Bruton's tyrosine kinase links the B cell receptor to nuclear factor kappaB activation

Abstract

The recognition of antigen by membrane immunoglobulin M (mIgM) results in a complex series of signaling events in the cytoplasm leading to gene activation. Bruton's tyrosine kinase (BTK), a member of the Tec family of tyrosine kinases, is essential for the full repertoire of IgM signals to be transduced. We examined the ability of BTK to regulate the nuclear factor (NF)-kappaB/Rel family of transcription factors, as the activation of these factors is required for a B cell response to mIgM. We found greatly diminished IgM- but not CD40-mediated NF-kappaB/Rel nuclear translocation and DNA binding in B cells from X-linked immunodeficient (xid) mice that harbor an R28C mutation in btk, a mutation that produces a functionally inactive kinase. The defect was due, in part, to a failure to fully degrade the inhibitory protein of NF-kappaB, IkappaBalpha. Using a BTK-deficient variant of DT40 chicken B cells, we found that expression of wild-type or gain-of-function mutant BTK, but not the R28C mutant, reconstituted NF-kappaB activity. Thus, BTK is essential for activation of NF-kappaB via the B cell receptor.

Figure 1

NF-κB/Rel DNA binding and translocation is decreased in anti-IgM–activated, btk-mutated, xid B cells. (A) Splenic B cells from wild-type and xid mice were cultured for 4 h in medium, F(ab′)₂ anti-IgM (10 μg/ml), or CD40 ligand (1:2 dilution), cells were harvested, nuclear extracts prepared, and EMSAs performed as described in Materials and Methods. This figure represents one of three experiments. Densitometry was performed on the upper band of the gel shift. Addition of a cold competitor (100-fold excess) demonstrated the specificity of NF-κB binding (data not shown), as shown previously 49. (B) Western analysis of nuclear c-rel expression (top panel) was conducted using 20 μg of extract prepared from wild-type or xid splenic B cells treated as described in A. Transcription factor SP-1 served as a nuclear loading control (bottom panel). Densitometry was performed. After normalization to the loading control, it was determined that anti-IgM treatment induced nuclear c-rel 4.8-fold in wild-type and 0.75-fold in xid B cells. This figure represents one of five experiments.

Figure 2

Anti-IgM induces normal c-rel transcript levels, and amounts of c-rel in xid B cells are normal. (A) Wild-type and xid B splenic cells were cultured for 4 h in medium, F(ab′)₂ anti-IgM (10 μg/ml), or CD40 ligand (1:2 dilution), and whole cell extracts were prepared. C-rel Western blot analysis was performed using extracts prepared from 5 × 10⁵ cells. β-actin served as a loading control. (B) Wild-type and xid splenic B cells were cultured for 4 h in medium, F(ab′)₂ anti-IgM (10 μg/ml), or PMA and ionomycin (30 nM and 1 μM, respectively), and total RNA was prepared. RT-PCR (top panel) analysis was conducted using primers specific for c-rel (or GαS, which served as a loading control). RNA samples from WEHI231 and T220 cells served as positive and negative controls, respectively, for this assay. Northern analysis (bottom panel) was conducted using 10 μg of each sample. Equal loading was determined by ethidium bromide staining of 18S and 28S ribosomal bands (data not shown). These figures represent one of three experiments.

Figure 3

Diminished IκBα degradation in anti-IgM–activated xid B cells. (A) Western blot analysis of IκBα expression (top panel) was performed with 25 μg of cytoplasmic extracts prepared from wild-type or xid splenic B cells cultured for 4 h in medium, F(ab′)₂ anti-IgM (10 μg/ml), or CD40 ligand (1:2 dilution). β-actin served as a loading control (bottom panel). This figure represents one of three experiments. (B) Wild-type and xid splenic B cells were cultured with ³⁵S-labeled cysteine and methionine, and the experiment was carried out as described (see text). Mean densitometry values from three independent experiments (shown as percent decrease in ³⁵S-labeled IκBα ± SD) were: wild-type, 55.7 ± 15.1 and xid, 19.0 ± 7.4.

Figure 5

The xid (R28C) mutation decreases BTK-dependent NF-κB activation. Transient transfections of BTK-deficient DT40 cells were conducted using 30 μg of vector (pApuro), 2.5, 5, or 10 μg of wild-type BTK, or 30 μg of BTK mutants, R28C, and E41K along with the NF-κB–driven luciferase reporter construct and the pRLTK internal control. Vector DNA was added when necessary to allow for equivalent micrograms of DNA to be transfected. Cells were cultured for 16 h and then stimulated for 4 h with medium, M4 anti-chicken IgM (10 μg/ml; black bars), or PMA and ionomycin (30 nM and 1 mM, respectively). Cells were lysed and assayed as described in Materials and Methods. For each sample, luciferase activity was normalized to the pRLTK internal control. The graph shows the fold induction of luciferase activity relative to the luciferase activity detected in the lysates from unstimulated BTK-deficient cells transfected with vector (= 1). The fold induction values are the mean of three experiments ± SD. PMA and ionomycin treatment of all cells resulted in similar fold induction values (data not shown). Western analysis for detection of BTK expression was performed using lysates (equivalent of 2.5 × 10⁶ cells) from this assay. The anti-BTK antibody recognizes both mouse and chicken BTK protein. Densitometry analysis was performed (see Results). This figure represents one of three Western blots.

Figure 4

Reconstitution of IgM-mediated NF-κB activity in BTK-deficient DT40 cells by ectopic expression of wild-type (WT) BTK. (A) Transient transfections of WT and BTK-deficient DT40 cells were conducted with NF-κB–driven luciferase reporter and pRLTK constructs, the latter serving as an internal transfection control for the assay. Cells were cultured for 16 h and then stimulated for 4 h with medium, M4 anti-chicken IgM (10 μg/m; light bars), or PMA and ionomycin (30 nM and 1 mM, respectively; dark bars). Cells were lysed and assayed as described in Materials and Methods. For each sample, luciferase activity was normalized to the pRLTK internal control. The graph shows the fold induction of luciferase activity relative to the luciferase activity detected in the lysates of cells cultured in medium only (= 1). This figure is representative of five experiments. (B) 10 μg of wild-type BTK or vector (pApuro) was transiently transfected together with reporter and internal control constructs as described above (A). Null + Vector lane shows background activity levels. PMA and ionomycin treatment of these cells resulted in similar induction (data not shown). Western blot analysis for detection of BTK expression was performed using lysates (equivalent of 2.5 × 10⁶ cells) from this assay. The anti-BTK antibody recognizes both mouse and chicken BTK protein. The reactivity of this antibody for the different BTK species is not known, and the fraction of cells transfected was not determined the expression levels in wild-type and transfected cells cannot be compared. The fold induction values are the mean of three experiments ± SD.