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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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Impairment of BCR-induced Ca2+ mobilization and tyrosine phosphorylation of PLCγ2 in the absence of Bam32. (A) Ca2+ mobilization in wild-type (WT) DT40 cells, and Bam32-deficient clones (M80–11, M82–5). Intracellular free calcium levels in Indo-1–loaded cells were monitored using a BD LSR system, after cells were stimulated with M4 mAb to chicken μ chain (2 μg/ml). (B) Intracellular Ca2+ release in the presence of EGTA. (C) Tyrosine phosphorylation of PLCγ2. At the indicated time points after BCR stimulation (M4, 5 μg/ml), immunoprecipitates with anti-PLCγ2 Ab were separated on a 10% SDS-PAGE gel, and were analyzed by Western blotting with biotinated anti-phosphotyrosine mAb (top, αpY) or anti-PLCγ2 Ab (bottom, αPLCγ2). (D) Densitometrical analyses of tyrosine phosphorylation of PLCγ2. The resulting values were expressed as the percentage in reference to that of maximal response in BCR-stimulated wild-type DT40 cells. The results were shown by average and SEM of three independent experiments.
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fig2: Impairment of BCR-induced Ca2+ mobilization and tyrosine phosphorylation of PLCγ2 in the absence of Bam32. (A) Ca2+ mobilization in wild-type (WT) DT40 cells, and Bam32-deficient clones (M80–11, M82–5). Intracellular free calcium levels in Indo-1–loaded cells were monitored using a BD LSR system, after cells were stimulated with M4 mAb to chicken μ chain (2 μg/ml). (B) Intracellular Ca2+ release in the presence of EGTA. (C) Tyrosine phosphorylation of PLCγ2. At the indicated time points after BCR stimulation (M4, 5 μg/ml), immunoprecipitates with anti-PLCγ2 Ab were separated on a 10% SDS-PAGE gel, and were analyzed by Western blotting with biotinated anti-phosphotyrosine mAb (top, αpY) or anti-PLCγ2 Ab (bottom, αPLCγ2). (D) Densitometrical analyses of tyrosine phosphorylation of PLCγ2. The resulting values were expressed as the percentage in reference to that of maximal response in BCR-stimulated wild-type DT40 cells. The results were shown by average and SEM of three independent experiments.

Mentions: As BCR-induced calcium mobilization is dramatically reduced in both PLCγ2-deficient DT40 cells and B cells from PLCγ2-deficient mice (10, 11), we evaluated BCR-induced calcium mobilization in Bam32−/− cells. In both Bam32−/− cells, this response was partially impaired compared with wild-type cells (Fig. 2 A). BCR-induced calcium response comprises an initial release phase and a subsequent sustained plateau phase due to release of Ca2+ from intracellular stores and Ca2+ influx across the plasma membrane, respectively (12). Pretreatment of cells with EGTA resulted in almost complete inhibition of the sustained phase of the calcium response. Under these conditions, the partial inhibition of BCR-induced calcium response in Bam32−/− cells was still observed, suggesting that Ca2+ release from intracellular stores is defective in Bam32−/− cells (Fig. 2 B). We also confirmed that thapsigargin-sensitive calcium stores are normal in Bam32−/− cells (data not shown). Tyrosine phosphorylation of PLCγ2 is required for an increase in its catalytic activity (13). Tyrosine phosphorylation of PLCγ2 after anti-IgM stimulation was modestly attenuated in Bam32−/− cells compared with wild-type cells (Fig. 2, C and D). These results suggest that Bam32 regulates the activation of PLCγ2, but is not essential for PLCγ2 activation upon BCR ligation in DT40 cells.


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)

Impairment of BCR-induced Ca2+ mobilization and tyrosine phosphorylation of PLCγ2 in the absence of Bam32. (A) Ca2+ mobilization in wild-type (WT) DT40 cells, and Bam32-deficient clones (M80–11, M82–5). Intracellular free calcium levels in Indo-1–loaded cells were monitored using a BD LSR system, after cells were stimulated with M4 mAb to chicken μ chain (2 μg/ml). (B) Intracellular Ca2+ release in the presence of EGTA. (C) Tyrosine phosphorylation of PLCγ2. At the indicated time points after BCR stimulation (M4, 5 μg/ml), immunoprecipitates with anti-PLCγ2 Ab were separated on a 10% SDS-PAGE gel, and were analyzed by Western blotting with biotinated anti-phosphotyrosine mAb (top, αpY) or anti-PLCγ2 Ab (bottom, αPLCγ2). (D) Densitometrical analyses of tyrosine phosphorylation of PLCγ2. The resulting values were expressed as the percentage in reference to that of maximal response in BCR-stimulated wild-type DT40 cells. The results were shown by average and SEM of three independent experiments.
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Related In: Results  -  Collection

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fig2: Impairment of BCR-induced Ca2+ mobilization and tyrosine phosphorylation of PLCγ2 in the absence of Bam32. (A) Ca2+ mobilization in wild-type (WT) DT40 cells, and Bam32-deficient clones (M80–11, M82–5). Intracellular free calcium levels in Indo-1–loaded cells were monitored using a BD LSR system, after cells were stimulated with M4 mAb to chicken μ chain (2 μg/ml). (B) Intracellular Ca2+ release in the presence of EGTA. (C) Tyrosine phosphorylation of PLCγ2. At the indicated time points after BCR stimulation (M4, 5 μg/ml), immunoprecipitates with anti-PLCγ2 Ab were separated on a 10% SDS-PAGE gel, and were analyzed by Western blotting with biotinated anti-phosphotyrosine mAb (top, αpY) or anti-PLCγ2 Ab (bottom, αPLCγ2). (D) Densitometrical analyses of tyrosine phosphorylation of PLCγ2. The resulting values were expressed as the percentage in reference to that of maximal response in BCR-stimulated wild-type DT40 cells. The results were shown by average and SEM of three independent experiments.
Mentions: As BCR-induced calcium mobilization is dramatically reduced in both PLCγ2-deficient DT40 cells and B cells from PLCγ2-deficient mice (10, 11), we evaluated BCR-induced calcium mobilization in Bam32−/− cells. In both Bam32−/− cells, this response was partially impaired compared with wild-type cells (Fig. 2 A). BCR-induced calcium response comprises an initial release phase and a subsequent sustained plateau phase due to release of Ca2+ from intracellular stores and Ca2+ influx across the plasma membrane, respectively (12). Pretreatment of cells with EGTA resulted in almost complete inhibition of the sustained phase of the calcium response. Under these conditions, the partial inhibition of BCR-induced calcium response in Bam32−/− cells was still observed, suggesting that Ca2+ release from intracellular stores is defective in Bam32−/− cells (Fig. 2 B). We also confirmed that thapsigargin-sensitive calcium stores are normal in Bam32−/− cells (data not shown). Tyrosine phosphorylation of PLCγ2 is required for an increase in its catalytic activity (13). Tyrosine phosphorylation of PLCγ2 after anti-IgM stimulation was modestly attenuated in Bam32−/− cells compared with wild-type cells (Fig. 2, C and D). These results suggest that Bam32 regulates the activation of PLCγ2, but is not essential for PLCγ2 activation upon BCR ligation in DT40 cells.

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