Phosphorylation of the multifunctional signal transducer B-cell adaptor protein (BCAP) promotes recruitment of multiple SH2/SH3 proteins including GRB2

Nicholas J Gay,Samer Halabi,Atul Udgata,Alex Bartram,Delphine De Sutter,David I Fisher,Sven Eyckerman,Johannes U Lauenstein

doi:10.1074/jbc.ra119.009931

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.

Highlights

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
We determined that phosphorylation is the basis of this phenomenon and identified that BCAP is the substrate of several kinases including tyrosine kinases BTK, SYK, and LYN
BCAP tyrosine phosphorylation is a central part of BCAP activation and has been observed downstream of Tolllike receptors (TLRs), IL-1R, and B-cell receptor (BCR) signaling complexes [16, 19, 20]

Summary

Introduction

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. 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). It was proposed that synergistic BCR/TLR signaling may be part of a supercomplex controlling oncogenic signaling in two major subtypes of diffuse large B-cell lymphomas (DLBCL) [9] These findings are substantiated by the fact that TLRs and BCR share a common pool of adaptor proteins and kinases. Most TLR receptors and several adaptor proteins are reported to be tyrosine phosphorylated, the kinases responsible and the functional importance remain unclear [13]

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