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

Bajpai UD, Zhang K, Teutsch M, Sen R, Wortis HH - J. Exp. Med. (2000)

Bottom Line: The recognition of antigen by membrane immunoglobulin M (mIgM) results in a complex series of signaling events in the cytoplasm leading to gene activation.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology and Program in Immunology, Tufts University School of Medicine and Sackler School of Graduate Biomedical Sciences, Boston, Massachusetts 02111, USA.

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.

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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.
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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.

Mentions: DT40, the chicken B cell line, has been a crucial tool in the delineation of the signal transduction events after antigen receptor cross-linking 295354. We used DT40 cells to further investigate the role of BTK in NF-κB activation. NF-κB–dependent luciferase reporter activity increased 25-fold after 4-h treatment of wild-type DT40 cells with anti-IgM (Fig. 4 A). The same luciferase reporter construct, but lacking the three NF-κB sites, was not induced under the same conditions (data not shown). BTK-deficient DT40 B cells failed to induce NF-κB activity after IgM stimulation (Fig. 4 A). Like wild-type cells, an increase of total protein tyrosine phosphorylation was detected after anti-IgM cross-linking of BTK-deficient DT40 cells. In BTK-deficient DT40 cells, NF-κB–dependent luciferase activity was induced by the pharmacological reagents phorbol ester and ionomycin. Expression of BTK in BTK-deficient cells restored the ability of membrane IgM to activate NF-κB (Fig. 4 B). Western blot analysis demonstrated that BTK protein was expressed in BTK-deficient cells in these assays (Fig. 4 B).


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

Bajpai UD, Zhang K, Teutsch M, Sen R, Wortis HH - J. Exp. Med. (2000)

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.
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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.
Mentions: DT40, the chicken B cell line, has been a crucial tool in the delineation of the signal transduction events after antigen receptor cross-linking 295354. We used DT40 cells to further investigate the role of BTK in NF-κB activation. NF-κB–dependent luciferase reporter activity increased 25-fold after 4-h treatment of wild-type DT40 cells with anti-IgM (Fig. 4 A). The same luciferase reporter construct, but lacking the three NF-κB sites, was not induced under the same conditions (data not shown). BTK-deficient DT40 B cells failed to induce NF-κB activity after IgM stimulation (Fig. 4 A). Like wild-type cells, an increase of total protein tyrosine phosphorylation was detected after anti-IgM cross-linking of BTK-deficient DT40 cells. In BTK-deficient DT40 cells, NF-κB–dependent luciferase activity was induced by the pharmacological reagents phorbol ester and ionomycin. Expression of BTK in BTK-deficient cells restored the ability of membrane IgM to activate NF-κB (Fig. 4 B). Western blot analysis demonstrated that BTK protein was expressed in BTK-deficient cells in these assays (Fig. 4 B).

Bottom Line: The recognition of antigen by membrane immunoglobulin M (mIgM) results in a complex series of signaling events in the cytoplasm leading to gene activation.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology and Program in Immunology, Tufts University School of Medicine and Sackler School of Graduate Biomedical Sciences, Boston, Massachusetts 02111, USA.

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.

Show MeSH
Related in: MedlinePlus