Abstract
Adaptor protein 3BP2, a c-Abl-Src homology 3 (SH3) domain-binding protein, is known to play a regulatory role in T-cell receptor-mediated transcriptional activation of nuclear factor of activated T cell and activator protein 1 by interacting with Syk/ZAP-70 protein-tyrosine kinase. We have previously demonstrated that aggregation of high affinity IgE receptor (FcepsilonRI) induces tyrosine phosphorylation of 3BP2, and overexpression of the 3BP2-SH2 domain suppresses antigen-induced degranulation in rat basophilic leukemia RBL-2H3 mast cell line. In this report, we attempt to analyze the biological relevance of 3BP2 tyrosine phosphorylation. By using the transient expression system in COS-7 cells, we have demonstrated that 3BP2 was predominantly phosphorylated on Tyr174, Tyr183, and Tyr446 when it was coexpressed with Syk. An in vitro binding study revealed that phosphorylation of Tyr446 by Syk was likely to create a binding site for the Lyn-SH2 domain in RBL-2H3 cells. In addition, proline-rich region of 3BP2 bound to the Lyn-SH3 domain. Conformational microscopic analysis showed that Lyn and 3BP2 are constitutively colocalized in RBL-2H3 cells. Overexpression of 3BP2 in RBL-2H3 cells resulted in an enhancement of Lyn autophosphorylation. These results suggest that the adaptor protein 3BP2 is a potential regulator of Lyn protein-tyrosine kinase as a ligand of its SH3/SH2 domains in FcepsilonRI-mediated signaling in mast cells.
Highlights
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 identified as a Syk kinase-interacting protein by the yeast two-hybrid screening [2]
These results suggest that the adaptor protein 3BP2 is a potential regulator of Lyn proteintyrosine kinase as a ligand of its SH3/SH2 domains in Fc⑀RI-mediated signaling in mast cells
Tyrosine Kinase Syk Phosphorylated Tyr174, Tyr183, and Tyr446 in 3BP2—We previously reported the Fc⑀RI-mediated tyrosine phosphorylation of the adaptor protein 3BP2 in RBL2H3 mast cells [5]. 3BP2 is rapidly tyrosine-phosphorylated and reached a peak 1 min after stimulation, suggesting that proximal nonreceptor type protein-tyrosine kinase (PTK) may contribute to tyrosine phosphorylation of 3BP2
Summary
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 identified as a Syk kinase-interacting protein by the yeast two-hybrid screening [2]. These results demonstrate that Tyr174, Tyr183, and Tyr446 in 3BP2 are the putative tyrosine phosphorylation sites by nonreceptor type PTKs, in particular Syk. 3BP2-Tyr446 Was a Major Binding Site to the Lyn-SH2 Domain—To examine the role of 3BP2 tyrosine phosphorylation, we utilized the RBL-2H3 cells in which the expression of HA3BP2 is induced by the addition of IPTG.
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