The Adaptor Protein Bam32 Regulates Rac1 Activation and Actin Remodeling through a Phosphorylation-dependent Mechanism

Abstract

The B cell adaptor molecule of 32 kDa (Bam32) is an adaptor that links the B cell antigen receptor (BCR) to ERK and JNK activation and ultimately to mitogenesis. After BCR cross-linking, Bam32 is recruited to the plasma membrane and accumulates within F-actin-rich membrane ruffles. Bam32 contains one Src homology 2 and one pleckstrin homology domain and is phosphorylated at a single site, tyrosine 139. To define the function of Bam32 in membrane-proximal signaling events, we established human B cell lines overexpressing wild-type or mutant Bam32 proteins. The basal level of F-actin increased in cells expressing wild-type or myristoylated Bam32 but decreased in cells expressing either an Src homology-2 or Tyr-139 Bam32 mutant. Overexpression of wild-type Bam32 also affected BCR-induced actin remodeling, which was visualized as increases in F-actinrich membrane ruffles. In contrast, Bam32 mutants largely blocked the BCR-induced increase in cellular F-actin. The positive and negative effects of Bam32 variants on F-actin levels were closely mirrored by their effects on the activation of the GTPase Rac1, which is known to regulate actin remodeling in lymphocytes. Bam32-deficient DT40 B cells showed decreased Rac1 activation and a failure of Rac1 to co-localize with the BCR, whereas cells overexpressing Bam32 had increased constitutive Rac1 activation. These results suggest that Bam32 regulates the cytoskeleton through Rac1. Bam32 variants also affected downstream signaling to JNK in a manner similar to that of Rac1, suggesting that the effect of Bam32 on JNK activation may be at least partially mediated through Rac1. Our results demonstrate a novel phosphorylation-dependent function of Bam32 in regulating Rac1 activation and actin remodeling.Signaling through antigen receptors expressed by T and B lymphocytes is critical for the induction and regulation of immune responses. These multi-subunit protein tyrosine kinaselinked receptors share many common signaling mechanisms with other protein tyrosine kinase-linked receptors, including the recruitment of the activated receptor to lipid rafts, the activation of several protein tyrosine kinases, and the use of adaptor proteins to link the kinase cascade to downstream amplification and effector pathways. The B cell antigen receptor (BCR)

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

“…One possible group is the Rho-family of GTPases such as Rac and Cdc42 which regulate a wide range of important cellular functions including actin polymerization, transcriptional activation, cell survival, and cell cycle entry (43). Interestingly, we found that Bam32 regulates BCR-induced activation of Rac1, suggesting that Bam32 regulates actin remodeling in B cells in part by a Rac-dependent mechanism (44). However, in contrast to a previous study (45), when we expressed a dominant-negative form of Rac1 in DT40 cells, we found that it had only a marginal effect on BCR internalization (data not shown).…”

“…An important clue to defining the molecules involved in this process is the fact that Bam32 needs to be tyrosine phosphorylated to regulate BCR internalization. Indeed, BCR-induced actin polymerization is impaired in BJAB cells expressing a point mutant (Y139F) of Bam32 (44). It is possible that tyrosine phosphorylation of Bam32 causes a conformational change which in turn facilitates the interaction of Bam32 with actin-related molecules.…”

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

“…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. anti-IgM).…”

The Pleckstrin Homology Domain Adaptor Protein Bam32/DAPP1 Is Required for Germinal Center Progression

“…Bam32 phosphorylation by Src family kinases is also PI3K-dependent (17,22). Bam32 has been implicated in BCR signaling processes, including activation of the GTPase Rac1, and MAPKs ERK and JNK (23)(24)(25). Bam32-deficient B cells develop normally, but show markedly impaired BCR-triggered proliferation in vitro; however, proliferation could be fully recovered with addition of anti-CD40 and/or IL-4 stimuli (23,26).…”