A Novel B Lymphocyte–Associated Adaptor Protein, Bam32, Regulates Antigen Receptor Signaling Downstream of Phosphatidylinositol 3-Kinase

Abstract

We have identified and characterized a novel src homology 2 (SH2) and pleckstrin homology (PH) domain–containing adaptor protein, designated Bam32 (for B cell adaptor molecule of 32 kD). cDNAs encoding the human and mouse Bam32 coding sequences were isolated and the human bam32 gene was mapped to chromosome 4q25–q27. Bam32 is expressed by B lymphocytes, but not T lymphocytes or nonhematopoietic cells. Human germinal center B cells show increased Bam32 expression, and resting B cells rapidly upregulate expression of Bam32 after ligation of CD40, but not immunoglobulin M. Bam32 is tyrosine-phosphorylated upon B cell antigen receptor (BCR) ligation or pervanadate stimulation and associates with phospholipase Cγ2. After BCR ligation, Bam32 is recruited to the plasma membrane through its PH domain. Membrane recruitment requires phosphatidylinositol 3-kinase (PI3K) activity and an intact PI(3,4,5)P3-binding motif, suggesting that membrane association occurs through binding to 3-phosphoinositides. Expression of Bam32 in B cells leads to a dose-dependent inhibition of BCR-induced activation of nuclear factor of activated T cells (NF-AT), which is blocked by deletion of the PH domain or mutation of the PI(3,4,5)P3-binding motif. Thus, Bam32 represents a novel B cell–associated adaptor that regulates BCR signaling downstream of PI3K.

A New Tyrosine Phosphorylation Site in PLCγ1: The Role of Tyrosine 775 in Immune Receptor Signaling

“…Additional work is required to distinguish whether phosphorylation alters the intramolecular conformation of PLC␥1 (24), its substrate recognition (12) or protein/ protein interactions that are required for activation (25). Furthermore, although we have shown that Y775 is phosphorylated after TCR engagement in human peripheral T cells and mouse CD4 ϩ T cells, the role that Y775 plays in PLC␥1 activation during T and B cell development and lymphocyte activation in vivo remains to be confirmed.…”

The B Lymphocyte Adaptor Molecule of 32 Kilodaltons (Bam32) Regulates B Cell Antigen Receptor Internalization

“…To visualize Bam32 in intact cells, we used a mammalian plasmid construct encoding Bam32 fused to the fluorescent marker protein, EGFP (14). This construct was transiently expressed in human BJAB cells, and the cells were stimulated with rhodamine-labeled F(abЈ) 2 anti-IgM sera to trigger BCR signaling and internalization (Fig.…”

“…BJAB cells were transfected with Bam32-enhanced GFP (EGFP) as described previously (14) and then stimulated for indicated time periods with 10 g/ml unlabeled or rhodamine-labeled goat anti-human IgM sera (10 g/ml) at 37°C. Cells were washed twice and fixed with 3.7% paraformaldehyde, permeabilized with PBS containing 1% BSA and 0.05% saponin (wash buffer), intracellularly stained with primary Abs for 30 min, and then washed in wash buffer.…”

“…PP1 was purchased from BIOMOL (Plymouth Meeting, PA). An anti-Bam32 serum and mAb (UW32) were described elsewhere (14).…”

“…The B lymphocyte adaptor molecule of 32 kDa, Bam32, also termed dual adaptor for phosphotyrosine and 3-phosphoinositides or 3Ј-phosphoinositide-interacting Src homology-containing protein (12,13), is an adaptor protein containing an N-terminal Src-homology 2 domain, one tyrosine phosphorylation site, and a C-terminal pleckstrin homology domain (14). Upon BCR cross-linking, Bam32 is tyrosine-phosphorylated and inducibly associated with phospholipase C (PLC)␥2 in human B cell lines (14). Moreover, activation of the PLC␥2-related signaling pathways is partially defective in Bam32 Ϫ/Ϫ DT40 cells (15).…”