The B Antigen Regulates the Transcriptional Activator β-Catenin Via C-Mediated Inhibition of Synthase Kinase-3 This information is current as of October 1, 2021. Sherri L. Christian, Peter V. Sims and Michael R. Gold J Immunol 2002; 169:758-769; ; doi: 10.4049/jimmunol.169.2.758 http://www.jimmunol.org/content/169/2/758 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2002 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

The B Cell Antigen Receptor Regulates the Transcriptional Activator ␤-Catenin Via C-Mediated Inhibition of Kinase-31

Sherri L. Christian, Peter V. Sims, and Michael R. Gold2

␤-Catenin is a transcriptional activator that is regulated by glycogen synthase kinase-3 (GSK-3). GSK-3 is constitutively active in unstimulated cells where it phosphorylates ␤-catenin, targeting ␤-catenin for rapid degradation. Receptor-induced inhibition of GSK-3 allows ␤-catenin to accumulate in the cytoplasm and then translocate to the nucleus where it promotes the of genes such as c- and D1. Wnt hormones, the best known regulators of ␤-catenin, inhibit GSK-3 via the Disheveled protein. However, GSK-3 is also inhibited when it is phosphorylated by Akt, a downstream target of 3-kinase (PI3K). We have previously shown that B cell Ag receptor (BCR) signaling leads to activation of PI3K and Akt as well as inhibition of GSK-3. Therefore, we hypothesized that BCR engagement would induce the accumulation of ␤-catenin via a PI3K/Akt/GSK-3 Downloaded from pathway. We now show that BCR ligation causes an increase in the level of ␤-catenin in the nuclear fraction of B cells as well as an increase in ␤-catenin-dependent transcription. Direct inhibition of GSK-3 by LiCl also increased ␤-catenin levels in B cells. This suggests that GSK-3 keeps ␤-catenin levels low in unstimulated B cells and that BCR-induced inhibition of GSK-3 allows the accumulation of ␤-catenin. Surprisingly, we found that the BCR-induced of GSK-3 on its negative regulatory sites, as well as the subsequent up-regulation of ␤-catenin, was not mediated by Akt but by the C-dependent activation of . Thus, the BCR regulates ␤-catenin levels via a /protein kinase C/GSK-3 http://www.jimmunol.org/ pathway. The Journal of Immunology, 2002, 169: 758–769.

ignaling by the B cell Ag receptor (BCR)3 can promote B phorylating the membrane phosphatidylinositol 4,5-

cell survival, proliferation, differentiation, , or bisphosphate (1, 2). Subsequent dephosphorylation of PIP3 yields anergy depending on the maturation state of the B cell and phosphatidylinositol 3,4-bisphosphate (PI(3,4)P ). Both PIP and S 2 3 the context provided by signals from other receptors. Although the PI(3,4)P2 act as anchors that recruit pleckstrin homology (PH) do- BCR activates multiple signaling pathways, the role of individual main-containing to the plasma membrane (7). BCR en- signaling pathways in mediating responses to BCR engagement is gagement has been shown to increase the levels of both PIP and 3 by guest on October 1, 2021 not completely understood. PI(3,4)P2 (8). This allows PH domain-containing signaling en- Activation of the phosphatidylinositol 3-kinase (PI3K) pathway zymes such as Btk, phospholipase C (PLC)-␥2, and Akt/protein is a key element in BCR signaling (1, 2). Upon BCR engagement, kinase B to be recruited to the plasma membrane where they are PI3K is recruited to the plasma membrane via the binding of its Src activated (1, 2). homology 2 domains to phosphotyrosine-containing sequences on We and others have shown that BCR engagement activates Akt membrane-associated scaffolding proteins such as CD19, Gab1, (9–14). Akt is the primary mediator of the anti-apoptotic signals BCAP, and Cbl (3–6). Once at the plasma membrane, PI3K gen- generated by PI3K (15), and recent work has shown that Akt ki- erates phosphatidylinositol 3,4,5-trisphosphate (PIP3) by phos- nase activity is essential for the survival of the DT40 chicken B cell line (16). Akt phosphorylates a number of proteins that regu- Department of Microbiology and Immunology, University of British Columbia, Van- late cell survival (17–21). In addition, Akt can also phosphorylate couver, British Columbia, Canada the / glycogen synthase kinase-3 (GSK-3)␣ Received for publication January 22, 2002. Accepted for publication May 9, 2002. and GSK-3␤ (22). The costs of publication of this article were defrayed in part by the payment of page GSK-3␣ and GSK-3␤ are constitutively active in resting cells, charges. This article must therefore be hereby marked advertisement in accordance but receptor-stimulated phosphorylation of GSK-3␣ at Ser21 or with 18 U.S.C. Section 1734 solely to indicate this fact. GSK-3␤ at Ser9 inhibits their enzymatic activity (23). These neg- 1 This work was supported by a grant from the Canadian Institutes for Health Re- ative regulatory sites on GSK-3␣ and GSK-3␤ can be phosphor- search (to M.R.G). S.L.C. was supported by graduate fellowships from the Canadian Institues for Health Research, the Michael Smith Foundation for Health Research, the ylated not only by Akt, but also by several other kinases including Natural Science and Engineering Research Council of Canada, and the University of the p90Rsk kinase, integrin-linked kinase, and several protein ki- British Columbia. nase C (PKC) isoforms (22, 24–26). In B cells, we have shown 2 Address correspondence and reprint requests to Dr. Michael R. Gold, Department of that BCR engagement induces the phosphorylation of GSK-3␣ and Microbiology and Immunology, University of British Columbia, 6174 University Boulevard, Vancouver, British Columbia, Canada V6T 1Z3. E-mail address: GSK-3␤ on these negative regulatory sites and inhibits the activity [email protected] of GSK-3␣ (9). 3 Abbreviations used in this paper: BCR, B cell Ag receptor; PI3K, phosphatidylino- An important target of GSK-3 is ␤-catenin (27), a transcriptional sitol 3-kinase; PIP3, phosphatidylinositol 3,4,5-trisphosphate; PI(3,4)P2, phosphati- coactivator that has important roles in early development (28, 29). dylinositol 3,4-bisphosphate; PH, pleckstrin homology; PLC, phospholipase C; GSK-3, glycogen synthase kinase-3; PKC, protein kinase C; LEF-1, lymphoid en- In unstimulated cells, GSK-3 constitutively phosphorylates ␤-cate- hancer factor-1; TCF, T cell factor; FKHR, Forkhead-related ; nin on N-terminal serine residues, targeting ␤-catenin for rapid PKD, protein kinase D; mER-Akt, myristoylated -Akt fusion pro- tein; ALLN, acetyl-leucine-leucine-norleucinol; PdBu, phorbol dibutyrate; 4-HT, ubiquitination and -mediated degradation (30, 31). In 4-hydroxytamoxifen; BIM I, bisindolylmaleimide I; DAG, diacylglycerol. immature progenitor cells of various lineages, including pro-B

Copyright © 2002 by The American Association of Immunologists, Inc. 0022-1767/02/$02.00 The Journal of Immunology 759

cells (32), Wnt hormones regulate developmental processes by in- B cell stimulation and preparation of cell lysates hibiting GSK-3 (33). This Wnt-induced inhibition of GSK-3 is To reduce basal signaling caused by serum components, WEHI-231 cells mediated by the Disheveled protein (29). Inhibition of GSK-3- were grown in complete medium with the FCS reduced to 1% for 12Ð18 h dependent phosphorylation of ␤-catenin allows ␤-catenin to accu- before stimulation while A20 cells were grown in complete medium with mulate in the cytoplasm and then translocate into the nucleus (34). 0.5 mg/ml BSA instead of FCS. The cells were washed once with modified ϫ 7 Once in the nucleus, ␤-catenin promotes transcription by binding HEPES-buffered saline (9), resuspended in this buffer at 1 10 and warmed to 37¡C for 10Ð30 min. Where indicated, the cells were incubated to members of the lymphoid enhancer factor-1 (LEF-1)/T factor with wortmannin (Biomol, Plymouth Meeting, PA), Ly294002 (Biomol), (TCF) family of DNA-binding proteins (35, 36). ␤-Catenin dis- acetyl-leucine-leucine-norleucinol (ALLN; Sigma-Aldrich, St. Louis, places Groucho/-like enhancer of split transcriptional re- MO), safingol (Calbiochem), U73122 (Biomol), or U73343 (Biomol) for pressors from LEF-1/TCF and provides a transactivation domain 20Ð30 min before stimulation. The cells were then stimulated with either anti-Ig Abs, phorbol dibutyrate (PdBu), 4-hydroxytamoxifen (4-HT) (Sig- that can recruit CBP/p300 and promote transcription (37–39). In ma-Aldrich), LiCl, or bisindolylmaleimide I (BIM I; Calbiochem) for the mammalian cells, ␤-catenin up-regulates the transcription of both indicated times. The reactions were terminated by adding ice-cold PBS

cyclin D1 and c-myc, genes whose products promote containing 1 mM Na3VO4 and then centrifuging the cells for 1 min at and proliferation (40–42). 1100 ϫ g in a cold microfuge. For cell lines, the cell pellets were solubi- Because the inhibition of GSK-3 kinase activity by the Wnt lized in Triton X-100 lysis buffer (20 mM Tris-HCl (pH 8), 1% Triton X-100, 137 mM NaCl, 2 mM EDTA, 10% glycerol, PMSF, 1 ␮g/ml apro- signaling pathway results in an increase in ␤-catenin levels, we ␮ ␤ tinin, 10 g/ml leupeptin, 1 mM Na3VO4,25mM -glycerophosphate). hypothesized that the inhibition of GSK-3 that occurs after BCR Splenic B cells were solubilized in buffer B (20 mM HEPES (pH 7.4), 0.42 engagement would also result in increased ␤-catenin levels. In this M NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 25% glycerol, 0.5 mM DTT, 1 ␮ ␮ report, we show that BCR signaling causes an increase in ␤-cate- mM PMSF, 1 g/ml aprotinin, 10 g/ml leupeptin, 1 mM Na3VO4,25mM ␤-glycerophosphate) containing 0.5% Igepal (ICN Pharmaceuticals, Costa Downloaded from nin levels in the nuclear fraction of B cells as well as an increase Mesa, CA). After 10 min on ice, insoluble material was removed by cen- in ␤-catenin-dependent transcription. Although Akt phosphory- trifugation and the protein concentrations determined using the bicincho- lates GSK-3 in other cell types (43, 44), we found that both BCR- ninic acid assay (Pierce, Rockford, IL). induced phosphorylation of GSK-3 and BCR-induced up-regula- Preparation of cytoplasmic and nuclear fractions tion of ␤-catenin were mediated primarily by PLC-dependent activation of PKC and not by Akt. Thus, the BCR regulates ␤-cate- Nuclear and cytoplasmic fractions were prepared as described by Dignam 6 ϫ ␮ http://www.jimmunol.org/ nin via a PLC-␥2/PKC/GSK-3 signaling pathway. et al. (50). After stimulation, 5 10 cells were resuspended in 200 l buffer A (10 mM HEPES (pH 7.4), 10 mM KCl, 1.5 mM MgCl2, 20% glycerol, 0.5 mM DTT, 1 mM PMSF, 1 ␮g/ml aprotinin, 10 ␮g/ml leu- ␤ peptin, 1 mM Na3VO4,25mM -glycerophosphate). After 15 min on ice, Materials and Methods the nonionic detergent Igepal was added to a final concentration of 0.5%. ϫ Antibodies The samples were then centrifuged for 1 min at 1100 g in a cold mi- crofuge. The supernatant was removed and used as the cytosolic fraction. Goat Abs against mouse IgM (␮-chain specific), mouse IgG (␥-chain spe- The pellets were rinsed once with buffer A and then extracted with 100 ␮l cific), and Armenian hamster Ig were purchased from Jackson Immuno- buffer B for 20 min on ice. The insoluble material was removed by cen- Research Laboratories (West Grove, PA). Goat anti-mouse-␬ L chain Abs trifuging for 3 min at full speed in a cold microfuge. The supernatant was were purchased from Southern Biotechnology Associates (Birmingham, collected and used as the nuclear fraction. Protein concentrations for the by guest on October 1, 2021 AL). The hybridoma producing the HM79-16 hamster anti-mouse Ig␤ cytosolic and nuclear fractions were determined using the bicinchoninic mAb was a gift from Dr. T. Nakamura (University of Tokyo, Tokyo, Ja- acid assay. pan) (45). The HM79-16 mAb was purified from the hybridoma superna- tant using a protein G-Sepharose column. Abs specific for Akt, Akt phos- Immunoblotting 473 473 308 phorylated on Ser (anti-P-Ser Akt), Akt phosphorylated on Thr Total cell extracts or cytoplasmic and nuclear fractions (20 ␮ 308 ␣ ␤ 21 9 (anti-P-Thr Akt), GSK-3 /GSK-3 phosphorylated on Ser and Ser , unless otherwise indicated) were separated on SDS-PAGE gels and then respectively (anti-P-GSK-3␣/GSK-3␤), the Forkhead-related transcription 256 transferred to nitrocellulose membranes. The membranes were blocked for factor (FKHR), and FKHR phosphorylated on Ser (anti-P-FKHR) were 1Ð2 h with 5% (w/v) skim milk powder in TBST and then incubated over- all purchased from Technologies (Beverly, MA). The anti- night at 4¡C with the primary Ab. All Abs were diluted in TBST/1% BSA, GSK-3 Ab was from Chemicon International (Temecula, CA). The mAb to with the exception of the Abs to PKD and Sam 68 which were diluted in ␤ -catenin was obtained from BD Transduction Laboratories (Lexington, TBST/1% skim milk powder. The membranes were then washed with KY). The anti-Sam 68 Ab (sc-733) and the anti-protein kinase D (PKD) Ab TBST and incubated with the appropriate HRP-conjugated secondary Ab (sc-639) were purchased from Santa Cruz Biotechnology (Santa (Bio-Rad, Hercules, CA) for1hatroom temperature. Immunoreactive Cruz, CA). bands were visualized using ECL (Amersham Pharmacia Biotech, Baie d’Urfe, Quebec, Canada). To reprobe the membranes, bound Abs were eluted by incubating the membrane in 10 mM Tris-HCl (pH 2), 150 mM B cell lines and murine splenic B cells NaCl for 30 min. The membranes were then reblocked and probed as The WEHI-231 and A20 murine B cell lines and the Ramos human B cell described above. To quantitate results, scans of ECL exposures were saved line were obtained from American Type Culture Collection (Manassas, as TIFF files and analyzed using ImageQuant 1.2 software (Molecular Dy- VA). The K40-B1 pro-B cell line (46) was a gift from Dr. A. DeFranco namics, Sunnyvale, CA). (University of California, San Francisco, CA). All cell lines were main- ␤-catenin pull-down assays using a GST-ECT fusion protein tained in RPMI 1640 supplemented with 10% heat-inactivated FCS, 50 ␮M 2-ME, 1 mM pyruvate, 2 mM glutamine, 15 U/ml penicillin, and 50 ␮g/ml A GST fusion protein containing the C-terminal portion of the cytoplasmic streptomycin (complete medium). WEHI-231 cells expressing a myristoy- domain of E-cadherin (GST-ECT) was used to precipitate unbound ␤-cate- lated Akt-estrogen receptor chimeric protein (mER-Akt) were maintained nin. The pGEX-4t1-ECT plasmid encoding this fusion protein (a gift from in complete medium supplemented with 0.25 ␮g/ml puromycin (Calbio- Drs. H. Aberle and R. Kemler, Max Planck Institute for Immunobiology, chem, La Jolla, CA). To generate this cell line, cDNA encoding the mER- Freiburg, Germany) (51) was transformed into the Escherichia coli strain Akt protein (a gift from Dr. R. Roth, Stanford University, Stanford, CA) DH5␣. Fusion protein production was induced by growing the bacteria in (47) was subcloned into the pMX-puro-IRES-EGFP retroviral vector, a the presence of 100 ␮M isopropyl-␤-D-thiogalactopyranoside for 12 h at derivative of pMX-puro (DNAX, Palo Alto, CA) (48). The resulting plas- 37¡C. The bacteria were then resuspended in sonication buffer (50 mM mid was transfected into the BOSC 23 packaging cell line and the viral Tris-HCl (pH 7.5), 150 mM NaCl, 1% Triton X-100, 1 mg/ml lysozyme, particles produced were used to infect WEHI-231 cells as described (49). 0.1 mg/ml DNase I, 10 ␮g/ml leupeptin, 10 ␮g/ml soybean trypsin inhib- Stable bulk populations of WEHI-231 cells expressing the mER-Akt pro- itor, 1 ␮g/ml aprotinin, 1 mM PMSF) and lysed by sonication. The lysate tein were selected using puromycin. Small resting B cells were isolated was centrifuged at 30,000 rpm for 45 min in the cold. The supernatant from the spleens of C57BL/6 mice by Percoll density centrifugation after containing the fusion protein was collected and stored as aliquots at Ab- and complement-mediated lysis of T cells (9). Ϫ70¡C. To precipitate ␤-catenin, 30 ␮l of bacterial lysate containing the 760 REGULATION OF ␤-CATENIN BY THE BCR

GST-ECT fusion protein was incubated with 15 ␮l packed glutathione- Results Sepharose beads (Amersham Pharmacia Biotech) for1hat4¡C. After BCR engagement causes an increase in ␤-catenin protein levels washing, the beads were mixed with Triton X-100 cell lysates (0.5 mg protein) for1hat4¡C. The beads were then pelleted and washed three GSK-3 is a constitutively active kinase that normally keeps ␤-cate- times with Triton X-100 lysis buffer. Bound proteins were eluted using nin levels low by phosphorylating ␤-catenin such that it becomes SDS-PAGE sample buffer. a target for -mediated degradation. Because BCR signal- Luciferase reporter assays ing inhibits GSK-3 activity (9), we hypothesized that BCR en- gagement would lead to an increase in ␤-catenin levels. To test The TOPtk and FOPtk plasmids were obtained from Dr. M. Waterman (University of California, Irvine, CA) (52). Transient transfection of this, we prepared cytosolic and nuclear fractions from WEHI-231 WEHI-231 cells was performed using the DMRIE-C reagent (Invitro- B lymphoma cells that had been stimulated with anti-IgM Abs for gen, Burlington, Ontario, Canada). Briefly, lipid:DNA complexes were various times (Fig. 1A). We found that BCR engagement caused an ␮ ␮ formed by adding the DMRIE-C lipid reagent (12 l) and the DNA (4 g) increase in ␤-catenin levels in both cellular compartments within to 1 ml of OPTI-MEM medium and incubating for 45 min at 21¡C. WEHI- ␤ 231 cells (4 ϫ 106) that had been resuspended in 0.2 ml complete medium 5Ð15 min. Because -catenin is a transcriptional activator, in all lacking antibiotics were added to this mixture. After4hat37¡C,2mlof subsequent experiments we focused on the levels of ␤-catenin in complete medium lacking antibiotics was added and the cells were grown the nuclear fraction of the cells. We found that BCR engagement for an additional 20 h at 37¡C. The transfected cells were then resuspended consistently caused an increase in the levels of ␤-catenin in the in fresh complete medium and divided into multiple wells of a 24-well dish. The cells were cultured with medium only, 10 ␮g/ml goat anti-mouse nuclear fraction of WEHI-231 cells, and that this increase persisted IgM Ab or 20 mM LiCl for4hat37¡C. After washing with PBS, the cells for at least 1 h (Fig. 1B). were lysed in Reporter Lysis Buffer (Promega, Madison, WI). Luciferase To show that the BCR-induced increase in the amount of ␤-catenin activity was determined using the Promega luciferase assay system (Pro- in the nuclear fractions of the cells was not due to contaminating Downloaded from mega). Readings were made using a MicroLumat Plus luminometer ␤ (EG&G Berthold, Bad Wildbad, Germany) set for a 10 s acquisition win- cytosolic -catenin, we validated our cell fractionation technique by dow. Relative luciferase units were derived by normalizing the luciferase reprobing the blots with Abs to the cytosolic protein PKD (53) and activity to the amount of protein in each sample. the nuclear protein Sam 68 (Fig. 1A, middle and lower panels). http://www.jimmunol.org/ by guest on October 1, 2021

FIGURE 1. BCR engagement increases ␤-catenin protein levels in WEHI-231 B lymphoma. A, WEHI-231 cells were stimulated with 40 ␮g/ml goat anti-mouse IgM Abs for the indicated times. Upper panel, Cytoplasmic and nuclear fractions (20 ␮g protein ϭ 5 ϫ 105 cell equivalents) were analyzed for ␤-catenin levels by immunoblotting with an Ab specific for ␤-catenin. Middle and lower panels, The membrane was reprobed with Abs to the cytosolic protein PKD and the nuclear protein Sam 68 to show that there was little cross-contamination of the nuclear and cytoplasmic fractions. Molecular mass standards (in kilodaltons) are indicated to the left. B, WEHI-231 cells were stimulated with 40 ␮g/ml goat anti-mouse IgM Abs for the indicated times. Nuclear fractions (20 ␮g protein ϭ 5 ϫ 105 cell equivalents) were analyzed for ␤-catenin levels by immunoblotting. As a loading control, the blot was reprobed with Abs to the nuclear protein Sam 68. The normalized amount of ␤-catenin for each sample was calculated by dividing the intensity of the ␤-catenin band by the intensity of the corresponding Sam 68 band. The relative ␤-catenin levels were calculated by setting the value for unstimulated cells to 1. Molecular mass standards (in kilodaltons) are indicated to the left of each panel. C, Sam 68 levels do not change significantly after BCR engagement (p Ͼ 0.09). The relative Sam 68 levels were determined by comparing the intensity of the Sam 68 band from the stimulated samples to the band from the unstimulated sample. For each point the mean Ϯ SEM of the relative level of Sam 68 (unstimulated ϭ 1.0) is shown. For each time point, the data represent results from 10 or more independent experiments except for the 60-min time point, which represents the data from five experiments. Significance was determined by Student’s t test. D, WEHI-231 cells were stimulated with 40 ␮g/ml goat anti-mouse IgM Abs for the indicated times and the relative levels of nuclear ␤-catenin were determined for all experiments performed. The mean Ϯ SEM for each time point is shown. n, The number of independent .p Ͻ 0.001 ,ءءء ;p Ͻ 0.005 ,ءء ;p Ͻ 0.05 ,ء :experiments. Significance as determined by Student’s t test The Journal of Immunology 761

PKD is a protein kinase while Sam 68 is a 68-kDa nuclear RNA- fore, may be part of a pro-BCR. Moreover, cross-linking this pu- binding protein that is phosphorylated by Src during mitosis (54, tative pro-BCR with anti-Ig␤ Abs induces a subset of the signaling 55). We found that the Sam 68 was entirely in the nuclear frac- reactions characteristic of the mature BCR (Ref. 56; S. L. Christian tions, while only a small amount of the total PKD was in the and M. R. Gold, unpublished observations). We found that engag- nuclear fractions. Thus, the nuclear fractions were only minimally ing the BCR on A20 cells resulted in an increase in the levels of contaminated with cytosolic proteins. Because the amount of ␤-catenin in the nuclear fractions of these cells (Fig. 2A). Simi- ␤-catenin in the nuclear fractions was equal to or greater than that larly, clustering the putative pro-BCR on K40-B1 cells with anti- in the fractions, neither the basal nor the BCR-stimulated Ig␤ Abs also caused an increase in nuclear ␤-catenin levels (Fig. levels of ␤-catenin in the nuclear fractions can be accounted for by 2B). To extend this analysis beyond murine B cell lines, we cytosolic contamination. Further analysis showed that the levels of showed that BCR engagement caused an increase in nuclear Sam 68 in the nuclear fractions increased slightly after BCR en- ␤-catenin levels in the Ramos human B cell line (Fig. 2C). Finally, gagement, but that this was not statistically significant as assessed to confirm that this response also occurs in normal B cells, we using Student’s t test ( p Ͼ 0.09) (Fig. 1C). Therefore, we used the showed that engaging the BCR on murine splenic B cells caused levels of Sam 68 as a loading control and calculated the relative an increase in ␤-catenin levels (Fig. 2D). Both the time course and levels of ␤-catenin in the nuclear fraction of each sample by di- the magnitude of the BCR-induced increases in ␤-catenin levels viding the intensity of the ␤-catenin band by the intensity of the were similar in all of the cells we examined (Figs. 1 and 2). The corresponding Sam 68 band for that sample (Fig. 1, B and D). Even up-regulation of ␤-catenin occurred within 2Ð5 min and was sus- if Sam 68 levels do increase slightly after BCR engagement, then tained for at least 30 min, with the maximal increases in the range we are underestimating the BCR-induced increases in the levels of of 2- to 3-fold. This response is similar in magnitude to the in- Downloaded from ␤-catenin in the nucleus. In any case, Fig. 1D shows that BCR crease in nuclear ␤-catenin levels caused by LPS in macrophages signaling caused a statistically significant 2- to 2.5-fold increase in (57). Thus, up-regulation of nuclear ␤-catenin levels is a consistent the levels of ␤-catenin in the nuclear fractions of WEHI-231 B characteristic of BCR signaling that occurs in normal B cells and lymphoma cells. in B cell lines representing multiple stages of B cell development. To determine whether up-regulation of ␤-catenin is a consistent characteristic of BCR signaling, we extended our analysis to in-

␤ http://www.jimmunol.org/ clude two other murine B cell lines as well as a human B cell line -catenin levels are regulated via proteasomal degradation in B and murine splenic B cells (Fig. 2). The A20 and K40-B1 murine cells B cell lines represent different stages of B cell development than In other cell types, receptor-induced increases in nuclear ␤-catenin WEHI-231 cells. Whereas WEHI-231 cells express membrane levels are a consequence of decreased degradation of ␤-catenin in IgM and represent immature/transitional B cells that are suscepti- the cytosol. In unstimulated cells, the constitutive phosphorylation ble to Ag-induced clonal deletion, A20 cells express surface IgG of ␤-catenin by GSK-3 keeps ␤-catenin levels low by targeting it and represent mature B cells that have undergone Ig class switch- for ubiquitination and proteasome-mediated degradation. How- ing. K40-B1 cells represent pro-B cells that express the Ig␣/Ig␤ ever, inhibition of GSK-3 by Wnt signaling, for example, allows ␤ heterodimer on their surface in the absence of Ig chains (46). The -catenin to accumulate in the cytosol and then rapidly translocate by guest on October 1, 2021 Ig␣/Ig␤ heterodimer on K40-B1 cells is associated with several into the nucleus. Because BCR engagement caused a rapid in- proteins including calnexin and Src family kinases (56); and there- crease in the levels of both cytosolic and nuclear ␤-catenin, we

FIGURE 2. BCR engagement increases ␤-catenin levels in B cell lines and in murine splenic B cells. A, A20 cells were stimulated with 40 ␮g/ml goat anti-mouse IgG Abs. B, K40-B1 pro-B cells were stimulated with 30 ␮g/ml of the HM79-16 anti-Ig␤ mAb plus 15 ␮g/ml goat anti-hamster Ig Abs. C, Ramos cells were stimulated with 40 ␮g/ml goat anti-human IgM Abs. Nuclear fractions of the cells (20 ␮g protein ϭ 5 ϫ 105 cell equivalents) were analyzed for ␤-catenin levels by immunoblotting. D, Splenic B cells were stimulated with 30 ␮g/ml goat anti-mouse ␬ L chain Abs. Total cellular extracts (1.6 ϫ 107 cell equivalents) were analyzed for ␤-catenin levels by immunoblotting. The blots were reprobed with Abs to the nuclear protein Sam 68 and relative levels of ␤-catenin were determined as in Fig. 1. Molecular mass standards (in kilodaltons) are indicated to the left of each panel. Each experiment was performed at least three times with similar results. 762 REGULATION OF ␤-CATENIN BY THE BCR asked whether this was also due to the inhibition of proteasome- proteins that can recruit ␤-catenin. Fig. 4 shows that cross-linking mediated degradation of ␤-catenin. To test this, we treated WEHI- the BCR on WEHI-231 cells caused a 2.2-fold increase in lucif- 231 cells with the proteasome inhibitor ALLN to allow ubiquiti- erase activity in cells transfected with the TOPtk plasmid, but did nated proteins to accumulate. We then used a GST fusion protein not cause a significant increase in luciferase activity in cells trans- containing the cytoplasmic domain of E-cadherin (GST-ECT) to fected with the FOPtk plasmid. Thus, BCR signaling can stimulate selectively pull down cytoplasmic ␤-catenin present in Triton ␤-catenin-dependent transcription driven by the LEF-1/TCF-bind- X-100 cell extracts. This GST-ECT fusion protein has been widely ing sites in the TOPtk promoter. This suggests that the BCR-in- used to selectively isolate free cytoplasmic ␤-catenin that can po- duced increase in ␤-catenin protein levels in B cells could result in tentially translocate into the nucleus, as opposed to ␤-catenin that increased transcription of genes that contain LEF-1/TCF binding is bound to cadherins, transmembrane proteins that function as sites. We also found that treating WEHI-231 cells with a GSK-3 adhesion molecules (51). inhibitor, LiCl, resulted in a 2-fold increase in luciferase activity We found that treating WEHI-231 cells with the proteasome (Fig. 4). As discussed in the following section, this indicates that inhibitor ALLN for 20 min was sufficient to cause a 4-fold increase inhibition of GSK-3 is sufficient to increase ␤-catenin-dependent in the levels of free ␤-catenin (Fig. 3, compare 0 min ethanol lane transcription in WEHI-231 cells. to 0 min ALLN lane). This argues that proteasome-mediated deg- ␤ radation normally prevents the accumulation of ␤-catenin in B GSK-3 regulates -catenin levels in B cells cells. The ALLN-induced increase in ␤-catenin levels was similar In Wnt-responsive cells, GSK-3 normally phosphorylates ␤-cate- in magnitude to that caused by anti-IgM in the ethanol-treated nin and targets it for degradation while Wnt signaling increases control cells (Fig. 3). Moreover, in the presence of ALLN, anti- ␤

-catenin levels by inhibiting GSK-3. Because BCR signaling in- Downloaded from IgM treatment did not cause a further increase in ␤-catenin levels hibits GSK-3 (9), the BCR may also regulate ␤-catenin via GSK-3. (Fig. 3). These results are consistent with the idea that the BCR- To determine whether GSK-3 normally targets ␤-catenin for deg- induced increase in ␤-catenin levels is due to inhibition of protea- radation in B cells, we asked whether inhibiting GSK-3 activity some-mediated degradation of ␤-catenin. When the cells were with LiCl would be sufficient to cause an increase in ␤-catenin treated with ALLN, we also observed slower migrating forms of levels. Lithium has been shown to specifically inhibit GSK-3 ki- ␤-catenin. These may be phosphorylated or ubiquitinated forms of nase activity by displacing the Mg2ϩ (58, 59). We found http://www.jimmunol.org/ ␤-catenin which are normally degraded very rapidly by that treating WEHI-231 cells with 20 mM LiCl for 15Ð30 min . resulted in an increase in nuclear ␤-catenin levels (Fig. 5A). As a specificity control, we showed that treating the cells with 20 mM ␤ BCR engagement increases -catenin-mediated transcription KCl did not increase ␤-catenin levels. Another inhibitor of GSK-3, Because ␤-catenin is a transcriptional coactivator, we examined BIM I (60), also increased the levels of ␤-catenin in the nuclear whether the BCR-induced increase in ␤-catenin protein levels in fraction of WEHI-231 cells (Fig. 5B). Thus, inhibition of GSK-3 is the nuclear fraction of B cells correlated with an increase in sufficient to allow the accumulation of ␤-catenin in the nuclear ␤-catenin-mediated transcription. In other cell types, ␤-catenin fraction of B cells. This indicates that GSK-3 normally prevents

promotes transcription by binding to LEF-1/TCF DNA-binding the accumulation of ␤-catenin in B cells and is consistent with the by guest on October 1, 2021 proteins and the p300/CBP coactivator. To assess ␤-catenin-de- idea that BCR up-regulates ␤-catenin by inhibiting GSK-3. pendent transcription, we performed reporter gene assays using the We also examined whether inhibition of GSK-3 activity is suf- TOPtk and FOPtk plasmids. The TOPtk plasmid contains multiple ficient to increase ␤-catenin-mediated transcription in WEHI-231 LEF-1/TCF binding sites, as well as the minimal thymidine kinase cells. We found that LiCl treatment caused a 2-fold increase in promoter, upstream of the luciferase gene (52). The binding of ␤-catenin-dependent transcription from the TOPtk promoter while ␤-catenin-LEF-1/TCF complexes to the TOPtk promoter has been having little or no effect on transcription driven by the control shown to stimulate transcription of the luciferase reporter gene. FOPtk promoter (Fig. 4). Thus, inhibition of GSK-3 is sufficient to The FOPtk plasmid is identical except that it contains mutated LEF-1/TCF binding sites; and therefore, does not bind LEF-1/TCF

FIGURE 3. ␤-catenin levels in B cells are regulated by proteasome- mediated degradation. WEHI-231 cells were pretreated with the protea- some inhibitor ALLN (25 ␮M) or an equivalent volume of ethanol for 20 min at 37¡C before stimulation with 40 ␮g/ml goat anti-mouse IgM for the indicated times. Triton X-100 cell extracts (1.5 ϫ 107 cell equivalents) FIGURE 4. BCR engagement increases ␤-catenin-dependent transcrip- were then incubated with glutathione-Sepharose beads preloaded with the tion. WEHI-231 cells were transiently transfected with the TOPtk or FOPtk GST-ECT fusion protein. ␤-catenin levels were analyzed by immunoblot- plasmids and then cultured for 20 h at 37¡C. Triplicate or duplicate samples ting. The slower migrating forms of ␤-catenin are indicated by the arrow- were then cultured for4hat37¡C with 10 ␮g/ml goat anti-mouse IgM, 20 heads. Note that in the absence of anti-IgM stimulation (0-min lanes), mM LiCl, or no addition (unstim). Luciferase activity assays were per- ALLN alone caused an increase in ␤-catenin levels, presumably by inhib- formed on cell extracts. Luciferase units were normalized to the protein iting proteasome-mediated degradation of ␤-catenin. Molecular mass stan- concentration for each sample. The values for unstimulated cells that had dards (in kilodaltons) are indicated to the left. Similar results were obtained been transfected with either the TOPtk or FOPtk plasmid were set to one in two independent experiments using the GST-ECT to pull down free and the values for the anti-IgM- and LiCl-treated samples are reported as ␤-catenin, and in two additional experiments in which ␤-catenin was im- “fold induction” relative to the corresponding unstimulated sample. The munoprecipitated with an anti-␤-catenin Ab. data represent the mean Ϯ SEM from four independent experiments. The Journal of Immunology 763

such as GSK-3, then the total 4-HT-induced Akt activation could be greater than the BCR-induced activation of endogenous Akt. To test whether 4-HT treatment of mER-Akt-expressing cells could stimulate the phosphorylation of known Akt substrates, we examined the phosphorylation of the FKHR, a protein that is found in both the cytoplasm and nucleus. Akt has been shown to phosphor- ylate FKHR on Ser253 and Thr32 (18). To assess FKHR phosphory- lation, we used an Ab that recognizes FKHR that is phosphorylated on Ser253. We found that FKHR appeared to be phosphorylated on this site even in unstimulated WEHI-231 cells (Fig. 6B). However, 4-HT treatment caused the appearance of a slower migrating form of FKHR (Fig. 6B). Because such changes in electrophoretic mobility fre- quently correlate with increased phosphorylation, this bandshift could reflect Akt-dependent phosphorylation of FKHR on Thr32. Note that BCR engagement caused the appearance of additional forms of FKHR that migrated even more slowly. This may reflect the phos- phorylation of FKHR on additional sites that are targeted by other BCR signaling pathways. Indeed, Ras-dependent phosphorylation of FIGURE 5. Inhibition of GSK-3 increases nuclear ␤-catenin levels in B FKHR has been reported (61). In any case, these data indicate that ␮ Downloaded from cells. A, WEHI-231 cells were treated with 40 g/ml goat anti-mouse IgM, 4-HT treatment of mER-Akt-expressing cells can stimulate Akt-de- 20 mM LiCl, or 20 mM KCl for the indicated times. B, WEHI-231 cells pendent phosphorylation events. were treated with 40 ␮g/ml goat anti-mouse IgM or 10 ␮M BIM I for the We then asked whether 4-HT-induced Akt activation could lead indicated times. Nuclear fractions were analyzed by immunoblotting with ␣ ␤ a ␤-catenin-specific Ab. The blots were reprobed with Abs to the nuclear to phosphorylation of GSK-3 /GSK-3 on their negative regula- ␣ ␤ 21 protein Sam 68 and relative levels of ␤-catenin were determined as in Fig. tory sites. Phosphorylation of GSK-3 and GSK-3 at Ser and 9 1. Molecular mass standards (in kilodaltons) are indicated to the left of Ser , respectively, was assessed using phosphorylation state-spe- http://www.jimmunol.org/ each panel. For each panel, similar results were obtained in three indepen- cific Abs. Fig. 6C shows that while BCR engagement caused a dent experiments. significant increase in the phosphorylation of both GSK-3␣ and GSK-3␤, 4-HT treatment caused only a very small increase in GSK-3 phosphorylation, even though it activated Akt to the same extent as BCR engagement (Fig. 6A). Thus, while Akt activation is increase both ␤-catenin protein levels and ␤-catenin-dependent sufficient to inhibit GSK-3 activity in muscle cells (43), in B cells transcription in B cells. the amount of Akt activation stimulated by the BCR does not cause significant phosphorylation of GSK-3 on the negative regulatory Akt does not mediate BCR-induced phosphorylation of the

sites. This indicates that BCR-induced phosphorylation and inhi- by guest on October 1, 2021 negative regulatory sites on GSK-3 bition of GSK-3 is mediated by a kinase other than Akt. Our next goal was to determine the mechanism by which the BCR Because the inhibition of GSK-3 is sufficient to increase ␤-cate- regulates GSK-3; and therefore, regulates ␤-catenin. Receptor-in- nin levels in WEHI-231 cells (Fig. 5), the inability of Akt activa- duced inhibition of GSK-3 kinase activity is due to phosphoryla- tion to cause significant phosphorylation of GSK-3 in these cells tion of Ser21 of GSK-3␣ or Ser9 of GSK-3␤ (23). The serine/ suggested that Akt activation would be unable to stimulate the threonine kinase Akt was a good candidate for BCR-induced up-regulation of ␤-catenin. Indeed, we found that 4-HT treatment GSK-3 phosphorylation since the BCR activates Akt (9) and Akt of mER-Akt-expressing WEHI-231 cells did not cause an increase has been shown to phosphorylate these negative regulatory sites on in ␤-catenin levels (Fig. 6D). Thus, Akt does not regulate the GSK-3␣/GSK-3␤ in -stimulated cells (44). GSK-3/␤-catenin pathway in B cells. To assess the contribution of Akt to the regulation of GSK-3 and ␤ ␤-catenin in B cells, we expressed a conditionally active form of The BCR regulates GSK-3 and -catenin via the PLC/PKC Akt (mER-Akt) in the WEHI-231 cell line. The mER-Akt protein pathway lacks the PH domain of Akt but contains a sequence In addition to Akt, several PKC isoforms can phosphorylate at the N terminus to localize it to the inner leaflet of the plasma GSK-3␤ in vitro (26). Moreover, treating cells with phorbol esters, membrane. This altered form of Akt is fused to a mutant form of compounds that activate both classical and novel PKC isoforms, the estrogen receptor that is responsive to the estrogen analog can inhibit GSK-3 activity (33). Therefore, we asked whether PKC 4-HT. In response to 4-HT, the estrogen receptor portion of the might link the BCR to the GSK-3/␤-catenin pathway. mER-Akt protein undergoes a that exposes First, we asked whether treating B cells with phorbol esters the Akt activation sites, allowing the mER-Akt protein to be phos- could stimulate GSK-3 phosphorylation. Fig. 7A shows that treat- phorylated and activated by PDK1 and PDK2 (47). We found that ing WEHI-231 cells with a low concentration (20 nM) of PdBu 4-HT treatment of WEHI-231 cells expressing the mER-Akt pro- caused significant phosphorylation of both GSK-3␣ and GSK-3␤. tein resulted in activation of this 90-kDa Akt fusion protein as This PdBu-induced GSK-3 phosphorylation was equal to or indicated by its phosphorylation on the key Akt regulatory sites greater than that caused by anti-IgM. Moreover, the PdBu-induced that correspond to Thr308 and Ser473 of wild-type Akt (Fig. 6A). In GSK-3 phosphorylation was not dependent on Akt, because this addition, the 60-kDa endogenous Akt was also activated when the concentration of PdBu did not activate Akt, as judged by phos- mER-Akt-expressing cells were treated with 4-HT (Fig. 6A), pos- phorylation of Akt on Ser473 (Fig. 7B), a very sensitive measure- sibly due to an interaction between the mER-Akt protein and the ment of Akt activation. endogenous Akt. The 4-HT-induced activation of the endogenous Akt Because PdBu treatment of WEHI-231 cells could stimulate the was similar in magnitude to that caused by BCR engagement (Fig. phosphorylation of GSK-3␣/GSK-3␤ on their negative regulatory 6A). If the mER-Akt can also phosphorylate cytoplasmic substrates sites, we asked whether it could also cause an increase in ␤-catenin 764 REGULATION OF ␤-CATENIN BY THE BCR Downloaded from http://www.jimmunol.org/

FIGURE 6. 4-HT-induced Akt activation does not lead to GSK-3 phosphorylation or up-regulation of ␤-catenin. WEHI-231 cells expressing the mER-Akt chimeric protein were stimulated for the indicated times with 40 ␮g/ml goat anti-mouse IgM or with 2 ␮M 4-HT to specifically activate the by guest on October 1, 2021 mER-Akt protein. A, Triton X-100 cell extracts were analyzed for the activation of mER-Akt and the endogenous Akt by immunoblotting with Abs that recognize Akt phosphorylated on Ser473 (anti-P-Ser473 Akt) (upper panel)orThr308 (anti-P-Thr308 Akt) (middle panel). Equal loading was analyzed by reprobing the membranes with an anti-Akt Ab (lower panel). A nonspecific band in the anti-P-Thr308 Akt blot (middle panel) is indicated by an asterisk. B, Triton X-100 cell extracts were analyzed by immunoblotting with an Ab to phosphorylated FKHR (anti-P-FKHR). The arrowhead indicates a basally phosphorylated form of FKHR that is present in unstimulated cells while the arrows indicate more highly phosphorylated forms of FKHR that are induced upon treating the cells with anti-IgM or 4-HT. Equal loading was analyzed by reprobing the membranes with an Ab recognizing FKHR. C, WEHI-231 cells expressing mER-Akt were stimulated with 40 ␮g/ml goat anti-mouse IgM or 2 ␮M 4-HT for the indicated times. Cytoplasmic fractions (5 ␮g protein) were analyzed for GSK-3 phosphorylation using an anti-P-GSK-3 Ab that recognizes GSK-3␣ phosphorylated on Ser21 and GSK-3␤ phosphorylated on Ser9. The membrane was then stripped and reprobed with an Ab recognizing all forms of GSK-3. D, WEHI-231 cells expressing mER-Akt were stimulated with 40 ␮g/ml goat anti-mouse IgM or 2 ␮M 4-HT for 15 min. Upper panel, Nuclear fractions were analyzed for ␤-catenin levels by immunoblotting. The blots were reprobed with Abs to the nuclear protein Sam 68 and relative levels of ␤-catenin were determined as in Fig. 1. Lower panel, The results from this experiment are presented graphically. Molecular mass standards (in kilodaltons) are indicated to the left of each panel. For each panel, similar results were obtained in at least three independent experiments. levels. Fig. 7C shows that low concentrations of PdBu (10Ð30 nM) similar extent (Fig. 8C). This is consistent with a model in which could increase the level of ␤-catenin in the nuclear fraction of GSK-3 phosphorylation and inactivation is responsible for the in- WEHI-231 cells to the same extent as anti-IgM treatment. Thus, crease in ␤-catenin levels. Moreover, these data argue that the PKC activation is sufficient to induce GSK-3 phosphorylation and BCR regulates GSK-3 and ␤-catenin via the PKC-mediated inhi- to increase nuclear ␤-catenin levels. Moreover, since the amount bition of GSK-3. In support of this idea, we found that inhibiting of PKC activation caused by anti-IgM treatment of WEHI-231 PKC activity with safingol completely blocked BCR-induced up- cells is similar to that caused by 10 nM PdBu (62), this suggests regulation of ␤-catenin protein levels in murine splenic B cells that PKC activation is sufficient to mediate the effects of BCR (Fig. 8D). engagement on GSK-3 and ␤-catenin. Our data suggest that the BCR regulates GSK-3 and ␤-catenin To test whether PKC activation is necessary for BCR-mediated via conventional (PKC-␣,-␤,-␥) or novel (PKC-␦,-⑀,-␩,-␪) PKC regulation of GSK-3 and ␤-catenin, we used safingol to inhibit isoforms. These PKC isoforms are inhibited by safingol and are PKC activity. Safingol functions by competitively inhibiting the activated by phorbol esters such as PdBu which mimic the action conserved diacylglycerol (DAG)-binding C1 domain of PKC en- of DAG. During BCR signaling, these PKC isoforms would be zymes. Treating WEHI-231 cells with safingol inhibited BCR-in- activated via the production of DAG by PLC-␥2 (63). Therefore, duced phosphorylation of GSK-3␣/GSK-3␤ (Fig. 8A) and the preventing the activation of PLC-␥2 should inhibit the ability of BCR-induced increase in nuclear ␤-catenin levels (Fig. 8B)toa the BCR to activate this putative PKC/GSK-3/␤-catenin pathway. The Journal of Immunology 765

clude a role for PI3K because PI3K regulates the membrane re- cruitment and activation of Btk. Phosphorylation of PLC-␥2by Btk is required for maximal activation of PLC-␥2 (63) and previ- ous work has shown that inhibition of PI3K reduces anti-IgM- induced production of PLC-␥2-derived second messengers by ϳ60% (64). Therefore, we would predict that inhibition of PI3K would partially block the ability of the BCR to regulate GSK-3 and ␤-catenin. Indeed, we found that two structurally distinct PI3K inhibitors, wortmannin and Ly294002, inhibited both the BCR- induced increase in GSK-3 phosphorylation and the BCR-induced increase in nuclear ␤-catenin levels by 60Ð75% (Fig. 10). Thus, PI3K does contribute to the ability of the BCR to regulate ␤-cate- nin, but this may reflect the role of PI3K in the activation of PLC-␥2 as opposed to Akt.

Discussion In this report we have shown for the first time that the transcrip- tional activator ␤-catenin is a target of BCR signaling. We show that BCR engagement increases ␤-catenin protein levels as well as Downloaded from ␤-catenin-dependent transcription. We also provide evidence that these responses are mediated by the BCR-induced inhibition of GSK-3 activity that we have described previously (9). Finally, we investigated the mechanism by which the BCR regulates GSK-3 activity. GSK-3 activity is inhibited when it is phosphorylated on negative regulatory sites, and we show in this study that the BCR- induced phosphorylation of GSK-3 on these sites is mediated by a http://www.jimmunol.org/ PI3K/PLC-␥2/PKC signaling pathway (Fig. 11). BCR signaling appears to regulate ␤-catenin levels by control- ling the rate at which ␤-catenin is degraded. Studies in several cell types have shown that ␤-catenin is rapidly degraded in unstimu- lated cells via a GSK-3-dependent mechanism (31). Free ␤-catenin FIGURE 7. PdBu can induce GSK-3 phosphorylation and up-regulate in the cytoplasm binds to a protein complex that contains GSK-3 ␤ -catenin in WEHI-231 cells. A and B, WEHI-231 cells were stimulated as well as a scaffolding protein called axin and the adenamatous with 40 ␮g/ml anti-IgM or 20 nM PdBu for the indicated times. Triton

␮ polyposis coli protein. Axin and adenamatous polyposis coli bind by guest on October 1, 2021 X-100 cell extracts (5 g protein) were analyzed for GSK-3 phosphory- ␤ lation using the anti-P-GSK-3␣/GSK-3␤ Ab (A) and for Akt phosphory- both GSK-3 and -catenin, facilitating the phosphorylation of ␤ ␤ lation using the anti-P-Ser473 Akt Ab (B). To ensure equal loading, the -catenin by GSK-3. Phosphorylation of -catenin by GSK-3 tar- membranes were stripped and reprobed with Abs against GSK-3 or Akt. C, gets it for ubiquitination and proteasome-mediated degradation. WEHI-231 cells were stimulated with 40 ␮g/ml anti-IgM, 10 nM PdBu, or Our finding that the proteasome inhibitor ALLN increases ␤-cate- 30 nM PdBu for the indicated times. Nuclear fractions were analyzed by nin levels in B cells argues that proteasome-mediated degradation immunoblotting with a ␤-catenin-specific Ab. The blots were reprobed keeps ␤-catenin levels low in unstimulated B cells. Wnt hormones, with Abs to the nuclear protein Sam 68 and relative levels of ␤-catenin the best-studied regulators of ␤-catenin, cause increases in ␤-cate- were determined as in Fig. 1. Molecular mass standards (in kilodaltons) are nin levels by inhibiting GSK-3. This allows ␤-catenin to accumu- to the left of each panel. For each panel, similar results were obtained in at late because it is no longer efficiently targeted for degradation. Our least two independent experiments. data suggest that the BCR also increases ␤-catenin levels by pre- venting the degradation of ␤-catenin. We found that BCR engage- ment did not cause a further increase in ␤-catenin levels in ALLN- Indeed, we found that treating WEHI-231 cells with U73122, an treated cells in which ␤-catenin degradation was already inhibited. inhibitor of PLC activity, blocked both the BCR-induced increase In addition, BCR signaling caused ␤-catenin levels to increase in GSK-3 phosphorylation (Fig. 9A) and the BCR-induced increase within 5Ð15 min, kinetics that are more consistent with inhibition in ␤-catenin levels (Fig. 9B). In contrast, an inactive structural of ␤-catenin degradation as opposed to an increase in transcription analog of U73122, U73343, had no effect on BCR-induced GSK-3 or translation. phosphorylation or ␤-catenin up-regulation (data not shown). The In epithelial cells and other cell types in which Wnt signaling finding that both PLC and PKC activities are required for the BCR has been studied, the constitutive phosphorylation of ␤-catenin by to regulate GSK-3 and ␤-catenin suggests that the BCR regulates GSK-3 keeps ␤-catenin levels low by targeting it for ubiquitination ␤-catenin via a PLC-␥2/PKC/GSK-3 pathway. and proteasome-mediated degradation. Our data indicate that GSK-3 kinase activity is also responsible for keeping ␤-catenin ␤ The BCR-induced increase in -catenin levels is partially levels low in unstimulated B cells. GSK-3 is constitutively active dependent on PI3K activity in B cells (9), and we found that two different inhibitors of GSK-3, We have previously shown that BCR-induced GSK-3 phosphory- LiCl and BIM I, both caused ␤-catenin levels to increase in B cells. lation, as well as the subsequent inhibition of GSK-3 activity, is LiCl inhibits GSK-3 kinase activity by displacing the Mg2ϩ co- dependent on PI3K (9). Because Akt is an important downstream factor (59), while BIM I competitively inhibits the ATP-binding target of PI3K, we had initially assumed that the BCR would reg- site of GSK-3 (60). Although BIM I also inhibits the activity of ulate GSK-3 via Akt. However, we have now shown that the BCR several PKC isoforms (65), its ability to increase ␤-catenin levels regulates GSK-3 via a PLC-␥2/PKC pathway. This does not pre- in B cells most likely reflects its inhibitory effect on GSK-3 since 766 REGULATION OF ␤-CATENIN BY THE BCR Downloaded from http://www.jimmunol.org/

FIGURE 9. PLC activity is required for the BCR to regulate GSK-3 and ␤-catenin. WEHI-231 cells were incubated with or without the PLC inhib- itor U73122 (10 ␮M) for 20 min at 37¡C. The cells were then stimulated for 15 min with 40 ␮g/ml goat anti-mouse IgM. A, Cytosolic fractions (5 ␮g protein) were analyzed for GSK-3 phosphorylation by immunoblotting

with the anti-P-GSK-3␣/GSK-3␤ Ab. The blots were then reprobed with by guest on October 1, 2021 anti-GSK-3 Abs. B, Nuclear fractions from the same samples were ana- lyzed by immunoblotting for ␤-catenin. The blots were reprobed with Abs to the nuclear protein Sam 68 and relative levels of ␤-catenin were determined as in Fig. 1. Similar results were obtained in two independent experiments.

inhibiting PKC activity blocks increases in ␤-catenin levels (Fig. 8). In any case, the use of these two different GSK-3 inhibitors indicates that GSK-3 is a central regulator of ␤-catenin levels in B cells. Inhibition of GSK-3 is thought to be the major mechanism by which receptor signaling increases ␤-catenin levels. Our data sug- gest that the BCR also regulates ␤-catenin via the inhibition of FIGURE 8. The BCR regulates GSK-3 and ␤-catenin via PKC. A–C, WEHI- ␮ GSK-3. We have previously shown that BCR signaling leads to the 231 cells were pretreated with 25 Msafingol or the equivalent volume of DMSO ␣ ␤ for 20 min at 37¡C. The cells were then stimulated with 40 ␮g/ml goat anti-mouse phosphorylation of GSK-3 /GSK-3 on their negative regulatory IgM for the indicated times. A, Cytosolic fractions (5 ␮g protein) were analyzed for sites and the concomitant inhibition of GSK-3 activity (9). As de- GSK-3 phosphorylation by immunoblotting with the anti-P-GSK-3␣/GSK-3␤ Ab. scribed above, we found that inhibition of GSK-3 is sufficient to The blots were then reprobed with anti-GSK-3 Abs. B, Nuclear fractions from the cause an increase in ␤-catenin levels in B cells. Moreover, we same samples were analyzed for ␤-catenin levels by immunoblotting. The blots found that agents that blocked the BCR-induced phosphorylation were then reprobed with anti-Sam 68 Abs as a loading control. C, right panel, The of GSK-3 on its negative regulatory sites also blocked the ability relative levels of ␤-catenin in this experiment were determined as in Fig. 1. Left of the BCR to increase ␤-catenin levels. Thus, it appears that the panel, The relative levels of phosphorylated GSK-3␤ were determined using Im- inhibition of GSK-3 is both necessary and sufficient for the BCR ␣ ageQuant Software. Note that anti-IgM-induced phosphorylation of GSK-3 was to up-regulate ␤-catenin. also inhibited by safingol treatment (A). Similar results were obtained in three A number of different kinases can phosphorylate the negative independent experiments. D, Splenic B cells were pretreated with the indicated regulatory sites on GSK-3 and inhibit GSK-3 activity. These in- amount of safingol or with DMSO for 20 min at 37¡C. The cells were then stim- Rsk ulated with 30 ␮g/ml goat anti-mouse ␬ L chain Abs for 15 min or left unstimu- clude Akt, the p90 kinase, the p70 S6 kinase, , lated (⅜). Total cellular extracts were analyzed for ␤-catenin levels by immuno- integrin-linked kinase, and the ␣, ␤I, ␤II, ␦, and ␨ isoforms of PKC blotting. The relative levels of ␤-catenin were determined as described in Fig. 1. (22, 24Ð26, 66Ð69). Thus, different receptors may regulate GSK-3 The results from a representative experiment are shown. Similar results were ob- via different signaling pathways (23). For example, insulin recep- tained in three independent experiments. tor-induced inhibition of GSK-3 is mediated by Akt (44), while the The Journal of Immunology 767

FIGURE 11. Proposed mechanism for the regulation of ␤-catenin by the BCR. The BCR regulates ␤-catenin levels primarily via a PLC-␥2/PKC/ GSK-3 pathway in which the activation of PLC-␥2 is partially dependent on PI3K. PKC activation leads to the inhibition of GSK-3. Because GSK-3 Downloaded from normally targets ␤-catenin for degradation, inhibition of GSK-3 allows ␤-catenin to accumulate. In contrast to PKC, Akt makes only a minor contribution to the inhibition of GSK-3 or the up-regulation of ␤-catenin by the BCR. http://www.jimmunol.org/ Thus, activation of a DAG-dependent PKC isoform is both nec- essary and sufficient for the BCR to stimulate the phosphorylation FIGURE 10. PI3K activity contributes to the ability of the BCR to reg- of GSK-3␣/GSK-3␤ and the accumulation of ␤-catenin. ulate GSK-3 and ␤-catenin. WEHI-231 cells were pretreated with 25 ␮M Ly294002 (Ly), 30 nM wortmannin (W) or an equivalent volume of Although other groups have shown that PKC can phosphorylate DMSO for 20 min at 37¡C. The cells were then stimulated with 40 ␮g/ml and inhibit GSK-3 (33, 70, 71), this is the first report showing that goat anti-mouse IgM for 5 or 15 min. A, Cytosolic fractions (5 ␮g protein) PKC-induced inhibition of GSK-3 is sufficient to cause the accu- were analyzed for GSK-3 phosphorylation by immunoblotting with the mulation of ␤-catenin. In epithelial cells, PKC activity is necessary anti-P-GSK-3␣/GSK-3␤ Ab. The blots were then reprobed with anti- for Wnt-induced accumulation of ␤-catenin but phorbol ester-in- by guest on October 1, 2021 GSK-3 Abs. B, Nuclear fractions from the 15-min samples were analyzed duced PKC activation is not sufficient to increase ␤-catenin levels by immunoblotting for ␤-catenin. The blots were then reprobed with anti- (70). This suggests that other signals are required for the up-reg- Sam 68 Abs as a loading control. Similar results were obtained in four ulation of ␤-catenin, but that these signals are already present in independent experiments. C, The relative levels of ␤-catenin were deter- Ϯ WEHI-231 cells. mined for each experiment and the mean SEM for each point is shown. ␣ ␤ ␦ ⑀ ␤ BCR engagement leads to the activation of PKC- ,- ,- ,-, Note that the PI3K inhibitors did not alter the level of -catenin in cells that ␨ were not treated with anti-IgM (data not shown). and - (72Ð76). Because each PKC isoform is likely to have a unique set of substrates, we are currently attempting to identify which PKC isoform is responsible for the BCR-induced phosphor- ␣1- inhibits GSK-3 via PKC-␨ (69). PKC ac- ylation and inhibition of GSK-3. Our data suggest that either a tivity is also required for Wnt-induced inhibition of GSK-3 (33). conventional PKC isoform (PKC-␣,-␤I, -␤II, -␥) or a novel PKC Although the BCR activates Akt (9), we found that selectively isoform (PKC-␦,-⑀,-␩,-␪) mediates GSK-3 phosphorylation be- activating the mER-Akt chimeric protein along with endogenous cause these PKC isoforms are responsive to increases in DAG. In Akt was not sufficient to induce significant GSK-3 phosphorylation addition, our finding that U73122 blocked BCR-induced phos- or up-regulation of ␤-catenin in WEHI-231 cells. In contrast, over- phorylation of GSK-3 and up-regulation of ␤-catenin supports the expressing a membrane-bound form of constitutively active Akt idea that these responses are mediated via a PLC-␥2/PKC path- does lead to the inhibition of GSK-3 in L6 cells way. We also found that PI3K contributes to the ability of the BCR (43). This suggests that the degree of coupling between Akt and to regulate GSK-3 and ␤-catenin. This presumably reflects the role GSK-3 could be cell type specific. of PI3K in the BCR-induced activation of PLC-␥2 (63). Whereas Akt does not play a major role in linking the BCR to ␤-Catenin is a transcriptional activator. During early develop- GSK-3 and ␤-catenin, our results indicate that the BCR regulates ment it regulates the expression of genes that determine cell fate GSK-3 and ␤-catenin primarily via PKC. Treating B cells with the while in differentiated cells it regulates the expression of genes that PKC activator PdBu was sufficient to induce GSK-3 phosphory- promote proliferation. Our data suggest that ␤-catenin can also lation and cause an increase in nuclear ␤-catenin levels. This was function as a transcriptional activator in B cells. We found that not dependent on Akt because PdBu did not stimulate the phos- BCR engagement could stimulate ␤-catenin-dependent transcrip- phorylation of Akt on sites that are required for its activation. In tion, as judged by a luciferase reporter gene assay. Moreover, treat- support of the idea that the BCR regulates GSK-3 and ␤-catenin ing WEHI-231 cells with the GSK-3 inhibitor LiCl was sufficient via PKC, we showed that the PKC inhibitor safingol blocked BCR- to stimulate ␤-catenin-dependent transcription to a similar extent induced GSK-3 phosphorylation and BCR-induced up-regulation as anti-IgM treatment. Thus, at least in WEHI-231 cells, other of ␤-catenin to the same extent. Safingol prevents the binding of BCR signaling pathways are not required to induce the expression DAG to the C1 domains of conventional and novel PKC isoforms. or activation of proteins that cooperate with ␤-catenin to drive 768 REGULATION OF ␤-CATENIN BY THE BCR transcription. 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Band. 1996. p120cbl is a major substrate of LEF-1/TCF proteins bind specific DNA sequences while ␤-catenin tyrosine phosphorylation upon B cell antigen receptor stimulation and interacts in vivo with Fyn and Syk tyrosine kinases, Grb2 and Shc adaptors, and the p85 provides a transactivation domain that can recruit CBP/p300. Al- subunit of phosphatidylinositol 3-kinase. J. Biol. Chem. 271:3187. though Wnt-responsive bone marrow pro-B cells express LEF-1/ 7. Lemmon, M. A., and K. M. Ferguson. 2000. Signal-dependent membrane tar- TCF family proteins, these proteins have not been detected in ma- geting by pleckstrin homology (PH) domains. Biochem. J. 350:1. 8. Gold, M. R., and R. Aebersold. 1994. Both phosphatidylinositol 3-kinase and ture B cells (32). WEHI-231 cells may express a novel member of phosphatidylinositol 4-kinase products are increased by antigen receptor signal- this family that can bind to the LEF-1/TCF sites in the TOPtk ing in B cells. J. 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