Tyrosine phosphorylation of 3BP2 regulates BCR-mediated activation of NFAT. (109.3)

Abstract

Abstract Adaptor protein c-Abl SH3 domain-binding protein-2 (3BP2, also referred to SH3BP2) regulates immune receptor-mediated signal transduction. In this report, we focused on the molecular mechanism of 3BP2 function in B cell receptor (BCR) signaling. Engagement of BCR induces tyrosine phosphorylation of 3BP2. Genetic analysis demonstrated that Syk is critical for BCR-mediated tyrosine phosphorylation of 3BP2. Mutational analysis of 3BP2 revealed that both Tyr183 and Src homology 2 (SH2) domain are necessary for 3BP2-mediated BCR-induced activation of nuclear factor of activated T cells (NFAT). Point mutation of Tyr183 or Arg486 in the SH2 domain of 3BP2 diminished BCR-mediated tyrosine phosphorylation of 3BP2. Endogenous 3BP2 forms complex with tyrosine phosphorylated cellular signaling molecules. Peptide binding experiments demonstrated that only phosphorylated Tyr183 in 3BP2 could form a complex with the SH2 domain(s) of phospholipase C (PLC)-γ2 and Vav1 from B cell lysates. These interactions were represented by using bacterial GST-PLC-γ2 or -Vav1 SH2 domain(s). Furthermore, pull down and far-western experiments showed that 3BP2-SH2 domain directly binds to B cell linker protein (BLNK) following BCR stimulation. These results demonstrated that 3BP2 induces protein complex with cellular signaling molecules through phosphorylation of Tyr183 and SH2 domain leading to the activation of NFAT in B cells.