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The B lymphocyte adaptor molecule of 32 kD (Bam32) regulates B cell antigen receptor signaling and cell survival.

Niiro H, Maeda A, Kurosaki T, Clark EA - J. Exp. Med. (2002)

Bottom Line: The B lymphocyte-associated adaptor protein 32 kD in size (Bam32) is expressed at high levels in germinal center (GC) B cells.Furthermore, Bam32(-/-) cells were more susceptible to BCR-induced death.Taken together, these findings suggest that Bam32 functions to regulate BCR-induced signaling and cell survival most likely in germinal centers.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, University of Washington, Seattle, WA 98195, USA.

ABSTRACT
The B lymphocyte-associated adaptor protein 32 kD in size (Bam32) is expressed at high levels in germinal center (GC) B cells. It has an NH(2)-terminal src homology 2 (SH2) domain which binds phospholipase C (PLC)gamma 2, and a COOH-terminal pleckstrin homology (PH) domain. Thus, Bam32 may function to integrate protein tyrosine kinase (PTK) and phosphatidylinositol 3-kinase (PI3K) signaling pathways in B cells. To further define the role Bam32 plays in B cells, we generated Bam32-deficient DT40 cells. These Bam32(-/-) cells exhibited lower levels of B cell antigen receptor (BCR)-induced calcium mobilization with modest decreases in tyrosine phosphorylation of phospholipase C (PLC)gamma 2. Moreover, BCR-induced activation of extracellular signal-regulated kinase (ERK), c-jun NH2-terminal kinase (JNK), and p38 mitogen-activated protein kinase (MAPK) pathways was impaired in Bam32(-/-) cells but not the activation of Akt-related pathways. Activation of downstream transcription factors such as nuclear factor of activated T cells (NF-AT) and nuclear factor of kappa binding (NF-kappa B) was also impaired in Bam32(-/-) cells. Furthermore, Bam32(-/-) cells were more susceptible to BCR-induced death. Taken together, these findings suggest that Bam32 functions to regulate BCR-induced signaling and cell survival most likely in germinal centers.

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Induction of apoptosis in wild-type and Bam32-deficient DT40 cells. (A) Wild-type cells (WT), Bam32-deficient clones (M80–11, M82–5), and Bam32-deficient clones expressing wild-type Bam32 were cultured with or without anti-μ (M4, 10 μg/ml) for 24 h, treated in hypotonic DNA staining solution containing 50 μg/ml propiduim iodide, and subjected to analysis by FACScan™. The percentage of fragmented nuclei is indicated. (B) The expression of chicken Bam32 transgene. Cell lysates were separated on a 12.5% SDS-PAGE gel, and analyzed by Western blotting with anti-Myc mAb (0.5 μg/ml).
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fig5: Induction of apoptosis in wild-type and Bam32-deficient DT40 cells. (A) Wild-type cells (WT), Bam32-deficient clones (M80–11, M82–5), and Bam32-deficient clones expressing wild-type Bam32 were cultured with or without anti-μ (M4, 10 μg/ml) for 24 h, treated in hypotonic DNA staining solution containing 50 μg/ml propiduim iodide, and subjected to analysis by FACScan™. The percentage of fragmented nuclei is indicated. (B) The expression of chicken Bam32 transgene. Cell lysates were separated on a 12.5% SDS-PAGE gel, and analyzed by Western blotting with anti-Myc mAb (0.5 μg/ml).

Mentions: BCR cross-linking leads to growth inhibition and apoptosis in immature B cell lines including DT40 cells. Treatment of wild-type DT40 cells for 24 h after anti-IgM stimulation results in a significant increase in apoptotic cells (Fig. 5) . In both PLCγ2- and IP3R-deficient DT40 cells, this BCR-induced apoptotic response is significantly decreased (22). In contrast, both Bam32−/− cell lines were much more susceptible to BCR-induced cell death (Fig. 5). As expected, transfection of chicken Bam32 cDNA into Bam32-deficient DT40 cells restored cells to a wild-type phenotype.


The B lymphocyte adaptor molecule of 32 kD (Bam32) regulates B cell antigen receptor signaling and cell survival.

Niiro H, Maeda A, Kurosaki T, Clark EA - J. Exp. Med. (2002)

Induction of apoptosis in wild-type and Bam32-deficient DT40 cells. (A) Wild-type cells (WT), Bam32-deficient clones (M80–11, M82–5), and Bam32-deficient clones expressing wild-type Bam32 were cultured with or without anti-μ (M4, 10 μg/ml) for 24 h, treated in hypotonic DNA staining solution containing 50 μg/ml propiduim iodide, and subjected to analysis by FACScan™. The percentage of fragmented nuclei is indicated. (B) The expression of chicken Bam32 transgene. Cell lysates were separated on a 12.5% SDS-PAGE gel, and analyzed by Western blotting with anti-Myc mAb (0.5 μg/ml).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2196019&req=5

fig5: Induction of apoptosis in wild-type and Bam32-deficient DT40 cells. (A) Wild-type cells (WT), Bam32-deficient clones (M80–11, M82–5), and Bam32-deficient clones expressing wild-type Bam32 were cultured with or without anti-μ (M4, 10 μg/ml) for 24 h, treated in hypotonic DNA staining solution containing 50 μg/ml propiduim iodide, and subjected to analysis by FACScan™. The percentage of fragmented nuclei is indicated. (B) The expression of chicken Bam32 transgene. Cell lysates were separated on a 12.5% SDS-PAGE gel, and analyzed by Western blotting with anti-Myc mAb (0.5 μg/ml).
Mentions: BCR cross-linking leads to growth inhibition and apoptosis in immature B cell lines including DT40 cells. Treatment of wild-type DT40 cells for 24 h after anti-IgM stimulation results in a significant increase in apoptotic cells (Fig. 5) . In both PLCγ2- and IP3R-deficient DT40 cells, this BCR-induced apoptotic response is significantly decreased (22). In contrast, both Bam32−/− cell lines were much more susceptible to BCR-induced cell death (Fig. 5). As expected, transfection of chicken Bam32 cDNA into Bam32-deficient DT40 cells restored cells to a wild-type phenotype.

Bottom Line: The B lymphocyte-associated adaptor protein 32 kD in size (Bam32) is expressed at high levels in germinal center (GC) B cells.Furthermore, Bam32(-/-) cells were more susceptible to BCR-induced death.Taken together, these findings suggest that Bam32 functions to regulate BCR-induced signaling and cell survival most likely in germinal centers.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, University of Washington, Seattle, WA 98195, USA.

ABSTRACT
The B lymphocyte-associated adaptor protein 32 kD in size (Bam32) is expressed at high levels in germinal center (GC) B cells. It has an NH(2)-terminal src homology 2 (SH2) domain which binds phospholipase C (PLC)gamma 2, and a COOH-terminal pleckstrin homology (PH) domain. Thus, Bam32 may function to integrate protein tyrosine kinase (PTK) and phosphatidylinositol 3-kinase (PI3K) signaling pathways in B cells. To further define the role Bam32 plays in B cells, we generated Bam32-deficient DT40 cells. These Bam32(-/-) cells exhibited lower levels of B cell antigen receptor (BCR)-induced calcium mobilization with modest decreases in tyrosine phosphorylation of phospholipase C (PLC)gamma 2. Moreover, BCR-induced activation of extracellular signal-regulated kinase (ERK), c-jun NH2-terminal kinase (JNK), and p38 mitogen-activated protein kinase (MAPK) pathways was impaired in Bam32(-/-) cells but not the activation of Akt-related pathways. Activation of downstream transcription factors such as nuclear factor of activated T cells (NF-AT) and nuclear factor of kappa binding (NF-kappa B) was also impaired in Bam32(-/-) cells. Furthermore, Bam32(-/-) cells were more susceptible to BCR-induced death. Taken together, these findings suggest that Bam32 functions to regulate BCR-induced signaling and cell survival most likely in germinal centers.

Show MeSH
Related in: MedlinePlus