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The Journal of Immunology, 2000, 164: 2311-2319.
Copyright © 2000 by The American Association of Immunologists

Identification of a Membrane Ig-Induced p38 Mitogen-Activated Protein Kinase Module That Regulates cAMP Response Element Binding Protein Phosphorylation and Transcriptional Activation in CH31 B Cell Lymphomas1

Jennifer M. Swart, Daniele M. Bergeron and Thomas C. Chiles2

Department of Biology, Boston College, Chestnut Hill, MA 02467


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The cAMP response element (CRE) binding protein (CREB) is emerging as a key regulatory factor of gene transcription in B lymphocytes; however, the postreceptor pathways that regulate CREB activity and CRE-dependent gene transcription remain largely undefined. We investigated B cell Ag receptor (BCR)-mediated phosphorylation and activation of CREB in the surface IgM+ CH31 B cell lymphoma, which undergoes Ag-dependent cell death. The activity of p38 mitogen-activated protein kinase (MAPK) was increased in response to BCR ligation. Phosphorylation of CREB on serine 133, a modification that positively regulates its trans-activation, was concomitantly increased. Inhibition of p38 MAPK by pretreating CH31 B cells with the highly specific bicyclic imidazole inhibitor, SB203580, reduced BCR-induced CREB phosphorylation. BCR cross-linking also led to increased MAPK-activated protein kinase-2 activity, an enzyme that lies immediately downstream from p38 MAPK; MAPK-activated protein kinase-2 immune complexes phosphorylated a peptide substrate containing the CREB serine 133 phosphoacceptor motif. Given the role of CREB in regulating junB gene expression in mature B lymphocytes, we examined whether p38 MAPK activity was necessary for CRE-dependent junB transcription in CH31 B cells. BCR ligation led to increased junB mRNA levels, which were significantly reduced in CH31 B cells pretreated with SB203580. Activation of a CRE-dependent junB promoter/chloramphenicol acetyltransferase (CAT) reporter gene by the BCR was also blocked by SB203580. Similarly, inhibition of p38 MAPK in surface IgM+ WEHI-231 B cell lymphomas resulted in reduced BCR-induced junB mRNA expression and junB promoter activation. The results implicate a p38 MAPK pathway in BCR-mediated CREB phosphorylation and junB transcriptional activation in B cell lymphomas.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The B cell Ag receptor (BCR)3 regulates cell-cycle progression in a manner dependent on the developmental stage of B lymphocytes. For example, BCR ligation on mature B cells can lead to G1 progression and S phase commitment, whereas immature B lymphocytes undergo apoptosis (1, 2, 3). The mechanism(s) by which the BCR regulates cellular physiology and the cell cycle is not completely understood. The earliest consequence of BCR cross-linking is the activation of src-protein tyrosine kinases (4, 5). Subsequent signal transmission occurs along four known pathways that include a phosphatidylinositol 3-kinase module, p21ras GTPase/Raf/extracellular signal-regulated kinase (ERK) cascade, phospholipase C{gamma}, and RhoGTPase (4, 5, 6, 7, 8). Although the biological function(s) of individual pathways remain unclear, it is recognized that signals must be transduced to the nucleus to alter gene expression. Some of the earliest known nuclear events include phosphorylation of Ets-1, induction of the immediate-early gene response, and synthesis of carbamoylphosphate synthetase-aspartate transcarbamylase-dihydroorotase and G1-cyclins (9, 10, 11, 12, 13, 14, 15, 16, 17).

It is now established that the 43-kDa phosphoprotein, cAMP response element (CRE) binding protein (CREB), is a major determinant in regulating gene transcription in B cells (18, 19, 20). For example, members of the CREB/activating transcription factor (ATF) family are involved in determining the activity of the 3' {kappa} enhancer in pre-B cell lines (18). CREB contributes to the activation of MHC class II promoters via X2 box sequences (21, 22). CREB also positively regulates bcl-2 gene transcription during B cell activation and rescue of immature B cells from apoptosis (23). It has been postulated that CREB/ATF family members may play a role in transcription dysregulation, given that several genes containing CRE are expressed aberrantly in malignant tumors and transformed cells (23, 24). A focal point of research at present is directed toward understanding the regulation of CREB trans-activation by B cell surface receptors. CREB is a member of the CREB/ATF bZip family of transcription factors that binds to nucleotide sequences homologous to the palindromic CRE, TGACGTCA (25, 26). In nonlymphoid cells, surface receptors regulate trans-activation of CREB primarily through protein kinase A (PKA), pp90rsk, mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK), Ca2+-dependent calmodulin kinase (CaMK) II, and CaMK IV pathways (25, 26, 27, 28). Phosphorylation on serine 133 located within the kinase-inducible domain does not affect its binding to the CRE site, but rather increases its association with adapter proteins, such as CREB-binding protein, leading to increased transcriptional activity from genes containing CRE (25, 26).

BCR cross-linking on mature B cells leads to increased phosphorylation of CREB on serine 133 (19, 20, 29, 30). Our laboratory demonstrated that BCR-stimulated transcription of the immediate-early response gene, junB, was mediated by a CRE-like sequence located between -194 and -42 bp (19, 30). This site is occupied by a protein heterodimer consisting of CREB/ATF-1. The relative importance of serine 133 phosphorylation to junB transcription was demonstrated in that a conserved serine-to-alanine substitution at amino acid 133 in CREB abrogated BCR-induced junB gene promoter activation (19). We recently provided evidence for a novel pathway controlling serine 133 phosphorylation in mature B cells that involves the opposing actions of PKA and an okadaic acid-sensitive serine/threonine protein phosphatase activity (30). In brief, CREB phosphorylation on serine 133 was achieved by a BCR-dependent decrease in the protein phosphatase activity without apparently affecting PKA-targeted serine 133 phosphorylation. Despite these observations, many of the intermediate steps that couple the BCR to CREB trans-activation remain poorly defined. In particular, it is not entirely clear to what extent signaling pathways activated by the BCR contribute to CREB phosphorylation. It is also recognized that little is known about the regulation of CREB activity in B cells other than mature B lymphocytes. We sought herein to identify intermediate signal transduction components that link the BCR to CREB phosphorylation and CRE-dependent transcription. Our experiments were conducted in surface IgM+ CH31 and WEHI-231 B cell lymphomas, which have been used as models for immature B cell tolerance (1).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture and reagents

The surface IgM+ murine B cell lymphomas, CH31 and WEHI-231, were kindly provided by Dr. David W. Scott (Department of Immunology, American Red Cross, Rockville, MD) (31, 32). Dr. Richard Asofsky (National Institutes of Health, Bethesda, MD) provided the mature Bal17 lymphoma (33). Cells were cultured in RPMI 1640 containing 10 mM HEPES, pH 7.5, 2 mM L-glutamine, 5 x 10-5 M 2-ME, and 10% FCS (BioWhittaker, Walkersville, MD). CH31 B cell lymphomas were grown in the presence of 1 mM sodium pyruvate and 1x RPMI 1640 amino acids (Sigma, St. Louis, MO). Cells were maintained in log phase growth in a humidified incubator at 37°C and 5% CO2. Mature splenic B lymphocytes were isolated from 8- to 12-wk-old BALB/c mice (The Jackson Laboratory, Bar Harbor, ME) as described (34). Mice were cared for and handled at all times in accordance with National Institutes of Health Institutional Guidelines. F(ab')2 of goat anti-mouse IgM (anti-Ig) was obtained from Jackson ImmunoResearch Laboratories (West Grove, PA). H-89, SB203580, PD98059, and KN-93 were purchased from CalBiochem-NovaBiochem (San Diego, CA); H-85 was purchased from Seikagaku (St. Petersburg, FL). Inhibitors were prepared in Me2SO at fold concentrations such that the final amount of Me2SO in culture medium was below 0.1%.

Western blotting

Whole-cell lysate protein was separated by electrophoresis through a 10% SDS polyacrylamide gel and transferred to Immobilon-P membrane (Millipore, Bedford, MA). The membrane was blocked in TBST (20 mM Tris, pH 7.6, 137 mM NaCl, and 0.05% Tween 20) containing 5% nonfat dry milk for 5 h and then incubated overnight (4°C) in TBST containing 1 µg/ml anti-p38 MAPK (Santa Cruz Biotechnology, Santa Cruz, CA), anti-phospho-p38 MAPK (New England BioLabs, Beverly, MA), anti-CREB, or anti-phospho(Ser133) CREB (Upstate Biotechnology, Lake Placid, NY) Abs. The membrane was washed several times in TBST and then incubated with a 1:2500 dilution of anti-rabbit IgG-coupled HRP Ab (Santa Cruz Biotechnology). After 90 min, the blot was washed several times with TBST and developed with enhanced chemiluminiscence reagents (Kirkegaard and Perry, Gaithersburg, MD).

MAPKAP kinase-2 assay

CH31 B cells were sonicated for 5 s in 1 ml lysis buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 5 mM EDTA, 1% Nonidet P-40, 20 mM Na3VO4, 1 mM NaF, 50 mM ß-glycerophosphate, 1 µg/ml aprotinin and leupeptin, 1 mM PMSF, and 0.7 µg/ml pepstatin) and freeze-thawed in a dry-ice/water bath (35, 36). Cellular debris was removed by centrifugation, and the supernatant was incubated with 5 µg isotype-matched rabbit IgG plus protein A-Sepharose for 2 h (4°C). The immune complexes were recovered by centrifugation, and the supernatant was incubated with 5 µg anti-MAPKAP kinase-2 Ab (Upstate Biotechnology) plus protein A-Sepharose for 2 h. The MAPKAP kinase-2 immune complexes were collected by centrifugation, washed six times with 1 ml lysis buffer, two times with 1 ml kinase buffer (25 mM HEPES, pH 7.4, 25 mM MgCl2, 0.5 mM Na3VO4, and 2 mM DTT), and then resuspended in 30 µl kinase buffer containing 25 µM ATP, 10 µCi [{gamma}-32P]ATP (6000 Ci/mmol; New England Nuclear, Boston, MA) and 5 µg recombinant heat shock protein (hsp) 25 substrate (StressGen Biotechnologies, Victoria, Canada). Kinase reactions were terminated after 30°C (30 min) by addition of 2x Laemmli sample buffer, separated by SDS-PAGE, and subjected to autoradiography.

In some assays, 1 µg of a peptide containing the serine 133 phosphoacceptor site of CREB (CREBtide = ILSRRPS133YRK, synthesis by WAJCSC Protein Chemistry Facility, Lake Placid, NY) was used as substrate (30). The amount of phosphorylated CREBtide was quantitated by spotting the reaction mixture on Whatman P81 phosphocellulose filters. The filters were dried at for 1 h and washed seven times with 40 ml of 0.75% H3PO4 and then with 20 ml acetone. The amount of 32P incorporated into the peptide substrate was quantitated by scintillation spectophotometry.

p38 MAPK assay

CH31 B cells were solubilized in 250 µl MAPK lysis buffer (20 mM Tris, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM ß-glycerophosphate, 1 mM Na3VO4, 1 mM PMSF, and 1 µg/ml leupeptin). The cell lysates were incubated for 18 h (4°C) with 1 µg anti-phospho(Thr180/Tyr182) p38 MAPK Ab (New England BioLabs)-bound protein A-Sepharose. The immune complexes were collected by centrifugation, washed twice with 1 ml MAPK lysis buffer, twice with 1 ml kinase buffer (25 mM Tris, pH 7.5, 2.5 mM ß-glycerophosphate, 2 mM DTT, 0.1 mM Na3VO4, and 10 mM MgCl2), and resuspended in 50 µl of kinase buffer containing 100 µM ATP and 1 µg of GST-ATF-2 fusion protein substrate. The kinase reactions were carried at 30°C (30 min), and p38 MAPK-mediated phosphorylation of GST-ATF-2 substrate was detected by immunoblotting with anti-phospho(Thr71) ATF-2 Ab (New England Biolabs).

mRNA isolation and Northern blot analysis

Total RNA was prepared using a MicroFastTrack kit (Invitrogen, Carlsbad, CA). Approximately 107 B cells were collected by centrifugation, washed in PBS, and lysed in a buffer containing 0.2 M Tris, pH 7.5, 0.2 M NaCl, 1.5 mM MgCl2, and 2% SDS. Insoluble material was removed by centrifugation at 4000 x g for 5 min, and then the lysate was adjusted to 0.5 M NaCl. Poly(A+) RNA was isolated by binding to oligo(dT) cellulose spin columns. Electrophoresis of RNA was conducted under denaturing conditions in a 1% agarose gel containing 0.2 M MOPS, pH 6.5, 50 mM sodium acetate, 5 mM EDTA, and 20% formaldehyde. Following electrophoresis, the gel was incubated in diethyl pyrocarbonate-treated water (15 min), 50 mM NaOH/150 mM NaCl (30 min), and 0.1 M Tris, pH 8.0/150 mM NaCl (30 min). RNA was transferred to nylon membrane (75 mm Hg for 45 min) using a Stratagene Pressure Blotter (Stratagene, La Jolla, CA) and then cross-linked to the membrane with a Stratalinker (Stratagene) at 1200 µJ, 254 nm. The membrane was prehybridized at 65°C for 1 h in 2 ml of hybridization buffer (0.5 M phosphate buffer, pH 7.0, 1 mM EDTA, 7% SDS, 1% BSA, 100 µg/ml salmon sperm DNA). A 32P-labeled cDNA probe specific for the murine junB gene was prepared using a DECAprime II kit (Ambion, Austin, TX) and allowed to hybridize overnight at 65°C. The membrane was then washed twice (15 min) in 50 ml of 40 mM phosphate buffer, 1 mM EDTA, 1% SDS, twice for 1 h (65°C), and then subjected to autoradiography at -70°C.

EMSA

Nuclei were isolated from CH31 B cells and extracted in a 450-mM NaCl buffer as described by Chiles and Rothstein (13). Binding reactions were conducted in a final volume of 15 µl and contained 1.5 µg nuclear extract protein, 1 µg poly(dI-dC), and 0.5 ng 32P-labeled oligodeoxynucleotide probe. After 20 min (23°C), the reaction products were electrophoresed through a 5% polyacrylamide/TBE gel and subjected to autoradiography. The oligodeoxynucleotide probe used to detect CRE binding activity of nuclear extracts corresponded to 5'-AGAGATTGCCTGACGTCAGAGAGCTAG-3'.

DNA transfection and transient expression

DNA transfection of B cell lymphomas was conducted as described by Sonenshein and coworkers 37 : B cells (2 x 107) were washed once in RPMI 1640 and resuspended in 1 ml of RPMI 1640 containing 20% FCS. Cells were then incubated on ice for 10 min and subsequently electroporated (240 V, 960 µF) in 250-µl aliquots containing 40 µg plasmid DNA using a Gene Pulser apparatus (Bio-Rad, Richmond, CA). B cells were then incubated sequentially on ice and at room temperature for 5 min, centrifuged, and resuspended at 2.5 x 106 cells/ml in RPMI 1640 medium. B cells were stimulated with 15 µg/ml of anti-Ig for 8 h, washed in PBS, resuspended in 0.2 M Tris, pH 8.0, and subjected to four freeze-thaw cycles. The lysates were incubated at 68°C for 15 min, and insoluble material was removed by centrifugation at 15,000 x g for 15 min. CAT assays were performed using 250 µg of cellular protein as described by Chiles and Rothstein (13). Reaction products were separated by TLC, and the resulting autoradiograms were analyzed by densitometry using a Molecular Dynamics Personal Densitometer equipped with ImageQuant software (Sunnyvale, CA). Reporter gene plasmid denoted as 3XTRE/CAT contains three tandem copies of the cis-acting tetradecanoyl phorbol acetate response element (TRE; 5'-TGACTCA-3') upstream of HSV-tk promoter in pBLCAT (38). The plasmids were kindly provided by Dr. Michael Karin (University of California at San Diego, La Jolla, CA).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
BCR engagement stimulates phosphorylation of endogenous CREB at Ser133 in CH31 B cells

To monitor CREB phosphorylation in response to BCR cross-linking, whole-cell extracts were prepared from CH31 B cells and the level of CREB phosphorylation on serine 133 was measured following SDS-PAGE by immunoblotting with an anti-phospho(Ser133)CREB Ab that specifically recognizes CREB phosphorylated on serine 133 (39). CH31 B cells treated with anti-Ig exhibited an increased level of CREB phosphorylation (~4.3-fold) in comparison to control cells, with maximal levels observed at the 40-min time point (Fig. 1GoA). It should be noted that the anti-phospho(Ser133)CREB Ab cross-reacts with ATF-1 phosphorylated on serine 63; ATF-1 is a 38-kDa bZIP family transcription factor that shares sequence homology surrounding the Ser133 phosphoacceptor motif (26, 39). These results indicate that a phospho-protein of ~38-kDa was detected in control CH31 B cells and its relative abundance increased in response to BCR ligation. The increased phospho(Ser133) CREB following BCR cross-linking did not arise from changes in the total amount of cellular CREB protein, as shown by immunoblotting with a polyclonal anti-CREB Ab (Fig. 1GoB).



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  		FIGURE 1. BCR-stimulated CREB phosphorylation on serine 133 is sensitive to pharmacological inhibitors. A, CH31 B cells were cultured in medium alone (M) or stimulated with 10 µg/ml anti-Ig for 10, 20, 40, and 90 min. The level of phospho(Ser133)CREB was determined by immunoblotting of cellular extracts with anti-phospho(Ser133)CREB Ab as described in Materials and Methods. pCREB and pATF1 denote location of the phosphorylated CREB and ATF-1, respectively. B, The membrane was stripped and immunoblotted with anti-CREB Ab to measure total CREB protein levels. C, Untreated CH31 B cells (None) or B cells treated with 10 µM KN93, 10 µM PD98059, or 20 µM SB203580 for 30 min were subsequently incubated in the absence (-) or presence (+) of 10 µg/ml anti-Ig for 40 min. Whole-cell lysates were immunoblotted with anti-phospho(Ser133)CREB Ab as described above. Densitometric analysis of the pCREB bands is summarized below each lane. The data are representative of three independent experiments.

 
Effects of kinase inhibitors on BCR-induced CREB serine 133 phosphorylation in CH31 B cells

To identify protein kinase pathways that mediate BCR-induced CREB phosphorylation, we evaluated the effect of several kinase inhibitors on anti-Ig-induced CREB serine 133 phosphorylation, including SB203580 (an inhibitor of p38 MAPK), PD98059 (an inhibitor of MEK), and KN93 (an inhibitor of CaMK II) (40, 41, 42). Of note, the concentrations of inhibitors used in these experiments have been previously reported to block surface receptor-mediated CREB and ATF-1 phosphorylation in several nonlymphoid cell types (27, 28). As shown in Fig. 1GoC, BCR-induced serine 133 phosphorylation of CREB (increased 3.1-fold over control B cells) was not significantly affected by pretreatment of CH31 B cells with 10 µM KN93. By contrast, pretreatment of CH31 B cells with 10 µM PD98059 reduced anti-Ig-induced CREB serine 133 phosphorylation to 2.6-fold. Pretreatment of CH31 B cells with 20 µM SB203580 completely abrogated anti-Ig-stimulated CREB phosphorylation on serine 133 (Fig. 1GoC). Of note, CH31 B cells incubated in the Me2SO solvent control exhibited levels of anti-Ig-stimulated CREB phosphorylation on serine 133 that were comparable to the control population of CH31 B cells treated with anti-Ig (data not shown). In control experiments, pretreatment of CH31 B cells with 20 µM SB203580 did not affect ERK1/2 and c-Jun NH2-terminal kinase (JNK) activities, whereas anti-Ig-induced p38 MAPK activity was reduced to a level equal to unstimulated cells, suggesting that SB203580 was specific for the p38 MAPK (data not shown) (41).

Evidence that BCR-regulated protein kinases involved in CREB phosphorylation on serine 133 in CH31 B cells differ from mature B lymphocytes

Previous reports have shown that BCR cross-linking in mature B lymphocytes induced rapid and transient phosphorylation of CREB on serine 133 (19, 20, 29). Additionally, anti-Ig stimulates p38 MAPK activity in B cells (42, 43, 44, 45, 46). Therefore, we sought to determine whether p38 MAPK activity was required for anti-Ig-induced CREB phosphorylation in splenic B lymphocytes and mature Bal17 B cells. Pretreatment of splenic B lymphocytes or Bal17 B cells with 20 µM SB203580 did not significantly affect the levels of CREB phosphorylation on serine 133 in response to BCR ligation (Fig. 2GoA, +Anti-Ig). Interestingly, SB203580 reduced the basal level of CREB phosphorylation on serine 133 in unstimulated cells (Fig. 2GoA, -Anti-Ig). This does not appear to reflect a toxic effect of the p38 MAPK inhibitor on unstimulated B cells, as cell viability was not affected by pretreatment with SB203580 (data not shown). Thus, we interpret these findings to mean that p38 MAPK activity may be required, at least in part, for CREB phosphorylation observed in unstimulated mature B cell populations; however, p38 MAPK activity does not appear necessary for BCR-induced CREB phosphorylation on serine 133.



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  		FIGURE 2. The p38 MAPK inhibitor, SB203580, blocks BCR-induced CREB serine 133 phosphorylation in CH31 B cells, but is not inhibitory in splenic B lymphocytes or Bal17 B cells. A, Splenic B lymphocytes and Bal17 B cells were preincubated in the absence (-) or presence (+) of 20 µM SB203580 for 60 min and then cultured in medium alone (M) or stimulated with 10 µg/ml anti-Ig for 40 min. Whole-cell extracts were prepared and phospho(Ser133)CREB levels were measured by immunoblotting with anti-phospho(Ser133)CREB Ab as described in Materials and Methods. B, CH31 B cells and BalI7 B cells were preincubated in the presence or absence of 10 µM H-89 or 10 µM H-85. After 20 min, cells were cultured in medium (M) or in medium containing 10 µg/ml anti-Ig for 40 min. Whole-cell extracts were prepared and phospho(Ser133)CREB content was evaluated as described in A. pCREB denotes the position of phosphorylated CREB. Densitometric analysis of the pCREB bands is summarized below each lane.

 
The observation that SB203580 blocked BCR-induced CREB phosphorylation in CH31 B cells, but was not inhibitory in Bal17 B cells and splenic B lymphocytes stimulated with anti-Ig, suggests that the protein kinase pathway(s) required for BCR-induced CREB phosphorylation on serine 133 may differ in CH31 B cells in comparison to mature B cells. To test this further, we examined whether PKA activity was required for BCR-induced CREB phosphorylation in CH31 B cells. Our laboratory has identified a requirement for PKA in BCR-induced CREB phosphorylation in mature B cells (19). Anti-Ig-induced CREB serine 133 phosphorylation in CH31 B cells was not inhibited by preincubating cells with 10 µM H-89, a potent inhibitor of cAMP-dependent PKA activity (Fig. 2GoB, CH31 B cells) (47). In parallel experiments, anti-Ig-stimulated CREB serine 133 phosphorylation was not affected in CH31 B cells preincubated with 10 µM H-85, a control agent that is not selective in producing inhibition of PKA activity (Fig. 2GoB). By contrast, anti-Ig stimulated CREB Ser133 phosphorylation in mature Bal17 B cells was effectively blocked by pretreatment with H-89, whereas H-85 was not inhibitory (Fig. 2GoB, Bal17 B cells), consistent with our previous reports in mature B lymphocytes (19, 30). The changes in phosphoCREB did not arise from differences in the total amount of cellular protein, based on immunoblotting with a polyclonal anti-actin Ab (data not shown). We interpret these data to mean that PKA activity is not required for BCR-induced CREB serine 133 phosphorylation in CH31 B cell lymphomas.

BCR cross-linking causes activation of p38 MAPK in CH31 B cells

The experiments with pharmacological inhibitors demonstrated that p38 MAPK mediates BCR-induced CREB phosphorylation in CH31 B cells. Therefore, we examined further the regulation of p38 MAPK in response to BCR cross-linking. Western blot analysis of whole-cell extracts prepared from control and anti-Ig-treated CH31 B cells revealed the presence of constitutively expressed p38 MAPK (Fig. 3GoA, p38 Blot). Immunoblotting with a highly specific anti-phospho(Thr180/Tyr182) p38 MAPK Ab revealed that anti-Ig-stimulation increased phosphorylation of p38 MAPK on conserved Thr180/Tyr182 in the TGY activation motif (Fig. 3GoA, phospho-p38 Blot). Maximal phosphorylation of p38 MAPK was detected at the 40-min time point. Additional support for the activation of p38 MAPK in response to BCR cross-linking was obtained by measuring the phosphotransferase activity of p38 MAPK in immune complexes using a recombinant GST-ATF-2 fusion protein substrate (48). Control CH31 B cells exhibited a relatively small amount of GST-ATF-2 phosphorylation on threonine 71 (Fig. 3GoA, p38 Activity). Anti-Ig treatment of CH31 B cells led to an increased phosphorylation of GST-ATF-2 fusion protein substrate that was maximal at 40 min (14-fold above untreated B cells).



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  		FIGURE 3. BCR cross-linking increases the activity of the p38 MAPK pathway in CH31 B cells. A, CH31 B cells were cultured in medium alone (M) or incubated with 10 µg/ml anti-Ig for 10–90 min. Whole-cell extracts were separated by SDS-PAGE and immunoblotted with an anti-phospho(Thr180/Tyr182) p38 MAPK Ab (phospho-p38 Blot). The membrane was also probed with an anti-p38 MAPK Ab to measure total cellular p38 MAPK levels (p38 Blot). Parallel cells were cultured in medium alone (M) or incubated with 10 µg/ml anti-Ig for the indicated times, and p38 MAPK immune complexes were then isolated and subjected to in vitro kinase assays using GST-ATF-2 fusion protein substrate as described in Materials and Methods (p38 Activity). Quantitation of phosphorylated GST-ATF-2 was conducted by scanning densitometry. B, CH31 B cells were cultured in medium alone (M) or with 10 µg/ml anti-Ig for 40, 60, and 90 min. Cell lysates were prepared and incubated with nonimmune IgG or anti-MAPKAP kinase-2 Ab. The immune complexes were then examined for phosphorylation of hsp25 substrate as described in Materials and Methods. The position of phosphorylated hsp25 is indicated by the arrow, and the Mr of a 29-kDa protein standard is shown on the left. The data are representative of five independent experiments. C, CH31 B cells were cultured in medium alone (0) or stimulated with anti-Ig for the indicated times. Lysates were then prepared and incubated with anti-MAPKAP kinase-2 Ab, and the resulting immune complexes were examined in vitro for kinase activity using CREBtide substrate. The data are representative of three independent experiments.

 
MAPKAP kinase-2 activity is increased in response to BCR cross-linking and mediates phosphorylation of the serine 133 phosphoacceptor site of CREB

To understand more fully how p38 MAPK regulates serine 133 site-specific phosphorylation of CREB, we evaluated the activity of MAPKAP kinase-2 in response to anti-Ig treatment of CH31 B cells. The decision to evaluate MAPKAP kinase-2 was prompted by recent reports demonstrating that MAPKAP kinase-2 is a substrate for p38 MAPK (36, 49). MAPKAP kinase-2 was immunoprecipitated from control and anti-Ig treated CH31 B cells, and the immune complexes were assayed for phosphorylation of recombinant hsp25 substrate. Untreated CH31 B cells exhibited detectable MAPKAP kinase-2 activity that was further increased following BCR ligation, with maximal levels observed at the 40-min time point (Fig. 3GoB). It is noteworthy that we consistently observed that the relative amount of phosphorylated hsp25 was somewhat lower at 60 and 90 min in comparison to control values. In parallel kinase assays, isotype matched rabbit IgG immune complexes recovered from cell lysates were devoid of hsp25 phosphotransferase activity (Fig. 3GoB).

The amino acid sequence defining the serine 133 phosphoacceptor site of CREB is homologous with the minimum consensus sequence (LXRXXSXX) required for efficient phosphorylation by MAPKAP kinase-2 (36, 39, 50). To test whether BCR-stimulated MAPKAP kinase-2 activity in CH31 B cells might be capable of phosphorylating the serine 133 phosphoacceptor site of CREB, MAPKAP kinase-2 was immunoprecipitated from whole-cell extracts and evaluated in vitro for phosphotransferase activity using the CREBtide substrate, a peptide corresponding to residues 127 to 136 of CREB and containing the conserved serine 133 phosphoacceptor site (30). MAPKAP kinase-2 immune complexes recovered from control CH31 B cells exhibited CREBtide phosphotransferase activity, consistent with the basal activity of MAPKAP kinase-2 (Fig. 3GoC). Phosphorylation of CREBtide substrate was increased in MAPKAP kinase-2 immune complexes from anti-Ig-stimulated CH31 B cells at 20 and 40 min, with CREBtide phosphorylation returning to control levels at the 90-min time point. Of note, MAPKAP kinase-2 immune complexes obtained from CH31 B cells pretreated with 20 µM SB203580 and then stimulated with anti-Ig for 40 min exhibited CREBtide phosphor-ylation equal to that of unstimulated CH31 B cells (data not shown).

BCR cross-linking on CH31 and WEHI-231 B cells increases junB mRNA levels in a p38 MAPK-dependent manner

Our results suggest that BCR cross-linking induces the phosphorylation of CREB on serine 133, at least in part, through a p38 MAPK pathway. A downstream target of CREB in mature B cells is the junB gene (19). Anti-Ig increases junB mRNA levels via transcriptional activation, an event that is dependent upon trans-activated CREB (19, 51). Therefore, we were interested in determining if BCR signals targeted chromatin-bound CREB. Nuclei were isolated from CH31 B cells and examined for CRE binding activity by EMSA. Both control and anti-Ig-treated CH31 B cells expressed constitutive CRE binding activity (Fig. 4GoA), consistent with previous findings in mature B cells (19). The specificity of nuclear extract binding was confirmed insofar as DNA binding activity was competed by including in the binding assays excess unlabeled CRE probe (Fig. 4GoA). Moreover, CRE binding activity from both control and anti-Ig-treated CH31 B cells was inhibited by incubating nuclear extracts with 0.5 µg anti-CREB Ab (Fig. 4GoA, CREB), whereas parallel binding assays containing 0.5 µg of isotype-matched rabbit IgG were not inhibited (Fig. 4GoA, NI). Incubation of nuclear extracts isolated from control CH31 B cells with 0.5 µg anti-phospho(Ser133) CREB Ab resulted in inhibition of CRE binding activity (Fig. 4GoA, pCREB). Importantly, a relatively greater percentage of total CRE binding activity was inhibited by the anti-phospho(Ser133)CREB Ab in nuclear extracts prepared from anti-Ig-stimulated CH31 B cells.



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  		FIGURE 4. BCR-induced junB mRNA levels are inhibited by SB203580. A, Nuclear extracts from control (M) and CH31 B cells stimulated with 10 µg/ml anti-Ig (Ig) for 40 min were examined by EMSA using a CRE probe as described in Material and Methods. Some nuclear extracts were preincubated with 0.5 µg/ml nonimmune rabbit IgG (NI), anti-CREB Ab (CREB), or anti-phospho(Ser133)CREB Ab (pCREB) before EMSA. Lane P is migration of the CRE probe in absence of nuclear extract. Nuclear extracts from anti-Ig-stimulated CH31 B cells were examined for binding to the CRE probe in the absence (C) and presence of 50-fold (50X) and 10-fold (10X) molar excess of unlabeled CRE probe. Arrow indicates the position of the CRE nucleoprotein complexes. B, Poly(A+) RNA was purified from CH31 or WEHI-231 B cells treated with 10 µg/ml anti-Ig for the indicated times. RNA was analyzed by Northern blotting for the expression of junB mRNAs as described in Materials and Methods. The blot was then stripped and reprobed for actin expression by a similar method. Poly(A+) RNA was also purified from CH31, WEHI-231, or Bal17 B cells pretreated in the absence (-) or presence (+) of 20 µM SB203580 for 60 min and then stimulated with 10 µg/ml anti-Ig for 0 (-) or 40 (+) min. RNA was analyzed by Northern blotting for the expression of junB and actin mRNAs. Densitometric analysis of the bands is represented as relative optical units, standardized to each autoradiographic film.

 
Because CREB regulates junB gene expression in mature B cells, we sought to determine whether anti-Ig increased junB mRNA levels in CH31 B cells. Poly(A+) RNA was prepared from untreated or anti-Ig-treated CH31 B cells and probed for junB mRNA expression by Northern blot analysis. BCR cross-linking increased the steady-state level of junB mRNA (Fig. 4GoB). The increase in junB mRNA levels was not attributed to a greater amount of total poly(A+) RNA in the anti-Ig lanes, as confirmed by reprobing of the membrane with a radiolabeled actin cDNA probe (Fig. 4GoB). Pretreatment of the CH31 B cells with SB203580 significantly reduced the level of junB mRNA expression in response to BCR cross-linking (Fig. 4GoB). Collectively, these data suggest that p38 MAPK activity is required for BCR-induced junB mRNA levels. Cross-linking the BCR on surface IgM+ WEHI-231 B cell lymphomas also led to increased junB mRNA levels (Fig. 4GoB). Pretreatment of WEHI-231 B cells with SB203580 led to ~50% reduction in anti-Ig-stimulated junB mRNA levels, suggesting that active p38 MAPK is required, at least in part, for junB mRNA expression by the BCR (Fig. 4GoB). BCR cross-linking in WEHI-231 B cells resulted in the activation of p38 MAPK (42, 44) and phosphorylation of CREB on serine 133 (data not shown). In comparison to CH31 and WEHI-231 B cells, anti-Ig-stimulated junB mRNA expression in Bal17 B cells was not inhibited by SB203580 (Fig. 4GoB). Note, we have previously characterized the regulation of junB mRNA expression by the BCR (13). These results are consistent with the notion that BCR-induced junB mRNA expression in Bal17 B cells is not dependent upon active p38 MAPK.

BCR cross-linking on CH31 and WEHI-231 B cells activates transcription of CRE-containing junB promoter constructs in a SB203580-sensitive manner

In an earlier report, we demonstrated that a CAT reporter gene plasmid containing 194 bp of 5'-flanking junB gene sequences (plasmid denoted JB194CAT5) was stimulated following BCR cross-linking in mature B cells (19). Reporter gene activity from the junB promoter is dependent upon a CRE site located between -135 and -128 bp (19). Therefore, this plasmid affords evaluation of the BCR-coupled signaling pathways leading to CREB-mediated transcriptional activation. To determine whether p38 MAPK activity was required for BCR-induced transcriptional activation of the CRE-dependent junB promoter/CAT reporter construct, CH31 B cells were transiently transfected with the JB194CAT5 plasmid, followed by anti-Ig stimulation in the presence and absence of SB203580. BCR cross-linking increased CRE-dependent junB/CAT reporter gene expression (Fig. 5GoA). Pretreatment of CH31 B cells with SB203580 markedly inhibited anti-Ig-stimulated junB promoter/CAT reporter gene activity under the control of the CRE. Anti-Ig-stimulated junB promoter activation in transiently transfected WEHI-231 B cells was also reduced by pretreatment with SB203580 (Fig. 5GoA). These findings demonstrate that p38 MAPK activity is required for BCR-induced junB promoter activation in CH31 and WEHI-231 B cell lymphomas.



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  		FIGURE 5. SB203580 blocks the transcriptional activation of the junB gene promoter in response to BCR cross-linking on CH31 and WEHI-231 B cells, but not on Bal17 B cells. A, Bal17, CH31, and WEHI-231 B cells were transiently transfected with 40 µg JB194CAT5 plasmid that contains a CAT reporter gene fused to a CRE-dependent junB gene promoter (19 ). Cells were incubated in the absence (-) or presence (+) of 20 µM SB203580 for 40 min and then cultured in media alone (-Anti-Ig) or with 15 µg/ml anti-Ig (+Anti-Ig) for 8 h. B, CH31 B cells were transiently transfected with 40 µg 3XTRE/CAT plasmid. Cells were incubated in the absence (-) or presence (+) of 20 µM SB203580 for 40 min and then cultured in media alone (-P/I) or with 100 ng/ml phorbol diester plus 300 ng/ml ionomycin (+P/I) for 8 h. Cell lysates were then prepared and CAT activity measured and quantitated as described in Materials and Methods. BCR-induced transcriptional activation is expressed as fold induction over the basal transcription in transiently transfected but unstimulated B cells. The results are representative of three independent experiments.

 
Based on these findings, we sought to determine whether activation of JB194CAT5 in a transfectable mature B cell line was dependent upon active p38 MAPK. The results in Fig. 5GoA demonstrate that anti-Ig-stimulated junB promoter/CAT reporter gene activity in transiently transfected Bal17 B cells. Pretreatment of a parallel population of Bal17 B cells with SB203580 did not inhibit anti-Ig-stimulated junB promoter/CAT activity. Taken together, these results suggest that p38 MAPK activity is not required for BCR-induced junB promoter activation in Bal17 B cells.

As a control for the specificity of SB203580 in blocking CRE-dependent transcription, experiments were conducted using chimeric plasmids containing three copies of a TRE sequence coupled to a HSV-tk promoter/CAT fusion gene (38). The TRE sequence binds members of the AP-1 family and in B cells has been shown to bind JunB/Fos and c-Jun/Fos heterodimers (13). AP-1 is activated by JNK family members (40, 48). CH31 B cells were transiently transfected with the 3XTRE/CAT plasmid, followed by phorbol diester plus ionomycin treatment to activate JNK. The results in Fig. 5GoB demonstrate that phorbol diester plus ionomycin increased TRE-dependent CAT reporter gene activity by ~15-fold above control B cells. In parallel CH31 B cells, pretreatment with SB203580 did not block phorbol diester/ionomycin-induced CAT reporter activity.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We have shown in this study that ligation of the BCR on CH31 B cell lymphomas leads to increased p38 MAPK activity. The significance of this BCR-regulated activity is underscored by the requirement for active p38 MAPK in anti-Ig-induced CREB trans-activation via site-specific phosphorylation on serine 133, junB mRNA expression, and junB promoter activation. Support is also provided for a role of p38 MAPK in anti-Ig-induced junB mRNA expression and junB promoter activation in WEHI-231 B cells. This is the first report demonstrating a role for p38 MAPK in regulating CREB activity and CRE-dependent gene transcription in response to Ag receptor cross-linking.

We do not mean to imply that p38 MAPK is the only functional pathway to couple the BCR to CREB serine 133 phosphorylation. Experiments using the MEK-1 inhibitor PD98059 suggest that the Ras pathway may contribute to BCR-inducible CREB serine 133 phosphorylation. It is not clear at this time if MEK-1 and p38 MAPK are functionally related or represent two separate and distinct pathways that regulate CREB phosphorylation on serine 133. In keeping with this observation, there is precedence for the existence of multiple signaling pathways (e.g., phosphatidylinositol 3-kinase, PKA, protein kinase C, ERK) that promote activation of a CREB kinase and concomitantly inhibit a Ser133-directed phosphatase activity (19, 25, 26, 27, 28, 29, 30). While both kinase and phosphatase activities regulate CREB phosphorylation, the relative contribution of each activity has not been established. Moreover, it is not known at present which pathways function to negatively regulate CREB phosphatase activity in response to surface receptor signals. In CH31 B cells, MEK-1 or other unknown BCR-coupled signaling pathways may contribute to the phosphorylation of CREB via negatively regulating a phosphoprotein phosphatase 1 or 2A activity (30).

Several experiments support the participation of p38 MAPK in BCR signaling. Foremost, anti-Ig led to a transient increase in the phosphorylation of p38 MAPK on Thr180/Tyr182 of the conserved TGY activation motif, an event necessary for activation of its serine/threonine kinase (40). BCR cross-linking also resulted in increased phosphorylation of recombinant GST-ATF-2 fusion protein substrate as directed by p38 MAPK immune complexes. Consistent with these observations, we found that anti-Ig stimulated a small and transient increase in MAPKAP kinase-2 activity, a substrate that lies immediately downstream from p38 MAPK (36, 52). The activation of p38 MAPK by the BCR in CH31 B cells agrees with recent studies in the immature WEHI-231 B cell lymphoma that showed a transient increase in p38 MAPK activity in response to anti-Ig (42, 44). Interestingly, anti-Ig also promotes activation of ERK2 and to a much lesser extent JNK in WEHI-231 B cells (44, 53, 54). A recent report by Purkerson and Parker (55) demonstrated that BCR and CD40 signals converge at MEK-1 to activate ERK. Though CH31 and WEHI-231 B cell lines are considered models for B cell tolerance (1), we were unable to detect substantial changes in the activities of JNK and ERK following BCR cross-linking in the CH31 B cell lymphoma (data not shown).

The mammalian p38 MAPK, an enzyme related to the HOG1 kinase from Saccharomyces cerevisiae, is activated by stress-inducing agents such as changes in osmotic strength, UV irradiation, and proinflammatory cytokines (40, 48, 56, 57, 58). p38 MAPK is also activated by CD40 and Fas in B and T cells (43, 59). The p38 MAPK family includes SB203580-inhibitable p38{alpha} and p38ß isoforms and p38{gamma} and p38{delta} isoforms that are activated by dual phosphorylation on TGY in kinase subdomain VIII by MAPK kinase (MKK) 3 and MKK6 (40, 41). Although the coupling of upstream MKKs, such as MKK3/6, to surface receptors remain to be completely defined, it is noteworthy that Clark and coworkers (60) recently showed, using Syk-/Lyn- double deficient DT40 B cells, that p38 MAPK was not efficiently activated following BCR ligation. By contrast, anti-Ig induced p38 MAPK activation in the corresponding Syk- or Lyn- deficient DT40 B cells. In a related study, Hashimoto et al. (61) demonstrated a requirement for Rac1 and phospholipase C{gamma}2 in BCR-induced p38 MAPK responses in DT40 B cells.

The cellular substrates and gene targets of p38 MAPK are likewise incompletely defined. It is recognized that a conserved serine/proline motif located in several transcription factors (e.g., ATF-2, CHOP/GADD153, and Elk-1) is phosphorylated by p38 MAPK signaling pathways (40). p38 MAPK activity is required for the transcriptional activation of c-fos in response to UV irradiation, TNF-{alpha}-induced cytokine production, and IL-1ß by LPS in monocytes (57, 62, 63). In the context of B lymphocytes, Craxton et al. (42) demonstrated that CD40-mediated NF-{kappa}B trans-activation is a target of p38 MAPK. Our findings indicate that CREB is a downstream target of the p38 MAPK pathway in CH31 B cell lymphomas. This is supported by the observation that the p38 MAPK inhibitor, SB203580, blocked BCR-stimulated CREB phosphorylation on serine 133. Our experiments also implicate MAPKAP kinase-2, as directly contributing to BCR-induced CREB serine 133 phosphorylation. We base this latter conclusion on the finding that MAPKAP-kinase 2 immune complexes from anti-Ig-treated CH31 B cells exhibited increased phosphorylation of a peptide substrate containing the kinase-inducible domain serine 133 phosphoacceptor site of CREB. Our findings agree with an earlier report by Tan et al. (39) in human neuroblastoma SK-N-MC cells that fibroblast growth factor increased CREB phosphorylation on serine 133 in a MAPKAP-kinase-2-dependent manner.

An important finding of this study concerns the diversity of intracellular protein kinases that regulate CREB trans-activation in B lymphocytes. Though we recognize the limited scope of these experiments, strong support is provided for the existence of distinct protein kinase pathways that mediate BCR-induced CREB phosphorylation on serine 133 in CH31 B cell lymphomas and mature B lymphocytes. This conclusion is based in part on previous findings in which CREB serine 133 phosphorylation in mature B cells stimulated with anti-Ig was blocked by PKA inhibitors (30). By contrast, experiments herein demonstrate that pretreatment of CH31 B cells with the PKA inhibitor, H-89, did not significantly reduce CREB phosphorylation on serine 133. Additionally, pretreatment of CH31 B cells with SB203580 prevented BCR-induced serine 133 phosphorylation of CREB, but did not affect the level of CREB phosphorylation stimulated by anti-Ig in the phenotypically mature Bal17 and splenic B lymphocytes. We interpret these findings to mean that p38 MAPK represents a critical pathway for the regulation of CREB trans-activation in CH31 B cells, whereas a PKA-dependent (SB203580 insensitive) pathway is required in mature B lymphocytes.

Perhaps most importantly, our data suggests that the p38 MAPK module serves to focus Ag-receptor signals to genes whose promoters contain CRE elements. EMSA experiments with anti-CREB and phospho(Ser133) anti-CREB Abs demonstrated that phosphorylated CREB assembles into CRE-containing nucleoprotein complexes. The significance of this finding to CREB-regulated gene expression was assessed by Northern blot analysis of junB mRNA levels. We found that anti-Ig increased endogenous junB mRNA levels in both CH31 and WEHI-231 B cells. Importantly, the increase in junB mRNA levels was reduced by pretreatment of cells with SB203580, suggesting that p38 MAPK plays a role in regulating junB gene expression in response to BCR ligation. The regulation of junB promoter activity by p38 MAPK was assessed using junB promoter-CAT reporter fusion gene constructs in transient transfection assays. The decision to examine the junB gene promoter is based on our previous observation that junB transcriptional activation in response to BCR ligation is dependent on a CRE located 5' to the transcriptional start site of the junB gene (19, 30). CREB binds the junB gene promoter CRE-like element, and serine 133 phosphorylation is necessary for BCR-stimulated junB promoter activation (19, 30). We found that anti-Ig-stimulated junB promoter/CAT reporter gene activity was significantly reduced by pretreating CH31 B cells with SB203580. In addition, transiently transfected WEHI-231 B cells exhibited anti-Ig-stimulated junB promoter/CAT reporter activity, which was sensitive to SB203580. By contrast, p38 MAPK activity does not appear to be required for anti-Ig-stimulated junB promoter activation in Bal17 B cells. These data point to a role for p38 MAPK in mediating BCR-induced junB promoter activation in both CH31 and WEHI-231 B cell lymphomas. Collectively, our data suggest a pathway for BCR-induced gene regulation that includes p38 MAPK->MAPKAP kinase-2->CREB->junB gene transcription.

An important question raised by these experiments concerns whether the requirement of p38 MAPK activity in BCR-regulated CREB activity is restricted to Ag-inhibited B cell lymphomas or reflects a developmental difference in BCR signaling between normal mature and immature B lymphocytes. Studies with primary immature B lymphocytes may provide insight into this important question. In addition, this study has not addressed the physiologic significance of p38 MAPK in Ag-induced apoptosis; however, p38 MAPK activity is required for BCR-induced apoptosis in the human B104 B cell line (46). Clark and coworkers (60) recently noted that in the DT40 cell line high doses of SB203580 were required to block BCR-mediated apoptosis. In data not shown, pretreatment of CH31 B cells with 20 µM SB203580 did not provide protection against BCR-induced apoptosis. We were unable to test higher concentrations of the inhibitor due to decreased cell viability in parallel CH31 B cell cultures pretreated with Me2SO solvent control. Thus, it is not known at present whether p38 MAPK may contribute to the induction of apoptosis in CH31 B cells following BCR cross-linking. Additional studies with dominant negative forms of p38 MAPK may provide insight into this important issue. Nevertheless, our findings are consistent with that of WEHI-231 B cells in which SB203580 failed to block anti-Ig-induced apoptosis (43).

In summary, experiments herein establish a role of the p38 MAPK module as a intracellular pathway of BCR-induced CREB serine 133 phosphorylation and CRE-mediated transcriptional activation in CH31 B cell lymphomas.


   Acknowledgments
 
We thank Dr. Gail Sonenshein (Department of Biochemistry, Boston University School of Medicine) for helpful discussions on DNA transfection methodology. We thank Dr. David W. Scott (Department of Immunology, Holland Laboratory for the Biomedical Sciences, American Red Cross, Rockville, MD) for providing the murine CH31 and WEHI-231 B cell lymphomas.


   Footnotes
 
1 This work was supported by the National Institutes of Health (AI34586). J.M.S. was supported by a Walsh Family Fellowship. D.B. was supported by a Research Experiences for Undergraduates from the National Science Foundation (MCB-9603784). Back

2 Address correspondence and reprint requests to Dr. Thomas C. Chiles, 411 Higgins Hall, Department of Biology, Boston College, Chestnut Hill, MA 02467. E-mail address: Back

3 Abbreviations used in this paper: BCR, B cell Ag-receptor; CREB, cAMP response element binding protein; ATF, activating transcription factor; anti-Ig, F(ab')2 fragment of goat anti-mouse IgM; CRE, cAMP response element; PKA, protein kinase A; JNK, c-Jun NH2-terminal kinase; MAPK, mitogen activated protein kinase; MAPKAP, MAPK-activated protein; ERK, extracellular signal-regulated kinase; CaMK, Ca2+-dependent calmodulin kinase; hsp, heat shock protein; CAT, chloramphenicol acetyltransferase; TRE, tetradecanoyl phorbol acetate response element; MEK, MAPK/ERK kinase; MKK, MAPK kinase. Back

Received for publication September 7, 1999. Accepted for publication December 14, 1999.


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 Introduction
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