Abstract
B-cell adaptor protein (BCAP) is a multimodular, multifunctional signal transducer that regulates signal transduction pathways in leukocytes, including macrophages, B-cells, and T-cells. In particular, BCAP suppresses inflammatory signaling by Toll-like receptors (TLRs). However, how BCAP itself is regulated and what its interaction partners are is unclear. Here, using human immune cell lines, including THP-1 cells, we characterized the complex phosphorylation patterns of BCAP and used a novel protein complex trapping strategy, called virotrap, to identify its interaction partners. This analysis identified known interactions of BCAP with phosphoinositide 3-kinase (PI3K) p85 subunit and NCK adaptor protein (NCK), together with previously unknown interactions of BCAP with Src homology 2 (SH2) and SH3 domain-containing adaptor proteins, notably growth factor receptor-bound protein 2 (GRB2) and CRK-like proto-oncogene, adaptor protein (CRKL). We show that the SH3 domain of GRB2 can bind to BCAP independently of BCAP phosphorylation status, suggesting that the SH2 domains mediate interactions with activated receptor tyrosine kinase complexes including the CD19 subunit of the B-cell receptor. Our results also suggested that the PI3K p85 subunit binds to BCAP via SH3 domains forming an inactive complex that is then activated by sequential binding with the SH2 domains. Taken together, our results indicate that BCAP is a complex hub that processes signals from multiple pathways in diverse cell types of the immune system.