(Received for publication, January 20, 1995; and in revised form, May 16, 1995)
From the
The molecular mechanism underlying the cAMP inhibition of
nuclear activation events in T lymphocytes is unknown. Recently, the
activation of fibroblasts and muscle cells are shown to be antagonized
by cAMP through the inhibition of mitogen-activated protein (MAP)
kinases signaling pathway. Whether a similar antagonism may account for
the late inhibitory effect of cAMP in T cell was examined.
Surprisingly, extracellular signal regulated kinase 2 (ERK2) activation
was resistant to cAMP inhibition in all the T lymphocytes tested.
Different isoforms (ERK1, ERK2, and ERK3) of MAP kinase were poorly
inhibited by cAMP. High concentration of cAMP also only weakly
antagonized Raf-1 in T cells. The resistance of ERK and Raf-1 to cAMP
clearly distinguishes T cells from fibroblasts. In contrast, another
MAP kinase homologue c-Jun N-terminal kinase (JNK) was inhibited by
cAMP in good correlation with that of IL-2 suppression. Moreover, JNK
was antagonized by a delayed kinetics which is characteristic of cAMP
inhibition. Despite that both ERK and JNK are essential for T cell
activation, selective inhibition by cAMP further supports the specific
role of JNK in T cell activation. MAP( An elevated intracellular cAMP inhibits T
cell activation(16) . cAMP inhibits TCR-coupled early
activation events such as calcium influx and phosphatidylinositol
breakdown in some T cells(17, 18) . The dominant
inhibitory effect of cAMP is also manifested in the suppression of
subsequent cytoplasmic and nuclear activation events such as IL-2 gene
expression(18, 19, 20) . This late inhibition
is characteristic by delayed kinetics. For example, cAMP has little
effect on early IL-2 secretion (21) , and NF-
Figure 1:
cAMP poorly antagonized ERK2 in T
lymphocytes. Three different types of T cells were examined: T cell
hybridoma 9C12.7 (A, B, and E), T lymphoma
EL4 (C), T lymphocytes isolated from spleen (D and F). T cells were activated either by Con A (A-D, 10 µg/ml) or by TPA (E and F, 10-20 ng/ml). A-D and F, in
the MAP kinase assay, lymphocytes were pretreated with dibutyryl cAMP
(Bt
A
minimal effect of cAMP was also found with TPA-induced ERK2 activation
in T lymphocytes. There was a much higher increase of ERK2 activity
(30-40-fold) in T cells upon TPA induction (Fig. 1F), which was accompanied by a slower migration
band on SDS-PAGE (Fig. 1E) due to the phosphorylation
of the specific threonine and tyrosine residues(33) .
Pretreatment of T cell hybridoma 9C12.7 with 2 mM Bt Even though ERK2 is the dominant
form of MAP kinase in T lymphocytes (2) , ERK1 and ERK3
activities could be detected upon TPA activation (Fig. 2, A and B). Since different isoforms of ERK may respond
differently to exogenous stimuli and regulation(34) , we have
also examined the sensitivity of TPA-induced ERK1 and ERK3 to cAMP.
Both ERK1 and ERK3 were little affected by a 4-h treatment of cAMP in T
lymphocytes (Fig. 2, A and B). Three different
isoforms of ERK were equally resistant to cAMP inhibition. In contrast,
a 15-min treatment of Bt
Figure 2:
Different isoforms of ERK were equally
resistant to cAMP in T cells, while ERK2 was effectively inhibited by
cAMP in A431. A and B, EL4 cells were pretreated with
forskolin at the concentrations indicated for 4 h before activation by
TPA (10 ng/ml). ERK was precipitated by anti-ERK1 antibody C-16 (A) or anti-ERK3 antibody D-23 (B) (Santa Cruz
Biotech) and assayed for kinase activity as described in Fig. 1. C, A431 was serum-starved for 24 h and then pretreated with
Bt
Figure 3:
cAMP did not effectively antagonize Raf-1
activation in T cells. A, EL4 cells were pretreated with
Bt
IL-2
production was much more sensitive to cAMP inhibition than Raf-1 kinase
activity in T cells (Fig. 3D). Therefore, the weak
antagonism of Raf-1 did not lead to a similar inhibition of ERK, and
Raf-1 inhibition was not correlated with the suppression of IL-2
synthesis by cAMP. It may be noted that resistance of ERK to cAMP has
also been observed in PC12 and Swiss 3T3
cells(25, 27) . Raf activation is suppressed by cAMP
in PC12 cells, and ineffectiveness of cAMP to inhibit ERK is due to the
presence of cAMP-independent pathway that activates ERK(38) .
Whether the partial inhibition of Raf-1 suggests the presence of a
similar mechanism in T cells remains to be determined.
Figure 4:
cAMP inhibited JNK with delayed kinetics. A, progressive inhibition of JNK activity in splenic T cells.
T lymphocytes freshly isolated from spleen were pretreated with
Bt
Figure 5:
The inhibition of JNK by cAMP was
prevented by cycloheximide and actinomycin D. A, splenic T
cells were pretreated with forskolin (FSK) at the
concentrations indicated for 15 min or for 2 h before activation with
TPA/A23187. 20 µg/ml each of cycloheximide (CHX) or
actinomycin D (Act.D) was added together with forskolin as
indicated. Cell extracts were prepared and JNK assays were performed as
described in Fig. 4A. B, the protein levels of
JNK were not affected by cAMP. Cell extracts were prepared from splenic
T lymphocytes pretreated with Bt
Figure 6:
Comparison between the JNK activity and
the IL-2 production in the presence of cAMP. JNK activity was
determined in Fig. 4. Activation-induced IL-2 secretion was
determined as described in Fig. 1. The fully activated JNK
activity (openbar) and IL-2 secretion (solidbar) are used as 100% (B). Each data point is
the average of duplicate. A, T lymphocytes from spleen; B, EL4.
The delayed
suppression of JNK in T cells follows kinetics similar to that for the
inhibition on the binding of NF- T cell activation events represent a full integration
of signals from different pathways. Raf-1 and JNK each define one of
the signaling pathways downstream of
Ras(3, 4, 7, 8, 9, 34) .
Both pathways are critical for T cell activation, as competitive
inhibition of either Raf-1 or JNK blocks IL-2 gene
activation(12, 14) . The induction of ERK2 by TPA
alone does not activate T cells, while a costimulatory signal is
required for JNK induction as well as for T cell
activation(12) . This is in contrast to the observation that
activation of JNK by TPA in fibroblasts does not require
A23187(12) . In the present study, cAMP is shown to inhibit JNK
but not MAP kinase in T lymphocytes, yet T cell activation is
effectively suppressed. This supports a specific role of JNK in T cell
activation, and is consistent with the suggestion that JNK activation
represents a stage at which different T cell activation signals are
integrated(12) . Hence, T cell activation can be blocked by
cAMP at this step without affecting another essential pathway (MAP
kinase cascade). Interestingly, JNK is stimulated by UV (7) or
tumor necrosis factor(8) , the stimulus that also activates
NF-
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
)
kinases and JNK define two distinct
activation pathways in T lymphocytes. Raf and MAP kinase are coupled to
p21
activation, and are induced by T cell
receptor (TCR)
engagement(1, 2, 3, 4) , IL-2 (5) or TPA treatment(2, 6) . Ras activation
also leads to induction of JNK(7, 8, 9) , an
MAP kinase analog (10, 11, 12, 13) .
However, TPA or TCR alone poorly activates JNK in T lymphocytes, and a
full JNK activity has to be induced by the combination of two signals
like Ca
ionophore/TPA or CD28/TCR(12) . Both
the induction of MAP kinase and JNK are essential for T cell
activation. Hence, IL-2 gene transcription is inhibited by dominant
negative mutant of Raf-1 (14) and by competitive inhibition of
JNK(12) , while overexpression of ERK enhances IL-2 expression
in T cells(15) .
B binding is
reduced only after 4 h of cAMP treatment (22) . The molecular
mechanism underlying the late inhibitory effects of cAMP remains
unclear. Recently, cAMP was shown to inhibit the activation of
fibroblasts, adipocytes, and muscle cells by antagonizing Raf-MAP
kinase
pathway(23, 24, 25, 26, 27, 28, 29) .
A similar antagonism in T cells may well explain the inhibitory
activity of cAMP. In this study we found that MAP kinases were
unexpectedly resistant to cAMP inhibition in T lymphocytes. Instead,
cAMP preferentially inhibited JNK in T lymphocytes. T cell activation
thus can be effectively inhibited by antagonizing a selective step
(JNK) without affecting other signaling pathway (ERK).
Reagents
A23187, TPA, N,2`-O-dibutyryladenosine 3`,5`-cyclic
monophosphate (Bt
cAMP), forskolin, Con A, and myelin basic
protein were purchased from Sigma. Epidermal growth factor (EGF) and
epidermoid carcinoma A431 (ATCC CRL 1555) were obtained from Dr.
Jaulang Huang (Institute of Molecular Biology, Academia Sinica, Taipei,
Taiwan). Anti-Raf antiserum (SP63) was a generous gift of Dr. Ulf Rapp
(National Cancer Institute, Frederick, MD). GST-c-Jun(1-79),
produced by Dr. Michael Karin, was obtained through Dr. Hsin-Fang Y.
Yen (Institute of Molecular Biology, Academia Sinica). Bacterially
expressed (His)
-MKK(K97M) was a generous gift of Dr.
Natalie Ahn (University of Colorado, Boulder, CO), and was purified
according to Mansor et al.(30) . Anti-ERK1 C-16,
anti-ERK2 C-14, anti-ERK3 D-23, anti-JNK2 N-19, and anti-JNK1,2 FL were
obtained from Santa Cruz Biotech (Santa Cruz, CA). N-19 reacted with
the 54-kDa JNK2 in T cells but not with the 46-kDa JNK1 claimed by
Santa Cruz Biotech.
T Cell Lines and T Cell Hybridomas
9C12.7 is a T
cell hybridoma that recognizes repressor cI 12-26 in the
context of I-A
(31, 32) . EL4 (ATCC TIB39)
was a gift of Dr. Nan-Shih Liao (Institute of Molecular Biology,
Academia Sinica). Splenic T lymphocytes were purified by nylon wool
(Polyscience, Warrington, PA) column and treated with J11d (anti-B
cell) followed by rabbit complement (Cedarlane, Ontario). IL-2 was
quantitated by the proliferation of IL-2-dependent cell line HT-2 (ATCC
CRL 1841) as described previously(31, 32) .
Protein Kinase Assay
Splenocytes (2-3
10
cells/sampling point), EL4 cell or T cell
hybridomas (2-3
10
cells/sampling point) were
pretreated or activated as indicated, and washed twice with
phosphate-buffered saline. The preparation of cell extracts,
precipitation of ERK and Raf-1 with the specific antibody, and assay of
the immune complex were performed according to Cook and
McCormick(24) . MAP kinase was assayed by the phosphorylation
of myelin basic protein, and the substrate for Raf kinase was
(His)
-MKK(K97M). The reaction products were resolved by 15%
SDS-PAGE for MAP kinase assay, and by 10% SDS-PAGE for Raf-1 kinase,
followed by autoradiography and quantitated by PhosphorImager
(Molecular Dynamics). Mobility shift of ERK2 and Raf-1 in Western blot
were performed according to Leevers and Marshall(33) . The
solid state JNK assay was performed by incubating cell extracts with
GST-c-Jun(1-79) GSH-agarose beads as described by Hibi et
al.(10) .
cAMP Poorly Antagonized ERK2 in T Cells
Even
though the effect of cAMP has been tested on MAP kinase by Nel et
al.(1) , the concentration used (BtcAMP, 100
nM) was not inhibitory for T cell activation. In the present
study, the effect of cAMP on ERK was examined at the range (100
µM to 2 mM of Bt
cAMP) known to
suppress T cell activation. Three different types of T cell were used:
T cell hybridomas 9C12.7 (Fig. 1, A, B, and E), T lymphoma EL4 (Fig. 1C), and T
lymphocytes freshly isolated from spleen (Fig. 1, D and F). Given the lag time (4 h) required for cAMP to inhibit
NF-
B binding(22) , T cells were preincubated with
Bt
cAMP for 15 min and for 4 h before Con A activation and
ERK2 activity quantitation. It was found that ERK2 activity was similar
between 15 min and 4 h of cAMP treatment. Only results from T cells
preincubated with cAMP for 4 h are thus presented. MAP kinase activity
was not affected by treatment with 0.5 mM Bt
cAMP (Fig. 1, A-D). This was distinct from the
profound inhibition of IL-2 secretion by cAMP. Bt
cAMP
inhibited IL-2 production at 100 µM (Fig. 1B) and largely suppressed IL-2 secretion at
0.5 mM (Fig. 1, B-D). Therefore, Con
A-activated IL-2 production was inhibited by Bt
cAMP at a
concentration (0.5 mM) that did not affect ERK2 in all 3
different T cells examined (Fig. 1, B-D). The
profound inhibition of IL-2 by cAMP apparently was not due to an
inhibition of ERK2 in T lymphocytes. It may be noted that a weak
inhibition of ERK2 by the higher concentration (2 mM) of
Bt
cAMP could be found in splenic T cells (Fig. 1D), but not in EL4 (Fig. 1C).
Thus, there is a small discrepancy in the sensitivity of ERK2 to high
concentrations of cAMP between different types of T lymphocytes.
cAMP) or forskolin (FSK) for 4 h before
activation. Cell lysates were prepared 10 min post-activation, and
200-400 µg of lysate was precipitated with 1 µg of
anti-ERK2 C-14 antibody (Santa Cruz Biotech) and 20 µl of protein
A-Sepharose. The kinase activity of the immune complexes was determined
by the phosphorylation of myelin basic protein (24) as resolved
on 15% SDS-PAGE and quantitated by PhosphorImager (Molecular Dynamics).
The relative reactivity represents the ratio of the activated kinase
activity over unstimulated kinase activity. B-D, for
IL-2 production, Bt
cAMP was added simultaneously with Con
A, and the IL-2 content was determined 8 h after activation. The fully
activated ERK2 activity (openbar) and IL-2 secretion (solidbar) are used as 100%. Each data point is the
average of three independent experiments. S.D. are expressed as error
bar, those not shown are too small in scale. E, the mobility
shift of the phosphorylated ERK2. The immunoprecipitated ERK2 was
resolved on 10% SDS-PAGE and transferred to polvinylidene difluoride
membrane (Millipore). ERK2 was detected by incubating with C-14 (0.375
µg/ml), followed with 1:1000 diluted alkaline
phosphatase-conjugated anti-rabbit Ig antibody, and developed by
Immunopure alkaline phosphatase substrate kit II
(Pierce).
cAMP had no effect on the mobility shift of ERK2 on
PAGE (Fig. 1E). Similarly, TPA-induced MAP kinase
activity was resistant to forskolin inhibition in T lymphocytes freshly
isolated from spleen (Fig. 1F). ERK2 can also be
activated by exogenous IL-2. The IL-2-induced ERK2 was similarly
tolerant to cAMP (data not shown).
cAMP resulted in an effective
suppression of the EGF-activated ERK2 in A431 cells (Fig. 2C). This is in accordance with the dominant
inhibition of ERK by cAMP in fibroblasts, adipocytes, and muscle
cells(23, 24, 25, 26, 27, 28, 29) .
cAMP (concentration indicated) for 15 min before EGF (20
ng/ml) activation(25) . Cell lysates were prepared 10 min after
activation, and the ERK2 activities were
determined.
Weak Inhibition of cAMP on Raf-1 Kinase in T
Lymphocytes
Since Raf-1 kinase mediates MAP kinase activation,
and cAMP inactivates Raf-1 in fibroblasts by PKA phosphorylation (23, 24, 25, 29) , we examined
whether cAMP blocks the activation of Raf-1 in T lymphocytes. The
phosphorylation of Raf-1 induced by TPA/A23187 resulted in a decreased
mobility when resolved on SDS-PAGE. Preincubation with 2 mM BtcAMP or 100 µM forskolin for 4 h had no
effect on the extent of mobility shift of Raf-1 (Fig. 3A). An identical observation on c-Raf by
Bt
cAMP in T cells has been reported
previously(35) . Because the mobility shift of Raf-1 does not
necessarily correlate with its kinase
activity(36, 37) , Raf-1 was also assessed directly by
the ability to phosphorylate MKK(K97M). Activation by TPA/A23187 led to
an immediate increase of Raf-1 kinase activity. There was a weak
inhibition of Raf-1 kinase by Bt
cAMP pretreatment (Fig. 3B), in which a 22% reduction at 0.5 mM Bt
cAMP and a 31% decrease at 2 mM Bt
cAMP were found with MKK phosphorylation. The extent
of inhibition demonstrated that Raf-1 was much more sensitive to cAMP
suppression than ERK2 at lower concentration of Bt
cAMP.
Despite the observed inhibition, cAMP did not effectively antagonize
Raf-1 at higher concentration. In contrast, activation-induced Raf-1
activity was completely suppressed by 0.5 mM
Bt
cAMP in 3T3 cells (Fig. 3C).
cAMP (2 mM) or forskolin (100 µM)
for 4 h followed by activation with TPA (10 ng/ml). Cell extracts were
prepared 10 min after activation, and then precipitated by anti-Raf
antiserum SP63. The mobility shift of Raf-1 was detected by antibody
blotting of immunoprecipitates resolved on 7.5% SDS-PAGE as described
in Fig. 1E. The bands corresponding to Raf-1 and
phosphorylated Raf-1 are marked. B and C, Raf-1
activity was inhibited by cAMP in 3T3 but not in 9C12.7. Before
activation with TPA (10 ng/ml) and A23187 (80 ng/ml), 9C12.7 was
pretreated with Bt
cAMP or 4 h (B), while 3T3 was
preincubated with Bt
cAMP for 15 min. The Raf-1 kinase
activity of the immune complexes was determined by the phosphorylation
of kinase-inactive MKK(K97M)(30) . The kinase assay mixture was
separated on 10% SDS-PAGE and quantitated by PhosphorImager. D, comparison of TPA/A23187-induced Raf-1 kinase activity and
IL-2 secretion in the presence of Bt
cAMP in 9C12.7. IL-2
production was determined as described in Fig. 1. The fully
activated Raf-1 activity (openbar) and IL-2
secretion (solidbar) are used as 100%. Each data
point is the average of three independent
experiments.
cAMP Inhibits JNK in Delayed Kinetics
The weak
inhibitory effect of cAMP on MAP kinase and Raf-1 clearly distinguishes
T cells from fibroblasts and other cells (Fig. 2C and
3C). The resistance of MAP kinase to PKA could in part reflect
the complicated nature of T lymphocyte activation, which involves
multiple signaling pathways downstream of T cell receptor
complex(39) . Because the newly identified JNK defines an
independent activation pathway in T lymphocytes(12) , we
examined whether it is similarly resistant to cAMP. JNK activity was
not affected when T lymphocytes isolated from spleen were treated with
BtcAMP for 15 min (Fig. 4A). Longer
incubation (30 min and 1 h) with cAMP resulted in a significant
suppression of JNK, in which a 40% decrease was found with 0.5
mM Bt
cAMP treatment. Two-hour incubation with 0.5
mM cAMP inhibited nearly 70% of JNK activity (Fig. 4A). Further incubation did not lead to additional
suppression (data not shown for 4-h treatment). Such a delayed
antagonism of JNK by cAMP is also illustrated by the progressive shift
of dose-inhibition curves (Fig. 4B). A nearly complete
inhibition of JNK was found with T lymphoma EL4 after 2-h incubation
with Bt
cAMP (Fig. 4C). Treatment with
forskolin for 2 h also led to a similar inhibition of JNK in splenic T
lymphocytes (Fig. 5A). The decrease in JNK activity was
well correlated with the reduction in IL-2 production by cAMP in both
splenic T lymphocytes and EL4 (Fig. 6).
cAMP at the indicated concentration for 15 min, 30 min, 1
h, and 2 h before activation with TPA (10 ng/ml) and A23187 (80 ng/ml).
Cell extracts were prepared 15 min after activation. Solid-phase JNK
assay was performed by incubating cell extracts with
GST-c-Jun(1-79) and GSH-agarose, followed by kinase reaction and
resolution on SDS-PAGE(10) . The phosphorylation of
c-Jun(1-79) was quantitated by PhosphorImager. B,
summary of dose-dependent inhibition curves at different time courses
of cAMP treatment. The Bt
cAMP incubation time was indicated
to the right. The fully activated JNK activity is used as
100%. Each data point is the mean of the two independent experiments. C, JNK was completely suppressed by cAMP in EL4. EL4 was
pretreated with different concentrations of Bt
cAMP for 2 h,
and JNK activity was determined as in A.
cAMP at the concentrations
indicated for 2 h, and were resolved on SDS-PAGE, blotted, and detected
with antibody specific for JNK2 (N-19, Santa Cruz Biotech). A
similar result was obtained with antibody reacted with both JNK1 and
JNK2 (FL, Santa Cruz Biotech) (not
shown).
B, a major transcriptional element
on the IL-2 promoter(22) . Together with an almost identical
correlation between the dose of Bt
cAMP and the extent of
suppression for both IL-2 and JNK (Fig. 6), it may be suggested
that the inhibition of IL-2 synthesis by cAMP is mediated by the
antagonism of JNK in T lymphocytes. The molecular mechanism underlying
the progressive inhibition of JNK remains unclear. JNK1 and JNK2 are
equally present in T cells(12) , and their protein levels were
not affected by 2-h treatment of Bt
cAMP (Fig. 5B for JNK2, data not shown for JNK1), suggesting the inhibition was
not due to a suppression of JNK synthesis by cAMP. In contrast, new RNA
and protein synthesis were apparently required for the inhibition of
JNK by cAMP, as demonstrated by the increased JNK activity and the
reduced sensitivity of JNK to cAMP inhibition in the presence of
actinomycin D or cycloheximide (Fig. 5A). Presumably,
the suppression of JNK was mediated by the newly synthesized
inhibitor(s) stimulated by cAMP. The slow inhibition of JNK is distinct
from the observation that a 15-min cAMP incubation is enough to
effectively suppress ERK2 in A431 cells (Fig. 2C). Such
rapid inhibition of MAP kinase cascade is ascribed to an inactivation
of Raf-1 by PKA phosphorylation in other type of cells (23, 24, 25, 29) . The requirement
of protein synthesis and the slow inhibitory kinetics may suggest a
lower likelihood of a direct phosphorylation of JNK by PKA in T cells.
It may be noted that the kinase cascade leading to the activation of
JNK has recently been identified in a number of
cells(8, 9) . It is possible that the stage inhibited
by cAMP is located upstream of JNK. Identification of the exact stage
that cAMP inhibits which results in JNK inhibition may provide clues to
our understanding of the nuclear suppressive effect of cAMP in T
lymphocytes.
B(40) . Both the binding of NF-
B (22) and
the activation of JNK (Fig. 4) are antagonized by cAMP in T
lymphocytes, and the inhibition time courses of NF-
B (22) and JNK (Fig. 4A) are strikingly similar.
Whether this implicates a direct involvement of JNK in the activation
of NF-
B is currently under investigation.
cAMP, N
,2`-O-dibutyryladenosine 3`,5`-cyclic
monophosphate; EGF, epidermal growth factor; ERK, extracellular signal
regulated kinase; JNK, c-Jun N-terminal kinase; MKK, MAP kinase kinase;
PKA, protein kinase A; TCR, T cell receptor; TPA,
12-O-tetradecanoylphorbol 13-acetate; PAGE, polyacrylamide gel
electrophoresis; IL, interleukin; Con A, concanavalin A.
We thank Dr. Michael Karin for the helpful suggestions
and for GST-c-Jun(1-79); Dr. Ulf Rapp for SP63 antiserum; Drs.
Sam Mansor and Natalie Ahn for MKK(K97M); and Drs. Ellen Rothenberg,
Hsiang-Fu Kung, and Sun-Yu Ng for the helpful discussions.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.