B cell antigen receptor (BCR) cross-linking activates three distinct families of nonreceptor protein tyrosine kinases (PTKs): src-family kinases, Syk, and Btk; these PTKs are responsible for initiating downstream events. BCR cross-linking in the chicken DT40 B cell line also activates three distinct mitogen-activated protein kinases (MAPKs): extracellular signal–regulated kinase (ERK)2, c-jun NH2-terminal kinase (JNK)1, and p38 MAPK. To dissect the functional roles of these PTKs in MAPK signaling, activation of MAPKs was examined in various PTK-deficient DT40 cells. BCR-mediated activation of ERK2, although maintained in Lyn-deficient cells, was abolished in Syk-deficient cells and partially inhibited in Btk-deficient cells, indicating that BCR-mediated ERK2 activation requires Syk and that sustained ERK2 activation requires Btk. BCR-mediated JNK1 activation was maintained in Lyn-deficient cells but abolished in both Syk- and Btk-deficient cells, suggesting that JNK1 is activated via a Syk- and Btk-dependent pathway. Consistent with this, BCR-mediated JNK1 activation was dependent on intracellular calcium and phorbol myristate acetate–sensitive protein kinase Cs. In contrast, BCR-mediated p38 MAPK activation was detected in all three PTK-deficient cells, suggesting that no single PTK is essential. However, BCR-mediated p38 MAPK activation was abolished in Lyn/Syk double deficient cells, demonstrating that either Lyn or Syk alone may be sufficient to activate p38 MAPK. Our data show that BCR-mediated MAPK activation is regulated at the level of the PTKs.

Bcells recognize and respond to foreign antigens through their surface-expressed B cell antigen receptor (BCR)1 complexes composed of antigen-binding membrane Ig and disulfide-linked heterodimer Igα/Igβ, which are the signaling subunits of the BCR (1, 2). The signaling cascade initiated by Igα/Igβ after BCR cross-linking can promote different biological outcomes, depending on the differentiation stage of the B cell and the nature of additional signals received by the cell. Immature B cells become anergized or undergo apoptosis upon antigen binding, whereas mature B cells enter the cell cycle after BCR ligation and can then be induced by other signals to differentiate into antibody-secreting plasma cells. A key early event of BCR signaling is the activation of protein tyrosine kinases (PTKs), which initiates most if not all of the subsequent signaling events (3, 4). Since the BCR has no intrinsic PTK activation, for signal transduction it uses three distinct families of nonreceptor cytoplasmic PTKs: Lyn, Blk, Fyn, Lck, and Fgr of the Src family, Syk of the Syk/ ZAP-70 family, and Btk of the Tec family (58).

Mitogen-activated protein kinases (MAPKs) play a central role in transducing extracellular signals from the cytosol to the nucleus (9, 10). Three structurally related MAPK subfamilies have been identified in mammalian cells: the p42 and p44 kinases (extracellular signal-regulated kinases [ERKs]; references 11, 12), the c-jun NH2-terminal kinases (JNKs)/stress activated protein kinases (SAPKs) (1315), and the p38 MAPK family (16, 17). Different patterns of activation of MAPKs may well direct B cells to different cell fates such as proliferation, differentiation, and apoptosis. In a human B cell line B104, activation of JNK and p38 MAPK, but not ERK, is correlated with induction of apoptosis by surface IgM stimulation (18). Both ERKs and JNK1 are activated during positive signaling in naive B cells, whereas only ERKs but not JNK1 are activated in tolerant cells upon BCR stimulation by the same ligand (19). However, the mechanisms that lead to the activation of these distinct MAPKs have not been elucidated.

The chicken DT40 B cell line expresses only specific members of the Src, Syk/ZAP-70, and Tec PTK families: Lyn of the Src-family, Syk of the Syk/ZAP70 family, and Btk of the Tec family (20, 21). Using Syk-, Lyn-, and Btk-deficient DT40 cells, we investigated the participation of PTKs in MAPK signaling. We report that BCR cross-linking in DT40 cells activates three distinct MAPKs: ERK2, JNK1, and p38 MAPK. Whereas Syk and Btk regulate the activation of ERK2 and JNK1, Lyn does not. Furthermore, either Syk or Lyn is sufficient for the activation of p38 MAPK upon BCR ligation, whereas Btk does not appear to regulate this MAPK family member. Thus, the BCR uses different PTKs to regulate members of distinct MAPKs in B cells.

Materials And Methods

Reagents.

The mouse anti–chicken IgM mAb M4 was prepared as previously described (22); PMA, ionomycin, and 4α-phorbol were purchased from Calbiochem, Inc. (San Diego, CA). Thapsigargin was purchased from Sigma Chemical Co. (St. Louis, MO). Polyclonal rabbit antisera against JNK1 (C-17), ERK2 (C-14), ERK1 (C-16), and p38 MAPK (C-20) were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Polyclonal rabbit antiphospho-MAPK (ERK2) was purchased from Promega Corp. (Madison, WI). Purified mouse anti–human JNK1 monoclonal antibody was purchased from PharMingen (San Diego, CA). Myelin basic protein (MBP) was purchased from Upstate Biotechnology, Inc. (Lake Placid, NY). Glutathione S-transferase (GST)-c-jun (5–89) and GST-activating transcription factor (ATF)2 were expressed in Escherichia coli, and the fusion proteins were purified as previously described (23). Protein A–sepharose was obtained from Amersham Pharmacia Biotech, Inc. (Piscataway, NJ). BAPTA-AM [the acetoxymethyl ester of bis-(o-aminophenoxy) ethane-N,N,N′,N′-tetraacetic acid] and Pluronic F-127 were purchased from Molecular Probes, Inc. (Eugene, OR).

Cell Lines.

The generation of Syk-, Lyn-, and Btk-deficient, and Lyn/Syk double deficient, DT40 cell lines was carried out as previously described (20, 21). DT40 wild-type and PTK-deficient cell lines were cultured in RPMI 1640 medium supplemented with 10% heat-inactivated FCS, 2 mM glutamine, 10 U/ml penicillin, 10 μg/ml streptomycin, 1 mM pyruvate and nonessential amino acids, 50 μM mercaptoethanol, and 1% heat-inactivated chicken serum. The cells were maintained below a density of 3 × 106 cells/ml.

Measurement of Intracellular Free Calcium.

Cells (2.5 × 107 cell per sample) were collected and resuspended to 107 cells/ml in fresh complete RPMI 1640 medium. After incubation at 37°C for 15 min, indo-1/AM (7 μg/ml final concentration) was added, and the cells were incubated for a further 45 min with resuspension every 15 min. The cells were subsequently centrifuged and resuspended in complete RPMI 1640 medium at 2 × 106 cells/ml. The fluorescence intensity of intracellular indo-1 was monitored and analyzed using a FACStar® plus flow cytometer (Becton Dickinson, Mountain View, CA) as previously described (24).

Western Blot Analysis.

Unstimulated cells or anti-IgM stimulated cells (5 × 106 cells per sample) were lysed in 100 μl RIPA lysis buffer (50 mM Tris pH 7.4, 150 mM NaCl, 10 mM Na4P2O7, 25 mM sodium β-glycerophosphate, 1 mM EDTA, 1% (wt/vol) NP-40, 0.5% (wt/vol) sodium deoxycholate, 0.1% (wt/ vol) SDS, 1 mM PMSF, 1 mM Na3VO4, 10 μM E-64, 1 μg/ml pepstatin, 10 μg/ml aprotinin, 10 μg/ml leupeptin). After incubation on ice for 15 min, cell lysates were centrifuged at 16,000 g for 15 min at 4°C. Lysates were subsequently denatured in an equal volume of 2× SDS sample buffer, resolved by 10% SDS-PAGE and electrotransferred to nitrocellulose membranes in non-SDS-containing transfer buffer (25 mM Tris, 0.2 M glycine, 20% methanol, pH 8.5). Western blotting was performed with the following primary antibodies and accompanying dilutions: antiphospho ERK2 (1:20,000), anti-MAPK (ERK1 or ERK2, 1:2,000), anti-p38 (1:1,000), and mouse monoclonal anti-JNK1 (1:500) followed by 1:2,000 anti–rabbit or anti–mouse horseradish peroxidase–conjugated IgG (Santa Cruz Biotechnology, Inc.) and were developed with an ECL Western blotting kit (Amersham Corp., Arlington Heights, IL).

Immunoprecipitation and In Vitro Kinase Assay.

For JNK1, ERK2, and p38 MAPK in vitro kinase assays, cells (5–10 × 106 cells per sample) were resuspended in complete RPMI 1640 medium to a density of 2 × 106 cells/ml and stimulated with either anti-IgM (10 μg/ml) or PMA (50 ng/ml) with or without ionomycin (250 ng/ml) for the indicated times. Incubations were terminated by addition of 10 vol of ice-cold PBS and were subsequently centrifuged at 500 g for 8 min at 4°C. The supernatants were aspirated and cells were lysed by resuspension in 0.5 ml RIPA lysis buffer. After incubation on ice for 15 min, the cell lysates were centrifuged at 16,000 g for 15 min at 4°C. For JNK1 and ERK2 in vitro kinase assays, supernatants were added to 20 μl packed protein A–sepharose beads and 0.5 μg polyclonal rabbit anti-JNK or anti-ERK2 antibody, followed by overnight incubation at 4°C. For p38 MAPK assays, the lysates were first added to 1 μg polyclonal rabbit anti-p38 MAPK antiserum with an additional 0.1% SDS. After constant mixing by inversion for 3 h or overnight at 4°C, the mixtures were added to 20 μl packed protein A-sepharose beads for another 1 h. The immune complexes were pelleted at 4°C by centrifugation at 16,000 g for 10 min. The beads were washed twice each with (a) 1 ml of RIPA buffer with 0.1 mM Na3VO4 and (b) 1 ml of p38 MAPK assay buffer (25 mM Hepes, pH 7.4, 25 mM sodium β-glycerophosphate, 25 mM MgCl2, 2 mM dithiothreitol, and 0.1 mM Na3VO4). The pellets were resuspended to 90 μl p38 MAPK assay buffer with 20 μM ATP containing 10 μCi γ-[32P]ATP and 50 μg/ml substrate (MBP for ERK2, GST–c-jun for JNK1, or GST-ATF2 for p38 MAPK). After incubation for 20 min at 30°C with mixing every 2 min, the reactions were terminated by adding 45 μl 2× SDS sample buffer. After boiling for 5 min followed by brief centrifugation, samples were resolved by SDS-PAGE (12% for JNK1 and p38 MAPK assays and 14 or 12% for ERK2 assays). The gels were stained for 30 min with 40% methanol, 10% acetic acid, and 0.005% Coomassie brilliant blue G-250, destained for 30 min with 40% methanol, 10% acetic acid, and 5% glycerol, and dried before autoradiography at −70°C for various times.

Results

BCR-mediated Activation of ERK2 Requires Syk and Btk.

Since engagement of the BCR complex strongly activates p42 ERK2 in other B cell lines and in normal B cells (25, 26), we tested whether BCR cross-linking in DT40 B cells could also lead to ERK2 activation. In vitro kinase assays were performed with anti-ERK2 immunoprecipitates to assess induction of ERK2 activation. Consistent with previous studies, ERK2 activation was detected shortly after BCR cross-linking, with a maximal fourfold activation compared with unstimulated cells evident at 2 min and sustained for at least 15 min (Fig. 1,A). To determine the PTK requirement for BCR-mediated ERK2 activation, we compared ERK2 activation in wild-type versus mutant DT40 cell lines deficient in Lyn, Syk, or Btk that expressed comparable levels of surface IgM (20, 21). BCR-mediated ERK2 activation was completely abolished in Syk-deficient cells even though ERK2 was activated by PMA to a similar extent compared with wild-type DT40 cells (Fig. 1,A); thus, Syk is required for BCR-mediated ERK2 activation. In contrast, BCR-mediated ERK2 activation was largely maintained in Lyn-deficient cells with a maximal threefold activation at 5 min (Fig. 1,A), indicating that ERK2 can be activated via the BCR in the absence of Lyn. In contrast to either Syk- or Lyn-deficient cell lines, BCR ligation in Btk-deficient cells led to a transient and weak ERK2 activation (less than twofold), suggesting that a Btk-independent ERK2 activation pathway exists and that Btk is required for sustained ERK2 activation (Fig. 1 A).

As an alternative assay for ERK2 activation, we measured the phosphorylation of ERK2 using Western blotting and a phospho-MAPK antiserum, which only detected phosphorylated ERK2 in DT40 cells (Fig. 1,B). Consistent with our in vitro kinase assays, phosphorylated ERK2 was detected in DT40 cells between 2 and 15 min after anti-IgM stimulation (Fig. 1,B). No BCR-mediated phosphorylation of ERK2 was detected in Syk-deficient cells; in Lyn-deficient cells phosphorylation of ERK2 was comparable with wild-type DT40 cells, whereas weak and transient ERK2 phosphorylation was observed in Btk-deficient cells (Fig. 1 B). In summary, BCR-mediated ERK2 activation requires Syk, whereas sustained ERK2 activation requires Btk.

BCR-mediated JNK1 Activation Requires both Syk and Btk but not Lyn.

JNKs are activated preferentially by a variety of extracellular stresses, and in most circumstances they are activated through a different pathway from ERKs (10, 13– 15). BCR cross-linking also led to JNK1 activation in DT40 cells as determined by its ability to phosphorylate GST–c-jun. JNK1 was maximally activated (fourfold) between 10 and 15 min and then decreased by 30 min (Fig. 2,A). To test whether BCR-mediated JNK1 activation has the same PTK requirement as BCR-mediated ERK2 activation, we compared the ability of the BCR to activate JNK1 in Syk-, Lyn-, and Btk-deficient DT40 cells. The expression levels of JNK1 were not affected either by the deletion of various PTK genes or anti-IgM stimulation (Fig. 2,B). BCR-mediated JNK1 activation was maintained in Lyn-deficient cells with a maximal fivefold JNK1 activation at 15 min (Fig. 2,A), suggesting that Lyn was not essential for BCR-mediated JNK1 activation. However, in both Syk- and Btk-deficient cells, the anti-IgM–induced JNK1 activation was completely abolished (Fig. 2 A), indicating that both Syk and Btk were essential for JNK1 activation. Thus, BCR-mediated JNK1 activation requires both Syk and Btk but not Lyn.

BCR-mediated p38 MAPK Activation Occurs via a Different Pathway from both ERK2 and JNK1.

As a relatively new member of the mammalian MAPKs, p38 MAPK has both a different substrate specificity and different upstream activating kinases (MKKs) and appears to have a distinct transducing pathway and function (9, 10, 16, 17). p38 MAPK activation was examined by the ability of anti-p38 MAPK immunoprecipitates to phosphorylate GST-ATF2. p38 MAPK reached a maximal fourfold activation at 15 min after anti-IgM stimulation, and, distinct from both ERK2 and JNK1 activation, persisted for at least 12 h (Fig. 3,A and data not shown). To investigate whether p38 MAPK has different requirements for upstream PTKs, we examined p38 MAPK activation in various PTK-deficient DT40 cells. There was no significant difference in p38 MAPK expression levels between the parental DT40 cells and various PTK-deficient DT40 cells including the Lyn/Syk double mutant cells (Fig. 3,B). In addition, the recovery of p38 MAPK after immunoprecipitation was also not significantly different in DT40 wild-type and mutant cell lines (data not shown). To our surprise, BCR-mediated p38 MAPK activation in Lyn-deficient cells (sixfold maximal activation), Syk-deficient cells (threefold), and Btk-deficient cells (threefold) was similar to that in wild-type DT40 cells (Fig. 3,A). The fold activations of p38 MAPK in Syk- and Btk-deficient cells were lower in part because these cells had a higher basal level of p38 MAPK activation (Fig. 3,A). Thus, these data suggest that none of these PTKs alone is essential for the BCR to activate p38 MAPK. Previous studies have shown that both Lyn and Syk associate with the BCR and account for most of the initial tyrosine phosphorylation including activation of Btk (21). Our data suggested either that there was another PTK required for p38 MAPK activation, or alternatively that Syk and Lyn had a redundant role in BCR-mediated p38 MAPK activation. To test these possibilities, a Lyn/Syk double deficient DT40 cell line was examined. BCR-mediated p38 MAPK activation was lost in Lyn/Syk double deficient cells (Fig. 3 A), indicating that either Lyn or Syk was sufficient for BCR-mediated p38 MAPK activation.

Activation of JNK1, but not ERK2 and p38 MAPK, via the BCR Is Dependent on Intracellular Calcium.

Anti-IgM stimulation leads to a rapid increase of intracellular calcium [Ca2+]i composed of both Ca2+ release from intracellular pools and extracellular calcium influx (27). Previous studies have demonstrated that loss of either Btk or Syk but not Lyn resulted in abrogation of calcium mobilization upon BCR cross-linking of DT40 cells (20, 21). Since the pattern of BCR-mediated JNK1 activation correlated precisely with that of BCR-mediated calcium mobilization, we hypothesized that a calcium signal may modulate BCR-mediated JNK1 activation. Furthermore, we could activate JNK1 only with a combination of both ionomycin and PMA (Fig. 2,A and data not shown), also supporting the idea that a calcium signal was required for BCR-mediated JNK1 activation. To define the requirement for calcium in BCR-mediated JNK1 activation and specifically determine whether intracellular or extracellular calcium was required, cells were treated with either BAPTA-AM, a cell permeable calcium chelator, or EGTA, which chelates extracellular calcium before anti-IgM stimulation. The concentrations of BAPTA-AM and EGTA were titrated to prevent calcium release and extracellular calcium influx, respectively (data not shown). BAPTA-AM pretreatment completely blocked BCR-induced JNK1 activation, whereas EGTA pretreatment did not (Fig. 4,A), suggesting that BCR-mediated JNK1 activation is dependent on intracellular rather than extracellular calcium. We also asked whether a calcium signal alone was sufficient for BCR- mediated JNK1 activation. To directly address this question, we pretreated DT40 mutant cells with thapsigargin, a pharmacological reagent that prevents reuptake of intracellular calcium into storage vesicles and thus mimics the receptor-dependent increase of [Ca2+]i while bypassing phosphatidylinositol 4,5-bisphosphate hydrolysis (28). The concentrations of thapsigargin were carefully titrated to give a similar peak calcium signal as anti-IgM stimulation (data not shown). JNK1 activation after BCR ligation was not reconstituted in either Syk- or Btk-deficient DT40 cells (Fig. 4 B), indicating that a calcium signal alone was not sufficient to activate JNK1. Other BCR-mediated signals, which require Syk and/or Btk, are also needed for JNK1 activation.

Since partial BCR-mediated ERK2 activation was detectable in Btk-deficient DT40 cells (Fig. 1), which are incapable of calcium mobilization after BCR ligation (21), it seemed unlikely that BCR-mediated ERK2 activation was calcium dependent. Indeed, pretreatment of DT40 cells with either BAPTA-AM or EGTA did not block BCR-mediated ERK2 activation (Fig. 4 A), indicating that a BCR-mediated calcium signal was not required for ERK2 activation.

Both Syk- and Btk-deficient DT40 cells maintained the ability to activate p38 MAPK via the BCR, suggesting that BCR-mediated p38 MAPK activation was independent of calcium. Indeed, neither BAPTA-AM nor EGTA pretreatment blocked BCR-mediated p38 MAPK activation, showing that BCR-mediated p38 MAPK activation was independent of calcium (Fig. 4,A). Furthermore, BAPTA-AM alone activated p38 MAPK (Fig. 4,A). Together with the observation that the Btk- and Syk-deficient DT40 cells, two mutants that lose their ability to mobilize calcium upon BCR ligation, also exhibited higher basal levels of p38 MAPK activation (Fig. 3), our results suggest that free calcium may play a negative role in p38 MAPK activation.

BCR-induced Activation of ERK2 and JNK1 Is, but p38 MAPK Is Not, PKC Dependent.

PMA treatment of wild-type DT40 cells leads to p38 MAPK and ERK2 activation, whereas both PMA and ionomycin are required for JNK1 activation (Fig. 1–3). Since both BCR cross-linking and PMA treatment lead to PKC activation, it was interesting to examine the role of PKCs in BCR-mediated MAPK signaling.

DT40 cells were pretreated for 24 h with PMA (which leads to depletion of PMA-dependent PKCs), an inactive derivative of PMA, 4α-phorbol, or medium, before stimulation with either anti-IgM or PMA. ERK2, JNK1, or p38 MAPK activation of the cells was then analyzed by in vitro kinase assays. Neither PMA nor 4α-phorbol pretreatment of DT40 cells had a significant effect on the expression levels of JNK1, p38 MAPK, and ERK2 (Figs. 2,B and 3,B and data not shown). BCR ligation did not significantly increase ERK2 activation above basal levels in PKC-depleted cells (Fig. 5,A), suggesting that BCR-mediated ERK2 activation is dependent on PMA-depletable PKCs. JNK1 activation was also strongly decreased in PKC-depleted cells (Fig. 5,B), suggesting that PMA-depletable PKCs are required in addition to a calcium signal (Fig. 4) for BCR-mediated JNK1 activation. The requirement of both a calcium signal and PKC activation is consistent with the observation that only a combination of PMA and ionomycin, but not PMA or ionomycin alone, could lead to significant JNK1 activation in DT40 cells (Fig. 5,B and data not shown). For p38 MAPK, significant BCR-dependent activation (greater than threefold increase) was still observed in PKC-depleted cells (Fig. 5 C), suggesting that BCR-mediated p38 MAPK activation is largely independent of PMA-depletable PKCs. However, our results cannot rule out the participation of other PMA-insensitive PKCs such as the atypical PKCζ in BCR-mediated p38 MAPK activation.

Discussion

In this report, we show that each of the three major mammalian MAPK family members activated through the BCR requires a different set of upstream protein tyrosine kinases (Figs. 13 and 6).

Although it has been reported that ERKs are activated upon anti-IgM stimulation via a Ras/Raf-1/MEK signaling pathway (26, 29), the participation of proximal PTKs in BCR-mediated MAPK activation is not well understood. Our data demonstrate that Syk but not Lyn is absolutely essential for BCR-mediated activation of ERK2 (Fig. 1). Supporting this notion, recent studies using the Lck-negative JCaM1.6 variant of the Jurkat T cell line have shown that Syk enabled the TCR to cause a full activation of ERKs in the absence of the Src family kinase Lck (30). In contrast, BCR-mediated ERK2 activation is only partially dependent on Btk, suggesting that Btk has a regulatory role rather than being an essential component in the ERK2 signaling pathway. This possibility is also supported by the fact that Syk is essential for phosphorylation of PLC-γ2, whereas the loss of Btk only reduces PLC-γ2 phosphorylation (20, 21). Previous studies suggest that BCR-mediated activation of Ras and PKCs may converge at the level of Raf-1 for activation of ERKs (26, 29, 31). In a parallel study (32), we found that BCR-mediated activation of ERK2 was incompletely blocked in PLC-γ2–deficient DT40 cells or in DT40 cells transfected with a dominant negative form of Ras (Ras N17). A complete block of ERK2 activation was seen in PLC-γ2–deficient cells transfected with Ras N17, indicating that both Ras- and PLC-γ2–regulated PKCs are involved in BCR-mediated ERK2 activation. Taken together, our data suggest that Syk is essential for both Ras-dependent activation of ERK2 and the PLC-γ2– and PKC-dependent, Btk-regulated pathway leading to ERK2 activation (Fig. 6). Lyn was reported to play a negative role in ERK activation (33), and Lyn-deficient DT40 cells showed augmented BCR-mediated PKC activation (34), leading to the speculation that Lyn-deficient DT40 cells have augmented ERK2 activation. The fact that we did not detect augmented ERK2 activation in Lyn-deficient cells may reflect different roles of Lyn and/or involvement of different PKCs for ERK activation at different stages of B cell development. Interestingly, a novel PKC isoform, PKCμ, which coprecipitates with both Syk and PLC-γ1/2 (35), showed the same pattern of BCR- mediated activation as did ERK2. PKCμ was activated in wild-type and Lyn-deficient DT40 cells but not in Syk- deficient DT40 cells, and was only partially activated in Btk-deficient cells. Furthermore, PKCμ is both calcium independent and PMA inducible, suggesting that PKCμ may participate directly in the activation of ERK2 upon BCR ligation. A possible role of PKCμ in MAPK signaling would not be surprising since other novel PKC isoforms are also capable of activating ERKs (36). Further studies are required to elucidate the precise role of PKCμ and other PKC isoforms in BCR-mediated ERK2 activation.

Both JNK and p38 MAPK are preferentially activated by inflammatory cytokines and cellular stresses instead of classic mitogenic stimuli (37, 38). The JNK activation pathway is well studied, and Rac1/cdc42, MEKK1, and MKK4/ MKK7 contribute to JNK activation (3941). Consistent with our finding that BCR-mediated JNK1 activation requires both a calcium signal and PMA-sensitive PKCs, JNK activation in a mouse B cell line also requires both PMA and ionomycin (42). In T lymphocytes, JNK activation requires engagement of both the TCR and CD28 or both PMA and ionomycin in contrast to the full activation of ERK1 and ERK2 by either PMA or TCR engagement alone (43). Our data also reveal that BCR-mediated JNK1 activation is dependent on Syk and Btk, which regulate PLC-γ2, but is not dependent on the Src family kinase Lyn (Fig. 6). Consistent with our data, a recent study showed that Syk but not Src family kinases in cooperation with Rac led to enhanced JNK activation in Jurkat T cells (44). Similarly, both a calcium signal and PKCs seem to account for the requirement of Syk for JNK activation. Since Syk- induced JNK activation is potentiated by anti-CD3, anti-CD28, or PMA, it is likely that the requirement of Syk could be at least partly due to a requirement for calcium (44); the fact that JNK activation is not solely activated by ionomycin or calcineurin, and that calcineurin does not synergize with Rac to enhance JNK activation, suggests that PKCs are the second component involved in JNK activation by Syk (45). We also found that PLC-γ2–deficient cells exhibited impaired JNK1 activation and, interestingly, a dominant negative Rac1 (Rac1 N17) blocked activation of JNK1, suggesting a model where cooperation of PKCs and Rac1 leads to JNK1 activation (32). Evidence from studies of Jurkat cells also supports this model. A recent study revealed that a particular PKC isoform, PKCθ, but not PKCα or PKCε, participated in JNK activation; moreover, specific cooperation between calcineurin and PKCθ converged on Rac leading to potent JNK activation (45).

Btk is of interest since Btk is essential for BCR-mediated JNK1 activation but not for p38 MAPK or ERK2 activation. A study using mast cells from Btk-defective mice also had a similar result: the lack of Btk only led to a loss of FcεRI-mediated JNK1 activation but not ERKs or p38 MAPK activation (46). Recently, a novel MKK kinase, MEKK4, which specifically activates the JNK pathway but not ERKs or p38 MAPK, was identified (47); MEKK4 has a Rac/cdc42 interacting binding motif, a putative pleckstrin homology (PH) domain, and a proline-rich region that could provide the binding site for the Src homology (SH)3 domain. Since Btk has a PH domain, a proline-rich region, and SH2 and SH3 domains (48, 49), and since PH domains can interact with PKCs (50), one attractive model is that the interaction of Btk and MEKK4 defines the specificity of JNK signaling pathway (Fig. 6).

Less is known concerning the p38 MAPK signaling pathway. p38 MAPK can be turned on independently of JNKs since both MKK3 and MKK6 activate p38 MAPK specifically (51, 52); however, the upstream activators of MKK3 and MKK6 are still not fully defined. One potential upstream activator may be a STE20/PAK homologue such as Mst1 (53, 54). It is also not clear at what point p38 MAPK and JNK pathways diverge and what the mechanisms are that lead to specific activation of p38 MAPK versus JNKs. Although our data do not entirely solve the puzzle, they do indicate that in DT40 cells p38 MAPK is activated by BCR ligation via a distinct pathway from JNK1: either Syk or Lyn is sufficient and Btk is not required for BCR-mediated p38 MAPK activation; in contrast, anti-IgM–induced JNK activation requires both Syk and Btk but not Lyn (Fig. 6). BCR-mediated p38 MAPK was only partially affected in Syk-deficient (Fig. 3,A) and PKC-depleted cells (Fig. 5,C), again suggesting that both Syk- and Lyn-dependent signals may contribute to p38 MAPK activation independently (Fig. 6). Just how the two p38 MAPK signaling pathways interact and are regulated remains to be determined.

Activation of MAPK family members may contribute to the induction of programmed cell death or apoptosis. Apoptosis plays an important role in B cell development and the generation of immune responses (55), and DT40 cells undergo apoptosis upon BCR cross-linking. Our studies using the B104 B cell line show that p38 MAPK plays a role in BCR-mediated apoptosis (56) and that activation of JNK1 correlates with induction of cell death (18). In DT40 cell lines, BCR-mediated apoptosis is blocked in both Syk- and Btk-deficient cells (57, 58), which also do not activate JNK1; cells in which JNK1 is activated via the BCR (wild-type and Lyn-deficient DT40 cells) also undergo apoptosis (53); in addition, BAPTA-AM blocks both apoptosis and JNK1 activation but not BCR-mediated ERK2 or p38 MAPK activation (Fig. 4 and data not shown). Thus, activation of JNK1 correlated best with the induction of apoptosis in DT40 cells. We also tested the role of p38 MAPK in apoptosis and found that only a relatively high dose of SB203580 (20 μM), a highly specific and cell-permeable inhibitor of p38 MAPK, completely blocked BCR-mediated apoptosis in DT40 cells (Jiang, A., A. Craxton, and E.A. Clark, unpublished data). Thus, it is not presently clear whether JNK1 alone or in cooperation with p38 MAPK may be needed to activate a death program in DT40 cells. Further work with knockout mice and primary B cells is needed to assess the function of distinct MAPKs in B cell development and activation.

Acknowledgments

We would like to thank M. Domenowske for preparation of figures, G. Shu and K.E. Draves for technical assistance, Y. Sun for reading the manuscript, and other members of the Clark laboratory for helpful discussions.

This work was supported by National Institutes of Health grants GM-42508 and GM-37905 (to E.A. Clark) and by grants from the Japanese Ministry of Education, Science, Sports and Culture (to T. Kurosaki).

Abbreviations used in this paper

     
  • ATF

    activating transcription factor

  •  
  • BAPTA-AM

    the acetoxymethyl ester of bis-(o-aminophenoxy) ethane-N,N,N′, N′-tetraacetic acid

  •  
  • BCR

    B cell antigen receptor

  •  
  • [Ca2+]i

    intracellular cytoplasmic free Ca2+ concentration

  •  
  • ERK

    extracellular signal-regulated kinase

  •  
  • GST

    glutathione S-transferase

  •  
  • JNK

    c-jun NH2-terminal kinase

  •  
  • MAPK

    mitogen-activated protein kinase

  •  
  • MBP

    myelin basic protein

  •  
  • PKC

    protein kinase C

  •  
  • PLC-γ2

    phospholipase C-γ2

  •  
  • PTK

    protein tyrosine kinase

References

References
1
Campbell
KS
,
Cambier
JC
B lymphocyte antigen receptors (mIg) are non-covalently associated with a disulfide linked, inducibly phosphorylated glycoprotein complex
EMBO (Eur Mol Biol Organ) J
1990
9
441
448
[PubMed]
2
Reth
M
Antigen receptors on B lymphocytes
Annu Rev Immunol
1992
10
97
121
[PubMed]
3
Bolen
JB
Protein tyrosine kinases in the initiation of antigen receptor signaling
Curr Opin Immunol
1990
7
306
311
[PubMed]
4
Sefton
BM
,
Campbell
MA
The role of tyrosine protein phosphorylation in lymphocyte activation
Annu Rev Cell Biol
1991
7
257
274
[PubMed]
5
Law
CL
,
Craxton
A
,
Otipoby
KL
,
Sidorenko
SP
,
Klaus
SJ
,
Clark
EA
Regulation of signalling through B-lymphocyte antigen receptors by cell–cell interaction molecules
Immunol Rev
1996
153
123
154
[PubMed]
6
Kurosaki
T
Molecular mechanisms in B cell antigen receptor signaling
Curr Opin Immunol
1997
9
309
318
[PubMed]
7
DeFranco
AL
The complexity of signaling pathways activated by the BCR
Curr Opin Immunol
1997
9
296
308
[PubMed]
8
Reth
M
,
Wienands
J
Initiation and processing of signals from the B cell antigen receptor
Annu Rev Immunol
1997
15
453
479
[PubMed]
9
Treisman
R
Regulation of transcription by MAP kinase cascades
Curr Opin Cell Biol
1996
8
205
215
[PubMed]
10
Su
B
,
Karin
M
Mitogen-activated protein kinase cascades and regulation of gene expression
Curr Opin Immunol
1996
8
402
411
[PubMed]
11
Boulton
TG
,
Nye
SH
,
Robbins
DJ
,
Ip
NY
,
Radziejewska
E
,
Morgenbesser
SD
,
DePinho
RA
,
Panayotatos
N
,
Cobb
MH
,
Yancopoulos
GD
ERKs: a family of protein-serine/theonine kinases that are activated and tyrosine phosphorylated in response to insulin and NGF
Cell
1991
65
663
675
[PubMed]
12
Ahn
NG
,
Seger
R
,
Bratlien
RL
,
Diltz
CD
,
Tonks
NK
,
Krebs
EG
Multiple components in an epidermal growth factor-stimulated protein kinase cascade. In vitro activation of a myelin basic protein/microtubule-associated protein 2 kinase
J Biol Chem
1991
266
4220
4227
[PubMed]
13
D'Erijard
B
,
Hibi
M
,
Wu
IH
,
Barrett
T
,
Su
B
,
Deng
T
,
Karin
M
,
Davis
RJ
JNK1: a protein kinase stimulated by UV light and Ha-Ras that binds and phosphorylates the c-Jun activation domain
Cell
1994
76
1025
1037
[PubMed]
14
Kyriakis
JM
,
Banerjee
P
,
Nikolakaki
E
,
Dai
T
,
Rubie
EA
,
Ahmad
MF
,
Avruch
J
,
Woodgett
JR
The stress-activated protein kinase subfamily of c-Jun kinases
Nature
1994
369
156
160
[PubMed]
15
Kallunki
T
,
Su
B
,
Tsigelny
I
,
Sluss
HK
,
D'Erijard
B
,
Moore
G
,
Davis
R
,
Karin
M
JNK2 contains a specificity-determining region responsible for efficient c-Jun binding and phosphorylation
Genes Dev
1994
8
2996
3007
[PubMed]
16
Han
J
,
Lee
JD
,
Bibbs
L
,
Ulevitch
RJ
A MAP kinase targeted by endotoxin and hyperosmolarity in mammalian cells
Science
1994
265
808
811
[PubMed]
17
Lee
JC
,
Laydon
JT
,
McDonnell
PC
,
Gallagher
TF
,
Kumar
S
,
Green
D
,
McNulty
D
,
Blumenthal
MJ
,
Heys
JR
,
Landvatter
SW
et al
A protein kinase involved in the regulation of inflammatory cytokine biosynthesis
Nature
1994
372
739
746
[PubMed]
18
Graves
JD
,
Draves
KE
,
Craxton
A
,
Saklatvala
J
,
Krebs
EG
,
Clark
EA
Involvement of stress-activated protein kinase and p38 mitogen-activated protein kinase in mIgM-induced apoptosis of human B lymphocytes
Proc Natl Acad Sci USA
1996
93
13814
13818
[PubMed]
19
Healy
JI
,
Dolmetsch
RE
,
Timmerman
LA
,
Cyster
JG
,
Thomas
ML
,
Crabtree
GR
,
Lewis
RS
,
Goodnow
CC
Different nuclear signals are activated by the B cell receptor during positive versus negative signaling
Immunity
1997
6
419
428
[PubMed]
20
Takata
M
,
Sabe
H
,
Hata
A
,
Inazu
T
,
Homma
Y
,
Nukada
T
,
Yamamura
H
,
Kurosaki
T
Tyrosine kinases Lyn and Syk regulate B cell receptor-coupled Ca2+mobilization through distinct pathways
EMBO (Eur Mol Biol Organ) J
1994
13
1341
1349
[PubMed]
21
Takata
M
,
Kurosaki
T
A role for Bruton's tyrosine kinase in B cell antigen receptor–mediated activation of phospholipase C-γ2
J Exp Med
1996
184
31
40
[PubMed]
22
Chen
CL
,
Lehmeyer
JE
,
Cooper
MD
Evidence for an IgD homologue on chicken lymphocytes
J Immunol
1982
129
2580
2585
[PubMed]
23
Berberich
I
,
Shu
G
,
Siebelt
F
,
Woodgett
JR
,
Kyriakis
JM
,
Clark
EA
Cross-linking CD40 on B cells preferentially induces stress-activated protein kinases rather than mitogen-activated protein kinases
EMBO (Eur Mol Biol Organ) J
1996
15
92
101
[PubMed]
24
Rabinovitch
PS
,
June
CH
,
Grossmann
A
,
Ledbetter
JA
Heterogeneity among T cells in intracellular free calcium responses after mitogen stimulation with PHA or anti-CD3. Simultaneous use of indo-1 and immunofluorescence with flow cytometry
J Immunol
1986
137
952
961
[PubMed]
25
Casillas
A
,
Hanekom
C
,
Williams
K
,
Katz
R
,
Nel
AE
Stimulation of B-cells via the membrane immunoglobulin receptor or with phorbol myristate 13-acetate induces tyrosine phosphorylation and activation of a 42-kDa microtubule-associated protein-2 kinase
J Biol Chem
1991
266
19088
19094
[PubMed]
26
Gold
MR
,
Sanghera
JS
,
Stewart
J
,
Pelech
SL
Selective activation of p42 mitogen-activated protein (MAP) kinase in murine B lymphoma cell lines by membrane immunoglobulin cross-linking. Evidence for protein kinase C–independent and –dependent mechanisms of activation
Biochem J
1992
287
269
276
[PubMed]
27
Wilson
HA
,
Greenblatt
D
,
Poenie
M
,
Finkelman
FD
,
Tsien
RY
Cross-linkage of B lymphocyte surface immunoglobulin by anti-Ig or antigen induces prolonged oscillation of intracellular ionized calcium
J Exp Med
1987
166
601
606
[PubMed]
28
Thastrup
O
,
Dawson
AP
,
Scharff
O
,
Foder
B
,
Cullen
PJ
,
Drobak
BK
,
Bjerrum
PJ
,
Christensen
SB
,
Hanley
MR
Thapsigargin, a novel molecular probe for studying intracellular calcium release and storage
Agents Actions
1989
27
17
23
[PubMed]
29
Tordai
A
,
Franklin
RA
,
Patel
H
,
Gardner
AM
,
Johnson
GL
,
Gelfand
EW
Cross-linking of surface IgM stimulates the Ras/Raf-1/MEK/MAPK cascade in human B lymphocytes
J Biol Chem
1994
269
7538
7543
[PubMed]
30
Williams
S
,
Couture
C
,
Gilman
J
,
Jascur
T
,
Deckert
M
,
Altman
A
,
Mustelin
T
Reconstitution of T cell antigen receptor-induced Erk2 kinase activation in Lck-negative JCaM1 cells by Syk
EurJ Biochem
1997
245
84
90
[PubMed]
31
Kawauchi
K
,
Lazarus
AH
,
Sanghera
JS
,
Man
GL
,
Pelech
SL
,
Delovitch
TL
Regulation of BCR- and PKC/Ca(2+)-mediated activation of the Raf1/MEK/MAPK pathway by protein-tyrosine kinase and -tyrosine phosphatase activities
Mol Immunol
1996
33
287
296
[PubMed]
32
Hashimoto
A
,
Okada
H
,
Jiang
A
,
Kurosaki
M
,
Greenberg
S
,
Clark
EA
,
Kurosaki
T
Involvement of guanosine triphosphatases and phospholipase C-γ2 in extracellular signal–regulated kinase, c-Jun NH2-terminal kinase, and p38 mitogen-activated protein kinase activation by the B cell antigen receptor
J Exp Med
1998
188
1287
1295
[PubMed]
33
Chan
VW
,
Meng
F
,
Soriano
P
,
DeFranco
AL
,
Lowell
CA
Characterization of the B lymphocyte populations in Lyn-deficient mice and the role of Lyn in signal initiation and down-regulation
Immunity
1997
7
69
81
[PubMed]
34
Katsuta
H
,
Tsuji
S
,
Niho
Y
,
Kurosaki
T
,
Kitamura
D
Lyn-mediated down-regulation of B cell antigen receptor signaling: inhibition of protein kinase C activation by Lyn in a kinase-independent fashion
J Immunol
1998
160
1547
1551
[PubMed]
35
Sidorenko
SP
,
Law
CL
,
Klaus
SJ
,
Chandran
KA
,
Takata
M
,
Kurosaki
T
,
Clark
EA
Protein kinase C mu (PKC mu) associates with the B cell antigen receptor complex and regulates lymphocyte signaling
Immunity
1996
5
353
363
[PubMed]
36
Schonwasser
DC
,
Marais
RM
,
Marshall
CJ
,
Parker
PJ
Activation of the mitogen-activated protein kinase/extracellular signal-regulated kinase pathway by conventional, novel, and atypical protein kinase C isotypes
Mol Cell Biol
1998
18
790
798
[PubMed]
37
Cano
E
,
Mahadevan
LC
Parallel signal processing among mammalian MAPKs
Trends Biochem Sci
1995
20
117
122
[PubMed]
38
Waskiewicz
AJ
,
Cooper
JA
Mitogen and stress response pathways: MAP kinase cascades and phosphatase regulation in mammals and yeast
Curr Opin Cell Biol
1995
7
798
805
[PubMed]
39
Moriguchi
T
,
Toyoshima
F
,
Masuyama
N
,
Hanafusa
H
,
Gotoh
Y
,
Nishida
E
A novel SAPK/JNK kinase, MKK7, stimulated by TNFalpha and cellular stresses
EMBO (Eur Mol Biol Organ) J
1997
16
7045
7053
[PubMed]
40
Minden
A
,
Lin
A
,
Claret
FX
,
Abo
A
,
Karin
M
Selective activation of the JNK signaling cascade and c-Jun transcriptional activity by the small GTPases Rac and Cdc42Hs
Cell
1995
81
1147
1157
[PubMed]
41
Coso
OA
,
Chiariello
M
,
Yu
JC
,
Teramoto
H
,
Crespo
P
,
Xu
N
,
Miki
T
,
Gutkind
JS
The small GTP-binding proteins Rac1 and Cdc42 regulate the activity of the JNK/SAPK signaling pathway
Cell
1995
81
1137
1146
[PubMed]
42
Sutherland
CL
,
Heath
AW
,
Pelech
SL
,
Young
PR
,
Gold
MR
Differential activation of the ERK, JNK, and p38 mitogen-activated protein kinases by CD40 and the B cell antigen receptor
J Immunol
1996
157
3381
3390
[PubMed]
43
Su
B
,
Jacinto
E
,
Hibi
M
,
Kallunki
T
,
Karin
M
,
Ben
N-Y
JNK is involved in signal integration during costimulation of T lymphocytes
Cell
1994
77
727
736
[PubMed]
44
Jacinto
E
,
Werlen
G
,
Karin
M
Cooperation between Syk and Rac1 leads to synergistic JNK activation in T lymphocytes
Immunity
1998
8
31
41
[PubMed]
45
Werlen
G
,
Jacinto
E
,
Xia
Y
,
Karin
M
Calcineurin preferentially synergizes with PKC-theta to activate JNK and IL-2 promoter in T lymphocytes
EMBO (Eur Mol Biol Organ) J
1998
17
3101
3111
[PubMed]
46
Kawakami
Y
,
Miura
T
,
Bissonnette
R
,
Hata
D
,
Khan
WN
,
Kitamura
T
,
Maeda
Y-M
,
Hartman
SE
,
Yao
L
,
Alt
FW
,
Kawakami
T
Bruton's tyrosine kinase regulates apoptosis and JNK/SAPK kinase activity
Proc Natl Acad Sci USA
1997
94
3938
3942
[PubMed]
47
Gerwins
P
,
Blank
JL
,
Johnson
GL
Cloning of a novel mitogen-activated protein kinase kinase kinase, MEKK4, that selectively regulates the c-Jun amino terminal kinase pathway
J Biol Chem
1997
272
8288
8295
[PubMed]
48
Tsukada
S
,
Saffran
DC
,
Rawlings
DJ
,
Parolini
O
,
Allen
RC
,
Klisak
I
,
Sparkes
RS
,
Kubagawa
H
,
Mohandas
T
,
Quan
S
et al
Deficient expression of a B cell cytoplasmic tyrosine kinase in human X-linked agammaglobulinemia
Cell
1993
72
279
290
[PubMed]
49
Vetrie
D
,
Vorechovsky
I
,
Sideras
P
,
Holland
J
,
Davies
A
,
Flinter
F
,
Hammarstrom
L
,
Kinnon
C
,
Levinsky
R
,
Bobrow
M
et al
The gene involved in X-linked agammaglobulinaemia is a member of the src family of protein- tyrosine kinases
Nature
1993
361
226
233
[PubMed]
50
Yao
L
,
Kawakami
Y
,
Kawakami
T
The pleckstrin homology domain of Bruton's tyrosine kinase interacts with protein kinase C
Proc Natl Acad Sci USA
1994
91
9175
9179
[PubMed]
51
D'Erijard
B
,
Raingeaud
J
,
Barrett
T
,
Wu
IH
,
Han
J
,
Ulevitch
RJ
,
Davis
RJ
Independent human MAP-kinase signal transduction pathways defined by MEK and MKK isoforms
Science
1995
267
682
685
[PubMed]
52
Han
J
,
Lee
JD
,
Jiang
Y
,
Li
Z
,
Feng
L
,
Ulevitch
RJ
Characterization of the structure and function of a novel MAP kinase kinase (MKK6)
J Biol Chem
1996
271
2886
2891
[PubMed]
53
Graves
JD
,
Gotoh
Y
,
Draves
KE
,
Ambrose
D
,
Han
DKM
,
Wright
M
,
Chernoff
J
,
Clark
EA
,
Krebs
EG
Caspase-mediated activation and induction of apoptosis by the mammalian Ste20-like kinase Mst1
EMBO (Eur Mol Biol Organ) J
1998
17
2224
2234
[PubMed]
54
Rudel
T
,
Zenke
FT
,
Chuang
TH
,
Bokoch
GM
p21-activated kinase (PAK) is required for Fas-induced JNK activation in Jurkat cells
J Immunol
1998
160
7
11
[PubMed]
55
VanParijs
L
,
Abbas
AK
Homeostasis and self-tolerance in the immune system: Turning lymphocytes off
Science
1998
280
243
248
[PubMed]
56
Graves
JD
,
Draves
KE
,
Craxton
A
,
Krebs
EG
,
Clark
EA
A comparison of signaling requirements for apoptosis of human B lymphocytes induced by the B cell receptor and CD95/Fas
J Immunol
1998
161
168
174
[PubMed]
57
Takata
M
,
Homma
Y
,
Kurosaki
T
Requirement of phospholipase C-γ2 activation in surface immunoglobulin M-induced B cell apoptosis
J Exp Med
1995
182
907
914
[PubMed]
58
Uckun
FM
,
Waddick
KG
,
Mahajan
S
,
Jun
X
,
Takata
M
,
Bolen
J
,
Kurosaki
T
BTK as a mediator of radiation-induced apoptosis in DT-40 lymphoma B cells
Science
1996
273
1096
1100
[PubMed]

Author notes

Address correspondence to Edward A. Clark, Department of Microbiology, Box 357242, University of Washington, Seattle, WA 98195. Phone: 206-543-8706; Fax: 206-685-0305; E-mail: eclark@bart.rprc.washington.edu