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

Petro JB, Rahman SM, Ballard DW, Khan WN - J. Exp. Med. (2000)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146, USA.

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 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.

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Related in: MedlinePlus

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).
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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).

Mentions: Prior studies have shown that RelA and c-Rel are the principle transactivator subunits of NF-κB in B cells 38. To analyze the nuclear contents of RelA and c-Rel in DT40 cells, immunoblotting experiments with DT40 nuclear extracts and Rel subunit–specific antibodies were performed. As shown in Fig. 2, nuclear accumulation of both RelA and c-Rel was evident in parental DT40 B cells after stimulation with anti-IgM antibodies (Fig. 2, lane 3, top and center panels), whereas this response was not apparent in DT40.BTK cells (Fig. 2, lane 4, top and center panels). In the absence of BCR stimulation, BTK-deficient B cells expressed lower basal levels of nuclear RelA relative to BTK-expressing controls (Fig. 2, lane 2, top and center panels). These differences could not be attributed to variations in nuclear extract integrity, because similar amounts of the constitutively expressed transcription factor SP1 were detected in all samples (bottom panel). Consistent with the results shown in Fig. 1 A, nuclear translocation of RelA and c-Rel in response to PMA/ionomycin was unaffected by the loss of BTK (Fig. 2, lanes 5 and 6). These findings indicate that the κB-specific DNA binding activity found in the nuclei of DT40 cells after BCR engagement involves BTK-dependent translocation of the RelA and c-Rel transactivating subunits of NF-κB to this subcellular compartment.


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

Petro JB, Rahman SM, Ballard DW, Khan WN - J. Exp. Med. (2000)

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).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2193161&req=5

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).
Mentions: Prior studies have shown that RelA and c-Rel are the principle transactivator subunits of NF-κB in B cells 38. To analyze the nuclear contents of RelA and c-Rel in DT40 cells, immunoblotting experiments with DT40 nuclear extracts and Rel subunit–specific antibodies were performed. As shown in Fig. 2, nuclear accumulation of both RelA and c-Rel was evident in parental DT40 B cells after stimulation with anti-IgM antibodies (Fig. 2, lane 3, top and center panels), whereas this response was not apparent in DT40.BTK cells (Fig. 2, lane 4, top and center panels). In the absence of BCR stimulation, BTK-deficient B cells expressed lower basal levels of nuclear RelA relative to BTK-expressing controls (Fig. 2, lane 2, top and center panels). These differences could not be attributed to variations in nuclear extract integrity, because similar amounts of the constitutively expressed transcription factor SP1 were detected in all samples (bottom panel). Consistent with the results shown in Fig. 1 A, nuclear translocation of RelA and c-Rel in response to PMA/ionomycin was unaffected by the loss of BTK (Fig. 2, lanes 5 and 6). These findings indicate that the κB-specific DNA binding activity found in the nuclei of DT40 cells after BCR engagement involves BTK-dependent translocation of the RelA and c-Rel transactivating subunits of NF-κB to this subcellular compartment.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146, USA.

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 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.

Show MeSH
Related in: MedlinePlus