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

Abstract

The B lymphocyte adaptor molecule of 32 kDa (Bam32) is an adaptor that plays an indispensable role in BCR signaling. In this study, we found that upon BCR ligation, Bam32 is recruited to the plasma membrane where it associates with BCR complexes and redistributes and internalizes with BCRs. BCR ligation induced colocalization of Bam32 with lipid rafts, clathrin, and actin filaments. An inhibitor of Src family protein tyrosine kinases (PTKs) blocked both BCR-induced tyrosine phosphorylation of Bam32 and BCR internalization. Moreover, BCR internalization is impaired in Bam32−/− and Lyn−/− cells, and expression of Bam32 with a mutation of its tyrosine phosphorylation site (Y139F) inhibited BCR internalization. These data suggest that Bam32 functions downstream of Src family PTKs to regulate BCR internalization. Bam32 deficiency does not affect tyrosine phosphorylation of clathrin or the association of clathrin with lipid rafts upon BCR cross-linking. However, BCR-induced actin polymerization is impaired in Bam32−/− cells. Collectively, these findings indicate a novel role of Bam32 in connecting Src family PTKs to BCR internalization by an actin-dependent mechanism.

Functional and structural requirements for the internalization of distinct BCR‐ligand complexes

“…This evidence could reconcile our results with recent reports that have stressed the role of lipid rafts and BCR signaling in BCR internalization, especially the recent report in which the authors highlight the importance of the protein Bam32 in anti-IgM-induced BCR internalization [34]. Interestingly, Bam32 is an adaptor protein that has been involved in Rac-mediated actin cytoske-leton remodeling at the cell membrane [45], suggesting that signaling-dependent cytoskeletal rearrangements are necessary for the internalization of more "complex" ligands (e.g.…”

“…In contrast to the analysis presented above, most published studies investigating the mechanisms of BCR internalization, including the role of signaling in BCR endocytosis, have relied on the use of polyclonal anti-heavy chain Ab as a BCR ligand [3,11,16,18,23,[32][33][34]. However, the nature of BCR ligation by the anti-IgM Ab is distinct from that of Ag.…”

“…In this regard, it is important to note that all of the published reports that suggest a role for signaling in BCR internalization were performed in B cell lines of unknown BCR specificity, thus obliging the use of polyclonal anti-Ig Ab as BCR ligands [15,16,23,34]. Thus, by analyzing the behavior of both PC-BSA (Ag) and polyclonal anti-IgM Ab in the same model system, it was determined that despite both ligands localize to coated pits to similar levels (Fig.…”

Requirement for Phosphoinositide 3-Kinase p110δ Signaling in B Cell Antigen Receptor-Mediated Antigen Presentation

“…The 2R.50 T cells express MHC class II (I-A d )-restricted TCR specific for rabbit IgG and were maintained and used as previously described (34,35). The LK35.2 B cell transfectant (HyHEL10), which expresses a hen egg lysozyme (HEL)-specific IgM BCR, and 2G7 T cells, which express a MHC class II (I-E k )-restricted TCR specific for HEL [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18] , were gifts from F. Batista (Medical Research Laboratory of Molecular Biology, Cambridge, U.K.) and were maintained and used as previously described (36).…”

“…Activation of Src family kinases, particularly Lyn, can regulate BCR internalization in some systems (7)(8)(9), perhaps via phosphorylation of clathrin H chain (10) and/or the adaptor protein Bam32 (11). Subsequent sorting of internalized BCR-Ag complexes into late endosomes and development of mature MHC class II-enriched compartment (MIIC) 3 (12)(13)(14) serve to quantitatively enhance generation of peptide-MHC complexes (15), and may promote expression of peptide-MHC complexes in a functional conformation capable of activating Ag-specific T cells and acting as a signal transduction unit for the B cell (16).…”