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Phospholipase C-γ2 Couples Bruton's Tyrosine Kinase to the NF-κB Signaling Pathway in B Lymphocytes

2001; Elsevier BV; Volume: 276; Issue: 3 Linguagem: Inglês

10.1074/jbc.m009137200

1083-351X

James B. Petro, Wasif N. Khan,

NF-κB Signaling Pathways

Mutations in the gene encoding Bruton's tyrosine kinase (BTK) interfere with B cell proliferation and lead to an X-linked immunodeficiency in mice characterized by reduced B cell numbers. Recent studies have established that BTK transmits signals from the B cell antigen receptor (BCR) to transcription factor NF-κB, which in turn reprograms a set of genes required for normal B cell growth. We now demonstrate that induction of NF-κB via this pathway requires the intermediate action of the -γ2 isoform of phospholipase C (PLC-γ2), a potential phosphorylation substrate of BTK. Specifically, pharmacologic agents that block the action of either PLC-γ2 or its second messengers prevent BCR-induced activation of IκB kinase. Moreover, activation of NF-κB in response to BCR signaling is completely abolished in B cells deficient for PLC-γ2. Taken together, these findings strongly suggest that PLC-γ2 functions as an integral component of the BTK/NF-κB axis following BCR ligation. Interference with this NF-κB cascade may account for some of the B cell defects reported forplc-γ2 −/− mice, which develop an X-linked immunodeficiency-like phenotype. Mutations in the gene encoding Bruton's tyrosine kinase (BTK) interfere with B cell proliferation and lead to an X-linked immunodeficiency in mice characterized by reduced B cell numbers. Recent studies have established that BTK transmits signals from the B cell antigen receptor (BCR) to transcription factor NF-κB, which in turn reprograms a set of genes required for normal B cell growth. We now demonstrate that induction of NF-κB via this pathway requires the intermediate action of the -γ2 isoform of phospholipase C (PLC-γ2), a potential phosphorylation substrate of BTK. Specifically, pharmacologic agents that block the action of either PLC-γ2 or its second messengers prevent BCR-induced activation of IκB kinase. Moreover, activation of NF-κB in response to BCR signaling is completely abolished in B cells deficient for PLC-γ2. Taken together, these findings strongly suggest that PLC-γ2 functions as an integral component of the BTK/NF-κB axis following BCR ligation. Interference with this NF-κB cascade may account for some of the B cell defects reported forplc-γ2 −/− mice, which develop an X-linked immunodeficiency-like phenotype. B cell antigen receptor X-linked immunodeficiency Bruton's tyrosine kinase phospholipase C-γ2 nuclear factor-κB IκB kinase protein kinase C phorbol 12-myristate 13-acetate bisindolylmaleimide I cyclosporin A 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid acetoxymethyl ester glutathione S-transferase electrophoretic mobility shift assays polyacrylamide gel electrophoresis mitogen-activated protein kinase The generation and survival of B lymphocyte subpopulations is contingent upon the expression of a functional B cell antigen receptor complex (BCR)1 (1Lam K.P. Kuhn R. Rajewsky K. Cell. 1997; 90: 1073-1083Abstract Full Text Full Text PDF PubMed Scopus (937) Google Scholar, 2Rajewsky K. Nature. 1996; 381: 751-758Crossref PubMed Scopus (1387) Google Scholar). BCR engagement directs B cell biological responses by initiating biochemical signaling cascades involving the cytoplasmic protein tyrosine kinases Lyn, Syk, and BTK (3Fruman D.A. Satterthwaite A.B. Witte O.N. Immunity. 2000; 13: 1-3Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar, 4Yang W.C. Collette Y. Nunes J.A. Olive D. Immunity. 2000; 12: 373-382Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar, 5Campbell K.S. Curr. Opin. Immunol. 1999; 11: 256-264Crossref PubMed Scopus (184) Google Scholar). BTK plays an integral role in transducing BCR-directed signals, because mutations in thebtk gene result in the B cell deficiencies X-linked agammaglobulinemia (XLA) in man and X-linked immunodeficiency (xid) in mice (6Vetrie D. Vorechovsky I. Sideras P. Holland J. Davies A. Flinter F. Hammarstrom L. Kinnon C. Levinsky R. Bobrow M.,. Smith C.I. Bently D.R. Nature. 1993; 361: 226-233Crossref PubMed Scopus (1253) Google Scholar, 7Tsukada S. Saffran D.C. Rawlings D.J. Parolini O. Allen R.C. Klisak I. Sparkes R.S. Kubagawa H. Mohandas T. Quan S. Belmont B.W. Cooper M.D. Conley M.E. Witte O.N. Cell. 1993; 72: 279-290Abstract Full Text PDF PubMed Scopus (1157) Google Scholar, 8Thomas J.D. Sideras P. Smith C.I. Vorechovsky I. Chapman V. Paul W.E. Science. 1993; 261: 355-358Crossref PubMed Scopus (573) Google Scholar, 9Rawlings D.J. Saffran D.C. Tsukada S. Largaespada D.A. Grimaldi J.C. Cohen L. Mohr R.N. Bazan J.F. Howard M. Copeland N.G. Jenkins N.A. Witte O.N. Science. 1993; 261: 358-361Crossref PubMed Scopus (777) Google Scholar, 10Khan W.N. Alt F.W. Gerstein R.M. Malynn B.A. Larsson I. Rathbun G. Davidson L. Muller S. Kantor A.B. Herzenberg L.A. Rosen F.S. Sideras P. Immunity. 1995; 3: 283-299Abstract Full Text PDF PubMed Scopus (640) Google Scholar). B cells from xid mice are defective in survival and proliferation, implicating BTK in these biological processes (10Khan W.N. Alt F.W. Gerstein R.M. Malynn B.A. Larsson I. Rathbun G. Davidson L. Muller S. Kantor A.B. Herzenberg L.A. Rosen F.S. Sideras P. Immunity. 1995; 3: 283-299Abstract Full Text PDF PubMed Scopus (640) Google Scholar, 11Anderson J.S. Teutch M. Dong Z. Wortis H.H. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 10966-10971Crossref PubMed Scopus (116) Google Scholar, 12Solvason N. Wu W.W. Kabra N. Lund-Johansen F. Roncarolo M.G. Behrens T.W. Grillot D.A. Nunez G. Lees E. Howard M. J. Exp. Med. 1998; 187: 1081-1091Crossref PubMed Scopus (69) Google Scholar). However, the molecular mechanisms by which BTK effects B cell proliferation and survival are not well understood. Like BTK, transcription factor NF-κB has been implicated in the regulation of genes essential for B cell responses including proliferation and survival (13Bendall H.H. Sikes M.L. Ballard D.W. Oltz E.M. Mol. Immunol. 1999; 36: 187-195Crossref PubMed Scopus (57) Google Scholar, 14Grumont R.J. Rourke I.J. O'Reilly L.A. Strasser A. Miyake K. Sha W. Gerondakis S. J. Exp. Med. 1998; 187: 663-674Crossref PubMed Scopus (208) Google Scholar, 15Kontgen F. Grumont R.J. Strasser A. Metcalf D. Li R. Tarlinton D. Gerondakis S. Genes Dev. 1995; 9: 1965-1977Crossref PubMed Scopus (641) Google Scholar). In resting cells, NF-κB is sequestered in the cytoplasmic compartment via its association with a family of inhibitory proteins, termed IκBs (16Baldwin Jr., A.S. Annu. Rev. Immunol. 1996; 14: 649-683Crossref PubMed Scopus (5578) Google Scholar). Recent studies have identified a cytokine-inducible IκB kinase complex (IKK) consisting of two catalytic (IKKα and IKKβ) and one regulatory subunit (IKKγ) (17Zandi E. Karin M. Mol. Cell. Biol. 1999; 19: 4547-4551Crossref PubMed Scopus (307) Google Scholar). In response to NF-κB activating signals, IKK phosphorylates and targets IκB for degradation (17Zandi E. Karin M. Mol. Cell. Biol. 1999; 19: 4547-4551Crossref PubMed Scopus (307) Google Scholar). We and others (18Petro J.B. Rahman S.M. Ballard D.W. Khan W.N. J. Exp. Med. 2000; 191: 1745-1754Crossref PubMed Scopus (259) Google Scholar, 19Bajpai U.D. Zhang K. Teutsch M. Sen R. Wortis H.H. J. Exp. Med. 2000; 191: 1735-1744Crossref PubMed Scopus (190) Google Scholar) have recently shown that BTK couples the BCR to IKK and NF-κB. However, the biochemical mechanism by which BTK activates NF-κB remains largely undefined. BTK, in concert with the protein tyrosine kinase Syk and the adaptor protein BLNK, has recently been demonstrated to phosphorylate and activate PLC-γ2 (22Fluckiger A.C. Li Z. Kato R.M. Wahl M.I. Ochs H.D. Longnecker R. Kinet J.P. Witte O.N. Scharenberg A.M. Rawlings D.J. EMBO J. 1998; 17: 1973-1985Crossref PubMed Scopus (358) Google Scholar, 23Takata M. Homma Y. Kurosaki T. J. Exp. Med. 1995; 182: 907-914Crossref PubMed Scopus (183) Google Scholar, 24Takata M. Kurosaki T. J. Exp. Med. 1996; 184: 31-40Crossref PubMed Scopus (426) Google Scholar). In response to BCR signals, PLC-γ2 catalyzes the hydrolysis of phosphatidylinositol 4,5-bisphosphate, generating inositol 1,4,5-trisphosphate and diacylglycerol. Inositol 1,4,5-trisphosphate induces the release of Ca2+ from intracellular stores, and diacylglycerol facilitates the activation of PKC isoenzymes (20Berridge M.J. Nature. 1993; 361: 315-325Crossref PubMed Scopus (6175) Google Scholar, 21Imboden J.B. Stobo J.D. J. Exp. Med. 1985; 161: 446-456Crossref PubMed Scopus (574) Google Scholar). Thus, BTK-dependent activation of PLC-γ2 is essential for BCR-initiated calcium fluxes (22Fluckiger A.C. Li Z. Kato R.M. Wahl M.I. Ochs H.D. Longnecker R. Kinet J.P. Witte O.N. Scharenberg A.M. Rawlings D.J. EMBO J. 1998; 17: 1973-1985Crossref PubMed Scopus (358) Google Scholar). However, the functional consequences of PLC-γ2 signaling in the activation of nuclear factors that direct B cell responses are not known. In this report, we provide two lines of evidence indicating that BCR-initiated activation of NF-κB is mediated by PLC-γ2. First, DT40 chicken B cells deficient for PLC-γ2 fail to translocate NF-κB to the nucleus upon BCR activation. Second, pharmacologic inhibition of PLC-γ2 or its second messengers prevents BCR-responsive activation of IKK and phosphorylation of IκBα in primary B cells. These biochemical findings provide a potential molecular explanation for the B cell defects recently reported forplc-γ2 −/− mice, which display an xid-like phenotype reminiscent of animals lacking functional BTK (10Khan W.N. Alt F.W. Gerstein R.M. Malynn B.A. Larsson I. Rathbun G. Davidson L. Muller S. Kantor A.B. Herzenberg L.A. Rosen F.S. Sideras P. Immunity. 1995; 3: 283-299Abstract Full Text PDF PubMed Scopus (640) Google Scholar). The chicken B cell line DT40, DT40 cells deficient for either BTK or PLC-γ2 (DT40.BTK, DT40.PLC-γ2), or mutant DT40 cells reconstituted with either human BTK or PLC-γ2 (DT40.BTKR, DT40.PLC-γ2R) were a kind gift of Dr. Tomohiro Kurosaki, Riken Cell Bank, Japan (23Takata M. Homma Y. Kurosaki T. J. Exp. Med. 1995; 182: 907-914Crossref PubMed Scopus (183) Google Scholar, 24Takata M. Kurosaki T. J. Exp. Med. 1996; 184: 31-40Crossref PubMed Scopus (426) Google Scholar). DT40 cells were maintained as described previously and were cultured in low serum media (RPMI with 0.5% FCS, 0.05% chicken serum) for 8–12 h prior to stimulation (18Petro J.B. Rahman S.M. Ballard D.W. Khan W.N. J. Exp. Med. 2000; 191: 1745-1754Crossref PubMed Scopus (259) Google Scholar). Splenocytes and primary B lymphocytes were isolated from spleens of C57Bl6 mice. For phospho-IκBα Western analyses, RBC-depleted splenocytes were cultured and stimulated as indicated. For IKK in vitro kinase assays, B cells were purified by a process of negative selection on an affinity chromatography column (Cedarlane, Ontario, Canada). The purity of B cells isolated in this manner was ∼90–95% as verified by fluorescence-activated cell sorter analysis using anti-B220 and anti-IgM antibodies (PharMingen). All purifications were performed at 4 °C, and primary cells were used immediately upon purification. All pharmacological reagents were purchased from Calbiochem. For inhibition of BCR signaling, cells were incubated with EGTA (5 mm), BAPTA-AM (20 nm), cyclosporin A (20 μg/ml), bisindolylmaleimide I (20 μm), or U-73122 (5 μm) for 30 min prior to and during stimulation. Except where indicated in the figure legends, DT40 B cells were either left unstimulated or stimulated with a 1:2 dilution of hybridoma supernatants containing anti-chicken IgM monoclonal antibody (M4) or PMA and ionomycin, 1 μm each. Purified B cells (3–5 × 106 cells per sample) were incubated with 10 μg/ml polyclonal goat anti-mouse IgM F(ab′)2 fragments (Jackson ImmunoResearch), 10 μg/ml anti-mouse CD40 (PharMingen), or with PMA and ionomycin (1 μm each) at a cellular density of 2 × 106/ml in culture media (RPMI 1640 supplemented with 10% serum). To monitor any effects of serum on the activation of NF-κB, cells that were not stimulated were also incubated in medium containing 10% serum for the duration of stimulation. Nuclear extracts were prepared and used in DNA-binding reactions as described previously (18Petro J.B. Rahman S.M. Ballard D.W. Khan W.N. J. Exp. Med. 2000; 191: 1745-1754Crossref PubMed Scopus (259) Google Scholar). For EMSAs, an [α-32P]CTP- and [α-32P]ATP-labeled double-stranded oligonucleotide probe derived from the κB enhancer element of the IL-2Rα receptor promoter (5′-CAACGGCAGGGGAATTCCCCTCTCCTT-3′) was used. To verify equal amounts and integrity of proteins in the nuclear extracts, a control oligonucleotide for NF-Y was used. DNA-binding reactions were resolved by PAGE and visualized by autoradiography. For Western blot analysis of RelA and c-Rel, nuclear extracts equivalent to 2 × 107 cells were denatured in Laemmli reducing buffer by boiling at 95 °C for 3 min, and the proteins were resolved by SDS-PAGE. Proteins were electrotransferred onto nitrocellulose membranes and subjected to immunoblotting with rabbit polyclonal antibodies against RelA, c-Rel, or SP1 as described previously (18Petro J.B. Rahman S.M. Ballard D.W. Khan W.N. J. Exp. Med. 2000; 191: 1745-1754Crossref PubMed Scopus (259) Google Scholar). For IκBα degradation assays, 4 × 106cells/sample were preincubated for 30 min in medium containing 50 μm cycloheximide and then stimulated as indicated. Cell extracts were resolved by SDS-PAGE, transferred onto nitrocellulose membranes, probed with antibodies against chicken IκBα (pp40; gift of C. Chen) and p38 MAPK (Santa Cruz Biotechnology), and detected using the ECL system. Western blot analyses of IκBα phosphorylation were performed as above and probed with antibodies against mouse IκBα (Santa Cruz Biotechnology) or phosphorylated Ser-32/Ser-36 IκBα (Santa Cruz Biotechnology). The κB reporter plasmid encoding firefly luciferase under the control of a promoter containing six consensus NF-κB binding sites (6κB) and a control vector containing a Renilla luciferase gene fused to a thymidine kinase promoter have been described previously (25Chen C.L. Yull F.E. Kerr L.D. Biochem. Biophys. Res. Commun. 1999; 257: 798-806Crossref PubMed Scopus (8) Google Scholar). The indicated DT40 cell lines were each cotransfected by electroporation (250 V, 960 microfarads, Bio-Rad Gene Pulser) with 5 μg of the 6κB reporter construct and 1 μg of theRenilla construct. 18 h post-transfection, cells were stimulated for 6 h with anti-IgM. Cells were harvested, and levels of both firefly and Renilla luciferase were determined using a Dual Luciferase Reporter Assay System (Promega). Levels of firefly luciferase expression were normalized against Renilla as a control for transfection efficiency. In vitro kinase assays were performed on the cytosolic fraction of 5 × 106 B cells as described previously (18Petro J.B. Rahman S.M. Ballard D.W. Khan W.N. J. Exp. Med. 2000; 191: 1745-1754Crossref PubMed Scopus (259) Google Scholar). Briefly, cell extracts from 0.5 × 106 cell equivalents were removed for Western blot analysis, and the remaining cell extract was subjected to immunoprecipitation with anti-IKKα plus anti-IKKβ antibodies (Santa Cruz Biotechnology). The immunocomplexes were then resuspended in 20 μl of kinase buffer (20 mm HEPES, pH 7.2, MgCl2 (2 mm), MnCl2 (2 mm), dithiothreitol (1 mm), ATP (20 μm)) containing 1.0 μCi of [γ-32P]ATP and 50 μg/ml wild type GST-IκBα substrate. The reaction was allowed to continue for 30 min at 30 °C under agitation and then was terminated by the addition of 4× SDS sample buffer. The samples were resolved by 8% SDS-PAGE and stained with Coomassie Brilliant Blue to visualize the GST-IκBα substrate. The gels were dried and exposed to x-ray film to visualize γ-32P-phosphorylated GST-IκBα. In prior studies, we established that BTK is required for nuclear translocation of NF-κB in BCR-stimulated B cells (18Petro J.B. Rahman S.M. Ballard D.W. Khan W.N. J. Exp. Med. 2000; 191: 1745-1754Crossref PubMed Scopus (259) Google Scholar). However, the molecular mechanism by which BTK facilitates NF-κB activation is poorly defined. Recent findings suggest that BCR-directed nuclear translocation of NF-κB requires the activation of the calcium-responsive phosphatase calcineurin (26Venkataraman L. Burakoff S.J. Sen R. J. Exp. Med. 1995; 181: 1091-1099Crossref PubMed Scopus (91) Google Scholar). To define further the mechanism employed by BTK to effect NF-κB activation, we investigated a role for calcium and calcineurin in BCR-responsive nuclear translocation of NF-κB in DT40 B cells. The DT40 B cell system is amenable to genetic manipulation and has thus proven invaluable for biochemical analysis of BCR-signaling events (27Kurosaki T. Annu. Rev. Immunol. 1999; 17: 555-592Crossref PubMed Scopus (370) Google Scholar). To determine whether calcium and calcineurin play a role in BCR-responsive activation of NF-κB in this cellular background, EMSA analyses were performed on nuclear extracts prepared from DT40 cells preincubated with pharmacological inhibitors of calcium, calcineurin, and PKCs. We used BAPTA-AM/EGTA to chelate intra- and extracellular calcium and cyclosporin A (CsA) to inhibit the calcium-responsive phosphatase calcineurin or bisindolylmaleimide (Bis I), a broad spectrum inhibitor of PKC isoenzymes (Fig.1). We also treated DT40 cells with PMA and ionomycin, pharmacological agents known to activate NF-κB via IKK, as a positive control (28Trushin S.A. Pennington K.N. Algeciras-Schimnich A. Paya C.V. J. Biol. Chem. 1999; 274: 22923-22931Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar). As expected, BCR cross-linking or PMA/ionomycin treatment resulted in the rapid nuclear accumulation of NF-κB (compare lane 1 with 2 and10). However, BCR-directed nuclear translocation of NF-κB was inhibited by treatment with BAPTA-AM/EGTA or CsA (lanes 3 and 4). Bis I treatment significantly, although not completely, inhibited this response (lane 5). However, preincubation with Bis in combination with either BAPTA-AM/EGTA or CsA resulted in a complete block in NF-κB nuclear translocation upon BCR activation (lanes 6 and 7). This result demonstrates that inhibition of either calcium or calcineurin and PKC completely abolishes BCR-directed activation of NF-κB in DT40 B cells. Upon BCR ligation, BTK activates a distinct set of signal transducers to initiate downstream signaling events (3Fruman D.A. Satterthwaite A.B. Witte O.N. Immunity. 2000; 13: 1-3Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar, 29Satterthwaite A.B. Li Z. Witte O.N. Semin. Immunol. 1998; 10: 309-316Crossref PubMed Scopus (158) Google Scholar). Of these, Akt, MAPK, and PLC-γ2 have the capability to activate NF-κB via IKK. Although both Akt and MAPK have been directly linked to IKK activation (30Ozes O.N. Mayo L.D. Gustin J.A. Pfeffer S.R. Pfeffer L.M. Donner D.B. Nature. 1999; 401: 82-85Crossref PubMed Scopus (1898) Google Scholar, 31Romashkova J.A. Makarov S.S. Nature. 1999; 401: 86-90Crossref PubMed Scopus (1667) Google Scholar), such a role has not been demonstrated for PLC-γ2. However, our finding that calcium and PKC are essential for nuclear translocation of NF-κB in BCR-stimulated DT40 cells implicates PLC-γ2 in this response (Fig. 1). Therefore, we next explored an involvement of PLC-γ2 in NF-κB nuclear translocation in B cells stimulated via the BCR. To determine whether PLC-γ2 is critical for BCR-directed nuclear translocation of NF-κB, we used mutant chicken DT40 B cells lacking PLC-γ2 (DT40.PLC-γ2) along with BTK-deficient (DT40.BTK) and parental DT40 B cells. Cells were induced via the BCR, and their nuclear NF-κB content was assessed by EMSA (Fig.2 A). Although BCR cross-linking leads to a marked increase in nuclear NF-κB in DT40 cells (Fig. 2 A, compare lanes 1 and4), both DT40.PLC-γ2 and DT40.BTK B cells failed to demonstrate this response (Fig. 2 A, compare lanes 2 and 5 and 3 and 6). However, PMA and ionomycin mobilized similar levels of nuclear NF-κB in all three cell types (Fig. 2 A, lanes 7–9). These results strongly suggest that like BTK, PLC-γ2 plays an essential role in the transmission of BCR signals to activate NF-κB. To ascertain whether the observed defect was due to delayed kinetics of NF-κB activation, we compared BCR-responsive nuclear translocation of NF-κB in DT40.PLC-γ2 cells with that in DT40 B cells over a period of 4 h (Fig. 2 B). Upon BCR cross-linking, DT40 B cells rapidly translocated NF-κB to the nucleus and maintained elevated levels up to 4 h after activation. In contrast, nuclear levels of NF-κB did not increase in DT40.PLC-γ2 B cells at any time point within that period (Fig. 2 B, compare lanes, 1, 3, 5, 7, and 9 with 2, 4, 6, and 8). To verify further that the NF-κB activation defect in DT40.PLC-γ2 B cells was due to PLC-γ2 deficiency, reconstitution experiments were performed. In response to BCR engagement, DT40.PLC-γ2 B cells expressing wild type human PLC-γ2 (DT40.PLC-γ2R (23Takata M. Homma Y. Kurosaki T. J. Exp. Med. 1995; 182: 907-914Crossref PubMed Scopus (183) Google Scholar)) were capable of NF-κB nuclear translocation as determined by EMSA and a NF-κB responsive luciferase reporter assay (Fig. 2, C andD). These data strongly suggest that PLC-γ2 is critical for transmission of BCR-dependent signals that lead to the nuclear translocation of NF-κB. Members of the NF-κB/Rel family of proteins include p50/NF-κB1, p52/NF-κB2, RelA, c-Rel, and RelB, which have the capacity to form either homo- or heterodimers (16Baldwin Jr., A.S. Annu. Rev. Immunol. 1996; 14: 649-683Crossref PubMed Scopus (5578) Google Scholar). NF-κB dimers containing the Rel family proteins RelA or c-Rel have been demonstrated to be the principal transactivating species activated in response to BCR engagement in B cells (35). We previously demonstrated that RelA and c-Rel fail to undergo nuclear translocation upon BCR stimulation in BTK-deficient B cells. To test whether BTK-mediated RelA and c-Rel nuclear translocation requires PLC-γ2, we compared the ability of DT40.PLC-γ2, DT40.BTK, and DT40 B cells to translocate these subunits to the nucleus upon BCR-cross-linking (Fig.3, A and B). Immunoblotting of nuclear extracts from unactivated (lanes 1–3), anti-IgM stimulated (lanes 4–6), and PMA/ionomycin treated (lanes 7–9) cells with Rel subunit-specific antibodies revealed that nuclear accumulation of both RelA and c-Rel occurs in DT40 B cells following BCR stimulation (Fig.3, A and B, lanes 1 and 4). In contrast, BCR-responsive nuclear translocation of RelA and c-Rel is not observed in either DT40.PLC-γ2 or DT40.BTK B cells. Treatment with PMA/ionomycin induced nuclear translocation of both Rel species (Fig. 3, A and B, lanes 7–9) in all three cell lines. Furthermore, the observed differences in Rel subunit translocation are not attributable to either variation in total protein content of the nuclear extracts or their integrity, because similar amounts of the constitutively expressed transcription factor SP1 are detectable in all samples (Fig. 3, A and B, lower panels). Thus, BCR-directed nuclear translocation of RelA and c-Rel is PLC-γ2-dependent. NF-κB dimers are found in the cytoplasm of quiescent cells, bound to members of a family of inhibitory molecules termed IκBs. BCR-induced nuclear translocation of NF-κB is contingent upon the phosphorylation and proteolytic degradation of IκBα, a process that requires BTK. We compared the ability of DT40.PLC-γ2 B cells with DT40.BTK and DT40 B cells to degrade IκBα in response to BCR activation. Cells were incubated with anti-IgM antibodies or PMA and ionomycin for indicated periods, and cytoplasmic extracts were immunoblotted for chicken IκBα (Fig. 4, upper panel). As expected, DT40 B cells rapidly degraded IκBα upon BCR activation. Consistent with the results shown in Fig. 2, DT40.PLC-γ2 B cells failed to degrade IκBα in response to BCR stimulation (Fig. 4, compare lanes 1–4 with 6–9and 11–14). All three cell lines efficiently degraded IκBα in response to treatment with PMA and ionomycin. Therefore, loss of PLC-γ2 does not affect the downstream components necessary for IκBα degradation. These results demonstrate that BCR-directed degradation of IκBα specifically requires PLC-γ2. Prior biochemical studies have identified several NF-κB agonists that converge on IKKα and IKKβ including TNF and IL-1 (17Zandi E. Karin M. Mol. Cell. Biol. 1999; 19: 4547-4551Crossref PubMed Scopus (307) Google Scholar). Additionally, we have recently established that BCR-initiated activation of NF-κB by BTK proceeds via IKK (18Petro J.B. Rahman S.M. Ballard D.W. Khan W.N. J. Exp. Med. 2000; 191: 1745-1754Crossref PubMed Scopus (259) Google Scholar). To extend our finding that PLC-γ2 is required for BCR-directed nuclear translocation of NF-κB, we explored a role for PLC-γ2 in IKK activation. We tested whether pharmacological agents that block PLC-γ2 and its second messengers could prevent BCR-induced activation of IKK in primary B cells (Fig.5 A). In response to activation signals via the BCR or CD40, or treatment with PMA, IKK enzymatic activity was significantly increased as determined by in vitro kinase assays using recombinant GST-IκBα as the substrate (Fig. 5 A, compare lane 1 with 2, 7, and 8). In contrast, incubation of B cells with either the PLC-γ-specific inhibitor (U-73122) or inhibitors of its second messengers (BAPTA-AM/EGTA, CsA, or Bis I) prior to BCR stimulation abolished this activity (Fig. 5 A, lanes 3–6). These data implicate PLC-γ2, calcium, calcineurin, and PKC in IKK activation upon BCR ligation. Moreover, they verify the role of these signaling molecules in BCR-responsive activation of IKK in a physiologically relevant background. To confirm this observation, we performed Western blot analyses of cytosolic fractions from BCR-, CD40-, or PMA-stimulated splenocytes using an antibody directed against Ser-32/Ser-36-phosphorylated IκBα (Fig. 5 B, upper panel). Stimulation via either the BCR or CD40 induced phosphorylation of IκBα (Fig. 5 B,compare lanes 1, 2, and 6). BCR-responsive IκBα phosphorylation was blocked by pretreatment with either BAPTA-AM/EGTA, CsA, or Bis I (Fig. 5 B, lanes 3–5). Also, PMA stimulation resulted in IκBα phosphorylation that was abrogated by pretreatment with the PKC inhibitor Bis I (Fig. 5 B, lanes 7 and 8). These observations implicate PLC-γ2, calcium, and PKC in BCR-responsive activation of IKK and phosphorylation of IκBα. Moreover, the observation that cells pretreated with CsA fail to activate IKK upon BCR cross-linking identifies calcineurin as a critical mediator of this response. This observation is consistent with the recent finding that calcineurin and PKCs synergize to induce IKK activation in T cells (28Trushin S.A. Pennington K.N. Algeciras-Schimnich A. Paya C.V. J. Biol. Chem. 1999; 274: 22923-22931Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar). Collectively, these data suggest that PLC-γ2 is likely to mediate BCR-responsive activation of IKK, phosphorylation of IκBα, and nuclear translocation of NF-κB. We have found that PLC-γ2 and its downstream signals are essential for BCR-directed activation of IKK and NF-κB. Prior studies in BCR-stimulated B cells have revealed that PLC-γ2 is activated via the concerted actions of BTK, Syk, and BLNK (3Fruman D.A. Satterthwaite A.B. Witte O.N. Immunity. 2000; 13: 1-3Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar, 32Rawlings D.J. Clin. Immunol. 1999; 91: 243-253Crossref PubMed Scopus (87) Google Scholar). Therefore, it is likely that PLC-γ2 is the principal BTK signal transducer for BCR-directed activation of IKK and NF-κB. Further investigation is required to determine whether additional BTK targets, including Akt and MAPK, synergize with PLC-γ2 to effect nuclear translocation of NF-κB in BCR-stimulated B cells. However, the placement of PLC-γ2 in the BCR/BTK/NF-κB signaling pathway provides the first potential molecular explanation for the similar xid-like B cell deficiencies displayed byplc-γ2 −/− andbtk −/− mice (10Khan W.N. Alt F.W. Gerstein R.M. Malynn B.A. Larsson I. Rathbun G. Davidson L. Muller S. Kantor A.B. Herzenberg L.A. Rosen F.S. Sideras P. Immunity. 1995; 3: 283-299Abstract Full Text PDF PubMed Scopus (640) Google Scholar, 33Wang D. Feng J. Wen R. Marine J.C. Sangster M.Y. Parganas E. Hoffmeyer A. Jackson C.W. Cleveland J.L. Murray P.J. Ihle J.N. Immunity. 2000; 13: 25-35Abstract Full Text Full Text PDF PubMed Scopus (408) Google Scholar). We thank David Strayhorn for technical advice and Drs. Dean Ballard, Eugene Oltz, Sebastian Joyce, and Jacek Hawiger for helpful discussions in the preparation of this manuscript.