3BP2 Deficiency Impairs the Response of B Cells, but Not T Cells, to Antigen Receptor Ligation

Abstract

The adapter protein 3BP2 is expressed in lymphocytes; binds to Syk/ZAP-70, Vav, and phospholipase C-␥ (PLC-␥); and is thought to be important for interleukin-2 gene transcription in T cells. To define the role of 3BP2 in lymphocyte development and function, we generated 3BP2-deficient mice. T-cell development, proliferation, cytokine secretion, and signaling in response to T-cell receptor (TCR) ligation were all normal in 3BP2 ؊/؊ mice. 3BP2 ؊/؊ mice had increased accumulation of pre-B cells in the bone marrow and a block in the progression of transitional B cells in the spleen from the T1 to the T2 stage, but normal numbers of mature B cells. B-cell proliferation, cell cycle progression, PLC-␥2 phosphorylation, calcium mobilization, NF-ATp dephosphorylation, and Erk and Jnk activation in response to B-cell receptor (BCR) ligation were all impaired. These results suggest that 3BP2 is important for BCR, but not for TCR signaling.3BP2 is a protein originally characterized as an Abl SH3-interacting protein (44). It consists of an N-terminal pleckstrin homology domain, a proline-rich central region, and a C-terminal SH2 domain. 3BP2 mRNA is expressed in hematopoietic tissues. 3BP2 protein is expressed in T cells, B cells, natural killer (NK) cells, monocytic cell lines, osteoclasts, and the rat basophilic leukemia cell line RBL-2H3 (11, 37). 3BP2 associates with 36). Association of 3BP2 with Syk was shown to require the SH2 domain of 3BP2 and the catalytic activity of Syk. The SH2 domain of 3BP2 has also been shown to bind to the adapter protein LAT in T cells and mast cells after T-cell receptor (TCR) and FcεRI ligation, respectively (11, 46). Phospholipase C-␥ (PLC-␥) and Vav were also identified as binding partners of 3BP2 (22). Other partners of 3BP2 include Grb2, Cbl, and Fyn (11,14,15).TCR stimulation induces a significant translocation of 3BP2 to the membrane and detergent-insoluble (cytoskeleton) fractions, suggesting a role for 3BP2 in TCR-mediated signal transduction (11). Transient overexpression of 3BP2 in Jurkat T cells induces transcriptional activation of the interleukin-2 (IL-2) gene promoter and its NF-AT and AP-1 elements and cooperates with TCR ligation and ionomycin in activating NF-AT/AP-1-driven transcription. Overexpression of 3BP2 resulted in calcineurin-dependent dephosphorylation of NF-ATc and stimulated AP-1 activity independently of NF-AT (11). The SH2 and PH domains of 3BP2, but not its proline-rich domain, are required for NF-AT activation. Furthermore, overexpression of the SH2 domain of 3BP2 inhibited TCRmediated NF-AT activation (11). Activation of NF-AT by 3BP2 in T cells required ZAP-70, because overexpression of 3BP2 in a ZAP-70-deficient Jurkat cell clone failed to activate NF-AT (11). Overexpression of 3BP2 enhances NK cell-mediated cytotoxicity (22). FcεRI ligation induces the phosphorylation of 3BP2 and its association with LAT in rat basophilic leukemia RBL-2H3 cells (46). Overexpression of the SH2 domain of 3BP2 in these cells suppresses FcεRI-induced signaling (46).Recent...

3BP2 Adapter Protein Is Required for Receptor Activator of NFκB Ligand (RANKL)-induced Osteoclast Differentiation of RAW264.7 Cells

“…Osteoclasts from DAP12 ϫ FcR␥-null mice exhibit defectiveNFATc1expressionasaconsequenceofimpairedPLC␥-dependent calcium signaling (6). Of note, B cells from 3BP2 Ϫ/Ϫ mice show impaired PLC␥ phosphorylation, calcium mobilization, and NFAT activation (24). Our observations that in myeloid cells 3BP2 forms a signaling complex with c-Src, Syk, Vav, and Cbl provide a mechanistic explanation of how 3BP2 might participate in RANK-mediated osteoclast formation.…”

“…Consistently, 3BP2 was found to positively regulate the activity of NFAT in T and B cells (21,23). In addition, studies in mouse deficient for 3BP2 expression have shown that 3BP2 regulates B cell development and BCR-mediated B cell activation and calcium mobilization (24,25). Finally, genetic evidence linking 3BP2 to the human genetic bone disease cherubism (31,32) indicates that 3BP2 also plays a crucial role during inflammation and bone remodeling.…”

“…These scaffolds transduce signals to the cytoplasm, cytoskeleton, and nucleus to activate lipid and calcium signaling, gene expression, and metabolic changes involved in leukocyte proliferation, differentiation, and motility. We and others have identified a regulatory role for 3BP2 (c-Abl SH3 domainbinding protein-2, also known as SH3BP2) in immunoreceptor signaling, including T (21,22), B (23)(24)(25)(26), NK (27), and mast (28) cells. Importantly, 3BP2 associates several signaling proteins such as Src/Syk kinases, Vav proteins, and PLC␥, involved in NFAT activation (reviewed in Refs.…”

Tyrosine Phosphorylation of 3BP2 Regulates B Cell Receptor-mediated Activation of NFAT

“…Anti-PLC-␥2, anti-CD19, anti-Vav1, and anti-BLNK antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA). Polyclonal anti-3BP2 antibody raised against an amino acid sequence from Leu 359 to Ser 462 of 3BP2, which is capable for the immunoprecipitation of 3BP2, was a gift from Dr. Raif S. Geha (Harvard Medical School) (32). Anti-glutathione S-transferase (GST) mAb was from Nacalai (Kyoto, Japan).…”

“…Suppression of the 3BP2 expression by small interfering RNA results in the inhibition of BCR and T cell receptor (TCR)-mediated activation of NFAT (25,31). Analysis of 3BP2-deficient mice demonstrated that 3BP2 is required for B cell proliferation and cell cycle progression (32,33). Lack of 3BP2 results in the abnormal phenotype in splenic marginal-zone B cells and peritoneal B1 B cells and diminished thymus-independent type 2 antigen response (33).…”