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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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BCR-induced activation of MAPK and Akt pathways in wild-type and Bam32-deficient DT40 cells. (A) Cells were stimulated with anti-μ (M4, 5 μg/ml) for indicated time periods. Cell lysates were subsequently separated on a 10% SDS-PAGE gel, and were analyzed by Western blotting with anti-phospho-ERK, anti-phospho-JNK, anti-phospho-p38 MAPK, anti-phospho-Akt, anti-phospho-GSK-3, and anti-Akt sera. (B) Densitometrical analyses of BCR-induced activation of MAPK. 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. (C) Cells were stimulated as described in A. Cell lysates were subsequently separated on a 10% SDS-PAGE gel, and were analyzed by Western blotting with anti-phospho-Raf-1 mAb, and anti-phospho-Mek sera.
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fig3: BCR-induced activation of MAPK and Akt pathways in wild-type and Bam32-deficient DT40 cells. (A) Cells were stimulated with anti-μ (M4, 5 μg/ml) for indicated time periods. Cell lysates were subsequently separated on a 10% SDS-PAGE gel, and were analyzed by Western blotting with anti-phospho-ERK, anti-phospho-JNK, anti-phospho-p38 MAPK, anti-phospho-Akt, anti-phospho-GSK-3, and anti-Akt sera. (B) Densitometrical analyses of BCR-induced activation of MAPK. 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. (C) Cells were stimulated as described in A. Cell lysates were subsequently separated on a 10% SDS-PAGE gel, and were analyzed by Western blotting with anti-phospho-Raf-1 mAb, and anti-phospho-Mek sera.

Mentions: Three structurally related MAPK subfamilies have been described in mammalian cells: the p42 and p44 kinases (ERKs), the JNKs, and the p38 MAPK family (14). Different patterns of activation of MAPKs may well direct B cells to different cell fates such as proliferation, differentiation, and apoptosis. In addition, PLCγ2 is required for full activation of all three MAPKs in DT40 B cells (15). Thus, we tested whether the phosphorylation of these MAPKs could be affected by the absence of Bam32 (Fig. 3 A). The kinetic profile of activation of the three MAPKs after anti-IgM stimulation of wild-type DT40 cells was consistent with our previous findings (15, 16). In Bam32−/− cells, the phosphorylation of JNK1 was consistently reduced by >85% compared with wild-type cells. In addition, the phosphorylation of both ERK2 and p38 MAPK was partially impaired in Bam32−/− cells. These findings are consistent with studies showing that JNK activation is drastically attenuated either by blocking intracellular calcium release of wild-type B cells treated with BAPTA-AM or in inositol 1,4,5-triphosphate receptor (IP3R)-deficient B cells (15, 16). The fact that the activation of all three MAPKs was affected suggests again a role for Bam32 in BCR-induced PLCγ2 activation.


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)

BCR-induced activation of MAPK and Akt pathways in wild-type and Bam32-deficient DT40 cells. (A) Cells were stimulated with anti-μ (M4, 5 μg/ml) for indicated time periods. Cell lysates were subsequently separated on a 10% SDS-PAGE gel, and were analyzed by Western blotting with anti-phospho-ERK, anti-phospho-JNK, anti-phospho-p38 MAPK, anti-phospho-Akt, anti-phospho-GSK-3, and anti-Akt sera. (B) Densitometrical analyses of BCR-induced activation of MAPK. 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. (C) Cells were stimulated as described in A. Cell lysates were subsequently separated on a 10% SDS-PAGE gel, and were analyzed by Western blotting with anti-phospho-Raf-1 mAb, and anti-phospho-Mek sera.
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Related In: Results  -  Collection

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fig3: BCR-induced activation of MAPK and Akt pathways in wild-type and Bam32-deficient DT40 cells. (A) Cells were stimulated with anti-μ (M4, 5 μg/ml) for indicated time periods. Cell lysates were subsequently separated on a 10% SDS-PAGE gel, and were analyzed by Western blotting with anti-phospho-ERK, anti-phospho-JNK, anti-phospho-p38 MAPK, anti-phospho-Akt, anti-phospho-GSK-3, and anti-Akt sera. (B) Densitometrical analyses of BCR-induced activation of MAPK. 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. (C) Cells were stimulated as described in A. Cell lysates were subsequently separated on a 10% SDS-PAGE gel, and were analyzed by Western blotting with anti-phospho-Raf-1 mAb, and anti-phospho-Mek sera.
Mentions: Three structurally related MAPK subfamilies have been described in mammalian cells: the p42 and p44 kinases (ERKs), the JNKs, and the p38 MAPK family (14). Different patterns of activation of MAPKs may well direct B cells to different cell fates such as proliferation, differentiation, and apoptosis. In addition, PLCγ2 is required for full activation of all three MAPKs in DT40 B cells (15). Thus, we tested whether the phosphorylation of these MAPKs could be affected by the absence of Bam32 (Fig. 3 A). The kinetic profile of activation of the three MAPKs after anti-IgM stimulation of wild-type DT40 cells was consistent with our previous findings (15, 16). In Bam32−/− cells, the phosphorylation of JNK1 was consistently reduced by >85% compared with wild-type cells. In addition, the phosphorylation of both ERK2 and p38 MAPK was partially impaired in Bam32−/− cells. These findings are consistent with studies showing that JNK activation is drastically attenuated either by blocking intracellular calcium release of wild-type B cells treated with BAPTA-AM or in inositol 1,4,5-triphosphate receptor (IP3R)-deficient B cells (15, 16). The fact that the activation of all three MAPKs was affected suggests again a role for Bam32 in BCR-induced PLCγ2 activation.

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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