©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
c-Jun N-terminal Kinase but Not Mitogen-activated Protein Kinase Is Sensitive to cAMP Inhibition in T Lymphocytes (*)

(Received for publication, January 20, 1995; and in revised form, May 16, 1995)

Yi-Ping Hsueh (1) (2) Ming-Zong Lai (1) (2)(§)

From the  (1)Graduate Institute of Microbiology and Immunology, National Yang-Ming University, Taipei 11221 and the (2)Institute of Molecular Biology, Academia Sinica, Taipei 11529, Taiwan, Republic of China

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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.


INTRODUCTION

MAP() 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) .

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-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).


MATERIALS AND METHODS

Reagents

A23187, TPA, N,2`-O-dibutyryladenosine 3`,5`-cyclic monophosphate (BtcAMP), 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) .


RESULTS AND DISCUSSION

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 BtcAMP) 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 BtcAMP 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 BtcAMP (Fig. 1, A-D). This was distinct from the profound inhibition of IL-2 secretion by cAMP. BtcAMP 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 BtcAMP 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 BtcAMP 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.


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 (BtcAMP) 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, BtcAMP 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).



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 BtcAMP 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).

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 BtcAMP 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) .


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 BtcAMP (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 BtcAMP 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 BtcAMP pretreatment (Fig. 3B), in which a 22% reduction at 0.5 mM BtcAMP and a 31% decrease at 2 mM BtcAMP 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 BtcAMP. 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 BtcAMP in 3T3 cells (Fig. 3C).


Figure 3: cAMP did not effectively antagonize Raf-1 activation in T cells. A, EL4 cells were pretreated with BtcAMP (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 BtcAMP or 4 h (B), while 3T3 was preincubated with BtcAMP 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 BtcAMP 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.



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.

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 BtcAMP 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 BtcAMP (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).


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 BtcAMP 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 BtcAMP 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 BtcAMP for 2 h, and JNK activity was determined as in A.




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 BtcAMP 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).




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-B, a major transcriptional element on the IL-2 promoter(22) . Together with an almost identical correlation between the dose of BtcAMP 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 BtcAMP (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.

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-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.


FOOTNOTES

*
This work was supported by a grant from Academia Sinica, Grant DOH83-HR-211 from the Department of Health, and Grant 83-0211-B001-003 from National Science Council, Taiwan, Republic of China. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Institute of Molecular Biology, Academia Sinica, Nankang, Taipei, 11529, Taiwan, R.O.C. Tel.: 886-2-7899236; Fax: 886-2-7826085.

The abbreviations used are: MAP, mitogen-activated protein; BtcAMP, 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.


ACKNOWLEDGEMENTS

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.


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