Adaptor Protein 3BP2 Is a Potential Ligand of Src Homology 2 and 3 Domains of Lyn Protein-tyrosine Kinase

Abstract

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...

Flotillin-1 Regulates IgE Receptor-Mediated Signaling in Rat Basophilic Leukemia (RBL-2H3) Cells

“…Cross-linking of FcRI by Ag triggers a series of protein phosphorylation reactions, phosphorylation of FcRI by Lyn, followed by the phosphorylation of Syk, Btk, and phospholipase C (22)(23)(24) (26 -28). Thus, we examined whether flotillin-1 affects phosphorylation of the MAPK cascade.…”

The Adaptor Protein 3BP2 Binds Human CD244 and Links this Receptor to Vav Signaling, ERK Activation, and NK Cell Killing

“…Recently, it has been reported that the chaperone protein 14-3-3 interacts with the proline-rich domains of 3BP2 and negatively regulates 3BP2 adaptor function in lymphocytes (3). In addition, 3BP2 binds to the SH2 and SH3 domains of the protein tyrosine kinase Lyn during Fc⑀RI-mediated signaling in mast cells (4). Besides mast cells, 3BP2 also has a key regulatory role in NK cells.…”

L’adaptateur 3BP2

“…Ce domaine paraît indispensable, car sa délétion réduit la capacité de 3BP2 à augmenter l'activité de facteurs nucléaires comme NFAT dans les cellules T et B [4,6]. Le domaine riche en proline, qui s'étend entre les domaines PH et SH2 sur plus de 200 acides aminés, contient de multiples motifs PxxP représentant des sites de fixation pour des protéines à domaine SH3, parmi lesquelles Abl [3], Vav [6], Fyn [4] et Lyn [7]. Le criblage de banques de peptides dégénérés par le groupe de Cantley a montré que la séquence de fixation optimale pour le domaine SH2 de 3BP2 se compose des acides aminés phosphoTyr-Glu-Asn (pYEN), un motif retrouvé dans plusieurs molécules de signalisation incluant les récepteurs tyrosine kinases Flt3/Flk2, EpoR, et G-CSFR [8].…”

“…L'interaction entre une forme recombinante du domaine SH2 de 3BP2 et un résidu phosphotyrosine du domaine intracytoplasmique du G-CSFR, connu pour lier Grb2 et Shc, a été récemment décrite, mais les conséquences fonctionnelles de cette interaction pour les cellules hématopoïétiques restent inconnues [9]. Le domaine SH2 de 3BP2 peut également se lier au motif YEN présent dans la partie intracytoplasmique de l'adapteur transmembranaire LAT exprimé par les cellules T et NK [4,10] [7,12]. Le résidu phospho-Tyr183 repré-sente un site de fixation du domaine SH2 de Vav1 [11], alors que le résidu phophoTyr446 interagit avec les domaines SH2 de Lyn et Lck [7,12].…”

“…Le domaine SH2 de 3BP2 peut également se lier au motif YEN présent dans la partie intracytoplasmique de l'adapteur transmembranaire LAT exprimé par les cellules T et NK [4,10] [7,12]. Le résidu phospho-Tyr183 repré-sente un site de fixation du domaine SH2 de Vav1 [11], alors que le résidu phophoTyr446 interagit avec les domaines SH2 de Lyn et Lck [7,12]. L'importance de ces événements est illustrée par le fait que la surexpression de formes mutées de 3BP2 sur les résidus Tyr183 ou Tyr446 réduit la signalisation par 3BP2 dans les cellules T ( [12] et Deckert, résultats non publiés) et NK [11].…”