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The Journal of Immunology, 1998, 161: 5901-5908.
Copyright © 1998 by The American Association of Immunologists

Convergence of CD19 and B Cell Antigen Receptor Signals at MEK1 in the ERK2 Activation Cascade1

Xiaoli Li* and Robert H. Carter2,*,{dagger},{ddagger}

Departments of * Medicine and {dagger} Microbiology, University of Alabama, Birmingham, AL 35294; and {ddagger} Birmingham Veterans Affairs Medical Center, Birmingham, AL 35294


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
CD19 plays a critical role in regulating B cell responses to Ag. We have studied the mechanism by which coligation of CD19 and the B cell Ag receptor, membrane Ig (mIg), augments signal transduction, including synergistic enhancement of release of intracellular Ca2+ and extracellular signal-regulated protein kinase 2 (ERK2) activation, in Daudi human B lymphoblastoid cells. The pathway leading to ERK2 activation was further dissected to determine how signals derived from CD19 and mIgM interact. The best-defined pathway, known to be activated by mIgM, consists of the sequential activation of the mitogen-activated protein kinase (MAPK) cascade that includes Ras, Raf, MAPK kinase 1 (MEK1), and ERK2. Ligation of CD19 alone had little effect on these. CD19-mIgM coligation did not increase activation of Ras or Raf beyond that induced by ligation of mIgM alone. In contrast, coligation resulted in synergistic activation of MEK1. Furthermore, synergistic activation of ERK2 occurred in the absence of changes in intracellular Ca2+, and was not blocked by inhibition of protein kinase C activity and represents a separate pathway by which CD19 regulates B cell function. Thus, the CD19-dependent signal after CD19-mIgM coligation converges with that generated by mIgM at MEK1. The intermediate kinases in the MAPK cascade leading to ERK2 integrate signals from lymphocyte coreceptors.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The functional complexity of lymphocytes requires the integration of multiple regulatory signals. CD19 is a component of a multimeric complex on mature B cells that includes CD21, CD81, and Leu-13 (1, 2). Coligation of this complex and membrane Ig (mIg)3 powerfully augments B cell activation. In vitro, such coligation results in enhanced release of intracellular calcium, activation of ERK2, induction of DNA synthesis, and Ab production (3, 4, 5, 6, 7, 8). In vivo, CD19 is required for normal germinal center formation and Ab responses to T-dependent Ags and for maintenance of the B1 subpopulation, and appears to act to amplify B cell Ag receptor (BCR)-driven responses (9, 10, 11). Purposeful coligation of CD21 and mIg in mice reduces by orders of magnitude the amount of Ag necessary to induce an Ab response (12).

Stimulation of B cells through the Ag receptor results in tyrosine phosphorylation of CD19 and binding of cytoplasmic signaling molecules, including phosphatidylinositol 3-kinase (PI3K) and Vav, to those residues (13, 14, 15, 16, 17). Ligation of CD19 or coligation of CD19 and mIgM results in enhanced tyrosine phosphorylation of Vav, a possible mechanism of enhancement of signaling (6, 16, 18, 19, 20). We have used Daudi human B lymphoblastoid cells expressing mutant variants of CD19 to determine which pathways linked to CD19 enhance mIg-induced signaling. Activation of ERK2 in these cells is similar to that in normal mature mouse and human B cells in that coligation of CD19 and mIgM results in synergistically enhanced activity (6, 17). In Daudi cells, ERK2 activity was maximal at 1 min and persisted for 30 min following such stimulation. The synergistic activation of ERK2 by coligation of CD19 and mIg was blocked by mutation of CD19 Y391 (6).

The current studies were designed to further our understanding of how synergy is produced between CD19 and mIg by determining the point at which their respective pathways leading to ERK2 intersect. ERK2 is the terminal kinase in the well-defined mitogen-activated protein kinase (MAPK) cascade that consists of Ras, Raf, MEK1, and ERK2. Activation of Ras and Raf by mIg has been reported previously, most likely through the Shc/SOS/Grb2 complex (21, 22, 23).

Where CD19 impacts on this series is unknown. CD19 might enhance Ras activation by enhanced formation of a Grb2-SOS-Shc complex (24, 25). Alternatively, CD19 may activate members of the Rac/cdc42 family (17, 19, 26), which in turn activate the Jun N-terminal kinase (JNK) MAPK cascade (27), members of which may phosphorylate MEK1 in the ERK2 cascade (28, 29, 30, 31). Finally, the increase in intracellular Ca2+ that results from CD19-mIg coligation could enhance the activity of enzymes such as protein kinase C{alpha} that phosphorylate Raf (21, 32, 33).

We find that ligation of mIgM alone, but not CD19 alone, results in activation of Ras, Raf, and MEK1. Neither Ras nor Raf activation is enhanced after coligation of CD19 and mIgM, relative to mIgM alone. In contrast, MEK1 is activated synergistically by CD19-mIgM coligation, and thus is an intersection point of the signal transduction pathways activated by these two receptor complexes. We also show that the CD19 component of the enhanced activation of ERK2 is predominantly independent of changes in intracellular calcium and PKC activity.

Beyond the relevance for CD19 itself, this provides a model for how signals for lymphocyte coreceptors are integrated in the multienzyme cascades. To our knowledge, this represents the first demonstration of synergistic activation of MEK1 by surface receptors. In addition to providing an amplification of a signal by a multienzyme cascade, individual members of the cascade may serve to integrate signals from different receptors.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Abs, reagents, plasmids, and cells

Polyclonal rabbit Abs to MEK1 (C18), Raf-1 (C12), and ERK2 (C14) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Murine [K97A]MEK1 (catalytically inactive) glutathione S-transferase (GST) agarose conjugate, mouse MEK1, mouse p42 MAP kinase-GST, ERK1-GST, and ERK1 [K71A]GST were purchased from Upstate Biotechnology (Lake Placid, NY). Peroxidase-coupled polyclonal mouse anti-rabbit IgG was purchased from Jackson ImmunoResearch (West Grove, PA). Streptavidin (Sigma, St. Louis, MO), myelin basic protein (MBP; Sigma), PMA (Sigma), indo-1-AM (Molecular Probes, Eugene, OR), 1,2-bis(2-aminophenoxylethane-N,N,N,N'-tetraacetic acid tetra(acetoxymethyl) ester (BAPTA-AM; Calbiochem, La Jolla, CA), bisindoylmaleimide I (Calbiochem), and bisindoylmaleimide V (Calbiochem) were purchased. Biotinylated F(ab')2 DA4.4 mouse anti-human IgM and biotinylated F(ab')2 ADF4.2 anti-CD19 were prepared as described previously (34). The pGEX-RBD plasmid was a gift from Dr. Stephen J. Taylor (Cornell University, Ithaca, NY). Daudi B lymphoblastoid cells were obtained from American Type Culture Collection (Manassas, VA).

Cell activation and immunoblotting

Daudi cells, 1–2 x 107/lane, were preincubated in HBSS containing 1 mg/ml BSA, 1 mM MgCl2, and 1 mM CaCl2 (except as described) with biotinylated F(ab')2 DA4.4, biotinylated F(ab')2 ADF4.2, or combinations of both Abs for 10 min and washed (preincubation with these Ab fragments had no significant effect on intracellular calcium concentration or ERK2 activity without the addition of a cross-linker (not shown)). The cells were stimulated with 5 µg/ml of streptavidin for 1 min. The cells were spun down, and the pellet was lysed with Triton X-100 lysis buffer, as previously described (6). Lysates were cleared by centrifugation at 14,000 rpm for 10 min at 4°C. Immunoprecipitations were prepared by addition of appropriate Abs (2 µg/ml), followed by protein A-Trisacryl (Pierce, Rockford, IL). The precipitates were washed five times with lysis buffer and then boiled in 2x Laemmli sample buffer for 5 min. Eluted proteins were resolved by SDS-PAGE, transferred to nitrocellulose, and blotted with polyclonal Abs (1:1,000). The Abs were detected by peroxidase-coupled anti-rabbit (1:10,000), followed by chemoluminescence (Amersham, Arlington Heights, IL).

Assay for detection of activated Ras

Activated Ras interaction assays were performed as described previously (35, 36, 37). A GST fusion protein containing the Ras-binding domain (RBD) of Raf (amino acids 1–149 of c-Raf-1), which binds only GTP-bound (activated) Ras, was prepared as described using the plasmid pGEX-RBD. For affinity precipitation of active Ras, 1–2 x 107 Daudi cells were stimulated for 1 min and lysed. Clarified lysates were incubated with GST-RBD immobilized on glutathione-agarose beads (25 µl packed beads containing 50 µg protein) for 30 min at 4°C with rocking. The GST-RBD beads were washed three times with lysis buffer. Bound proteins were eluted by boiling in SDS-PAGE sample buffer, resolved on 10% acrylamide gels, and transferred to nitrocellulose. Affinity-precipitated Ras was detected by immunoblotting with anti-pan Ras (Transduction Labs, Lexington, KY).

Raf1 immunoprecipitation cascade kinase assay

Lysates of Daudi cells (1 x 107/lane) were clarified by centrifugation at 10,000 x g for 10 min at 4°C. Raf1 kinase in the supernatants was immunoprecipitated with 2 µg/ml of polyclonal anti-c-Raf-1 or normal rabbit IgG for 1 h at 4°C and 20 µl of protein A-Trisacryl beads (Pierce) for 1 h at 4°C. The beads were washed three times with lysis buffer and two times with Raf-1 kinase assay buffer (20 mM MOPS, pH 7.2, 25 mM ß-glycerophosphate, 5 mM EGTA, 1 mM Na3VO4, 1 mM DTT, 0.167 mM ATP, and 25 mM MgCl2). Raf-dependent activation of inactive GST-ERK2 was measured using a c-Raf1 immunoprecipitation kinase cascade assay (33) (Upstate Biotechnology). Except where indicated, the kinase cascade reactions were conducted in a mixture containing 30 µl of kinase assay buffer, immunoprecipitated Raf1, 0.4 µg inactive MEK1, and 1 µg inactive GST-ERK2 at 30°C for 30 min in a shaking incubator. Phosphorylation of MBP by activated GST-ERK2 was conducted in a mixture containing 4 µl of the above mixture, 15 µl ERK2 kinase assay buffer (20 mM HEPES, pH 7.6, 20 mM MgCl2, 20 mM ß-glycerophosphate, 20 mM p-nitrophenyl phosphate, 0.1 mM Na3VO4, and 2 mM DTT), 20 µg MBP, and 10 µCi [{gamma}-32P]ATP at 30°C for 10 min. The reaction was terminated by adding 20 µl of 2x Laemmli sample buffer and boiling for 5 min. Samples were resolved on a 10% SDS-polyacrylamide gel. The gels were analyzed for incorporation of 32P into MBP by autoradiography and by phosphor imager (Fujix BAS1000) analysis. Proteins in the assay mixture were immunoblotted with anti-c-Raf1 polyclonal antisera to confirm similar kinase equivalents in all samples. The results indicated the presence of Raf1 protein at equivalent levels in all Raf1 immunoprecipitates (data not shown).

Raf1 kinase activity assay

Raf1 kinase activity was determined as described by Niculescu et al. (38). Raf1 immunoprecipitates were prepared from Daudi cells (1 x 107/lane), as described above. The immunoprecipitates were washed three times with lysis buffer and twice with cold 50 mM Tris-HCl, pH 7, and suspended in 50 µl 20 mM PIPES reaction buffer, pH 7, containing 10 mM MnCl2, 10 µCi [{gamma}-32P]ATP, and 0.5 µg [K97A]MEK1-GST as a Raf-1-specific substrate. The samples were incubated for 30 min at 30°C in a shaking incubator. The reaction was terminated by adding 50 µl of 2x Laemmli sample buffer and boiling for 5 min. Samples were resolved on a 10% SDS-polyacrylamide gel and analyzed by autoradiography.

MEK1 immunoprecipitation cascade kinase assay

MEK1 was immunoprecipitated from lysates of Daudi cells (1 x 107/lane). The immunoprecipitates were suspended in 30 µl assay buffer (20 mM HEPES, pH 7.6, 20 mM ß-glycerophosphate, 20 mM p-nitrophenyl phosphates, 0.1 mM Na3VO4, 20 mM MgCl2, and 2 mM DTT) containing 10 µCi of [{gamma}-32P]ATP, 0.4 µg of inactive GST-ERK2, and 20 µg of MBP, except as indicated. The reaction was conducted at 30°C for 30 min in a shaking incubator. The reaction was terminated by adding 30 µl of 2x Laemmli sample buffer and boiling for 5 min. Samples were resolved on a 10% SDS-polyacrylamide gel. The phosphorylation of MBP was analyzed by autoradiography and by phosphor imager analysis.

ERK2 immune complex kinase assay

In vitro protein kinase assays of ERK2 immunoprecipitates were performed as described previously (6). Incorporation of 32P into MBP was measured by autoradiography and by phosphor imager analysis. The amount of ERK2 in the immunoprecipitates was shown to be similar by immunoblotting using anti-ERK2 antisera. The data shown in Fig. 4Go are representative; blots are not shown in other figures.



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  		FIGURE 4. Activation of MEK1 by CD19-mIgM coligation. A, Specific activation of MEK1. Activity of MEK1 was determined in an immunoprecipitation kinase cascade assay using GST-ERK2 and MBP as substrates. Daudi cells (1 x 107/lane) were incubated with buffer only (PBS), biotinylated anti-CD19 mAb (5 µg/ml) alone, biotinylated anti-IgM (1 µg/ml) alone, or both biotinylated anti-IgM (1 µg/ml) and anti-CD19 mAb (5 µg/ml) at 25°C for 10 min. Cells were stimulated by the addition of avidin (5 µg/ml) for 1 min and lysed. Immunoprecipitates formed with anti-MEK1 (2 µg/ml) or normal rabbit Ig (2 µg/ml) were analyzed for the ability to activate ERK2 by measuring incorporation of 32P into MBP, as described in Materials and Methods. Gels were analyzed by autoradiography and by phosphor imager. The fold increase in incorporation of radioactivity into MBP for each stimulation condition in anti-MEK1 immunoprecipitates, relative to that in cells stimulated with PBS only, is shown. B, Requirement for GST-ERK2 for MEK1 assay. Immunoprecipitates formed with anti-MEK1 were analyzed for kinase activity in the presence or absence of inactive GST-ERK2, as indicated. One immunoprecipitate formed with anti-ERK2 was assayed for kinase activity as a positive control. Gels were analyzed as in A.

 
Flow cytometric analysis of intracellular Ca2+

Daudi cells (5 x 106/ml) were loaded with 1 µM indo-1 AM at 37°C for 40 min. BAPTA-AM (5–25 µM final concentration, from stock in DMSO) or DMSO was added during the indo-1 loading. DMSO was added to all samples to yield equal final DMSO concentrations. Cells pretreated with DMSO vehicle only were washed and resuspended in HBSS containing 0.1% BSA, 1 mM MgCl2, and 1 mM CaCl2. Cells pretreated with BAPTA-AM were washed and resuspended in HBSS containing 1 mM EGTA, 0.1% BSA, 1 mM MgCl2, and no Ca2+. Changes in indo-1 fluorescence were monitored by flow cytometry (FACS Vantage; Becton Dickinson, San Jose, CA). After a 20-s baseline, polyclonal goat anti-human IgM (20 µg/ml) was added, and analysis continued for a total of 4 min.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
CD19-mIgM coligation does not result in enhanced Ras activation

Activation of Ras by stimulation with anti-mIgM leads to the rapid activation of the Raf/MEK/ERK cascade in human B lymphocytes (22). The synergistic activation of ERK2 by coligation of CD19 with mIgM might result from enhanced activation of Ras. To test this hypothesis, Daudi B lymphoblastoid cells were incubated with biotinylated F(ab')2 anti-CD19 or biotinylated Fab anti-IgM, or the combination of the two, washed, stimulated with avidin for 1 min, and lysed. Active (GTP-bound) Ras was detected using an assay based on the known specificity of the interaction between Ras-GTP and the RBD of Raf1 (35, 36, 37). GTP-Ras is affinity precipitated with GST-RBD and detected by immunoblotting using anti-Ras antisera. Active Ras increased by 2.9-fold following ligation of mIgM for 1 min (Fig. 1GoA, top). CD19 ligation alone induced no increase in active Ras. The level of active Ras was no greater following coligation of CD19 and mIgM than that induced by ligation of mIgM alone.



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  		FIGURE 1. Activation of Ras after CD19-mIgM coligation. A, Comparison of Ras and ERK2 activation. Daudi B lymphoblastoid cells (1 x 107/lane) were incubated with buffer only (PBS), biotinylated (F(ab')2 ADF4.2 anti-CD19 mAb (5 µg/ml) alone, or biotinylated Fab DA4.4 anti-IgM (1 µg/ml) alone, or both biotinylated DA4.4 anti-IgM (1 µg/ml) and biotinylated ADF4.2 anti-CD19 mAb (5 µg/ml), as indicated, at 25°C for 10 min. The cells were stimulated by the addition of avidin (5 µg/ml) for 1 min and lysed. Clarified lysates were subjected to affinity precipitation with GST-RBD. Affinity-precipitated GTP-Ras was detected by immunoblotting with anti-Ras antisera (top). The blot was scanned and analyzed for the density of Ras bands. The fold increase in the density of Ras bands for each stimulation condition, relative to that in cells stimulated with PBS only on the same gel, is shown. Replicate aliquots of the same lysates were subjected to immunoprecipitation with anti-ERK2 antisera. The immunoprecipitates were analyzed for kinase activity by incubation with MBP in the presence of [{gamma}-32P]ATP. The mixture was resolved by SDS-PAGE, and the portion of the gel containing MBP was exposed to film (bottom). Equivalent loading of kinase and substrate was confirmed as described in Materials and Methods. The gel used to produce the autoradiogram shown in ERK2 assay was further analyzed for 32P by phosphor imager, and the fold increase in incorporation of radioactivity into MBP for each stimulation condition, relative to that in cells stimulated with PBS only on the same gel, is shown. B, Combined results of six independent assays of Ras activation. The data were obtained by densitometric analysis and shown as the average fold enhancement ± SD in the density of Ras bands for each stimulation condition relative to that in cells stimulated with PBS only on the same gel.

 
To verify that ERK2 was synergistically activated under the stimulation conditions described, ERK2 immunoprecipitates from aliquots of the same lysates as were used in the active Ras assay were assayed for ERK2 kinase activity (Fig. 1GoA, bottom). The results show synergistic enhancement of ERK2 following CD19-mIgM coligation occurred in the same lysates used to test Ras activation.

Similar results were obtained in six experiments. For each of these, the fold increase in GTP-Ras in stimulated cells relative to cells treated with PBS only was calculated. The mean ± SD of the fold increase for each condition in the six experiments is shown (Fig. 1GoB). Coligation of CD19 and mIgM does not result in greater activation of Ras than ligation of mIgM alone. These results demonstrate that while ERK2 activation following ligation of mIgM alone correlates with an increase in GTP-Ras, the further, enhanced activation of ERK2 by CD19-mIgM coligation does not, suggesting that the CD19-dependent component of the synergistic interaction is not mediated by Ras.

CD19-mIgM coligation does not result in synergistic Raf1 activation

Raf1 is the most upstream protein kinase in the ERK MAPK cascade, and its activation is sufficient for ERK activation. The synergistic activation of ERK2 might result from synergy between CD19 and mIgM in activation of Raf1. We measured the activity of Raf1 after stimulation with PBS only or by ligation of CD19, mIgM, or both together under conditions shown to induce synergistic ERK2 activation. Immunoprecipitates formed with anti-Raf1 or control Abs were incubated with purified MEK1 and GST-ERK2. The incorporation of 32P into MBP, a substrate of ERK2, was analyzed by autoradiography and phosphor imager analysis. As shown in Fig. 2GoA, while anti-CD19 alone induced a small increase in Raf1 activation, stimulation with anti-mIgM resulted in a 3.5-fold increase in Raf1 activation. In three independent experiments, levels of Raf1 activation after CD19-mIgM coligation were 11–14% higher than that induced by anti-mIgM alone, less than the sum of the increases induced by CD19 alone and mIgM alone. The measured kinase activity was specifically related to Raf1, as when normal rabbit IgG was used as a negative control for the immunoprecipitates, little kinase activity was detected (Fig. 2GoA).



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  		FIGURE 2. Activation of Raf1 by CD19-mIgM coligation. A, Specific activation of Raf1. Activity of Raf-1 was determined in an immunoprecipitation kinase cascade assay using MEK1, ERK2, and MBP as substrates. Daudi cells (1 x 107/lane) were incubated with buffer only (PBS), biotinylated anti-CD19 mAb (5 µg/ml) alone, biotinylated anti-IgM (1 µg/ml) alone, or both biotinylated anti-IgM (1 µg/ml) and anti-CD19 mAb (5 µg/ml) at 25°C for 10 min. Cells were stimulated by the addition of avidin (5 µg/ml) for 1 min and then lysed. Immunoprecipitates formed from the lysates with anti-Raf1 antisera (2 µg/ml) or normal rabbit IgG (2 µg/ml) were analyzed for the ability to activate MEK1 and ERK2, measured by incorporation of 32P into MBP, as described in Materials and Methods. Gels were analyzed by autoradiography and with the phosphor imager. The fold increase in incorporation of radioactivity into MBP for each stimulation condition in anti-Raf1 immunoprecipitates, relative to that in cells stimulated with PBS only on the same gel, is shown. B, Comparison of activation of ERK2 and Raf1. Aliquots of lysates of Daudi cells stimulated as in A were subjected to ERK2 and Raf1 immunoprecipitation and immunocomplex kinase assays. Gels were analyzed by autoradiography and with the phosphor imager. Relative activation of ERK2 (dark bars) and Raf1 (light bars) is represented as the fold increase in incorporation of 32P into MBP for each stimulation condition, relative to that in cells stimulated with PBS only.

 
In a separate experiment, we compared the activation of Raf1 and ERK2 in the same lysates following stimulation of Daudi cells with PBS only or by ligation of either CD19 alone or mIgM alone or by coligation of CD19 and mIgM (Fig. 2GoB). The activity of ERK2 was synergistically enhanced following CD19-mIgM coligation (3.38-fold, compared with 1.28 with IgM alone, relative to the activity in cells stimulated with PBS only). In contrast, the activity of Raf1 precipitated from the same lysates was increased by 1.83-fold following ligation of mIgM alone and 1.99-fold after CD19-mIgM coligation. The small increase in Raf1 activity is unlikely to account for the large increase in ERK2 activity following CD19-mIgM coligation. Experiments in which the cells were lysed 30 s after stimulation gave similar results (not shown).

Additionally, Raf1 activation under similar stimulation conditions was determined in a Raf1 immunocomplex kinase assay using [K97A]MEK1-GST (lacking kinase and autophosphorylation activities due to mutation of lysine 97 to alanine in the ATP binding site) as a substrate (38). Fig. 3Go shows the mean (±SD) of the fold increase in Raf1 kinase activity for each stimulation condition, relative to activity in PBS-treated cells, in four separate experiments. The results parallel those observed in the immunoprecipitation cascade assay. mIgM alone induced a 2.4-fold increase in Raf1 kinase activity. CD19-mIgM coligation resulted in only a minimal further increase (to 2.5-fold).



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  		FIGURE 3. Analysis of Raf activation by in vitro kinase assay. Daudi cells (1 x 107/lane) were incubated with buffer only (PBS), biotinylated anti-CD19 mAb (5 µg/ml) alone, biotinylated anti-IgM (1 µg/ml) alone, or both biotinylated anti-IgM (1 µg/ml) and anti-CD19 mAb (5 µg/ml) at 25°C for 10 min. Cells were stimulated by the addition of avidin (5 µg/ml) for 1 min and lysed. Immunoprecipitates formed from the lysates with anti-Raf1 antisera (2 µg/ml) were analyzed for kinase activity, using [K97A]MEK1-GST as a substrate. For each stimulation condition, the fold increase, relative to PBS only, was calculated. The mean ± SD fold increase from four independent experiments is shown.

 
MEK1 was synergistically activated in response to CD19-mIgM coligation

MEK1 is phosphorylated and activated by Raf (39) and in turn phosphorylates and activates ERK2 (40, 41). To determine whether MEK1 functions as a convergent point for distinct signaling pathways mediated by CD19-mIgM coligation, MEK1 activation was measured in Daudi cells under the same stimulation conditions shown to induce synergistic ERK2 activation. To monitor MEK1 activation, we performed an immunoprecipitation kinase cascade assay measuring the ability of MEK1 to activate a downstream substrate, ERK2. Immunoprecipitates formed with either anti-MEK1 or control Ab were incubated with GST-ERK2, MBP, and [{gamma}-32P]ATP. The activity of the GST-ERK2 in incorporating 32P into MBP was measured. In the absence of precipitated MEK1, the GST-ERK2 was minimally active (Fig. 4GoA, rabbit IgG lanes). Anti-CD19 stimulation by itself had little effect on MEK1 activity (Fig. 4GoA, anti-MEK1 lanes). Anti-mIgM induced an increase in specific MEK1 activity toward ERK2. CD19 and mIgM coligation resulted in synergistic activation of MEK1 (in replicate experiments, 3–3.5-fold by anti-IgM alone versus 5.3–7.3-fold by anti-CD19 plus anti-IgM). The level of synergistic activation of MEK1 is comparable with that of ERK2 by CD19-mIgM coligation (Figs. 1GoA, 2B, and 4A).

MEK1 is a dual kinase that acts on ERK with narrow specificity. The substrate specificity of the MEK1 assay was examined by assessing the requirement for the GST-ERK2. MBP was phosphorylated in the presence of immunoprecipitated MEK1 and added GST-ERK, or with directly precipitated ERK2, from cells stimulated by CD19-mIgM coligation. In the absence of GST-ERK2, however, little kinase activity was detected in MEK1 immunoprecipitate from cells stimulated by CD19-mIgM coligation, demonstrating that the phosphorylation of MBP required ERK2 and thus reflected MEK1 activity (Fig. 4GoB). Experiments were also performed comparing the activity measured after addition of either unmutated or kinase-dead [K71A]ERK. Both the Raf and MEK (Fig. 5Go) cascade assays required activatible ERK, further demonstrating the specificity of the assays as well as the synergistic activation of MEK, but not Raf.



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  		FIGURE 5. Requirement for ERK kinase activity in Raf and MEK cascade assays. Daudi cells (1 x 107/lane) were incubated with buffer only (PBS), biotinylated anti-CD19 mAb (5 µg/ml) alone, biotinylated anti-IgM (1 µg/ml) alone, or both biotinylated anti-IgM (1 µg/ml) and anti-CD19 mAb (5 µg/ml) at 25°C for 10 min. Cells were stimulated by the addition of avidin (5 µg/ml) for 1 min and then lysed. Immunoprecipitates formed from the lysates with either anti-Raf1 antisera (2 µg/ml) or anti-MEK1 (2 µg/ml) were analyzed for the ability to activate either MEK1 and ERK1 ("Raf assay") or ERK1 ("MEK1 assay"), respectively, measured by incorporation of 32P into MBP, as described in Materials and Methods. For both assays, replicate samples were incubated with either unmutated ("Erk1" in figure) or kinase-dead ERK1 ("Erk1 [K71A]"). Gels were analyzed by autoradiography and by phosphor imager. The fold increase in incorporation of radioactivity into MBP for each stimulation condition, relative to that in cells stimulated with PBS only, is shown for the assays that received unmutated ERK.

 
Synergistic activation of ERK2 by CD19-mIgM coligation does not require increased intracellular Ca2+ or PKC activity

CD19-mIgM coligation induces a synergistic increase in intracellular free Ca2+ (34), which may in turn stimulate activity of certain kinases, including some isoforms of PKC, including PKC{alpha}, which may regulate ERK2 activation by the BCR (32, 42). To begin to understand the mechanism by which CD19 up-regulates the ERK MAPK pathway, we asked whether the intracellular Ca2+ flux is required for synergistic ERK2 activation. Daudi cells were pretreated with 20 µM BAPTA-AM, an intracellular Ca2+ chelator, or DMSO vehicle only, washed, and then resuspended in HBSS with 1 mM Mg2+ and either 1 mM EGTA or 1 mM Ca2+, respectively. In preliminary experiments, we found that the calcium flux induced by stimulation with polyclonal anti-IgM was partially suppressed by 5 µM BAPTA-AM and completely suppressed by 10 µM BAPTA-AM under these conditions (not shown).

Daudi cells were stimulated with anti-CD19 or anti-mIgM or the combination of both, and ERK2 activity in these cells was measured. ERK2 activity in cells stimulated by ligation of mIgM alone was diminished, but not blocked by BAPTA-AM + EGTA. Although the absolute level of ERK2 activity after CD19-mIgM coligation was diminished to a similar degree by BAPTA-AM + EGTA, the ability of CD19 coligation to enhance ERK2 activation was preserved in BAPTA-AM + EGTA-treated cells (Fig. 6GoA, top). The relative enhancement or suppression by BAPTA-AM + EGTA, compared with DMSO only, of the increase in ERK2 activity was calculated for mIgM alone, relative to PBS, and for CD19-mIgM coligation, relative to the sum of the increase over PBS observed with CD19 alone and mIgM alone. For three experiments, the average change in the increase over PBS induced by mIgM alone in the BAPTA-AM + EGTA samples was 0.59 ± 0.13 relative to DMSO samples. The average synergistic change induced by CD19-mIgM coligation was 1.29 ± 0.33 in the BAPTA-AM + EGTA samples, relative to DMSO samples. Thus, the stimulation of ERK2 by mIgM was reduced by 41% by chelation of calcium, but the relative enhancement of ERK2 activation by CD19 was unchanged. Pretreatment of the cells with BAPTA-AM did not alter the amount of ERK2 immunoprecipitated from the cells (Fig. 6GoA, bottom). The concentration of BAPTA-AM used in these experiments (20 µM) was shown to be sufficient to prevent an increase in intracellular free Ca2+ with the Ab concentrations used for the ERK2 assays (Fig. 6GoB). A synergistically enhanced Ca2+ flux was observed in cells treated with DMSO and stimulated by coligation of CD19 and IgM. No increase in calcium was observed after any stimulation in cells treated with 20 µM BAPTA-AM + EGTA. The synergistic activation of ERK2 after CD19-mIgM coligation, therefore, occurred under conditions that blocked the increase in intracellular Ca2+.



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  		FIGURE 6. Analysis of the requirement for an increase in intracellular Ca2+ in the synergistic activation of ERK2 by CD19-mIgM coligation. A, Requirement for intracellular Ca2+ in ERK2 activation. Daudi cells (1 x 107/lane) were pretreated with either 20 µM BAPTA-AM or an equal volume of DMSO vehicle only at 37°C for 40 min, washed, and resuspended in HBSS containing either 1 mM EGTA, 0.1% BSA, 1 mM MgCl2, and no Ca2+ (BAPTA-AM-treated cells), or 0.1% BSA, 1 mM MgCl2, and 1 mM CaCl2 (DMSO only cells). The cells were then incubated with buffer only (PBS), anti-CD19 mAb (5 µg/ml) alone, anti-IgM (1 µg/ml) alone, or both anti-IgM (1 µg/ml) and anti-CD19 mAb (5 µg/ml) at 25°C for 10 min. Cells were stimulated by the addition of avidin (5 µg/ml) for 1 min and lysed. Immunoprecipitates formed with anti-ERK2 were analyzed for kinase activity. Gels were analyzed by autoradiography and with the phosphor imager (top). The fold increase in incorporation of 32P into MBP for each stimulation condition, relative to that in cells stimulated with PBS only, is shown. Equivalent kinase loading in all samples was confirmed by immunoblotting with anti-ERK2 antisera (bottom). B, Pretreatment with 20 µM BAPTA-AM blocks Ca2+ flux induced by coligation of CD19 and mIgM. Daudi cells (5 x 106/ml) were treated with 20 µM BAPTA-AM or an equal volume of DMSO and loaded with indo-1 AM (1 µM) at 37°C for 40 min. Cells were washed into HBSS containing either 1 mM EGTA, 0.1% BSA, 1 mM MgCl2, and no Ca2+ (BAPTA-AM-treated cells), or 0.1% BSA, 1 mM MgCl2, and 1 mM CaCl2 (DMSO only cells). The cells were then incubated with buffer only (PBS), anti-CD19 mAb (5 µg/ml) alone, anti-IgM (1 µg/ml) alone, or both anti-IgM (1 µg/ml) and anti-CD19 mAb (5 µg/ml) at 25°C for 10 min, washed, and monitored for changes in intracellular free calcium concentration by flow cytometry. After a 20-s baseline, cells were stimulated by the addition of avidin (5 µg/ml).

 
The inhibition of increased calcium would block activation of calcium-dependent isoforms of PKC, but other isoforms are calcium independent. Therefore, we performed a similar analysis in cells treated with either bisindoylmaleimide I, which inhibits all isoforms of PKC tested, or bisindoylmaleimide V, a related compound that serves as a control (43). The results were similar to those observed following inhibition of the increase in calcium. The activation of ERK2 following ligation of mIgM alone was partially suppressed (1.3- versus 1.9-fold) by pretreatment with bisindoylmaleimide I (Fig. 7Go). However, the ability of CD19 to enhance activation when coligated with mIgM was predominantly intact (1.93- versus 2.05-fold in the presence of control versus active inhibitor). Activation of ERK2 by PMA was blocked by bisindoylmaleimide I, demonstrating effective inhibition of PKC. The activity of bisindoylmaleimide I against PKCµ, which is activated by CD19 (44), has not been reported. Therefore, we also tested the ability of another PKC inhibitor, Gö 6976, which is active against this isoform, and again no suppression of the CD19 component was observed (not shown).



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  		FIGURE 7. Analysis of the requirement for PKC activity in the synergistic activation of ERK2 by CD19-mIgM coligation. Daudi cells (1 x 107/lane) were pretreated with 10 µg/ml of either bisindoylmaleimide I (BIM) or bisindoylmaleimide V (BIM-V) for 1 h. The cells were then incubated with buffer only (PBS), anti-CD19 mAb (5 µg/ml) alone, anti-IgM (1 µg/ml) alone, or both anti-IgM (1 µg/ml) and anti-CD19 mAb (5 µg/ml) at 25°C for 10 min. Cells were stimulated by the addition of avidin (5 µg/ml) for 1 min and lysed. Immunoprecipitates formed with anti-ERK2 were analyzed for kinase activity. Gels were analyzed by autoradiography and with the phosphor imager (top). The fold increase in incorporation of 32P into MBP for each stimulation condition, relative to that in cells stimulated with PBS only, is shown. Equivalent kinase loading in all samples was confirmed by immunoblotting with anti-ERK2 antisera (bottom).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Stimulation through the BCR elicits signal transduction events that affect fundamental biologic responses of the B cell, including proliferation, differentiation, and apoptosis. The complex responses of B cells to various environmental stimuli occur by the integration of signals from the BCR and its coreceptors. Currently, three paradigms for coreceptor modulation of B cell activation have been described. The B cell-specific membrane protein CD22 forms a complex with the tyrosine phosphatase Src homology 2-containing protein tyrosine phosphatase (SHP-1) upon ligation of mIgM, which negatively regulates signaling and activation of ERK2 by the BCR (8, 45). Similarly, coligation of BCR with the FcR for IgG (Fc{gamma}RII) inhibits B cell activation and proliferation by activating SHP-1 and/or Src homology 2-containing inositol phosphatase (SHIP) (46). Coligation of Fc{gamma}RII with mIgM has also been shown to disrupt Shc-Grb2 interactions, which blocks Ras activation (47). Thus, CD22 and Fc{gamma}RII function in negative signaling, including inhibition of MAPK activation, by modifying signals from the BCR. In contrast, CD40, a transmembrane glycoprotein expressed on several types of APCs, mediates signaling in B cells through BCR-independent pathways. CD40 engagement alone induces rapid and sustained increase in the tyrosine phosphorylation of distinct signaling proteins; activates the transcription factor, nuclear factor-{kappa}B; and promotes B cell survival and proliferation (48, 49). In addition, CD40 ligation alone induces ERK activation through a PKC-independent pathway, distinct from the mechanism of induced by mIgM (42, 50). Although the CD19/CD21/CD81 complex also may serve other functions (51, 52), CD19 positively amplifies BCR signaling, but in a BCR-dependent manner. Ligation of mIgM results in tyrosine phosphorylation in the intracellular domain of CD19, which recruits cytoplasmic signaling molecules that enhance the activation of downstream pathways such as the MAPK pathways (6, 16, 17, 24). The association of PI3K with CD19 is required for optimal signaling following ligation of mIgM alone (53). In contrast, CD19 Y391 is critical for the enhanced calcium release and ERK2 activation that follow coligation of CD19 and mIgM (6, 17).

The MAP kinase pathway links many extracellular stimuli to transcriptional regulation and is critically involved in mediating cellular proliferation and differentiation. The convergence of the BCR and BCR/CD19-derived signaling pathways in activation of ERK is likely to play an important role in B cell function. ERK kinases are capable of phosphorylating and activating a number of transcription factors, including c-Myc and p62TCF (54, 55, 56), and may augment the transcription of early genes, including c-fos (55). Transcription of the early gene egr-1 in immature B cells after mIgM ligation is mediated by activation of the Ras-MAP kinase pathway (57). Therefore, the purpose of this study is to use ERK2 activation by CD19-mIgM coligation as a model of how the additional pathways that are activated by CD19 integrate with signals generated by mIgM alone.

We have provided evidence that, in Daudi cells, ligation of mIgM alone, but not CD19 alone, results in activation of the Ras/Raf1/MEK1 cascade. Minimal further activation of Ras and Raf is detected after CD19-mIgM coligation. This conclusion is supported by three different assays: analysis of association of Ras-GTP with the Ras-binding domain of Raf, the kinase activity of Raf for its substrate MEK, and the ability of Raf to activate the terminal components of this cascade. Synergistic activation of ERK was demonstrated in the same lysates in which little CD19 had little effect on Ras or Raf, indicating that the coligation enhances ERK2 activation through a pathway separate from Ras.

In contrast, MEK1 is synergistically activated in response to CD19-mIgM coligation. Thus, the distinct signals from the two receptor complexes converge at MEK1 to provide synergy in stimulating ERK2 activation. These findings are consistent with our previous observations, using the selective MEK inhibitor PD98059, that synergistic activation of ERK2 acts through MEK1 (6).

Chelation of intracellular calcium with BAPTA-AM, under conditions that blocked the increase in calcium induced by coligation, reduced the absolute level of activation of ERK2 following coligation of CD19 and mIgM, but did not reduce the fold increase induced by coligation relative to individual ligation of CD19 and mIgM alone. We conclude that the CD19 component of synergy is not dependent on calcium-dependent kinases. This would include PKC{alpha}, which phosphorylates and activates Raf (32), a possible intermediary in this pathway. This is consistent with the lack of effect of coligation on Raf. A similar result was observed with bisindoylmaleimide I, a more general inhibitor of PKC isoforms, and with Gö 6976, which inhibits PKCµ. Again, the overall ERK2 activity induced by coligation was reduced in cells treated with bisindoylmaleimide I, compared with the bisindoylmaleimide V control. However, while the increase in ERK2 activity induced by ligation of mIgM alone, relative to unstimulated cells, was reduced by 66% in Fig. 7Go, the further increase induced by coligation, relative to cells stimulated with anti-IgM alone, was reduced by only 5%. Thus, while the activation of Raf by ligation of mIgM alone is highly dependent on intracellular calcium and PKC activity, the CD19 component of the synergistic activation appears to be largely independent of these.

The linkage between CD19 and MEK remains to be determined. Our current data suggest that the mechanism involves factors that act distal to Ras and Raf. We have shown previously that, in transfected Daudi cells, the synergy is blocked by mutation of CD19 tyrosine 391, the site of Vav association (6). Vav is a GTP exchange factor for the Rac1/Cdc42 family of GTP-binding proteins, which have been implicated in the activation of p21-activated kinase and MEK1 kinase (MEKK1) (19). MEKK1 may only phosphorylate one of two critical residues on MEK1, but in the presence of a second signal, derived through Raf, this may up-regulate MEK1 activation (28, 29, 30, 58). p21-activated kinase 1 also acts synergistically with Raf to activate MEK1 by direct phosphorylation (30, 31, 59). However, in mice, disruption of the Vav gene did not block synergistic activation of ERK2 (17). In preliminary experiments, Raf activation was analyzed in five preparations of normal human B cells, prepared from tonsils, in which synergy was observed in activation of ERK2 after coligation of CD19 and mIgM. In four such preparations, no such synergy was detected in Raf activity, but synergy was observed in one. Thus, we conclude that the Raf-independent activation of ERK2 by CD19 occurs in normal cells. However, the one exception, and the small but consistent decrease in synergy following treatment with PKC inhibitors, leads us to suspect that there may be some redundancy. At present, we do not exclude the possibility that an alternative signaling molecule(s) regulated by CD19 is involved in the enhanced activation of MEK1 by CD19. In addition, the interaction between Vav, PI3K, PKC, Ras, and Rac/cdc42 is likely to be complex (20, 60).

The most important conclusion from these findings is that optimal ERK activation requires at least two signals. One signal, activated by mIgM alone, targets Ras and leads to an intermediate level of ERK activity. The second signal, generated upon CD19-mIgM coligation, acts through a separate pathway that converges at MEK1 and induces enhanced ERK2 activation. Our findings provide a model for how signal input from a coreceptor is integrated in a multienzyme cascade. Intermediate members of the MAPK pathways can serve as integration sites for modification of signals, derived from ligation of Ag receptors, by cell surface coreceptors that modulate lymphocyte activation in response to the context in which Ag is recognized. Convergence of distinct, coreceptor-dependent pathways feeding into the ERK cascade during BCR signaling would provide multiple levels of regulation and allow more effective and precise control of B cell activation.


   Acknowledgments
 
We thank Stephen Taylor for providing pGEX-RBD, and Dr. Taylor, Jennifer Swantek, and Mark Coggeshall for helpful comments.


   Footnotes
 
1 This work was supported by the National Institutes of Health and the Office of Research and Development, Medical Research Service, Department Of Veterans Affairs. Back

2 Address correspondence and reprint requests to Dr. Robert H. Carter, University of Alabama, 409 LHRB, 701 South 19th Street, Birmingham, AL 35294. Back

3 Abbreviations used in this paper: mIg, membrane immunoglobulin; BAPTA-AM, 1,2-bis(2-aminophenoxylethane-N,N,N,N'-tetraacetic acid tetra(acetoxymethyl) ester; BCR, B cell antigen receptor; ERK, extracellular signal-regulated protein kinase; GST, glutathione S-transferase; MAPK, mitogen-activated protein kinase; MBP, myelin basic protein; MAPK kinase; PI3K, phosphatidylinositol 3-kinase; PKC, protein kinase C; RBD, Ras-binding domain. Back

Received for publication April 17, 1998. Accepted for publication July 27, 1998.


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 Discussion
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M. Fujimoto, J. C. Poe, M. Hasegawa, and T. F. Tedder
CD19 Amplification of B Lymphocyte Ca2+ Responses. A ROLE FOR Lyn SEQUESTRATION IN EXTINGUISHING NEGATIVE REGULATION
J. Biol. Chem., November 21, 2001; 276(48): 44820 - 44827.
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