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Originally published In Press as doi:10.1074/jbc.M402793200 on May 11, 2004

J. Biol. Chem., Vol. 279, Issue 29, 30123-30132, July 16, 2004
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B Cell Receptor-induced cAMP-response Element-binding Protein Activation in B Lymphocytes Requires Novel Protein Kinase C{delta}*

Joseph T. Blois{ddagger}, Jennifer M. Mataraza{ddagger}, Ingrid Mecklenbraüker§, Alexander Tarakhovsky§, and Thomas C. Chiles{ddagger}

From the {ddagger}Department of Biology, Boston College, Chestnut Hill, Massachusetts 02467 and §Laboratory of Lymphocyte Signaling, The Rockefeller University, New York, New York 10021

Received for publication, March 11, 2004 , and in revised form, May 6, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The cAMP-response element-binding protein (CREB) is activated by phosphorylation on Ser-133 and plays a key role in the proliferative and survival responses of mature B cells to B cell receptor (BCR) signaling. The signal link between the BCR and CREB activation depends on a phorbol ester (phorbol 12-myristate 13-acetate)-sensitive protein kinase C (PKC) activity and not protein kinase A or calmodulin kinase; however, the identity and role of the PKC(s) activity has not been elucidated. We found the novel PKC{delta} (nPKC{delta}) activator bistratene A is sufficient to induce CREB phosphorylation in murine splenic B cells. The pharmacological inhibitor Gö6976, which targets conventional PKCs and PKCµ, has no effect on CREB phosphorylation, whereas the nPKC{delta} inhibitor rottlerin blocks CREB phosphorylation following BCR cross-linking. Bryostatin 1 selectively prevents nPKC{delta} depletion by phorbol 12-myristate 13-acetate when coapplied, coincident with protection of BCR-induced CREB phosphorylation. Ectopic expression of a kinase-inactive nPKC{delta} blocks BCR-induced CREB phosphorylation in A20 B cells. In addition, BCR-induced CREB phosphorylation is significantly diminished in nPKC{delta}-deficient splenic B cells in comparison with wild type mice. Consistent with the essential role for Bruton's tyrosine kinase and phospholipase C{gamma}2 in mediating PKC activation, Bruton's tyrosine kinase- and phospholipase C{gamma}2-deficient B cells display defective CREB phosphorylation by the BCR. We also found that p90 RSK directly phosphorylates CREB on Ser-133 following BCR cross-linking and is positioned downstream of nPKC{delta}. Taken together, these results suggest a model in which BCR engagement leads to the phosphorylation of CREB via a signaling pathway that requires nPKC{delta} and p90 RSK in mature B cells.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Signaling through the B cell antigen receptor (BCR)1 is required throughout B cell development and peripheral maturation (1, 2). BCR signaling requires the activities of multiple nonreceptor protein-tyrosine kinases, including Syk, Bruton's tyrosine kinase (Btk), and Src family kinases and the lipid kinase, phosphatidylinositol 3-kinase (3). Btk plays an integral role in transducing BCR signals, because mutations in the gene encoding btk result in the B cell deficiencies, X-linked immunodeficiency in the mouse and X-linked agammaglobulinemia in humans (reviewed in Ref. 4). The X-linked immunodeficiency phenotype results from a single amino acid substitution (R28C) in the pleckstrin homology domain of Btk and is characterized by a near-complete absence of peritoneal B-1a cells, defective responses in vivo to immunization with thymus-independent type II antigen, and a block at the transitional-to-mature B cell checkpoint (4–7).

Much genetic and biochemical data support the concept that B cell activation by the BCR occurs through a signalosome in which the adaptor protein, B cell linker protein (BLNK), allows protein-tyrosine kinases access to signal transduction molecules (8, 9). It is envisaged in this model that BLNK is phosphorylated by Syk following BCR ligation (9–12). Btk and phospholipase C{gamma}2 (PLC{gamma}2) are recruited to BLNK via their Src homology 2 domains, thereby allowing Syk and Btk to fully phosphorylate and activate PLC{gamma}2 (9–14). PLC{gamma}2 catalyzes the hydrolysis of phosphatidylinositol 4,5-bisphosphate, generating diacylglycerol and inositol 1,4,5-trisphosphate (15). Inositol 1,4,5-trisphosphate functions in part to mobilize calcium from intracellular compartments and by promoting extracellular calcium influx, whereas diacylglycerol binds to and activates conventional (Ca2+-dependent) protein kinase C (cPKC) and novel (Ca2+-independent) PKC (nPKC) isoforms (15, 16).

The cAMP-response element-binding protein (CREB) is a 43-kDa protein belonging to the CREB/ATF bZip family of transcription factors. It binds to a consensus TGANNTCA cAMP-response element (CRE) as a homo- or heterodimer with other CREB/ATF and activator protein-1 transcription factor members (17). Phosphorylation of CREB on Ser-133 increases its association with the CREB-binding protein, leading to activation of the basal transcriptional machinery (17, 18). CREB phosphorylation and CRE-dependent gene expression occurs in response to BCR cross-linking of resting B cells (19–21). Most interesting, a recent report (22) showing that CREB binding activity is induced following BCR cross-linking is negatively regulated by interferon-{gamma}.

Accumulating evidence suggests that CREB plays an important role in B cell growth and survival. Phosphorylation of CREB is necessary for activation of the BCR-dependent c-fos/jun-B immediate-early growth response program (20, 23). In particular, transgenic mice expressing a mutant CREB containing a serine-to-alanine substitution at position 133 exhibit impaired proliferation and survival in response to BCR cross-linking (24). In regard to the latter, CREB phosphorylation is necessary for bcl-2 gene expression in human B cells (25). In addition, CREB has been implicated in the regulation of numerous gene promoters involved in B cell function, including the 3' {kappa} enhancer, major histocompatibility complex class II promoter, OCA-B promoter, and human I{gamma}1 proximal promoter (26–30).

Despite the importance of CREB phosphorylation in B cell function, there remain many fundamental questions concerning the mode of regulation of CREB by the BCR. One of these questions related to the role of PKC in linking the BCR to CREB phosphorylation. Nearly a decade ago a phorbol ester (PMA)-sensitive PKC activity was shown to be required for BCR-induced CREB phosphorylation in B cells (21, 31). In contrast, several known CREB kinases, including inducible protein kinase A (PKA) and calmodulin kinase II (CaMK II) activities, do not contribute to CREB phosphorylation in mature B cell responses to BCR cross-linking (31). Although these observations suggest an important contribution of PKC to CREB phosphorylation, the identity of the PKC isoform(s) has not been established. Furthermore, the signaling components that link PKC activity to CREB phosphorylation remain to be defined. In the work described herein, we have made use of pharmacological activators and inhibitors of c/nPKC isoforms, nPKC{delta}-deficient mice, A20 B cells expressing a kinase-inactive nPKC{delta}, and DT40 B cell lines deficient in signalosome components to define the signaling pathways that regulate CREB phosphorylation in mature B cells. Our results show that BCR cross-linking mediates CREB phosphorylation via a signaling pathway involving nPKC{delta} and p90 RSK.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents and Antibodies—Anti-CREB antibodies (Abs) were purchased from Upstate Biotechnology (Lake Placid, NY). Anti-phospho-(Ser-32/36) I{kappa}B{alpha}, anti-nPKC{delta} (Thr-505), and anti-p90 RSK (Thr-573) Abs were purchased from Cell Signaling Technology (Beverly, MA). Anti-nPKC{delta} (C-17), anti-PKC{zeta} (C-20), anti-PKC{alpha},{beta},{gamma} (MC5), anti-rabbit, and anti-mouse IgG-coupled horseradish peroxidase Abs were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-PKC{epsilon} (P9103-20C) was from United States Biological (Swampscott, MA). The anti-chicken IgM (M4) Ab and chicken serum were obtained from Southern Biotechnology Associates (Birmingham, AL). Bryostatin 1, U73122 [GenBank] , and rottlerin were obtained from Biomol (Plymouth Meeting, PA). Protease inhibitor mixture, phorbol 12-myristate 13-acetate (PMA), and 4{alpha}-PMA were obtained from Sigma. Inhibitors were prepared in Me2SO at fold concentrations such that the final amount of Me2SO in culture medium was below 0.1%. F(ab')2 fragments of goat anti-mouse IgM or IgG (anti-Ig) were obtained from Jackson Immuno-Research (West Grove, PA). Gö6976 was purchased from Calbiochem-Novabiochem. The Immobilon-P membrane was from Millipore (Bedford, MA). Enhanced chemiluminescence reagents were purchased from Kirkegaard & Perry Laboratories (Gaithersburg, MD). The rabbit complement and Lympholyte M were purchased from Accurate Chemicals (Westbury, NY).

Cell Lines and Preparation of Murine B Lymphocytes—The murine sIgM+ Bal17 B lymphoma was obtained from Dr. Richard Asofsky (National Institutes of Health, Bethesda). The murine A20 B lymphoma was kindly provided by Dr. Jeffery V. Ravetch (Rockefeller University, New York). Stable A20 cell lines were generated by culturing at a density of 5 x 105 cells/1 ml RPMI 1640 medium (Sigma) containing 3 µg of plasmid DNA/6 µl of LipofectAMINE 2000 (Invitrogen) according to the manufacturer's protocol. A20 cells were stably transfected with pMTH plasmids constitutively expressing either wild type nPKC{delta} or a mutant nPKC{delta} containing a substitution (K376R) in the ATP-binding site that resulted in a kinase-inactive nPKC{delta} as described previously (32). Stable transfectants were selected in culture medium containing G418 (Mediatech, Herndon, VA). The murine B cell lines were maintained in RPMI 1640 medium containing 5% heat-inactivated fetal calf serum (Atlanta Biologicals, Norcross, GA) in a 37 °C humidified incubator at 5% CO2.

The chicken DT40 B cell lines were provided by Drs. E. A. Clark (Department of Microbiology, University of Washington, Seattle, WA) and T. Kurosaki (Riken Cell Bank, Japan) and maintained in RPMI 1640 medium containing 5% fetal calf serum, 1% chicken serum, 10 mM Hepes, pH 7.5, 2 mM L-glutamine, and 5 x 10–5M 2-mercaptoethanol (11, 12).

CBA/CaJ and BALB/c mice were purchased from The Jackson Laboratories (Bar Harbor, ME) and housed at Boston College. Mice were cared for and handled at all times in accordance with National Institutes of Health and institutional guidelines. Splenic B cells were purified by depletion of T cells with anti-Thy-1.2 plus rabbit complement; macrophages (and other adherent cells) were removed by plastic adherence (20). Red blood cells and nonviable cells were removed by sedimentation on Lympholyte M. Splenic B lymphocytes were purified from nPKC{delta}–/– mice as described previously (33); the generation of nPKC{delta}-null mice on C57Bl/6 background has been described (33). The resulting B cell populations were cultured in RPMI 1640 medium supplemented with 10 mM Hepes, pH 7.5, 2 mM L-glutamine, 5 x 10–5 M 2-mercaptoethanol, 100 units/ml penicillin, 100 µg/ml streptomycin, 0.25 µg/ml fungizone, and 10% heat-inactivated fetal calf serum. Splenic B cells were maintained in a 37 °C humidified incubator at 5% CO2.

Western Blotting—B cells were solubilized in modified RIPA buffer (20 mM Tris, pH 7.4, 100 mM NaCl, 1% sodium deoxycholate, 1% Nonidet P-40, and 1% SDS) containing 1 mM phenylmethylsulfonyl fluoride, 1 mM NaF, 1 mM Na3VO4 and supplemented with protease inhibitor mixture. Lysate protein was separated by electrophoresis through an SDS-10% polyacrylamide gel and transferred to Immobilon-P membrane. The membrane was blocked in TBS-T (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) with primary Ab at 1 µg/ml in TBS-T. The membrane was washed several times in TBS-T and then incubated with a 1:2500 dilution of anti-rabbit or anti-mouse IgG-coupled horseradish peroxidase Abs for 90 min and developed by enhanced chemiluminescence. Autoradiograms were analyzed by densitometry using a Bio-Rad GS-800 Calibrated Densitometer and Quantity One software (Bio-Rad).

In Vitro Kinase Assay—Approximately 2.0 x 107 B cells were collected by centrifugation, washed once in 1x phosphate-buffered saline, and solubilized in a lysis buffer containing 20 mM Tris, pH 7.5, 150 mM NaCl, 1 mM EGTA, 1 mM EDTA, 1% Triton X-100, 1 mM {beta}-glycerophosphate, 1 mM Na3VO4, 1 µg/ml aprotinin, 1 µg/ml leupeptin, and 0.2 mM phenylmethylsulfonyl fluoride. Lysates were then incubated for 18 h at 4 °C with anti-phospho-p90 RSK (Ser-380) Ab that recognizes activated p90 RSK (Cell Signaling Technology). Immune complexes were collected with a 40-µl slurry of protein A/G-agarose beads and washed six times with lysis buffer. Kinase reactions were carried out at 30 °C for 30 min in 20 µl of kinase buffer (20 mM Hepes, pH 7.2, 5 mM MnCl2, 200 µM Na3VO4, 10 µM ATP, and 2 µg of recombinant MBP-CREB fusion protein as substrate (BIOSOURCE International, Camarillo, CA)). The reactions were terminated by adding SDS-PAGE sample buffer and the products resolved by SDS-PAGE. CREB phosphorylated on Ser-133 was detected by Western blotting with anti-phospho-CREB (Ser-133) Ab as described above.

In vitro nPKC{delta} kinase activity was measured as described above with the following modifications. Anti-nPKC{delta} (C-17) immune complexes obtained from B cell extracts were used to phosphorylate 1 µgof histone H1 substrate in 30 µl of kinase buffer supplemented with 5 µCi of [{gamma}-32P]ATP for 15 min at 30 °C. The kinase reactions were terminated by the addition of SDS-PAGE sample buffer, boiled (5 min), and separated by SDS-PAGE. The phosphorylated histone H1 was detected by autoradiography.

Electrophoretic Mobility Shift Assay—Nuclei were isolated by hypotonic lysis and extracted in a 450 mM NaCl buffer as described previously (20). Binding reactions were carried out in a final volume of 15 µl and contained 1.5 µg of nuclear protein (extracted with 450 mM NaCl), 0.5 µg double-stranded poly(dI-dC), and 10,000 cpm of DNA labeled probe. DNA probes were labeled with [{gamma}-32P]ATP by T4 polynucleotide kinase (New England Biolabs, Beverly, MA). After 20 min (23 °C), the reaction products were electrophoresed through a 5% polyacrylamide/TBE gel and subjected to autoradiography. For the analysis of nuclear extract binding to the bcl-2 promoter CRE site, the wild type and mutant probes corresponded to 5'-GAACCGTGTGACGTTACGCA-3' and 5'-GAACCGTGTGAATTTACGCA-3', respectively. B cell-derived nuclear extracts containing CREB or purified recombinant CREB bind the wild type bcl-2 promoter CRE site, whereas the mutant bcl-2 promoter CRE site fails to bind CREB/ATF proteins (25). Supershift analysis was performed by including in the nuclear extract binding reactions ~1 µg of anti-CREB, anti-phospho-(Ser-133)CREB, or nonimmune Abs.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
BCR-induced Phosphorylation of CREB Is Dependent on c/nPKCs—We initially sought to demonstrate the requirement for a c/nPKC isoform(s) in BCR-induced CREB phosphorylation on Ser-133. The PKC isoforms expressed in mouse B cells and activated by BCR-induced diacylglycerol production include calcium-dependent cPKC-{alpha},-{beta}I/II, and -{gamma} and calcium-independent nPKC -{delta}, -{epsilon}, and -{eta} (33, 35, 36). Splenic B cells were pretreated for 18 h with 100 ng/ml PMA in order to deplete cells of c/nPKC isoforms and then stimulated with anti-Ig or PMA. As a control for the specificity of PMA, B cells were also pretreated for 18 h with 100 ng/ml of the inactive PMA analog, 4{alpha}-PMA. Treatment of B cells with PMA (18 h) led to a block in BCR-induced CREB phosphorylation (Fig. 1A, lanes pCREB, compare M with {alpha}Ig, PMA o/n). Addition of fresh PMA to parallel B cell cultures failed to increase CREB phosphorylation (Fig. 1A, lanes pCREB, compare M with P, PMA o/n). The lack of CREB phosphorylation cannot be attributed to depletion of cellular CREB by prolonged PMA treatment (Fig. 1A, lanes CREB, PMA o/n). By contrast, CREB phosphorylation was induced in 4{alpha}-PMA-pretreated B cells subsequently stimulated with anti-Ig or PMA (Fig. 1A, lanes pCREB, 4{alpha}PMA o/n). To assess the efficacy of PMA as it pertains to PKC down-regulation, we found that the cellular levels of nPKC{delta} and nPKC{epsilon} were depleted by PMA, but not 4{alpha}-PMA pretreatment (Fig 1A, lanes nPKC{delta} and nPKC{epsilon}). Similar results were obtained for the cPKC-{alpha}, -{beta} and -{gamma} (data not shown).



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  		FIG. 1.
nPKC{delta} activity is required for BCR-induced CREB phosphorylation. A, splenic B cells were cultured in medium (M) with 100 ng/ml PMA or 100 ng/ml 4{alpha}-PMA for 18 h (o/n). Cells were then washed and cultured in medium alone or stimulated with 12 µg/ml anti-Ig ({alpha}Ig) or 300 ng/ml PMA (P) for 30 min. Splenic B cells were also pretreated with Gö6976 for 2 h (B) or rottlerin for 30 min (C) at the indicated concentrations. Cells were then cultured in medium or stimulated with 12 µg/ml anti-Ig for 30 min. The inset in C shows anti-nPKC{delta}- or nonimmune IgG-containing immune complexes isolated from parallel anti-Ig-stimulated B cells pretreated with 10 µM rottlerin. The immune complexes were assayed for phosphorylation of histone H1 as described under "Experimental Procedures." D, splenic B cells were incubated in medium containing the absence (0) or presence of 50 nM bistratene A for the indicated times. Whole cell detergent extracts were prepared and Western-blotted with anti-phospho-CREB(Ser-133), anti-CREB, anti-phospho-I{kappa}B{alpha}(Ser-32/36), or anti-{beta}-actin Abs as indicated. To evaluate depletion of nPKCs, the membrane in A was stripped and reprobed with anti-nPKC{delta} or anti-nPKC{epsilon} Abs. Numerical values below C represent densitometric analysis of the pCREB bands and are represented as fold induction above medium in relative optical units, standardized to each autoradiographic film. The data are representative of three independent experiments.

 
Effects of nPKC{delta} Inhibitors and Activators on BCR-induced CREB Phosphorylation in Splenic B Cells—To identify the PKC isoform(s) contributing to BCR-induced CREB phosphorylation, several cell-permeable inhibitors of PKCs were evaluated for their ability to inhibit CREB phosphorylation. Gö6976 is a specific inhibitor of cPKCs (ID50 = 2–6nM) and PKCµ (ID50 = 20 nM) (37, 38). Pretreatment of splenic B cells with Gö6976 ranging from 5 to 25 nM did not measurably inhibit anti-Ig-induced CREB phosphorylation under the conditions used (Fig. 1B, lanes pCREB). Similar results were obtained with the cPKC inhibitor Gö6850 (data not shown). The efficacy of Gö6976 was confirmed in that it blocked anti-Ig-induced I{kappa}B{alpha} phosphorylation on Ser-32/36, an event dependent, at least in part, on cPKCs (Fig. 1B, lanes pI{kappa}B{alpha}) (39). Taken together with the data in Fig. 1A, these findings point to a possible role for nPKCs in linking the BCR to CREB activation.

Of the known nPKC isoforms expressed in B lymphocytes, nPKC{delta} exhibits the highest level of expression and is phosphorylated on regulatory residues, Tyr-311 and Thr-505 in response to BCR cross-linking (33, 40). To determine whether nPKC{delta} activity is necessary for BCR-induced CREB phosphorylation, splenic B cells were preincubated with rottlerin, a specific inhibitor of nPKC{delta} (41). At concentrations ranging between 10 and 20 µM, rottlerin blocked BCR-induced CREB phosphorylation in primary splenic B cells isolated from either BALB/c or CBA/CaJ mice (Fig. 1C, lanes pCREB). As a control, we found that BCR-induced nPKC{delta} activity was inhibited by pretreatment of splenic B cells with 10 µM rottlerin (Fig. 1C, inset). These findings suggest that nPKC{delta} activity is required for BCR-induced CREB phosphorylation.

To obtain further evidence for a role of nPKC{delta} in CREB phosphorylation, we evaluated whether the highly selective activator of nPKC{delta}, bistratene A (Bis A), was sufficient to promote CREB phosphorylation. Bis A is a cyclic polyether toxin isolated from Lissoclinum bistratum and has been shown to selectively activate nPKC{delta} in vivo at concentrations between 50 and 100 nM (42). Treatment of splenic B cells with 50 nM Bis A led to a rapid and transient increase in CREB phosphorylation (Fig. 1D, lanes pCREB). Bis A did not alter the cellular levels of {beta}-actin protein (Fig. 1D, lanes {beta}-actin). Bis A also induced CREB phosphorylation in splenic B cells from CBA/CaJ mice (data not shown). Thus, BCR-induced CREB phosphorylation is blocked by the nPKC{delta} inhibitor, rottlerin, and can be induced by selective activation of nPKC{delta} in the absence of BCR cross-linking.

Bryostatin 1 Selectively Protects nPKC{delta} from PMA Depletion Concomitant with BCR-induced CREB Phosphorylation—To corroborate the findings above, we evaluated the effects of bryostatin 1, a non-phorbol ester activator of PKCs, on anti-Ig-stimulated CREB phosphorylation. Numerous studies have shown that exposure of mammalian cells to bryostatin 1 at concentrations between 100 nM and 1 µM results in the selective protection of nPKC{delta}, but not other c/nPKC, from PMA-induced depletion when coapplied (43, 44). To test whether bryostatin 1 was capable of protecting BCR-induced CREB phosphorylation following PMA depletion of c/nPKCs, splenic B cells were pretreated with PMA alone or with the combination of PMA and bryostatin 1. In agreement with the data in Fig. 1A, pretreatment of splenic B cells with PMA alone led to a near complete block in anti-Ig-induced CREB phosphorylation at the time points examined (Fig. 2, lanes pCREB). By contrast, anti-Ig stimulated CREB phosphorylation was induced in B cells coincubated with the combination of PMA and bryostatin 1. In data not shown, we also found that bryostatin 1 effectively protected BCR-induced CREB phosphorylation from PMA in the mature B cell lines, A20 and Bal17. The cellular level of nPKC{delta} in B cells treated with the combination of PMA plus bryostatin 1 was significantly greater in comparison to PMA alone, indicating that nPKC{delta} levels were protected from PMA-induced depletion (Fig. 2, lanes nPKC{delta}). Furthermore, the cellular levels of aPKC{zeta}, which are not responsive to PMA depletion, were not altered by PMA alone or the combination of PMA and bryostatin 1 (Fig. 2, lanes aPKC{zeta}). The cellular levels cPKCs-{alpha},-{beta}, and -{gamma} in PMA plus bryostatin 1-treated B cells were actually lower in comparison to PMA alone, suggesting that these cPKC isoforms were not protected by bryostatin 1 (Fig. 2, lanes cPKC{alpha}{beta}{gamma}). These findings provide further evidence in support of a role for nPKC{delta} in BCR-induced CREB phosphorylation.



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  		FIG. 2.
Bryostatin 1 protects nPKC{delta} from PMA-induced depletion, coincident with recovery of anti-Ig-stimulated CREB phosphorylation. Splenic B cells were cultured in medium containing 0.5 µM PMA alone or in combination with 1.0 µM bryostatin 1 for 18 h as described (37, 43). Cells were then stimulated in the absence (0) or presence of 12 µg/ml anti-Ig ({alpha}Ig) for 15–60 min. Whole cell detergent extracts were prepared and immunoblotted with anti-phospho-CREB-(Ser-133) or anti-CREB Abs. The cellular levels of individual c/nPKCs were monitored by immunoblotting with anti-cPKC-{alpha}, -{beta}, and -{gamma} Ab that recognizes a shared epitope (amino acids 292–317), anti-nPKC{delta},or anti-aPKC{zeta} Abs (lower band denoted by arrow). The data are representative of four independent experiments.

 
BCR-induced CREB Phosphorylation Is Diminished in nPKC{delta}-deficient Splenic B Cells—To determine directly if nPKC{delta} is important in BCR-mediated phosphorylation of CREB on Ser-133, we compared CREB phosphorylation in anti-Ig-stimulated splenic B cells from wild type and nPKC{delta}–/– mice (33). Cross-linking of the BCR on nPKC{delta}-deficient B cells resulted in a significantly diminished level of CREB phosphorylation in comparison to wild type B cells (Fig. 3A, lanes pCREB). The impaired CREB phosphorylation in nPKC{delta}-deficient B cells cannot be attributed to a decrease in the amount of CREB protein in nPKC{delta}–/– B cells relative to wild type (Fig. 3A, lanes CREB). It is noteworthy that recent reports (33, 45) demonstrated induction of NF-{kappa}B and NF-IL6 in response to BCR cross-linking in nPKC{delta}-null B cells that is comparable with wild type. Thus, impaired phosphorylation of CREB following BCR ligation in nPKC{delta}-null B cells does not reflect a general defect in BCR signaling to nuclear transcription factor activation.



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  		FIG. 3.
BCR-induced CREB phosphorylation is impaired in nPKC{delta}-deficient B cells. A, splenic B cells from nPKC{delta}–/– and wild type mice (nPKC{delta}+/+) were cultured in medium alone (0) or stimulated with 12 µg/ml anti-Ig ({alpha}Ig) for the indicate times (15–60 min). B, parental A20 B cells (A20), A20 B cells stably expressing a wild type (nPKC{delta}WT), or kinase-inactive recombinant nPKC{delta} (nPKC{delta}DN) were cultured in medium alone (M) or stimulated with 10 µg/ml F(ab')2 fragments of goat anti-mouse IgG ({alpha}IgG) or 100 ng/ml PMA for 30 min. Whole cell detergent extracts were prepared and immunoblotted with anti-phospho-CREB(Ser-133) or anti-CREB Abs as indicated. C, Western blot analysis indicating the level of expression of endogenous nPKC{delta} and recombinant {epsilon}-tagged nPKC{delta} (nPKC{delta}-tagged) in the parental A20 cell line (A20) and the A20 B cells stably expressing a wild type (WT), or kinase-inactive (DN) recombinant nPKC{delta} (32). Numerical values represent densitometric analysis of the pCREB bands and represent as fold induction above medium in relative optical units, standardized to each autoradiographic film.

 
To obtain further support for nPKC{delta} in the regulation of CREB phosphorylation by the BCR, we generated mature A20 B cell lines that stably express either wild type nPKC{delta} (nPKC{delta}WT) or a kinase-inactive nPKC{delta} (nPKC{delta}DN). The nPKC{delta}DN recombinant protein contains an amino acid substitution (K376R) in the catalytic domain, which is necessary for ATP binding as described previously (32). Stable expression of wild type nPKC{delta} in A20 B cells cultured in medium alone resulted in an elevated level of CREB phosphorylation in comparison to the parental A20 B cells (Fig. 3B, lanes M, compare A20 and nPKC{delta}WT). Nonetheless, stimulation of wild type nPKC{delta} expressing A20 B cells with anti-Ig or PMA resulted in increased CREB phosphorylation (Fig. 3B, nPKC{delta}WT, lanes {alpha}IgG and PMA, respectively). In contrast, stable expression of nPKC{delta}DN in A20 B cells resulted in a near-complete loss of PMA- and anti-Ig-induced CREB phosphorylation (Fig. 3B, nPKC{delta}DN). The levels of total cellular CREB were relatively equal between the A20 cell lines (Fig. 3B, lanes CREB). These data support a critical role for nPKC{delta} in linking the BCR to CREB phosphorylation.

BCR-induced Phosphorylation of CREB Requires Btk and PLC{gamma}2—Little is known about the regulation of CREB phosphorylation by signaling components proximal to the BCR. The results above suggest that CREB phosphorylation is dependent on nPKC{delta}. It might be expected that PLC{gamma}2 is necessary for CREB phosphorylation, given that PLC{gamma}2 activation results in the production of diacylglycerol that binds to and activates Ca2+-independent nPKCs. However, nPKC{delta} can be activated by alternative pathways, some of which may occur independent of membrane translocation and PLC{gamma}2 (46, 47). To test whether PLC{gamma}2 is necessary for BCR-induced CREB phosphorylation, we made use of chicken DT40 B cells deficient in PLC{gamma}2 (34, 48). PLC{gamma}2–/– DT40 B cells exhibited impaired CREB phosphorylation in response to BCR cross-linking in comparison to the parental DT40 B cell line (Fig. 4A, compare wt and PLC{gamma}2–/–). In agreement with this finding, pretreatment of ex vivo splenic B cells with 1 µM U73122 [GenBank] , a highly specific PLC{gamma} inhibitor, resulted in ~50% reduction in BCR-induced CREB phosphorylation in comparison to nontreated anti-Ig-stimulated B cells (Fig. 4B). We also found that DT40 B cells deficient in Btk, a key upstream activator of PLC{gamma}2, failed to induce CREB phosphorylation in response to anti-Ig (Fig. 4A, compare wt and Btk–/–). DT40 B cells deficient in Syk, which contributes to PLC{gamma}2 activation, also failed to phosphorylate CREB in response to BCR cross-linking (Fig. 4A, compare wt and Syk–/–). It should be noted that these results cannot be attributed to decreased cellular CREB or differences in protein loads for SDS-PAGE as the levels of CREB and hsp90 were similar in each of the DT40 B cell types (Fig. 4A).



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  		FIG. 4.
PLC{gamma}2 and Btk are necessary for BCR-induced CREB phosphorylation. DT40 B cell lines were cultured in the absence (M) or presence of 4 µg/ml anti-chicken IgM Ab ({alpha}Ig) for 30 min. Whole cell extracts were prepared and Western-blotted with anti-phospho-CREB-(Ser-133), anti-CREB, and anti-HSP90 Abs (A) or anti-phospho-nPKC{delta}(Thr-505) and anti-nPKC{delta} Abs (C) as indicated. B, splenic B cells were pretreated with 1 µM U73122 [GenBank] for 30 min; the cells were then cultured in medium alone or stimulated with 12 µg/ml anti-Ig for 30 min. Whole cell extracts were prepared, and phosphorylation of CREB on Ser-133 and CREB levels were determined by Western blot. Numerical values represent densitometric analysis of the pCREB bands and are represented as fold induction above medium in relative optical units, standardized to each autoradiographic film.

 
Because phosphorylation at residues in the activation loop of nPKC{delta} is thought to play a role in determining catalytic activation and its regulation by the B cell signalosome (33, 46), we examined whether BCR-induced phosphorylation of nPKC{delta} at the activation loop site Thr-505 was impaired in mutant DT40 B cells. As shown in Fig. 4C, BCR cross-linking on the parental DT40 B cell line induced an increase in nPKC{delta} phosphorylation at Thr-505, consistent with a previous report in mouse splenic B cells (33). In contrast, DT40 B cells deficient in Btk, PLC{gamma}2, or Syk exhibited a complete absence of BCR-induced phosphorylation of nPKC{delta} on Thr-505 (Fig. 4C).

Role for p90 RSK in Linking nPKC{delta} to BCR-mediated CREB Phosphorylation—We next sought to define signaling components that function to link nPKC{delta} to CREB phosphorylation. Several protein kinases have been identified that function as CREB kinases, including MSK-1, PKA, p90 RSK, CaMK II/IV, and MAPKAP-K2/3 (reviewed in Refs. 49 and 51–53). Previous reports (20, 21, 31) ruled out direct participation of inducible PKA activity and CaMKII in mediating CREB phosphorylation in mature B lymphocytes following BCR cross-linking. We found that stimulation of Bal17 or splenic B cells with anti-Ig led to an increase in MSK-1 phosphorylation on the activation residue Ser-376; however, the increase in phosphorylation was not dependent on nPKC{delta} activity (data not shown). In addition, we were unable to detect activation of MAPKAP-K2 in response to anti-Ig stimulation of either splenic B cells or the Bal17 B cell lymphoma (data not shown) (50). With this in mind, we evaluated the activation of p90 RSK in splenic B cells and Bal17 B cell lymphomas following BCR cross-linking.

Phosphorylation of p90 RSK on the activation residue Thr-573, which has been shown to correlate with catalytic activity (54), was observed at 5, 15, and 30 min following BCR ligation in Bal17 B cells (Fig. 5A, Bal-17). Similar results were obtained using splenic B cells (Fig. 5A, Balb/c). To determine whether p90 RSK phosphorylation is dependent on nPKC{delta}, Bal17 B cells or splenic B cells were pretreated with rottlerin, and then p90 RSK phosphorylation on Thr-573 was monitored by Western blot. As shown in Fig. 5B, rottlerin pretreatment completely blocked BCR-induced p90 RSK phosphorylation at the time point examined in both splenic and Bal-17 B cells. In addition, pretreatment of B cells with PMA (for 18 h), which serves to deplete cellular c/nPKCs (refer to Fig. 1A), blocked BCR-induced p90 RSK phosphorylation (Fig. 5B, lanes PMA). Of note, the basal level of p90 RSK phosphorylation was elevated in splenic B cells cultured overnight in PMA. Pretreatment with the inactive PMA analog, 4{alpha}-PMA had no effect on anti-Ig-induced CREB phosphorylation in splenic B cells, although it reduced the level of anti-Ig-stimulated CREB phosphorylation in the Bal-17 B cell lymphoma (Fig. 5B, 4{alpha}PMA, lanes Balb/c and Bal-17, respectively). Consistent with these data, A20 B cells stably expressing a kinase-inactive nPKC{delta} failed to phosphorylate p90 RSK in response to BCR engagement or PMA (Fig. 5C, lanes nPKC{delta}DN), whereas parental A20 B cells, whereas wild type nPKC{delta} expressing A20 B cells exhibited inducible p90 RSK phosphorylation (Fig. 5C, A20 and nPKC{delta}WT). We note that stable expression of wild type nPKC{delta} in A20 B cells cultured in medium alone resulted in an elevated level of p90 RSK phosphorylation in comparison to the parental A20 B cells



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  		FIG. 5.
p90 RSK activation is dependent on nPKC{delta} and contributes to CREB phosphorylation in response to BCR cross-linking. A, Bal17 B cell lymphomas or splenic B cells were cultured in the absence (M) or presence of anti-Ig ({alpha}IgM) for the indicated times. B, Bal17 B cell lymphomas and splenic B cells were pretreated with PMA or 4{alpha}-PMA for 18 h as described in Fig. 1A or with 10 µM rottlerin for 30 min. Cells were then cultured in medium (M) or stimulated with anti-Ig for 15 min. C, A20 B cells (A20), A20 B cells stably expressing a wild type (nPKC{delta}WT), or kinase-inactive nPKC{delta} (nPKC{delta}DN) were cultured in medium alone or stimulated with 10 µg/ml F(ab')2 fragments of goat anti-mouse IgG or 100 ng/ml PMA for 30 min. Whole cell extracts were prepared and Western-blotted with anti-phospho-p90 RSK(Thr-573) and anti-p90 RSK Abs as indicated. D, B cells were cultured in medium alone or stimulated with 10 µg/ml anti-Ig for the indicated times (5, 15, and 30 min). B cells were also pretreated with 10 µM rottlerin for 30 min and then stimulated with 10 µg/ml anti-Ig for 15 min (R). Whole cell extracts were then prepared and immunoprecipitated with isotype-matched rabbit IgG or anti-p90 RSK Ab ({alpha}-p90 RSK). The immune complexes were assayed for phosphorylation of MBP-CREB fusion protein (CREB) on Ser-133 as described under "Experimental Procedures." Numerical values below (C) represent densitometric analysis of the bands and represented as fold induction above medium in relative optical units, standardized to each autoradiographic film.

 
We next determined whether endogenous p90 RSK was capable of directly phosphorylating CREB on Ser-133. For these experiments, p90 RSK-containing immune complexes were isolated from anti-Igstimulated Bal-17 B cells or splenic B cells and assayed for phosphorylation of a recombinant MBP-CREB fusion protein in vitro. p90 RSK-containing immune complexes isolated from B cell cultures in medium alone were essentially devoid of MBP-CREB kinase activity, whereas p90 RSK-immune complexes isolated from anti-Ig-stimulated B cells phosphorylated MBP-CREB on Ser-133 (Fig. 5D, lanes {alpha}-p90 RSK). Isotype-matched nonimmune IgG did not exhibit MBP-CREB kinase activity (Fig. 5D, lanes normal rabbit IgG). Most important, pretreatment of B cells with rottlerin effectively blocked p90 RSK-mediated CREB phosphorylation (Fig. 5D, lanes R). Taken together, these data suggest that p90 RSK phosphorylates CREB on Ser-133 and functions downstream of nPKC{delta} in mature B cells.

Requirement for nPKC{delta} in BCR-induced CREB Phosphorylation at the bcl-2 Promoter CRE Site—The bcl-2 gene is important for B cell survival and is regulated by CREB that binds to a canonical CRE site in the bcl-2 gene promoter (25). The signaling pathway linking BCR-induced bcl-2 gene expression via CREB has not been defined; however, activation of the bcl-2 gene promoter in response to phorbol diester stimulation of B cells suggests that a c/nPKC activity is necessary (25). We sought to determine whether nPKC{delta} contributes to BCR-induced CREB phosphorylation bound to the bcl-2 gene promoter CRE site. In initial experiments, nuclear extracts were isolated from control and anti-Ig-stimulated B cells, and nucleoprotein binding activity specific to a probe containing the bcl-2 gene promoter CRE was examined by electrophoretic mobility shift assay (EMSA). As shown in Fig. 6A, a single nucleoprotein complex was detected with the bcl-2 promoter CRE probe. The nuclear extract binding was specific in that EMSA with a mutant bcl-2 promoter CRE probe, which fails to bind CREB/ATF proteins, was devoid of nuclear extract binding activity (Fig. 6A, inset, lane Mut) (25). Supershift analysis with anti-CREB Ab using nuclear extracts from anti-Ig-stimulated B cells indicate the presence of CREB in the nucleoprotein complex (Fig. 6B). Nuclear extract binding activity was supershifted by the anti-phospho-(Ser-133)CREB Ab (Fig. 6B). As a control, inclusion of a nonimmune Ab did not inhibit or supershift the nucleoprotein complex.



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  		FIG. 6.
nPKC{delta} is required for BCR-induced phosphorylation of CREB at the bcl-2 promoter CRE site. A, nuclear extracts were prepared from splenic B cells culture in media (M) alone and stimulated with anti-Ig ({alpha}Ig) for the indicated times. EMSA using a 32P-labeled probe corresponding to the bcl-2 promoter CRE site was performed. P and NC denote the migration of the probe alone and the specific nucleoprotein complex, respectively. The inset denotes nuclear extract binding activity by EMSA using wild type (WT) or mutant (Mut) bcl-2 promoter CRE site containing probes. B, supershift analysis was performed by carrying out binding reactions with nuclear extracts from anti-Ig-stimulated B cells (30 min) in the presence of anti-CREB (CREB), anti-phospho-(Ser-133)CREB (pCREB), or nonimmune (NI) Abs. C, splenic B cells were cultured in media alone or pretreated with the indicated concentrations of rottlerin (30 min). Cells were then stimulated with 12 µg/ml anti-Ig for 30 min. D, splenic B cells were culture in media alone or in the presence of 100 nM bistratene A (Bis A) for the indicated times. Binding reactions with a 32P-labeled bcl-2 promoter CRE site probe were carried out in the presence of anti-CREB or anti-phospho-(Ser-133)CREB Abs with CREB and pCREB denoting the corresponding supershifts, respectively. NC denotes migration of the unsupershifted nucleoprotein complex. The data are representative of three independent experiments. E, splenic B cells were cultured in the absence (–) or presence (+) of 10 µM rottlerin for 30 min. Cells were then cultured in medium alone (– {alpha}Ig) or stimulated with 12 µg/ml anti-Ig (+ {alpha}Ig) for 8 h. Whole cell extracts were prepared and Western-blotted with anti-bcl-2 or anti-{beta}-actin Abs.

 
To establish whether nPKC{delta} was required for phosphorylation of CREB at the bcl-2 promoter CRE site, ex vivo splenic B cells were preincubated with varying concentrations of rottlerin and then cultured in medium alone or stimulated with anti-Ig. Treatment of B cells with rottlerin resulted in a concentration-dependent inhibition of Ser-133 phosphorylation of CREB at the bcl-2 promoter CRE site, as judged by a decrease in the relative amount of anti-phospho-(Ser-133)CREB Ab-supershifted nucleoprotein complex (Fig. 6C, lanes pCREB). The decrease in CREB phosphorylation cannot be attributed to reduced levels of CREB, as the amount of CREB bound to the bcl-2 promoter CRE was not decreased by rottlerin at the concentrations used (Fig. 6C, lanes CREB). To demonstrate further a role for nPKC{delta}, B cells were treated with Bis A alone for various times, and then nuclear extracts were prepared and analyzed by EMSA. The data in Fig. 6D indicate that Bis A is sufficient to promote Ser-133 phosphorylation of CREB bound to the bcl-2 promoter CRE site. Most important, Bis A did not increase the total amount of CREB bound to the bcl-2 promoter CRE site (Fig. 6D, CREB). Finally, we observed that inhibition of nPKC{delta} by pretreatment with 10 µM rottlerin blocked the basal level and BCR-induced bcl-2 expression in splenic B cells (Fig. 6E). These results are consistent with the inhibition by rottlerin of CREB Ser-133 phosphorylation bound at the bcl-2 gene promoter CRE (Fig. 6C).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Activation of CREB by phosphorylation on Ser-133 represents an early event in the signaling pathway triggered by BCR cross-linking. Phosphorylation of CREB is important for the transcription of numerous genes involved in B cell function and survival (e.g. bcl-2) (19–31). Despite the critical role that CREB plays in B cell responses, the molecular pathway(s) that link CREB Ser-133 phosphorylation to the BCR remain unknown. A notable exception is an earlier report that prolonged treatment of B cells with PMA abrogated BCR-induced CREB phosphorylation, suggesting a contribution of c/n PKC activity (21). Most interesting, in contrast to nonlymphoid cells, this BCR-induced phosphorylation was determined to be independent of PKA or CaMK activities (20, 21, 31). In the current study, we have investigated the signaling pathway responsible for CREB phosphorylation in ex vivo splenic B cells. Evidence is provided consistent with a model wherein BCR-induced CREB phosphorylation on Ser-133 is mediated by a pathway that requires the novel isoform nPKC{delta}. The validity of this model is supported by several experimental findings. The chemical nPKC{delta} inhibitor rotterlin blocks anti-Ig-stimulated CREB phosphorylation. Congruent with this observation, BCR signaling was shown to activate nPKC{delta} (this work and see Ref. 40). We also find that bryostatin 1, a macrocyclic lactone that is used in combination with PMA to elucidate the cellular role of endogenous nPKC{delta}, protects anti-Ig-stimulated CREB phosphorylation from PMA-induced down-regulation concomitant with protection of nPKC{delta} from depletion (43, 44). Consistent with these findings, bistratene A, a highly selective activator of nPKC{delta}, when added to B cells in the absence of BCR cross-linking is sufficient to stimulate CREB phosphorylation.

These findings are also supported by data indicating that expression of a kinase-inactive nPKC{delta} in mature A20 B cells completely blocks BCR-induced CREB phosphorylation on Ser-133. We also find that BCR-induced CREB phosphorylation is significantly diminished in splenic B cells isolated from pkc{delta}-deficient mice in comparison to wild type mice. That CREB phosphorylation in nPKC{delta}-deficient splenic B cells is not completely abrogated following BCR cross-linking suggests the existence of a second pathway that contributes, albeit minor, to CREB phosphorylation. Whether this pathway exists in normal B cells or alternatively represents a compensatory up-regulated pathway in pkc{delta}-deficient mice is not known. On this point, BCR-induced CREB phosphorylation was not completely abolished by rottlerin pretreatment of normal splenic B cells. It would seem unlikely that a cPKC activity is responsible for the residual CREB phosphorylation given that the cPKC/PKCµ inhibitor Gö6976 did not block BCR signaling to CREB phosphorylation. Most important, the efficacy of Gö6976 was demonstrated insofar as BCR-mediated I{kappa}B{alpha} phosphorylation was blocked by this inhibitor, an event dependent on cPKC activity (39, 51). nPKC{epsilon} but not nPKC{delta} has been shown to mediate {beta}c cytokine receptor-induced CREB phosphorylation in myeloid cells (52). These findings raise the possibility that nPKC{epsilon} may contribute, albeit minor, to BCR-induced CREB phosphorylation in mature B cells. Most interesting, expression of a kinase-inactive nPKC{delta} in A20 B cells resulted in a complete loss of BCR-induced CREB phosphorylation, suggesting that CREB phosphorylation is entirely dependent upon a nPKC{delta} pathway in A20 B cells. The discrepancy in CREB phosphorylation between A20 B cells and pkc{delta}-deficient splenic B cells (wherein some CREB phosphorylation occurs in response to BCR ligation) remains unexplained, although this may reflect differences in the cell types used (i.e. primary culture (splenic B cells) and transformed (A20) B cells). Notwithstanding, the data collectively point to a critical requirement for nPKC{delta} in linking the BCR to CREB Ser-133 phosphorylation.

The nuclear targets of nPKC{delta} in B cells are poorly characterized; however, a recent study has shown that nPKC{delta} mediates NF-AT-dependent gene expression in response to BCR cross-linking (53). Wilson et al. (25) demonstrated that phosphorylation of CREB represents a necessary signaling event in the activation of the bcl-2 gene promoter, and that promoter activation is dependent upon a PKC activity. We find that when ex vivo splenic B cells are pretreated with rottlerin, there is a concentration-dependent reduction in the amount of Ser-133-phosphorylated CREB bound to the bcl-2 gene promoter CRE site. Moreover, bistratene A pretreatment of splenic B cells is sufficient to induce Ser-133 phosphorylation of CREB bound to the bcl-2 promoter CRE site. In agreement with these observations, inhibition of nPKC{delta} with rottlerin blocks BCR-induced bcl-2 expression. These results provide the first evidence that nPKC{delta} plays a role in CREB-dependent bcl-2 gene expression in mature B cells. Additional studies are currently underway to understand more fully the regulation of bcl-2 gene expression by nPKC{delta} in the context of BCR signaling.

Our results also implicate for the first time p90 RSK in BCR-mediated CREB phosphorylation in mature B cells. In particular, we show that endogenous p90 RSK-containing immune complexes are capable of directly phosphorylating an MBP-CREB fusion protein on Ser-133. Evidence positioning p90 RSK downstream of nPKC{delta} is provided by experiments in which treatment of splenic B cells with rottlerin blocks BCR-induced p90 RSK phosphorylation on the activation residue Thr-573 (54). It should be noted that although rottlerin has been implicated in the inhibition of cPKCs, much higher concentrations of rottlerin are required in comparison to nPKCs (46). Studies have shown that rottlerin blocks the kinase activity of nPKC{delta} and nPKC{theta}; however, among these two nPKCs, only nPKC{delta} is expressed in B cells (55). Although we cannot exclude a possible involvement of other rotterlin-sensitive pathways in BCR-induced p90 RSK activation, we find that expression of a kinase-inactive nPKC{delta} in A20 B cells results in a complete loss of BCR-induced p90 RSK phosphorylation on Thr-573. It is noteworthy that members of the p90 RSK family have been identified as direct CREB kinases (56). In particular, the importance of the p90 RSK member RSK-2 in CREB phosphorylation is demonstrated by the finding that CREB phosphorylation is impaired in human fibroblasts isolated from Coffin-Lowry syndrome patients, which carry mutations in the gene encoding RSK-2 (57). Of note, the molecular link(s) between nPKC{delta} and p90 RSK was not evaluated in this study. Earlier reports in mammalian cell types demonstrated that extracellular signal-regulated kinase activation results in phosphorylation of RSK2 in an nPKC{delta}-dependent manner (56, 58, 59). A recent report by Khan and co-workers (53) suggests that nPKC{delta} may link the BCR to NF-AT activation by inducing extracellular signal-regulated kinase activity.

Although our results do not allow us to definitively rule out the contribution of other known CREB kinases in linking nPKC{delta} to CREB phosphorylation following BCR cross-linking, experimental results not described herein fail to support a role for MSK-1 in this capacity. Moreover, previous work has excluded the involvement of inducible PKA and CaMK II activities in the BCR-mediated CREB phosphorylation in mature B cells (20, 21, 31). It should be mentioned that p38 MAP kinase has also been implicated in linking nPKC{delta} to gene transcription (55). The action of p38 MAPK kinase is thought to be mediated, in part, by its downstream kinase MAPKAP kinase-2, which in turn directly phosphorylates CREB on Ser-133 (60). Given the lack of inducible phosphorylation of MAPKAP kinase-2 on the activation residue Thr-334 (this study) and our previous finding that the p38 MAPK inhibitor SB203580 does not block BCR-induced CREB phosphorylation in splenic B cells, we do not believe that components of the p38 MAPK kinase pathway contribute to CREB phosphorylation in mature B cells stimulated via the BCR (50). This contrasts with the regulation of CREB phosphorylation in CH31 B cell lymphomas, a model for BCR-induced growth arrest and apoptosis in immature B cells, wherein CREB phosphorylation is dependent on the p38 MAPK pathway (50). These results, taken together with the data herein, suggest the interesting possibility that the BCR directs activation of CREB via different signaling pathways in developmentally distinct B cell subsets. This situation contrasts with that of nerve growth factor-treated PC12 cells wherein both RSK and p38 MAPKAP kinase-2 pathways contribute to CREB Ser-133 phosphorylation (61).

We have also provided the first insights into the requirement for individual components of the signalosome in mediating BCR-induced CREB phosphorylation in mature B cells. The absence of detectable CREB phosphorylation in PLC{gamma}2-deficient DT40 B cells together with inhibition of BCR-induced CREB phosphorylation in normal B cells treated with the PLC{gamma} inhibitor U73122 [GenBank] suggest that PLC{gamma}2 is essential for BCR-mediated CREB phosphorylation. Congruent with these observations, a recent report (40) demonstrated that U73122 [GenBank] prevents cytosol-to-membrane translocalization of nPKC{delta} in response to BCR cross-linking in B cells. Activation of PLC{gamma}2 upon BCR ligation requires membrane recruitment and tyrosine phosphorylation by Btk (11–14, 48, 62). In agreement with the role of Btk in regulating PLC{gamma}2, we find that Btk-deficient DT40 B cells do not exhibit BCR-induced CREB phosphorylation in comparison to wild type DT40 B cells. We also observed that BCR-induced phosphorylation of nPKC{delta} on Thr-505 is impaired in each of the mutant DT40 B cells. These findings establish that the functional integrity of the Btk/PLC{gamma}2 signaling block is crucial for the induction of CREB phosphorylation by the BCR.

In summary, the experiments herein establish a role for nPKC{delta} in BCR signaling to CREB. The data are consistent with a model in which BCR engagement on mature B lymphocytes leads to the phosphorylation of CREB via a signaling pathway that requires Btk/PLC{gamma}2, nPKC{delta}, and p90 RSK.


   FOOTNOTES
 
* This work was supported by Public Health Service Grant AI34586. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

To whom correspondence should be addressed: Dept. of Biology, Boston College, 414 Higgins Hall, Chestnut Hill, MA 02467. Tel.: 617-552-0840; Fax: 617-552-3130; E-mail: ChilesT{at}bc.edu.

1 The abbreviations used are: BCR, B cell antigen receptor; Ab, antibody; anti-Ig, F(ab')2 fragments of anti-mouse IgM; BLNK, adaptor protein B cell linker protein; Btk, Bruton's tyrosine kinase; cPKC, conventional PKC; CRE, cAMP-response element; CREB, cAMP-response element-binding protein; EMSA, electrophoretic mobility shift assay; MAPK, mitogen-activated protein kinase; MSK-1, mitogen-activated stress kinase 1; nPKC, novel PKC; nPKC{delta}, novel PKC{delta}; PKC, protein kinase C; PLC{gamma}, phospholipase C{gamma}; PMA, phorbol 12-myristate 13-acetate; RSK, ribosomal S6 kinase; CaMK II, calmodulin kinase II; MBP, maltose-binding protein; Bis A, bistratene A. Back


   ACKNOWLEDGMENTS
 
We thank Dr. Dianne Watters (Queensland Institute of Medical Research, Herston, Brisbane, Australia) for generously providing the bistratene A. We also thank Miguel E. Martinez and Carrie Wardle for assistance with some of the experiments.



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 DISCUSSION
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