From the Kennedy Institute of Rheumatology, Hammersmith, London W6
8LH, United Kingdom, the § Virginia Mason Research Center,
Seattle, Washington 98101, the ¶ Department of Immunology,
University of Washington, Seattle, Washington 98195, the
Department of Immunology, Graduate School and Faculty of
Medicine, University of Tokyo, Hongo-7-3-1, Bunkyo-ku, Tokyo 113, Japan, and the
Picower Institute for
Medical Research, Manhasset, New York 11030
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ABSTRACT |
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We have shown recently that interleukin (IL)-2
activates the mitogen-activated protein (MAP) kinase family members p38
(HOG1/stress-activated protein kinase II) and p54 (c-Jun N-terminal
kinase/stress-activated protein kinase I). Furthermore, the p38 MAP
kinase inhibitor SB203580 inhibited IL-2-driven T cell proliferation,
suggesting that p38 MAP kinase might be involved in mediating
proliferative signals. In this study, using transfected BA/F3 cell
lines, it is shown that both the acidic domain and the
membrane-proximal serine-rich region of the IL-2R T cell clonal proliferation upon antigenic challenge plays an
essential role in mounting an effective immune response. Interleukin (IL)1-2 is a potent T cell
mitogen that plays a key role in driving this process (1). The
intracellular signal transduction pathways activated by IL-2 and the
relative roles of these pathways in mediating the mitogenic signal have
been extensively studied but have yet to be fully elucidated.
IL-2 exerts its cellular effects through binding to specific cell
surface receptors. The high affinity IL-2 receptor (IL-2R) is a
heterotrimeric complex consisting of IL-2 activates two other major signaling pathways: the
phosphatidylinositol 3-kinase pathway and the p42/44 MAP kinase
pathway. The activation of phosphatidylinositol 3-kinase (20, 21) and the subsequent activation of protein kinase B/Akt (22) and
p70S6 kinase (23, 24), has been associated with the
serine-rich region of the IL-2R We have now examined further the role of these kinases in IL-2
proliferative signaling by mapping the regions of the IL-2R Cell Culture and Reagents--
The murine
IL-2-dependent T cell line CT6 (kindly provided by
Genentech, S. San Francisco, CA) was maintained in
glutamine-supplemented RPMI 1640 (BioWhittaker, Verviers, Belgium) with
5% fetal bovine serum (Sigma, Poole, Dorset, UK), 1 unit/ml
penicillin/streptomycin (BioWhittaker), and 50 µM
2-mercaptoethanol (ICN, Thame, Oxon, UK) with the addition of 5 ng/ml
recombinant human IL-2 (generously provided by Dr. P Lomedico, Roche
Inc., Nutley, NJ). The stable transformant clones F7, S25, and A15
previously described (4) were initially derived from the BAF-B03 clone
of the IL-3-dependent BA/F3 cell line and were maintained
in RPMI 1640 supplemented with 5% fetal bovine serum, 1% WEHI-3B
conditioned medium (as a source of IL-3) and 0.2 µg/ml G418
(Calbiochem-Novabiochem Ltd., Nottingham, UK). The parental BA/F3 cell
line was also transfected with versions of human IL-2R
The tetravalent guanylhydrazone CNI-1493 was synthesized as described
previously (41). SB203580 was from Calbiochem-Novabiochem Ltd., and
PD098059 was from New England Biolabs (Hitchin, Herts, UK). Rabbit
antisera to p54 (SAK10) and p38 (SAK7) MAP kinases were provided by
Prof. J Saklatvala (Kennedy Institute of Rheumatology, London) (44).
Antibody to p42 MAP kinase/extracellular signal-regulated kinase 2 was
from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Antibodies to
phosphorylated p42/44, p38, and p54 MAP kinases were from New England
Biolabs. Antibodies to Shc and Grb2 and the anti-phosphotyrosine
antibody PY-20 were from Transduction Labs (Lexington, KY). 4G10
anti-phosphotyrosine antibody was from Upstate Biotechnology,
Inc. (Lake Placid, NY). The cDNA for histidine-tagged MAPK-activated protein kinase 2 was the generous gift of Prof. M. Gaestel (Max Delbruck Center for Molecular Medicine, Berlin, Germany).
Proliferation Assays--
Cellular DNA synthesis was measured by
[3H]thymidine incorporation as described previously
(45).
Immunoprecipitation--
p38 MAP kinase and c-Jun N-terminal
kinase/stress-activated protein kinase were immunoprecipitated from
cleared cell lysates as described previously (40). Shc
immunoprecipitations were performed as described previously (33).
In VitroKinase Assays--
In vitro kinase assays for
p54 and p38 MAP kinase activity were performed on precipitated immune
complexes. p54 MAP kinase assays were performed as described previously
using glutathione S-transferase-activating transcription
factor 2 (GST-ATF2) as a substrate (40). In vitro kinase
assays for p38 MAP kinase were performed using
His6-MAPK-activated protein kinase 2 as a substrate.
Immunoprecipitates were incubated with 30 µl of kinase assay buffer
(25 mM HEPES, pH 7.5, 25 mM MgCl2,
25 mM Western Immunoblotting--
p42/44 MAP kinase (extracellular
signal-regulated kinase 1/2) and phosphotyrosine Western blotting was
performed as described previously (34). Western blotting for Shc, Grb2,
and phosphorylated forms of p42/44, p38, and p54 MAP kinases was
performed according to the antibody manufacturer's instructions.
Both the Serine-rich and the Acidic Domains of the IL-2R IL-2 Mediates Activation of p38 and p54 MAP Kinase Activation in
BA/F3 Cells through Recruitment of Shc to the IL-2R IL-2-driven Proliferation of BA/F3 Cells Is Suppressed by the p38
MAP Kinase Inhibitor SB203580--
The observation that the IL-2R CNI-1493 Inhibits the IL-2 Activation of p38 MAP and p54 MAP
Kinases but Has No Effect on IL-2-driven Proliferation in the Murine T
Cell Line CT6, BA/F3 Cell Lines, or Primary T Cells--
It is
possible that the discrepancy between the data presented here and our
previous studies is due to cell type differences in IL-2 signaling
between the CT6 T cell line used previously and the pro-B, BA/F3 cell
lines used here. In order to address this possibility, we have made use
of a second synthetic inhibitor. CNI-1493, a tetravalent
guanylhydrazone compound, has been shown to inhibit the production of
proinflammatory cytokines by monocytic/macrophage cells in response to
lipopolysaccharide (46). The inhibition of proinflammatory cytokine
production appears to operate at a post-transcriptional level (47), and
there is some preliminary evidence to suggest that CNI-1493 is able to
inhibit activation of p38 MAP kinase (48). We have shown that CNI-1493
does inhibit the lipopolysaccharide-induced phosphorylation and
activation of p38 MAP kinase in both human monocytes and the murine
macrophage cell line RAW 264.7, and in addition we have observed that
both p42 MAP kinase/extracellular signal-regulated kinase 2 and p54 MAP
kinase are similarly
inhibited.2 In CT6 cells, 1 µM CNI-1493 was found to completely ablate both p54 and
p38 MAP kinase activation but to have no effect on p42/extracellular signal-regulated kinase phosphorylation induced by IL-2 (Fig. 5). The ability of CNI-1493 to inhibit
p54 and p38 MAP kinase activation provided us with a tool with which to
reassess the role of the MAP kinases in proliferative responses of T
cells. CNI-1493 had no effect on IL-2-driven proliferation of CT6 cells or activated primary human T cells at doses that inhibit p38 and p54
MAP kinase activation in CT6 cells (Figs. 5 and
6A). Combinations of CNI-1493
and the MAP kinase kinase 1/2 inhibitor PD098059 (49), which inhibited
the activation of all three MAP kinases, also failed to suppress the
proliferation of CT6 cells, indicating that in T cells there is no
redundant usage of MAP kinases in IL-2-driven proliferation (Fig.
7). This observation confirms the data
obtained from the A15 and Y338F BA/F3 cell lines, which proliferate
normally in response to IL-2 but in which no IL-2-stimulated MAP kinase
activation was detected. The addition of CNI-1493 over 5 days failed to
prevent cell propagation, suggesting that p38 and p54 MAP kinases are
also not required for long term cell survival (data not shown). Similar
effects on MAP kinase activation of CNI-1493 were observed in BA/F3 F7
cells (Fig. 5), although at 5-10 µM CNI-1493, inhibition
of p42 MAP kinase phosphorylation was observed, an effect that was not
seen in CT6 cells (data not shown). CNI-1493 was seen to have no effect
on IL-2-driven BA/F3 cell proliferation, even in the CNI-1493 Blocks p38 and p54 MAP Kinase Activation at a Point Distal
to Shc and Grb2--
Because receptor mutagenesis studies indicated a
role for Shc in p38 and p54 MAP kinase activation, we examined the
effects of CNI-1493 on IL-2-stimulated Shc phosphorylation in both
BA/F3 cells and CT6 cells by immunoprecipitation of Shc and
phosphotyrosine Western blotting. CNI-1493 has no effect on
IL-2-induced Shc tyrosine phosphorylation in either cell type, and
subsequent Western blotting for Grb2 reveals that the interaction
between Shc and Grb2 is also unaffected by this compound (Fig.
8). This observation suggests that the
target of this drug must lie downstream of Shc and Grb2 and is
consistent with the fact that CNI-1493 has no effect on p42/44 MAP
kinase activation. However, in the This study has shown that the IL-2 activation of p38 and p54 MAP
kinases, like that of p42 MAP kinase, requires both the acidic and the
serine-rich regions of the IL-2R SB203580 was found to inhibit IL-2-driven proliferation of BA/F3 cells
with an IC50 of around 4 µM, similar to that
observed in IL-2-stimulated T cells, regardless of whether p38 MAP
kinase was activated upon IL-2 stimulation. This observation raises
doubts as to the specificity of the inhibitor in the cells used here. We have shown previously in CT6 cells that SB203580 is capable of
inhibiting the IL-2 activation of MAPK-activated protein kinase 2, the
immediate downstream kinase substrate of p38 MAP kinase, with an
IC50 of 0.3-0.5 µM. In contrast, the
inhibitory effects of SB203580 on the IL-2-driven proliferation of
these cells are seen at drug concentrations approximately 1 order of
magnitude higher. We originally suggested that this discrepancy may be
due to the differences in the nature of the proliferation and kinase assays. Two very recent papers have demonstrated that at higher concentrations SB203580 directly inhibits the activity of the p54 MAP
kinase isoform c-Jun N-terminal kinase 2 (50, 51). However, as we have
shown here, p54 MAP kinase activation is not a requirement for BA/F3
cell proliferation; therefore, the inhibition of this kinase is
unlikely to be responsible for the effects of SB203580 observed.
Therefore, it must be concluded that at higher drug doses other
target(s) are affected and that it is through its effect on this
secondary target that the antiproliferative effects of SB203580 are
mediated. This conclusion has wider implications. Since the discovery
of SB203580, many cellular functions have been ascribed to p38 MAP
kinase purely on the basis of their inhibition by SB203580 at doses
well in excess of 1 µM. It should now be considered that
unless the effects of SB203580 on a particular physiological response
can be seen at the doses at which p38 MAP kinase signaling is inhibited
(0.1-0.5 µM), no definite conclusions can be drawn as to
the involvement of p38 MAP kinase in mediating the effect.
In order to compare the mechanism of activation of MAP kinases and
their role in proliferation in pro-B cells with that in T cells, we
have made use of a tetravalent guanylhydrazone inhibitor of p38 MAP
kinase phosphorylation, CNI-1493 (48). CNI-1493 inhibits the production
of proinflammatory cytokines by monocytes/macrophages but has no effect
on The effects of CNI-1493 on MAP kinase activation and studies with
receptor mutants raise a number of interesting questions about the
architecture of the upstream signaling pathways responsible for MAP
kinase activation by IL-2. The requirement for Tyr338, the
Shc binding site, for the activation of p38 and p54, as well as p42,
MAP kinases indicates that Shc may be a common origin for signaling to
all three. A role for Shc in the activation of p38 and p54 MAP kinases
is supported by the fact that the expression of an IL-2R Although we have demonstrated that Tyr338 and Shc
recruitment are required for p38 and p54 MAP kinase activation in BA/F3
cells, whether this is also the case in T cells is not known. We have shown previously that another T cell mitogen, IL-7, is capable of
activating p38 and p54 MAP kinases in T cells (40) and have evidence to
suggest that IL-4 is also able to activate these kinases in CT6
cells.3 However, neither IL-7
nor IL-4 is able to activate p42/44 MAP kinases, and no phosphorylation
of Shc can be detected upon stimulation with either of these cytokines
(34). This suggests that in T cells either the recruitment of Shc is
not involved in the activation of p38 and p54 MAP kinases or that the
activation of these kinases in the CT6 cell line proceeds through
different pathways depending on the mitogen used. Transfection of
granulocyte-macrophage colony-stimulating factor-IL-2R In summary, we have shown that the activation of p38 and p54 MAP
kinases by IL-2 in BA/F3 cells, like that of p42/44 MAP kinase, requires the presence of both the acidic and the serine-rich region of
the IL-2R chain are required
for p38 and p54 MAP kinase activation and that, as for p42/44 MAP
kinase, this activation requires the Tyr338 residue
of the acidic domain, the binding site for Shc. It is well established
that the acidic domain of the IL-2R
chain is dispensable for
IL-2-driven proliferation, and thus our observations suggest that
neither p38 nor p54 MAP kinase activation is required for IL-2-driven
proliferation of BA/F3 cells. In addition, the tetravalent
guanylhydrazone inhibitor of proinflammatory cytokine production,
CNI-1493, can block the activation of p54 and p38 MAP kinases by IL-2
but has no effect on IL-2-driven proliferation of BA/F3 cells,
activated primary T cells, or a cytotoxic T cell line. Furthermore, our
observations provide evidence for the existence of an additional,
unknown target of the p38 MAP kinase inhibitor SB203580, the activation
of which is essential for mitogenic signaling by IL-2.
INTRODUCTION
Top
Abstract
Introduction
References
-,
-, and
c-subunits, the
c subunit being shared
with the receptors for the other T cell mitogens, IL-4, IL-7, IL-9, and
IL-15 (2). The
-subunit is responsible for conferring high affinity
cytokine binding, while the
- and
c-subunits recruit
cytoplasmic molecules, thereby transducing the proliferative signal.
The
-subunit has the larger cytoplasmic tail, consisting of
subdomains previously identified as the membrane-proximal serine-rich,
acidic, and distal proline-rich regions (3, 4). The IL-2R
chain
contains six cytoplasmic tyrosine residues: Tyr338,
Tyr355, Tyr358, and Tyr361, which
lie in the acidic region, and Tyr392 and
Tyr510, which lie within the proline-rich region. The
presence of at least one of the tyrosines Tyr338,
Tyr392, and Tyr510 appears to be sufficient to
allow IL-2-driven proliferation (5, 6). Studies in transfected BA/F3
cells have shown that loss of the IL-2R
acidic region has no effect
on proliferation (4) or the expression of Myc and Bcl-2, factors
essential for proliferation and cell survival (7). The tyrosine kinases
p56Lck, p72Syk, Jak1, and Jak3 are recruited to the
IL-2R and activated upon IL-2 binding (8-11). However, of these
only the activation of Jak3, which associates with the
IL-2R
c chain, appears to be an absolute requirement for
IL-2-driven proliferation (8, 10, 12-16). It has been suggested that
Jak3 mediates IL-2 proliferative signaling through activation of
another tyrosine kinase, Pyk2 (17). Signal transducer and activator of
transcription (STAT) 5 is also activated as a result of Jak activation,
but its role in proliferation is unclear (6, 18, 19).
chain (25), and the activation of
these factors results in the phosphorylation of Rb, suggesting a key role for these kinases in proliferation (26). IL-2-induced activation of the p42/44 MAP kinase (extracellular signal-regulated kinase) pathway proceeds through the activation of p21ras, Raf, and MAP
kinase kinase 1/2 (27-29) and requires both the acidic and the
serine-rich regions of the IL-2R
chain (30). Tyr338,
within the acidic region of the IL-2R
chain, is responsible for the
recruitment of Shc (31) and the subsequent assembly of the
p21ras-activating complex along with Grb2 and SOS (31-33).
However, loss of p42/44 MAP kinase signaling does not appear to prevent
IL-2-driven cell cycle progression (30, 31, 34). Two further MAP kinase family members, p54 (stress-activated protein kinase I/c-Jun N-terminal kinase) and p38 (stress-activated protein kinase II/HOG1), were typically thought to be activated by cellular stress and
proinflammatory stimuli (35-39). However, we have recently
demonstrated that p38 MAP kinase and p54 MAP kinase are activated by
IL-2 in T cells (40). We observed that an inhibitor of p38 MAP kinase
function, SB203580 (39), was able to inhibit T cell proliferation
induced by IL-2, suggesting that p38 MAP kinase activation may be
required for cell cycle progression.
chain
required for p38 and p54 MAP kinase activation. We observe that both
the serine-rich and the acidic regions are required for the activation
of p54 and p38 MAP kinases by IL-2 in BA/F3 cells and that the
activation of these kinases, like that of p42/44 MAP kinase, is
dependent on the presence of Tyr338 in the IL-2R
chain
and proceeds through the recruitment of Shc. Since the acidic region of
the IL-2R
chain is dispensable for BA/F3 cell proliferation, our
observations indicate that the activation of p38 and p54 MAP kinase is
not required for IL-2-driven cell cycle progression. This conclusion is
supported by studies using the tetravalent guanylhydrazone
CNI-1493 (41), which inhibited IL-2-induced p38 and p54 MAP kinase
activation but had no effect on IL-2-driven proliferation of either
BA/F3 cells or T cells. Our data therefore suggest that the previously
observed inhibition of IL-2-induced proliferation by the inhibitor
SB203580 is likely to be due to its action on a target(s) other than
p38 MAP kinase, which is required for cell cycle progression.
EXPERIMENTAL PROCEDURES
chain bearing
the mutations
355, Y338F,
355:Y338F, and
325-Shc described
previously (42) under control of the human
-actin promoter in an
expression vector also encoding neomycin phosphotransferase. Stably
transfected subclones were derived through selection at limiting
dilution in the presence of G418 and were maintained in RPMI 1640 supplemented with 5% fetal bovine serum, 1% WEHI-3B conditioned
medium, and 0.8 µg/ml G418. Human peripheral T cells were isolated as
described previously (43). All cells were washed in cytokine-free
medium and deprived of cytokine supplements for 16 h prior to
experimental use.
-glycerophosphate) containing 50 µg/ml
recombinant His6-MAPK-activated protein kinase 2, 30 µM ATP, and 0.5 µCi of [
-32P]ATP
(Amersham International, Little Chalfont, Buckinghamshire, UK) for 25 min at room temperature. Reactions were terminated by the addition of
gel sample buffer and boiling for 5 min. All substrates were separated
by SDS-polyacrylamide gel electrophoresis. Gels were dried, and
phosphorylated substrates were visualized using a Fuji FLA-2000 Imager
(Raytek Scientific Ltd, Sheffield, UK) and by autoradiography at
70 °C .
RESULTS
Chain
Are Required for MAP Kinase Activation by IL-2 in BA/F3
Cells--
IL-3-dependent BA/F3 cells normally express
both the
- and the
c-chains, but not the
-chain,
of the IL-2R. When transfected with the IL-2R
subunit, they become
responsive to IL-2 (4). We have used BA/F3 cell lines expressing either
the wild type IL-2R
chain (F7) or mutant forms of the IL-2R
chain, lacking either the serine-rich region (S25) or the acidic region
(A15) (Fig. 1A), to examine
the role of the IL-2R
chain subdomains in the activation of p38 and
p54 MAP kinase. The serine-rich and the acidic domains of the
-chain
are essential for p42/44 MAP kinase activation (30); this is confirmed
in Fig. 2A by detection of the
phosphorylated kinase by Western blotting using a phosphospecific p42/44 MAP kinase antibody. Using immunokinase assays (for p38 and p54;
Fig. 2B) or Western blotting for the phosphorylated, activated form (p38 only, Fig. 2A) to measure p38 and p54
MAP kinase activation, we have shown that these kinases are stimulated by IL-2 in F7 cells (Fig. 2, A and B). However,
IL-2 was unable to activate either p38 or p54 MAP kinase in the absence
of the serine-rich (S25) or acidic (A15) regions, indicating that both regions are required for kinase activation. IL-3 activated p42, p38,
and p54 MAP kinases in all three cell lines and is included as a
positive control. Kinetic experiments examining p38 and p54 MAP kinase
activation over a 2-h period have established that the activation of
these kinases by IL-2 is not simply retarded in the S25 and A15 cell
lines (data not shown).
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Fig. 1.
Schematic diagram of the
IL-2R chain mutants expressed in the stably
transfected BA/F3 cell lines used in this study.
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Fig. 2.
The serine-rich and acidic domains of
the IL-2R chain are required for activation of
p38 and p54 MAP kinases by IL-2. F7, S25, and A15 cells were
stimulated with IL-2 (20 ng/ml) or IL-3 (5% WEHI supernatant) or were
left untreated (Un). MAP kinase activity was assayed as
described under "Experimental Procedures" as follows. A,
Western blotting. The top panels show activation
of p42/44 MAP kinase determined by Western blotting using a
phosphospecific antibody, and the second set
of panels show the same blots reprobed for total
p42/44 MAP kinase to demonstrate equal protein loading. The
third set of panels show
p38 MAP kinase phosphospecific Western blots, and the bottom
panels show the same blots reprobed for total p38 MAP
kinase. p54 MAP kinase phosphorylation could not be detected using a
phosphospecific anti-stress-activated protein kinase/c-Jun N-terminal
kinase antibody in these cells. B, kinase assays.
Immunoprecipitated p38 MAP kinase activity was determined using
His6-MAPK-activated protein kinase 2 as a substrate.
Immunoprecipitated p54 MAP kinase activity was determined using
GST-ATF2 as a substrate.
Chain Tyrosine
Tyr338--
This similarity in the
-chain subdomain
requirements led us to examine whether, as for the activation of p42
MAP kinase, Shc is involved in the activation of p38 and p54 MAP
kinases. A second panel of BA/F3 clones expressing mutant forms of the IL-2R
chain was used (Fig. 1B). In the cell line
355,
a truncation of the
-chain at amino acid 355 removes the
proline-rich region and part of the acidic region to eliminate all of
the cytoplasmic tyrosine residues except the Tyr338 residue
required for Shc binding. IL-2 is nonetheless able to activate both p38
and p54 MAP kinases in this cell line (Fig. 3A). However, in another BA/F3
cell line expressing a full-length IL-2R
chain with a point mutation
of Tyr338 to phenylalanine (Y338F), IL-2 fails to activate
p38 and p54 MAP kinases, as determined by kinase assay and
phosphospecific Western blotting (Fig. 3A). Curiously, there
is still a very slight residual activation of all three MAP kinases by
IL-2 when the Y338F mutation is combined with the
355 truncation in
the BA/F3 cell line
355:Y338F (Fig. 3, A and
B). An additional BA/F3 line,
325-Shc, bears a version of
the IL-2R
chain in which the entire acidic and proline-rich regions,
including all cytoplasmic tyrosines of IL-2R
, are replaced with a
covalently tethered Shc molecule to specifically reconstitute
Shc-mediated signals, as described previously (42). IL-2 promoted p38
and p54 MAP kinase activation in these
325-Shc cells, suggesting
that Shc may mediate the activation of p38 and p54 MAP kinases by IL-2
(Fig. 3A). However, the presence of the receptor fusion
protein appears to compromise the ability of IL-3 to activate p38 and
p54 MAP kinases. This may indicate that the activation of these kinases
by IL-3 also occurs through Shc if the IL-2R
-Shc fusion protein is
somehow inhibiting a functional interaction between endogenous Shc and
the IL-3 receptor. Fig. 3B shows p42 MAP kinase Western
blots, confirming the role of the IL-2R
chain tyrosine
Tyr338 and Shc in p42 MAP kinase phosphorylation and
activation.
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Fig. 3.
Tyr338 in the
IL-2R chain and Shc are required for
activation of p38 and p54 MAP kinases by IL-2.
355, Y338F,
355:Y338F, and
325-Shc cells were stimulated with IL-2 (20 ng/ml)
or IL-3 (5% WEHI supernatant) or left unstimulated (Un) for
10 min. MAP kinase activation was determined by Western blotting for
phosphorylation and by kinase assays as described under "Experimental
Procedures." A, the top panels show
p38 MAP kinase activation determined by kinase assay using
His-MAPK-activated protein kinase 2 (His-MAPKAPK-2) as a
substrate, the corresponding Western blots for tyrosine/threonine
phosphorylated-p38 MAP kinase are shown below, and the same
blots reprobed for total p38 MAP kinase are shown to demonstrate even
sample loading. p54 MAP kinase activation was determined by kinase
assay using GST-ATF2 as a substrate, the corresponding Western blots
for tyrosine/threonine phosphorylated-p54 MAP kinase are shown
below, and the same blots reprobed for total p54 MAP kinase
are shown in the bottom panels. B, p42
MAP kinase phosphorylation was determined by electrophoretic mobility
shift on p42 MAP kinase Western blots.
chain acidic region is dispensable for IL-2-driven proliferation but
that both the acidic and the serine-rich regions must be present for
p38 and p54 MAP kinase activation in BA/F3 cells indicates that neither
kinase is required for proliferation. This conflicts with our previous suggestion (40), based on studies using the p38 inhibitor SB203580, that p38 MAP kinase activation is required for IL-2-driven T cell proliferation. We therefore examined whether proliferation of the
IL-2-responsive BA/F3 cell lines was also sensitive to this drug. Of
the cell lines used, only two did not proliferate in response to IL-2:
the S25 cell line, lacking the entire serine-rich region (4), and the
355:Y338F cell line, in which the IL-2R
chain lacks all three of
the tyrosine residues of which at least one is required for
proliferation (5, 42). Treatment of the cell line F7, A15, or Y338F
with SB203580 (0.1-30 µM) resulted in the inhibition of
IL-2-driven DNA synthesis with an IC50 of ~2-6
µM (Fig. 4). Proliferation
of the other cell lines was similarly inhibited in each case (data not
shown). The IC50 of SB203580 on IL-2-driven proliferation
observed in these cell lines was comparable with that observed
previously in T cells (40). However, since no activation of MAP kinases
could be detected in either the A15 or the Y338F cells, in these cell
lines the effect of SB203580 on proliferation could not be due to the
inhibition of p38 MAP kinase and must instead reflect an effect of
SB203580 on an unknown target.
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Fig. 4.
SB203580 inhibits IL-2- and IL-3-driven
proliferation of BA/F3 cell lines. Inhibition of IL-2 (20 ng/ml)
induced [3H]thymidine incorporation by F7 ( ), A15
(
), and Y338F (
) cells, and inhibition of IL-3 (5% WEHI
supernatant) induced [3H]thymidine incorporation by F7
cells (
) by the indicated concentrations of SB203580. A
proliferative response of 100% was defined for each cell line as
follows: [3H]thymidine incorporation by stimulated cells
(cpm)
[3H]thymidine incorporation by unstimulated
cells (cpm) in the absence of inhibitor. Unstimulated cell values were
subtracted from each data point, and the results were expressed as
percentage proliferative responses. Data points represent the mean of
triplicate cultures ± S.E.
355 cell line,
in which Tyr338 is the only intracellular tyrosine residue
present and proliferation is Tyr338-dependent
(Fig. 6B). This observation indicates that these kinases are
not involved in proliferative signaling even as a functionally redundant pathway only required in the absence of proliferative signals
from the other intracellular tyrosines Tyr392 and
Tyr510.
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Fig. 5.
The inhibitor CNI-1493 blocks activation of
p38 and p54 MAP kinases by IL-2 in CT6 cells. Effect of
pretreatment (1 h) with indicated concentrations of CNI-1493 on MAP
kinase activation by IL-2 (20 ng/ml; 10 min) in CT6 cells and BA/F3 F7
cells. p54 and p38 MAP kinase activities were determined by
immunokinase assays using GST-ATF2 and His6-MAPK-activated
protein kinase 2 (His-MAPKAPK-2) as substrates,
respectively. p42 MAP kinase activation was determined by
electrophoretic mobility shift on p42 MAP kinase Western blots.
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Fig. 6.
The inhibitor of p38 and p54 MAP kinase
activation, CNI-1493, has no effect on IL-2-driven proliferation.
Effect of the indicated concentrations of CNI-1493 on
[3H]thymidine incorporation in IL-2 (20 ng/ml)-stimulated
CT6 cells ( ) and anti-CD3 (OKT-3, 35 ng/ml)-activated human
peripheral blood mononuclear cells (
) (A) and in IL-2 (20 ng/ml)-stimulated F7 (
), A15 (
),
355(
), and Y338F (
)
BA/F3 cell lines (B). Results are expressed as percentage
proliferative responses (as defined in Fig. 4), and data points
represent the means of triplicate cultures ± S.E.
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Fig. 7.
Blockade of all three MAP kinase pathways has
no effect on IL-2-driven proliferation of CT6 cells. CT6 cells
were treated with 10 µM PD098059 and the indicated
concentration of SB203580 ( ) or CNI-1493 (
) and then stimulated
with IL-2 (20 ng/ml). The effect on [3H]thymidine
incorporation is shown. Results are expressed as percentage
proliferative responses (as defined in Fig. 4), and data points
represent the mean of triplicate cultures ± S.E.
325-Shc cell line the inhibitory
effects of CNI-1493 on kinase activation are lost (data not shown).
This result may indicate that the receptor-bound Shc can provide an
alternative to the CNI-1493-sensitive component of the p38/p54 MAP
kinase activation pathway. Alternatively, the loss of sensitivity to
CNI-1493 may simply be a result of the overexpression of the
IL-2R
-Shc fusion, a hypothesis that is supported by the
observation that the inhibition of p42 MAP kinase phosphorylation by
high doses of CNI-1493 is also lost in this cell line despite the fact
that p42 MAP kinase phosphorylation is Shc-dependent.
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Fig. 8.
CNI-1493 does not inhibit IL-2-induced Shc
phosphorylation or Grb2 recruitment. CT6 (a) and BA/F3
F7 (b) cells were treated for 1 h with CNI-1493 as
shown and stimulated for 10 min with IL-2 (20 ng/ml) or IL-3 (5% WEHI
supernatant) as indicated. Cell lysates were incubated with an anti-Shc
antibody or a nonimmune control antibody (NI) and protein
G-Sepharose for 3 h at 4 °C. Immunoprecipitates were washed
(lysis and wash buffers used are described in Ref. 33) and separated by
SDS-polyacrylamide gel electrophoresis. Western blotting for
phosphotyrosine (upper panels), Grb2
(center panels), and Shc (lower
panels) was performed as described under "Experimental
Procedures."
DISCUSSION
chain. In particular, activation of
these kinases requires the IL-2R
chain Tyr338 and
appears to involve the recruitment of Shc. Since it has been well
established that the acidic region of the IL-2R
chain is not
required for IL-2-induced proliferation, our results indicate that p38
MAP kinase and p54 MAP kinase are dispensable for this IL-2 function.
This finding contradicts our previous suggestion (40) that p38 MAP
kinase was required for IL-2-induced proliferation of T cells. This
conclusion was drawn from the antiproliferative activity of the p38 MAP
kinase inhibitor SB203580. A possible explanation for these conflicting
data is that there are different requirements for p38 MAP kinase in the
proliferative response of T cells, used in the original study, and in
the BA/F3 cell lines used here. However, such a possibility was not
supported by studies with a second inhibitor of p38 MAP kinase
activation, CNI-1493, which had no effect on the proliferation of
either T cells or BA/F3 cells.
CD3/
CD28-induced cytokine production in T cells (52). We have
demonstrated that not only is this compound capable of inhibiting both
the phosphorylation and activation of p38 MAP kinase induced by
lipopolysaccharide in monocytes and macrophages but that it can also
inhibit the activation of p54 and p42 MAP kinases by lipopolysaccharide
in these cells as well (data not shown). In this study, we have
demonstrated that in CT6 and BA/F3 cells the IL-2 activation of p38 and
p54 MAP kinases is inhibited by CNI-1493. In contrast, p42/44 MAP
kinase activation is not inhibited by the same doses of CNI-1493,
although we have observed inhibition of p42 MAP kinase activation in
BA/F3 cells with higher concentrations of the drug (5-10
µM). We have shown that CNI-1493, unlike SB203580, has no
effect on proliferation in either CT6 cells, primary human T cells or
in the IL-2-responsive BA/F3 cell lines, confirming that neither p38
nor p54 MAP kinase activation is essential for either T cell or BA/F3
cell proliferation. The actual function of these kinases in these cell
types and the significance of their activation in IL-2 signaling remain
unknown and are not addressed in this study. However, CNI-1493 has been used successfully to inhibit the toxic side effects of IL-2 in anti-tumor therapy, and it is suggested that this may be the result of
its ability to inhibit tumor necrosis factor and NO production (53). An
implication of our results is that CNI-1493 will not prevent
IL-2-driven expansion of T cell clones with anti-tumor activity while
simultaneously preventing the release of toxic macrophage products.
-Shc fusion
protein in BA/F3 cells was able to compensate for the loss of the
Tyr338 and restore the IL-2-induced activation of p38 and
p54 MAP kinases. The fact that CNI-1493 is able to discriminate between
p42 and p38/54 MAP kinases indicates that there must be a bifurcation of the activation pathways of these kinases at a point distal to Shc
itself, with the target of CNI-1493 lying downstream of Shc on the
p38/p54 MAP kinase arm of the activation pathway. From Shc, the
activation of p42 MAP kinase follows an established pathway requiring the formation of a complex of Grb2 and Sos with
phosphorylated Shc (31-33) and the subsequent activation of
p21ras (27) and Raf (28, 29). The failure of CNI-1493 to affect the phosphorylation of Shc or the subsequent recruitment of Grb2 in
either CT6 or BA/F3 cells is consistent with its lack of effect on p42
MAP kinase activation.
subunit
chimeras into CT6 cells or CTLL cells as described recently (42) will
allow this question to be addressed.
chain and, in particular, is dependent on the presence of
Tyr338 and the recruitment of Shc. It is also shown that
the activation of these kinases is not required for IL-2-driven
proliferation in either T cells or BA/F3 cells. Our data suggest
the existence of a non-MAP kinase target for SB203580, which is
affected at doses of 1-10 µM and which plays a critical
role in mitogenic signaling by IL-2. This latter point has important
general implications for the interpretation of data from studies using
SB203580 in a number of systems.
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ACKNOWLEDGEMENTS |
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We thank Prof. T. Taniguchi for providing the F7, S25, and A15 BA/F3 cell lines and Prof. J. Saklatvala and Prof. M. Feldmann for critical reading of the manuscript.
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FOOTNOTES |
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* This work was supported in part by the Arthritis Research Campaign (UK) and the Wellcome Trust (UK).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.
Supported by the Biotechnology and Biological Sciences Research
Council (UK) and Rhône-Poulenc Rorer (UK) Ltd.
§§ To whom correspondence should be addressed: Kennedy Institute of Rheumatology, 1 Aspenlea Rd., London W6 8LH, United Kingdom. Tel.: 44 181 383 4444; Fax: 44 181 383 4499; E-mail: b.foxwell{at}cxwms.ac.uk.
** Present address: The Burnham Institute, La Jolla Cancer Center, La Jolla, CA 92037.
2 A. E. Hunt and B. M. J. Foxwell, unpublished observation.
3 A. E. Hunt, F. V. Lali, J. D. Lord, B. H. Nelson, T. Miyazaki, K. J. Tracey, and B. M. J. Foxwell, manuscript in preparation.
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ABBREVIATIONS |
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The abbreviations used are: IL, interleukin; IL-2R, interleukin-2 receptor; MAP, mitogen-activated protein; MAPK, MAP kinase; GST-ATF2, glutathione S-transferase-activating transcription factor 2.
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REFERENCES |
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