins and more particularly proteins with
molecular mass of 38, 42, 56, and 70 kD in Jurkat T cells.1
Owing to the well-established effect of thrombin on p42-44 MAPK in
different cell lines,11-14 it is likely that the two former
proteins corresponded to members of the MAP kinase family.
The MAP kinases refer to a family of apparented
serine/threonine kinases, including p42-44 MAPK encoded by the Erk2 and
Erk1 gene that are activated by tyrosine and threonine phosphorylation in response to mitogenic stimuli15-17 and the JNK and p38
MAPK, which are also activated by tyrosine and threonine
phosphorylation after stress or triggering of heterotrimeric
G-protein-coupled seven-transmembrane-spanning
receptors.18,19 These latters included both receptors that
couple to Gi or to Gq, or both. In this line,
it has been proposed that Gq -mediated MAPK activation is PKC
dependent and p21ras independent,20 whereas the
Gi-coupled pathway is G  mediated, p21ras
dependent, and PKC-independent.21,22 In addition, thrombin has been found to increase tyrosine kinase activity of src kinases in
human platelets and hamster CCL39 fibroblasts.23,24 It is also well established that Src family members are involved in the
activation of the Ras signaling pathway through adaptors molecules such
as Shc24 or p36 in T cells.25 Furthermore, it
was recently proposed that thrombin induced a transient rise in both
p38 MAPK tyrosine phosphorylation and activity in platelets, suggesting a role for this MAPK in thrombin-mediated signaling events in platelets.26
This study took advantage of the availability of different Jurkat cell
lines deficient in either the tyrosine kinase p56Lck or the tyrosine
phosphatase CD45 to investigate the role of components of the T-cell
receptor (TCR) signaling pathway on thrombin responses in Jurkat cell
lines. We found that the tyrosine kinase p56Lck exerted a negative
regulation on thrombin-induced [Ca2+]i
release and p38 MAPK activation in Jurkat T cells.
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MATERIALS AND METHODS |
Cells and reagents.
Jurkat leukemic cell lines JCaM1, J45.01, JE6.1, and J45/CD45 (clone
LB3.3-3) were kindly provided by Arthur Weiss (University of
California, San Francisco) and have been described
elsewhere.27 JCaM1/Lck were obtained from Jean Philippe
Breittmayer (INSERM U343, Nice, France). Cells were maintained in RPMI
1640 medium/5% fetal calf serum (FCS) at 37°C, as previously
described.2 Biotin-conjugated (4G10) antiphosphotyrosine
antibody was purchased from UBI (Upstate Biotechnology,
Lake Placid, NY), phospho-p38 MAPK (Tyr 182) or (Thr 180, Tyr 182), and
phospho-p44/42 MAPK (Tyr 204) antibodies from New England Biolabs
(Beverly, MA), anti-p56Lck from Santa Cruz Biotechnology (Santa Cruz,
CA). Peroxidase-conjugated secondary antibody was
purchased from DAKO. P38 MAPK and JNK antibodies were kind gifts of
Benoit Derijeard (Centre de Biochimie CNRS-INSERM, Nice, France). ATAP2
MoAb was a generous gift from Lawrence F. Brass (University of
Pennsylvania, Philadelphia). Bovine thrombin was obtained from Sigma
and the highly potent thrombin receptor agonist peptide
Ala-pfluoro-Phe-Arg-Cha-homo-Arg-Tyr-NH228 was
purchased from Neosystem (Strasbourg, France).
Flow cytometry.
Cells were first incubated with the antithrombin receptor MoAb ATAP2
(1/200, 30 minutes, 4°C) followed by a biotin-conjugated goat
anti-mouse IgG secondary antibody (1/500, 30 minutes, 4°C) and by
streptavidin-phycoerythrin (1/500, 30 minutes 4°C). Analyses were
performed by flow cytometry on a FACScan flow cytometer (Becton Dickinson, Mountain View, CA).
Cytosolic-free Ca2+ measurements.
Cytoplasmic free calcium levels were determined using the fluorescent
dye indo-1 and an ATC 3000 cytofluorograph, as previously described.29-31 Briefly, cells were incubated for 1 hour
with 4 µmol/L indo-1 in a buffer containing 140 mmol/L NaCl, 5 mmol/L KCl, 0.7 mmol/L MgCl2, 0.7 mmol/L CaCl2, 20 mmol/L HEPES, 10 mmol/L glucose, and 0.1% bovine serum albumin (BSA)
(pH 7.4) (calcium buffer) at a final concentration of 5 × 106 cells/mL. After a fivefold dilution in the
same medium, the mean violet/blue ratio of 3,000 cells was determined
every 15 seconds after the addition of effectors.
Tyrosine protein phosphorylation and immunoblotting analysis.
Jurkat cells (3 × 106 cells/condition) were
stimulated without or with either 100 nmol/L thrombin or 2.5 µmol/L
thrombin receptor agonist peptide for the indicated times at 37°C.
Stimulation was terminated by chilling rapidly the cells in liquid
nitrogen. Cells were solubilized in lysis buffer containing 50 mmol
HEPES, pH 7.4, 150 mmol/L NaCl, 20 mmol/L EDTA, 10 mmol/L sodium
orthovanadate, 100 mmol/L NaF, 1% Nonidet P-40 (NP-40), and a cocktail
of protease inhibitors (5 µg/mL aprotinin, 1 mmol/L
phenylmethylsulfonyl fluoride, 1 µmol/L pepstatin) for 30 minutes on
ice and then centrifuged at 4°C for 15 minutes at 13,000 rpm.
Supernatants were analyzed on sodium dodecyl sulfate
(SDS)-polyacrylamide gels and transferred to Immobilon membrane
(Millipore, Bedford, MA). Membranes were then blocked for 2 hours at
room temperature with 3% (wt/vol) BSA and probed overnight with
biotin-conjugated (4G10) antiphosphotyrosine antibody or
phospho-specific p38 MAPK antibody or phospho-specific p44/42 MAPK
antibodies. Blots were further incubated with horseradish peroxidase
(HRP)-conjugated secondary antibody and immunoreactivity was detected
by enhanced chemiluminescence.
P38 MAPK, p42-44 MAPK, JNK, and p56Lck activity assays.
Cell lysates were prepared as described earlier. After centrifugation,
supernatants were incubated at 4°C for 18 hours with anti-p38 MAPK,
anti-JNK, anti-p42/44 MAPK, or anti-p56Lck antibodies preadsorbed to
protein A-Sepharose. Immune complexes were washed four times with lysis
buffer and once with kinase buffer A (20 mmol/L HEPES, pH 7.4, 10 mmol/L MgCl2, 1 mmol/L dithiothreitol, 10 mmol/L
p-nitrophenyl phosphate). Beads were finally resuspended in 40 µL
kinase buffer containing 5 µg of recombinant ATF2 (p38 MAPK and JNK)
or 5 µg of MBP (p42-44 MAPK) and [-32P]ATP (50 µmol/L, 5 µCi). Reactions were initiated by addition of ATP. After
incubation at 30°C for 30 minutes, assays were terminated by the
addition of 4 µL of 10× Laemmli sample buffer. The samples were
heated at 95°C for 5 minutes and analysed by SDS/12%
polyacrylamide gel electropheresis (PAGE). The gels were dried and
subjected to autoradiography. P56Lck immunoprecipitates were performed
as described earlier, except that the incubation was performed in buffer B (50 mmol/L HEPES, pH 6.8, 5 mmol/L MnCl2, 5 mmol/L
MgCl2) containing 10 µg of enolase.
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RESULTS |
Cell-surface thrombin receptor expression.
Expression of the thrombin receptor on different Jurkat cell lines was
assessed by flow cytometry, using the monoclonal antibody (MoAb) ATAP2.
Thrombin receptor was observed on the surface of the different Jurkat
cell lines JE6.1 (parental) and variant clones JCaM1, JCaM1/Lck,
J45.01, and J45.01/CD45 with a similar level of expression
(Fig 1).
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| Fig 1.
Thrombin receptor expression in different Jurkat cell
clones. Cells were first stained with the antihuman thrombin receptor ATAP2 MoAb (black area) or an isotype matched control antibody (white
area), followed by biotinylated goat antimouse antibody and
phycoerythrin-streptavidin. Stained cells were analyzed with a flow
cytometer FACScan (Becton Dickinson) gated to eliminate nonviable
cells.
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Thrombin and thrombin receptor agonist peptide induced a rapid
increase in cellular protein tyrosine phosphorylation in different
Jurkat cell lines.
Parental cell line JE6.1, p56Lck-deficient cell line JCaM1, and
CD45-deficient cell line J45.01 were incubated for different times at
37°C with either 100 nmol/L thrombin or 2.5 µmol/L of the highly
potent thrombin receptor agonist peptide
Ala-pFluoro-Phe-Arg-Cha-homo-Arg-Tyr-NH2.28 Cell lysates were immediately prepared and analysed by immunoblotting with the antiphosphotyrosine antibody 4G10.
Figure 2A shows that thrombin induced a
rapid and significant increase in the tyrosine phosphorylation of
proteins with apparent molecular weights of 38 and 36 kD. Tyrosine
phosphorylation of these proteins was observed within 15 seconds and
was maintained for at least 1 minute. Significant differences in the
level of tyrosine phosphorylation of these proteins as well as other
(more particularly in the 60-kD region) were, however, observed in the
three cell lines. Indeed, stimulation of p38 phosphorylation was only
observed in JCaM1 and J45.01 cell lines, whereas phosphorylation of a
p42/44-kD protein was observed to various extent in the three cell
lines. Moreover, the phosphorylation of a 36-kD protein increased
drastically in both deficient clones as compared with JE6.1. The same
results were obtained when these different cell lines were stimulated
by the thrombin receptor agonist peptide (Fig 2B).
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| Fig 2.
Induction of tyrosine phosphorylation of cellular
proteins by thrombin and thrombin receptor agonist peptide in different Jurkat cell clones. Cells were stimulated for the indicated times with
100 nmol/L thrombin (A) or 2.5 µmol/L thrombin receptor agonist peptide (B). Proteins from cell lysates were separated by SDS-PAGE and
transferred to Immobilon membranes for Western blotting with biotin-conjugated 4G10 antibody. Blots were further incubated with
horseradish peroxidase-conjugated secondary antibody and immunoreactivity was detected by enhanced chemiluminescence.
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Characterization of the low-molecular-weight
thrombin-stimulated tyrosine-phosphorylated proteins.
Several thrombin-stimulated tyrosine-phosphorylated proteins have been
identified in different studies, including p42-44 MAPK11 and p38 MAPK in human platelets.32 To identify the proteins with apparent molecular weight of 38 and 42 to 44 kD, we used specific
antiphosphotyrosine antibodies, that specifically recognized tyrosine
phosphorylated p42-44 MAPK and p38 MAPK.
Figure 3 shows tyrosine phosphorylation of
p42-44 MAPK after thrombin and thrombin receptor agonist peptide
stimulation. Thrombin (100 nmol/L) and thrombin receptor agonist
peptide (2.5 µmol/L) poorly stimulated tyrosine phosphorylation of
p42-44 MAPK in JE6.1. By contrast, thrombin induced a significant
increase in p42-44 MAPK tyrosine phosphorylation in J45.01 and in
JCaM1 (Fig 3A). P42-44 MAPK was phosphorylated on tyrosine residues
within 15 seconds. Identical results were obtained with thrombin
receptor agonist peptide (Fig 3B), even though the ratio of p44 versus p42 phosphorylation was different in thrombin receptor agonist peptide-stimulated cells versus thrombin-treated cells.
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| Fig 3.
Effect of thrombin and thrombin receptor agonist peptide
on p42-44 MAPK tyrosine phosphorylation in different Jurkat clones. Cells were stimulated for the times indicated with 100 nmol/L thrombin
(A) or 2.5 µmol/L thrombin receptor agonist peptide (B). Proteins
from cell lysates were separated by SDS-PAGE and transferred to
Immobilon membranes for Western blotting with phospho-specific p42-44
MAPK antibody. Development was performed as described in Fig 1.
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Tyrosine phosphorylation of p38 MAPK after thrombin or thrombin
receptor agonist peptide treatment was also assessed using highly
specific antiphosphotyrosine p38 MAPK antibody. Thrombin (Fig 4A) and thrombin receptor agonist
peptide (Fig 4B) induced a significant increase in p38 MAPK tyrosine
phosphorylation in both JCaM1 and J45.01 cells after a 1-minute
incubation. As expected from the results of Fig 2, p38 MAPK tyrosine
phosphorylation was barely detectable in JE6.1 after thrombin or
thrombin receptor agonist peptide stimulation. Increase in the
phosphorylation of p38 MAPK cannot be accounted for by differences in
the level of p38 MAPK, as approximatively the same amounts of p38 MAPK
were detected by Western blot in each condition (Fig 4C). Densitometric scanning of the p38 phosphorylated band in the different Jurkat clones
is shown in Fig 4D. There was a significant increase in p38MAPK
phosphorylation in thrombin and agonist peptide induced JCaM1 and
J45.01 clones, as compared with JE6.1 cells, even though basal p38MAPK
phosphorylation was higher in agonist peptide-treated cells. Thrombin
induced a rapid activation of p42-44 MAPK, P38 MAPK, and JNK in Jurkat
cell lines.
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| Fig 4.
Effect of thrombin and thrombin receptor agonist peptide
on p38 MAPK tyrosine phosphorylation. Cells were stimulated for the times indicated with 100 nmol/L thrombin (A) or 2.5 µmol/L thrombin receptor agonist peptide (B). Proteins from cell lysates were subjected
to SDS-PAGE as described above and transferred to Immobilon membranes
for Western blotting using phospho-specific p38 MAPK antibody. Equal
amounts of p38 MAPK were detected in each condition (C). Densitometric
scanning of the p38 phosphorylated band present in (A)
(thrombin-stimulated Jurkat clones) and (B) (thrombin receptor agonist
peptide stimulated Jurkat clones) are shown. Results are representative
of three experiments.
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Thrombin induced a rapid increase in p42-44 MAPK activity in JCaM1 and
JE6.1 within 15 seconds of stimulation. In J45.01, basal level of
p42-44 MAPK activity was elevated, and thrombin failed to stimulate
Erk1/Erk2 further (Fig 5A). Surprisingly, although p42-44 tyrosine phosphorylation was poorly detected upon thrombin treatment of JE6.1 cells (Fig 3), kinase activity was significantly increased (Fig 5A). However, this increase in MAP kinase
activity could reflect phosphorylation on Thr of p42-44 MAPK, as the
antibody used in the experiment presented in Fig 3 recognized
specifically the tyrosine-phosphorylated form of p42-44 MAPK. In JCaM1,
p42-44 MAPK tyrosine phosphorylation correlated with the kinase
activity. However, we did not find a strict correlation between p42-44
MAPK phosphorylation and activity in both J45.01 and JE6.1 clones. Here
again, phosphorylation on threonine residues might explain some
discrepancies observed between tyrosine phosphorylation of p42-44 MAPK
and kinase activity, but the simplest explanation for these differences
is likely to come from the different features of the antibodies used in
each type of experiment. Nevertheless, variations in kinase activity
were not caused by differences in the amount of proteins loaded on the
gels (Fig 5A, bottom).
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| Fig 5.
Effect of thrombin on p42/44, p38 MAPK, and Jun kinase
activities in different Jurkat clones. Cells were stimulated with 100 nmol/L thrombin for the times indicated. Cell lysates were prepared as
described in Materials and Methods and incubated overnight with
anti-p42/44 MAPK (A) , anti-p38 MAPK (B), or anti-JNK antibodies (C)
preadsorbed to protein A-Sepharose. Immune complexes were washed and kinase activities were determined using myelin basic protein
for p42-44 MAPK and ATF2-GST for p38 MAPK and JNK. Equal amounts of
p42/44 MAPK and p38 MAPK were immunoprecipitated in each condition (A
and B, bottom).
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We then looked for p38 MAPK activity in thrombin-stimulated Jurkat
cells. Basal p38 MAPK activity was higher in J45.01 cells than in JE6.1
or JCaM1 cells (Fig 5B). This higher basal activity could perfectly be
explained by the higher level of phosphorylated p38 MAPK in J45. 01 cells (Fig 4A). Thrombin failed to induce P38 MAPK activity in JE6.1
according to the fact that p38 MAPK was not phosphorylated in these
cells (Figs 2 and 4). Conversely, thrombin induced a significant
increase in p38 MAPK activity in both deficient cell lines.
Because p38 MAPK is known to be activated by proinflammatory cytokines
and environmental stress,33 we also determined JNK activity
after thrombin treatment of Jurkat cell variants. As shown in Fig 5C,
thrombin was found to increase JNK activity significantly, whatever the
clones used.
P56Lck exerts a negative control on thrombin-induced p38 MAPK
activation in Jurkat cells.
As p56Lck is critical for signaling via the TcR34 and TcR
triggering induced a total loss of thrombin response,1 we
sought to analyze the effect of thrombin on p56Lck phosphorylation and activity. The level of p56Lck phosphorylation was visualized after a
1-minute thrombin stimulation after immunoprecipitation, followed by
Western blotting with a rabbit polyclonal anti-p56Lck antibody. Thrombin stimulated p56Lck tyrosine phosphorylation in J45.01 cells
within 15 seconds, whereas level of p56Lck phosphorylation remained
unchanged in JE6.1 (Fig 6A). As expected,
p56Lck phosphorylation was undectectable in JCaM1 cells (Fig 6A). Basal
p56Lck phosphorylation was approximatively twofold lesser in J45.01
cells as compared with JE6.1 cells. Furthermore, thrombin-induced
phosphorylation of p56Lck was accompanied by a significant decrease in
p56Lck autophosphorylation (50% to 80%) and kinase activity (40% to
50%) in J45.01 cells, as judged by the phosphorylation of enolase (Fig 6B). Again inhibition of p56Lck kinase activity in J45.01 cells did not
reflect differences in the amount of immunoprecipitated p56Lck (Fig
6C).
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| Fig 6.
p56Lck tyrosine phosphorylation and activity in
different Jurkat cell clones. Cells were stimulated with 100 nmol/L
thrombin for the times indicated. (A) Cell lysates were
immunoprecipitated with 4G10 antibody preadsorbed on protein
A-Sepharose. Immunoprecipitated proteins were subjected to
SDS-PAGE and transferred to Immobilon membranes for Western blotting
with anti-p56Lck antibody. Development was performed
as described above. (B) Cell lysates were immunoprecipitated with
anti-p56Lck antibody preadsorbed to protein A-Sepharose. After
extensive washing, p56Lck activity was determined with enolase as
exogenous substrate. (C) Equal amounts of p56Lck were
immunoprecipitated in JE6.1 and J45.01 cells. Note the lack
of p56Lck and p56Lck activity in JCaM1.
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Thrombin-induced [Ca2+]i release is
increased in J45.01 and JCaM1 cells.
Figure 7 depicts a time course of
[Ca2+]i release after thrombin and thrombin
receptor agonist peptide stimulation. Thrombin induced a transient
increase in [Ca2+]i, with a peak at 45 seconds, in the three cell lines. This rapid increase in
[Ca2+]i after thrombin stimulation has been
ascribed solely to the release from internal stores1,2 and
is mediated by the heterotrimeric pertussis toxin insensitive Gq
protein.1 However, the peak and amount of
[Ca2+]i released were drastically increased
(twofold to threefold) in J45.01 and JCaM1 cells as compared with
parental cell lines JE6.1 (Fig 7A). Maximal stimulation was observed
for 20 nmol/L thrombin, whatever the cell lines (Fig 7B). The kinetics
and dose-response curves for thrombin receptor agonist peptide-induced
[Ca2+ ]i release in the three cell types were
identical to those observed in the presence of thrombin (Fig 7C and D).
However, [Ca2+]i increase was always
significantly higher in J45.01 cells, as compared with JCaM1 cells (Fig
7A through D).
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| Fig 7.
Effect of thrombin and thrombin receptor agonist peptide
on [Ca2+]i in Jurkat clones deficient in
either the tyrosine kinase p56Lck or tyrosine phosphatase CD45. The
cell lines used were the leukemic cell line Jurkat (clone JE6.1,  )
and two variants lacking p56Lck (JCaM1,  ) or the CD45 molecule
(J45.01, timesb). Cells were loaded with indo-1 as described in Materials
and Methods. Thrombin 100 nmol/L or thrombin receptor agonist peptide
(2.5 µmol/L) was added at 0 time and fluorescence monitored as a
function of time. (A and C) Time course of thrombin and thrombin
receptor agonist peptide effects. (B and D) Dose-response curves for
thrombin (2 to 100 nmol/L) and thrombin receptor agonist peptide (0.1 to 2 µmol/L) effects. Results are representative of three different experiments.
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Reconstitution of JCaM1 and J45.01 cells.
We then looked for thrombin-induced [Ca2+]i
response and p38 MAPK phosphorylation after retransfection of CD45 and
Lck-deficient Jurkat cell lines. Consistent with the results depicted
in Fig 7, J45.01 and JCaM1 cell lines exhibited a huge increase in
thrombin-induced [Ca2+]i response, as
compared with JE6.1 cells (Fig 8A). This
response was lost in cells stably retransfected with CD45 and Lck.
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| Fig 8.
Effect of thrombin on CD45 and Lck reconstituted
deficient clones. (A) Thrombin-induced
[Ca2+]i responses. JE6.1 ( ), J45.01
( ), J45.01/CD45 ( ), JCaM1 (), or JCaM1/Lck ( ) were
stimulated with 100 nmol/L thrombin and fluorescence analyzed as a
function of time. (B and C) Thrombin-mediated phosphorylation of
p38MAPK. Cells were stimulated with 100 nmol/L thrombin for the times
indicated. Proteins from cell lysates were subjected to SDS-PAGE as
described in the legend to Fig 4 and were transferred to Immobilon
membranes for Western blotting using phospho-specific p38 MAPK
antibody. Equal amounts of p38 MAPK were detected in each condition
(not shown).
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Moreover, there was a good correlation between thrombin-induced
[Ca2+]i response and p38 MAPK
phosphorylation. Indeed, reconstitution of J45.01 and JCaM1-deficient
cell lines with either CD45 or p56Lck was accompanied by loss of
thrombin-mediated p38MAPK phosphorylation (Fig 8B and C).
| |
DISCUSSION |
Thrombin effects on platelets, fibroblasts, and vascular smooth muscle
cells have been well documented.35 Thrombin plays a central
role on platelets, causing aggregation and secretion of granules and on
fibroblasts, eliciting mitogenic responses. On vascular smooth muscle
cells, thrombin enhances endothelial permeability and induces the
production of both inflammatory factors36 and growth
factors.37 Although thrombin also causes chemotaxis and
adhesion of monocytes and neutrophiles,38 its action on components of the immune system has remained poorly documented. The
recent characterization of the thrombin receptor on T lymphocytes or
T-leukemic cell lines underscores the potential role of thrombin at
sites of hemostatic stress and inflammation.1,2,10,39
We have examined signaling events after thrombin stimulation of Jurkat
cell lines either deficient in the p56Lck tyrosine kinase or the CD45
tyrosine phosphatase. We report here that thrombin activated the three
MAPK pathways in Jurkat cells. P42-44 MAPK activation is a
well-characterized mechanism for G-protein-coupled receptor.20,40,41 Accordingly, we show that thrombin
induced tyrosine phosphorylation and activation of p42-44 MAPK in
Jurkat T-cell lines. As previously reported in platelets,11
thrombin responses are rapid and transient, maximal within 15 seconds
after stimulation with the protease or the thrombin receptor agonist peptide. P42-44 MAPK and JNK activation were observed to various extent
both in p56Lck and CD45-deficient cell lines and in parental cell
lines, suggesting that thrombin-induced p42-44 MAPK and JNK activity
might be largely independent on p56Lck or CD45 expression.
Conversely, phosphorylation and activation of p38 MAPK was drastically
higher in p56Lck and CD45-deficient cell lines, suggesting that one or
more components of the TcR signaling pathway exert a negative control
on thrombin-induced p38 MAPK activation in Jurkat cells. Furthemore,
thrombin stimulation of Jurkat cells resulted in an increase in
[Ca2+]i release, which was drastically
enhanced in cell lines deficient in either the p56Lck or the CD45
molecule. In addition, thrombin and thrombin receptor agonist
peptide-induced [Ca2+]i release was
significantly higher in J45.01 cells than in JCaM1 cells. The reasons
for which J45.01 cells exibited higher levels of
[Ca2+]i than JCaM1 cells in response to
thrombin or thrombin receptor agonist peptide is unknown. This effect
cannot be accounted for by differences in thrombin receptor expression
in JE6.1, JCaM1, JCaM1/Lck, J45.01, or J45.01/CD45 clones. However, one
may supposed that besides p56Lck other signaling molecules regulated by
the CD45 tyrosine phosphatase may exert a negative control on thrombin responses in Jurkat cells, a good candidate for this type of effect being p59Fyn. Finally, we also found that a first treatment of JE6.1
cells with an anti-CD3 MoAb (OKT3) totally impaired further stimulation
by thrombin or the thrombin receptor agonist peptide2 (not
shown), whereas after a prior stimulation with OKT3, JCaM1, and J45.01
cells were still able to respond fully to thrombin and thrombin
receptor agonist peptide (not shown). Taken together, these findings
suggest a cross-talk between signaling components of the TcR and those
of the thrombin receptor, controling at least [Ca2+]i release and p38 MAPK activation.
Interestingly, reconstitution of JCaM1 and J45.01 cells with either
p56Lck or CD45 was sufficient to restore normal thrombin responses (ie,
identical to those observed in JE6.1 cells).
Activation of the p38 MAP kinase cascade by seven-transmembrane
receptors is a recently described mechanism of intracellular signaling
by this family of receptors.32,42 On the basis of our
findings on an enhanced response to thrombin in deficient cell lines,
we hypothesized that the increase in thrombin-induced p38 MAPK
activation and [Ca2+]i release could be due
to the lack of p56Lck expression and to a decrease of p56Lck activity
in JCaM1 and J45.01, respectively. Accordingly, we found that thrombin
stimulated tyrosine phosphorylation of p56Lck in J45.01, but not in
JE6.1 cells. This increase in p56Lck tyrosine phosphorylation was
accompanied by a significant decrease in kinase activity. Weiss and
Littman34 recently proposed that the basal state of
activation of the TcR signaling pathway is an equilibrium between
positive and negative regulatory signals. Thus it appears that
inhibition of p56Lck kinase activity could be the mechanism by which
thrombin induced [Ca2+]i release and p38 MAPK
activation in Jurkat cells. Consistently, lack of p56Lck or decreased
p56kinase activity upon thrombin stimulation of J45.01 cells was
accompanied by an increase in [Ca2+]i release
and p38 MAPK activation. However, other components of the TcR signaling
pathway such as p59Fyn may also be important in thrombin signaling,
especially in view of the higher thrombin response in J45.01 cells.
In this study we also observed tyrosine phosphorylation of a 36-kD
protein after thrombin and thrombin receptor agonist peptide stimulation. Phosphorylation of p36 paralleled that of p38 MAPK and was
also strongly increased in JCaM1 and J45.01 cell lines. Several
observations support the notion that p36/Lnk mediates interaction
between Grb2 and PLC1 and links the TcR to the ras pathway.43 Moreover, in HEL cells and platelets, a 36-kD
molecule associates with and is phosphorylated by Csk.44
This association is thought to relocate the cytosolic kinase to the
particulate fraction in contact with members of the src family of
tyrosine kinases. If the 36-kD protein phosphorylated on tyrosine upon thrombin and thrombin receptor agonist peptide stimulation corresponds to p36/Lnk, an attractive hypothesis would be that phosphorylated p36
can bind and relocate Csk to the plasma membrane where it could in turn
inactivate p56Lck by phosphorylation. This would bring a good
explanation for the decrease in p56Lck activity observed in J45.01
cells treated with thrombin. Accordingly, preliminary results indicate
that thrombin induced relocation of Csk in Jurkat T cells (not shown).
In summary, our data demonstrate a cross-talk between components of the
TcR and the thrombin receptor signaling pathways in Jurkat T cells. We
also show that the tyrosine kinase p56Lck is likely to exert a negative
regulation on thrombin-induced [Ca2+]i
release and p38 MAPK activation in T lymphocytes. This model may have
important physiological implications more particularly concerning
thrombin responses in preactivated or anergized T cells on sites of
hemostatic stress and inflammation. Thus thrombin receptor expression
in T cells may be particularly relevant to a role in preactivated
or anergized T lymphocytes, where TcR signaling is
impaired. This hypothesis is in line with the apparent
activation-induced regulation of thrombin receptor mRNA expression in
peripheral blood lymphocytes.1
| |
NOTE ADDED IN PROOF |
While this manuscript was in revision, Joyce et al reported an enhanced
thrombin receptor signaling in a TCR-negative T-cell line and T-cell
lines deficient in either p56Lck or CD45.45
| |
FOOTNOTES |
Submitted April 28, 1997;
accepted January 14, 1998.
Supported by the Ligue Nationale contre le Cancer, by the
Féderation Nationale des Entreprises Françaises dans la
Lutte contre le Cancer, and by Grant No. 6684 from the Association pour la Recherche Contre le Cancer.
Address reprint requests to Dr Patrick Auberger, CJF
INSERM 96.05, Faculté de Médecine, Av de Valombrose, 06107 Nice Cédex 02, France.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
| |
ACKNOWLEDGMENT |
We thank Jean-François Peyron for helpful discussion and Aurore
Grima for illustration work. JE6.1, JCaM1, J45.01, and J45.01 reconstituted cells (LB3.3-3) were kindly provided by Arthur Weiss (University of California, San Francisco). We are undebted to Lawrence
F. Brass for the kind gift of human thrombin receptor antibodies.
| |
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Abstract
Introduction
Abstract