Adaptor protein 3BP2 was originally isolated as a proteintyrosine kinase c-Abl-Src homology 3 (SH3) 1 domain-binding protein of unknown function (1). 3BP2 was also identified as a Syk kinase-interacting protein by the yeast two-hybrid screening (2). Transient overexpression of 3BP2 resulted in a transcriptional activation of nuclear factor of activated T cell and activator protein 1, which is induced by T-cell receptor aggregation. Ser 225 and Ser 277 of 3BP2 were identified as essential sites for interacting with 14-3-3 to negatively regulate the function of 3BP2 in lymphocytes (3). Moreover, infection of 3BP2 wild type into NK cells by vaccinia virus enhanced cell cytotoxicity. An in vitro binding study suggested that phosphorylation of Tyr 183 of 3BP2 could be associated with Vav and phospholipase C-␥ (4). In mast cells, overexpression of the 3BP2-SH2 domain suppressed high affinity IgE receptor (Fc⑀RI)-mediated tyrosine phosphorylation of phospholipase C-␥, Ca 2ϩ mobilization, and degranulation (5). These findings have demonstrated that 3BP2 plays a critical role in hematopoietic cells.To propagate the immunoreceptor signal, adaptor proteins contribute to protein-protein and protein-lipid interactions through multiple domains and/or specific phosphotyrosine-containing sequences. Tyrosine phosphorylation of 3BP2 was observed in NK cells and mast cells by cross-linking Fc␥R and Fc⑀RI, respectively (4, 5). To elucidate the function of 3BP2, it is necessary to determine the protein-tyrosine kinase (PTK) that phosphorylates 3BP2 and its binding partner to assemble a signaling complex through specific phosphotyrosine-containing motifs in 3BP2.The Src family PTK Lyn is associated with Fc⑀RI␤. Upon aggregation of Fc⑀RI, Lyn is critical for phosphorylating Fc⑀RI␤ and -␥ subunits on Tyr residues within the immunoreceptor tyrosine-based activating motif (ITAM) (6 -8). By analogy with studies on Hck, Lyn is thought to be activated by the disassembly of the closed intramolecular interaction by (i) CD45-mediated dephosphorylation of C-terminal regulatory Tyr residue, (ii) binding to SH3 and SH2 ligands, and (iii) autophosphorylation of Tyr in the activation loop (9). What is the binding ligand of the SH3 and SH2 domains of Lyn in Fc⑀RI signaling pathway? Pull-down experiments using glutathione S-transferase (GST)-Lyn-SH2 fusion protein indicated that there were multiple phosphoproteins interacting with the Lyn-SH2 after the antigen stimulation of RBL-2H3 cells (10). In addition, although the displacement of intramolecular SH3 interaction is not well understood, it seems likely that some aggregation-induced change in an associated molecule provides a higher affinity SH3 ligand that binds to the Lyn-SH3 domain (11). The SH3 domain is directed toward the proline-rich region, but such a ligand has not been identified yet in the Fc⑀RI signaling.We isolated nonreceptor type PTK Syk from porcine spleen (12). Syk is expressed in hematopoietic, epithelial, and endothelial cells (13)(14)(15)(16). When the ITAM of Fc⑀RI␥ subunits is...

Background : Recent studies have demonstrated that c-Cbl functions as a ubiquitin-protein ligase toward immune receptors and non-receptor protein-tyrosine kinase Syk by facilitating their ubiquitination and subsequent targeting to proteasomes. However, it was not clear whether Src family kinase Lyn is regulated by the Cbl family of ubiquitin-protein ligases.

Molecular adaptors/scaffolds have indispensable roles in the activation of lymphocytes. In this report, we have demonstrated the role of tyrosine phosphorylation of an adaptor protein 3BP2 (c-Abl-SH3 domain binding protein-2, also known as SH3BP2) in T cell receptor (TCR)-mediated activation of transcription factor. Short interfering RNA for 3BP2 suppresses the expression level of endogenous 3BP2 and inhibits TCR-mediated activation of interleukin (IL)-2 promoter and nuclear factor of activated T cells (NFAT) element. Engagement of TCR induces tyrosine phosphorylation and lipid raft translocation of 3BP2. The overexpression studies reveal that substitution of 3BP2-Tyr(183), Tyr(446), or Arg(486) in the SH2 domain suppresses TCR-mediated activation of NFAT. Point mutations of 3BP2 cannot affect the translocation of 3BP2 into the lipid raft. Phosphorylation of Tyr(183) is required for the interaction with Vav1, the guanine nucleotide exchanging factor of Rac1. In fact, overexpression of dominant-negative form of Rac1 inhibits TCR-mediated activation of NFAT. Phosphorylation of Tyr(446) recruits the SH2 domain of Lck for the optimal activation of transcription factors. Furthermore, point mutation of Arg(486) in the 3BP2-SH2 domain that couples ZAP-70 to LAT dramatically reduces NFAT activation. These results suggest that the site-directed functions of 3BP2 induce the activation of transcription factors.