Bruton's tyrosine kinase is required for activation of IkappaB kinase and nuclear factor kappaB in response to B cell receptor engagement

Abstract

Mutations in the gene encoding Bruton's tyrosine kinase (btk) cause the B cell deficiency diseases X-linked agammaglobulinemia (XLA) in humans and X-linked immunodeficiency (xid) in mice. In vivo and in vitro studies indicate that the BTK protein is essential for B cell survival, cell cycle progression, and proliferation in response to B cell antigen receptor (BCR) stimulation. BCR stimulation leads to the activation of transcription factor nuclear factor (NF)-kappaB, which in turn regulates genes controlling B cell growth. We now demonstrate that a null mutation in btk known to cause the xid phenotype prevents BCR-induced activation of NF-kappaB. This defect can be rescued by reconstitution with wild-type BTK. This mutation also interferes with BCR-directed activation of IkappaB kinase (IKK), which normally targets the NF-kappaB inhibitor IkappaBalpha for degradation. Taken together, these findings indicate that BTK couples IKK and NF-kappaB to the BCR. Interference with this coupling mechanism may contribute to the B cell deficiencies observed in XLA and xid.

Figures

Figure 1

Defective NF-κB activation in BTK-deficient (DT40.BTK) B cells upon BCR stimulation. (A) EMSA analysis of nuclear NF-κB in DT40 and DT40.BTK B cells. Cells were incubated for 2 h in the presence or absence of anti-IgM (lanes 1–4) or PMA/ionomycin (lanes 5 and 6), and nuclear extracts equivalent to 4 × 106 cells were added in a reaction containing a 32P-labeled oligonucleotide probe derived from the κB enhancer element of the IL-2Rα promoter. DNA–nucleoprotein complexes were resolved on nondenaturing polyacrylamide gels. Binding of nucleoprotein complexes to an NF-Y probe 34 was also performed to verify the integrity and the concentration of proteins in the nuclear extracts (bottom panel). (B) Kinetics of NF-κB activation in DT40 and DT40.BTK B cells upon IgM stimulation. Nuclear extracts were prepared and subjected to EMSA as described in A. (C) NF-κB nucleoprotein complexes in DT40 B cells bind specifically to a consensus NF-κB site. EMSAs were conducted with the same nuclear extracts as in A. Nuclear extracts were either preincubated with 100-fold excess of unlabeled wild-type (lanes 3 and 6), mutant NF-κB probe (Mut; lanes 4 and 7; reference 32), or without the unlabeled probes (lanes 1, 2, and 5) before addition of a 32P-labeled wild-type probe. Protein integrity and concentration in the nuclear extracts was verified with NF-Y probe (bottom panel). (D) Ectopic expression of BTK restores BCR-directed NF-κB activation in DT40.BTK B cells. DT40 and DT40.BTK B cells were transiently cotransfected with an expression vector encoding wild-type BTK (pMMP.BTK), an NF-κB reporter plasmid (6κB), and a Renilla luciferase plasmid to normalize for transfection efficiency. Control transfections were performed with blank vector (pMMP). After 24-h culture, cells were stimulated with anti-IgM for 6 h and then assayed for reporter gene activity as described in Materials and Methods. Results for these rescue experiments are reported as the mean fold induction of NF-κB–directed transcription relative to unstimulated DT40 cells.

Figure 1

Defective NF-κB activation in BTK-deficient (DT40.BTK) B cells upon BCR stimulation. (A) EMSA analysis of nuclear NF-κB in DT40 and DT40.BTK B cells. Cells were incubated for 2 h in the presence or absence of anti-IgM (lanes 1–4) or PMA/ionomycin (lanes 5 and 6), and nuclear extracts equivalent to 4 × 106 cells were added in a reaction containing a 32P-labeled oligonucleotide probe derived from the κB enhancer element of the IL-2Rα promoter. DNA–nucleoprotein complexes were resolved on nondenaturing polyacrylamide gels. Binding of nucleoprotein complexes to an NF-Y probe 34 was also performed to verify the integrity and the concentration of proteins in the nuclear extracts (bottom panel). (B) Kinetics of NF-κB activation in DT40 and DT40.BTK B cells upon IgM stimulation. Nuclear extracts were prepared and subjected to EMSA as described in A. (C) NF-κB nucleoprotein complexes in DT40 B cells bind specifically to a consensus NF-κB site. EMSAs were conducted with the same nuclear extracts as in A. Nuclear extracts were either preincubated with 100-fold excess of unlabeled wild-type (lanes 3 and 6), mutant NF-κB probe (Mut; lanes 4 and 7; reference 32), or without the unlabeled probes (lanes 1, 2, and 5) before addition of a 32P-labeled wild-type probe. Protein integrity and concentration in the nuclear extracts was verified with NF-Y probe (bottom panel). (D) Ectopic expression of BTK restores BCR-directed NF-κB activation in DT40.BTK B cells. DT40 and DT40.BTK B cells were transiently cotransfected with an expression vector encoding wild-type BTK (pMMP.BTK), an NF-κB reporter plasmid (6κB), and a Renilla luciferase plasmid to normalize for transfection efficiency. Control transfections were performed with blank vector (pMMP). After 24-h culture, cells were stimulated with anti-IgM for 6 h and then assayed for reporter gene activity as described in Materials and Methods. Results for these rescue experiments are reported as the mean fold induction of NF-κB–directed transcription relative to unstimulated DT40 cells.

Figure 2

BTK-deficient B cells fail to translocate RelA and c-Rel to the nucleus in response to BCR stimulation. Western blot analysis of RelA and c-Rel in nuclear extracts of DT40 or DT40.BTK B cells. Nuclear extracts were prepared from cells that were either not stimulated (lanes 1 and 2) or stimulated either with anti-IgM (lanes 3 and 4) or with PMA/ionomycin (lanes 5 and 6). Equal amounts of nuclear extracts (2 × 107 cell equivalents per lane) were resolved by SDS-PAGE and transferred to nitrocellulose membranes. The relative amounts of RelA and c-Rel were then determined by immunoblotting using antisera directed against either RelA (top panel) or c-Rel (center panel) and visualized by enhanced chemiluminescence detection. The blots were stripped and reprobed for SP1, a constitutively expressed transcription factor to ensure protein integrity and the loading equivalent amounts in each lane (bottom panel).

Figure 3

A requirement for BTK in B cells for the degradation of IκBα in response to BCR cross-linking. Western blot analysis of IκBα in cytoplasmic extracts from DT40 (lanes 1–5) or DT40.BTK (lanes 6–10). Cells were pretreated with cycloheximide (50 μM) for 30 min to arrest translation. Translational arrest cells were stimulated with anti-IgM or PMA/ionomycin for indicated time periods. Equal amounts of cytosolic extracts (4 × 106 cell equivalents per lane) were subjected to immunoblot analysis as described in Fig. 2 with an antiserum against chicken IκBα (pp40). Blots were stripped and reprobed for a constitutively expressed protein, p38 MAPK, to verify protein integrity and the loading amount of protein in each lane (bottom panel).

Figure 4

Impaired NF-κB nuclear translocation in btk−/− primary B cells in response to BCR stimulation. EMSA analyses for κB binding activity in the nuclear extracts from anti-IgM– and PMA/ionomycin-stimulated B cells. Purified splenic B cells from btk−/− or C57Bl6 (WT) mice (WT, 93%; btk−/−, 85%; B220+/IgM+ B cell) were either left unstimulated (lanes 1 and 2) or stimulated for 1 h with anti–mouse F(ab′)2 (lanes 3 and 4) or with PMA and ionomycin (lanes 5 and 6). Equal amounts of nuclear extracts (3 × 106 cell equivalents per lane) were used in each DNA binding reaction. NF-Y binding was used as a control to verify the integrity and concentration of proteins in the nuclear extracts. Based on quantitative analysis of three separate experiments, the mean fold induction of NF-κB DNA binding activity normalized by their NFY controls in WT versus btk−/− cells was 1.7 ± 0.3 and 1.0 ± 0.1, respectively.

Figure 5

btk−/− B cells fail to activate IKKα and IKKβ in response to BCR stimulation. (A) primary B cells from spleens of btk−/− and C57Bl6 (WT) mice were purified and stimulated as in Fig. 4. Cells were either left unstimulated (lanes 1 and 2) or stimulated for 30 min with anti–mouse IgM F(ab′)2 (lanes 3 and 4) or with PMA and ionomycin (lanes 7 and 8). Cytosolic extracts from 3 × 106 cells per sample were immunoprecipitated with antibodies directed against IKKα (Santa Cruz Biotechnology), and the resulting immunocomplexes were subjected to in vitro kinase assay containing γ-[32P]ATP and 1.0 μg of GST-IκBα as the substrate. To determine the specificity of the kinase activity on the IκBα substrate, a mutant GST-IκBα in which Ser-32 and Ser-36 were replaced with Ala (SS/AA) was used (lanes 5 and 6). The kinase assays were resolved by SDS-PAGE and visualized by autoradiography. The immunoblots with anti-IKKα (A, bottom panel) were performed on 10% of the cell extract that was used in in vitro kinase assay to monitor the integrity and the steady state levels of IKKα protein. (B) IKKβ activity was also determined on the same cytosolic extracts as in A, and both the IKKβ kinase activity (B, top panel) and the IKKβ protein are shown (B, bottom panel).