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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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Disruption of the Bam32 gene in chicken DT40 B cells. (A) Partial restriction map of chicken Bam32 locus and expected structure of the targeted Bam32 alleles. The restriction endonuclease cleavage sites are abbreviated: X, XbaI; E, EcoRI. (B) Southern analysis of genomic DNA from wild-type DT40 cells (lane 1), neo-targeted cells (+/−, lane 2), and neo- and hisD-targeted cells (−/−, lane 3 and 4). Xba I-digested genomic DNA separated on an agarose gel was blotted and hybridized with a chicken Bam32 DNA probe shown in A. Wild-type (WT) and targeted alleles show 15.5- or 5.0-kb fragments, respectively. (C) Northern analysis using a chicken cDNA probe for Bam32 (top) and β-actin (bottom). Wild-type (WT) DT40 cells contain a hybridizable RNA species of ∼3,000 nucleotides. (D) Cell surface expression of BCR. BCR expression on the surface of wild-type (WT) and two independent Bam32-deficient (M80–11, M82–5) DT40 cells was analyzed by flow cytometry. Unstained cells were used as negative controls.
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fig1: Disruption of the Bam32 gene in chicken DT40 B cells. (A) Partial restriction map of chicken Bam32 locus and expected structure of the targeted Bam32 alleles. The restriction endonuclease cleavage sites are abbreviated: X, XbaI; E, EcoRI. (B) Southern analysis of genomic DNA from wild-type DT40 cells (lane 1), neo-targeted cells (+/−, lane 2), and neo- and hisD-targeted cells (−/−, lane 3 and 4). Xba I-digested genomic DNA separated on an agarose gel was blotted and hybridized with a chicken Bam32 DNA probe shown in A. Wild-type (WT) and targeted alleles show 15.5- or 5.0-kb fragments, respectively. (C) Northern analysis using a chicken cDNA probe for Bam32 (top) and β-actin (bottom). Wild-type (WT) DT40 cells contain a hybridizable RNA species of ∼3,000 nucleotides. (D) Cell surface expression of BCR. BCR expression on the surface of wild-type (WT) and two independent Bam32-deficient (M80–11, M82–5) DT40 cells was analyzed by flow cytometry. Unstained cells were used as negative controls.

Mentions: A lamda Zap DT40 cDNA library was screened by human Bam32 cDNA. The chicken Bam32 gene exhibited ∼80% identity to human counterpart at the amino acid level. Using the isolated cDNA of chicken Bam32, a chicken DNA library was screened to obtain genomic clones. After subcloning, the targeting vectors pBam32-Neo and pBam32-HisD were made by replacing the genomic fragment containing exons corresponding to amino acid residues 106–148 with neo- and hisD-cassette (Fig. 1 A). These cassettes were flanked by 2.5 and 1.8 kb of chicken Bam32 genomic sequence on the 5′ and 3′ sides, respectively. Targeting constructs were sequentially transfected into wild-type DT40 cells by electroporation to obtain mutants. Selection for drug-resistant clones was performed using G418 (2 mg/ml) and histidinol (1 mg/ml), and clones were screened by Southern analysis. Evidence for DT40 mutants was demonstrated by Northern analysis using the probe containing an entire coding region (Fig. 1 C). No truncated transcripts were observed. Wild-type chicken Bam32 cDNA with a COOH-terminal flanking myc epitopic tag was cloned into the pApuro vector (8). Bam32-deficient DT40 cells were transfected with this myc-tagged cDNA construct and selected in the presence of 0.5 μg/ml puromycin (Calbiochem). Transfection was performed by electroporation at 350V, 960 μF.


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)

Disruption of the Bam32 gene in chicken DT40 B cells. (A) Partial restriction map of chicken Bam32 locus and expected structure of the targeted Bam32 alleles. The restriction endonuclease cleavage sites are abbreviated: X, XbaI; E, EcoRI. (B) Southern analysis of genomic DNA from wild-type DT40 cells (lane 1), neo-targeted cells (+/−, lane 2), and neo- and hisD-targeted cells (−/−, lane 3 and 4). Xba I-digested genomic DNA separated on an agarose gel was blotted and hybridized with a chicken Bam32 DNA probe shown in A. Wild-type (WT) and targeted alleles show 15.5- or 5.0-kb fragments, respectively. (C) Northern analysis using a chicken cDNA probe for Bam32 (top) and β-actin (bottom). Wild-type (WT) DT40 cells contain a hybridizable RNA species of ∼3,000 nucleotides. (D) Cell surface expression of BCR. BCR expression on the surface of wild-type (WT) and two independent Bam32-deficient (M80–11, M82–5) DT40 cells was analyzed by flow cytometry. Unstained cells were used as negative controls.
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Related In: Results  -  Collection

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fig1: Disruption of the Bam32 gene in chicken DT40 B cells. (A) Partial restriction map of chicken Bam32 locus and expected structure of the targeted Bam32 alleles. The restriction endonuclease cleavage sites are abbreviated: X, XbaI; E, EcoRI. (B) Southern analysis of genomic DNA from wild-type DT40 cells (lane 1), neo-targeted cells (+/−, lane 2), and neo- and hisD-targeted cells (−/−, lane 3 and 4). Xba I-digested genomic DNA separated on an agarose gel was blotted and hybridized with a chicken Bam32 DNA probe shown in A. Wild-type (WT) and targeted alleles show 15.5- or 5.0-kb fragments, respectively. (C) Northern analysis using a chicken cDNA probe for Bam32 (top) and β-actin (bottom). Wild-type (WT) DT40 cells contain a hybridizable RNA species of ∼3,000 nucleotides. (D) Cell surface expression of BCR. BCR expression on the surface of wild-type (WT) and two independent Bam32-deficient (M80–11, M82–5) DT40 cells was analyzed by flow cytometry. Unstained cells were used as negative controls.
Mentions: A lamda Zap DT40 cDNA library was screened by human Bam32 cDNA. The chicken Bam32 gene exhibited ∼80% identity to human counterpart at the amino acid level. Using the isolated cDNA of chicken Bam32, a chicken DNA library was screened to obtain genomic clones. After subcloning, the targeting vectors pBam32-Neo and pBam32-HisD were made by replacing the genomic fragment containing exons corresponding to amino acid residues 106–148 with neo- and hisD-cassette (Fig. 1 A). These cassettes were flanked by 2.5 and 1.8 kb of chicken Bam32 genomic sequence on the 5′ and 3′ sides, respectively. Targeting constructs were sequentially transfected into wild-type DT40 cells by electroporation to obtain mutants. Selection for drug-resistant clones was performed using G418 (2 mg/ml) and histidinol (1 mg/ml), and clones were screened by Southern analysis. Evidence for DT40 mutants was demonstrated by Northern analysis using the probe containing an entire coding region (Fig. 1 C). No truncated transcripts were observed. Wild-type chicken Bam32 cDNA with a COOH-terminal flanking myc epitopic tag was cloned into the pApuro vector (8). Bam32-deficient DT40 cells were transfected with this myc-tagged cDNA construct and selected in the presence of 0.5 μg/ml puromycin (Calbiochem). Transfection was performed by electroporation at 350V, 960 μF.

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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