Systems‐wide analysis of BCR signalosomes and downstream phosphorylation and ubiquitylation

Abstract

B-cell receptor (BCR) signaling is essential for the development and function of B cells; however, the spectrum of proteins involved in BCR signaling is not fully known. Here we used quantitative mass spectrometry-based proteomics to monitor the dynamics of BCR signaling complexes (signalosomes) and to investigate the dynamics of downstream phosphorylation and ubiquitylation signaling. We identify most of the previously known components of BCR signaling, as well as many proteins that have not yet been implicated in this system. BCR activation leads to rapid tyrosine phosphorylation and ubiquitylation of the receptor-proximal signaling components, many of which are co-regulated by both the modifications. We illustrate the power of multilayered proteomic analyses for discovering novel BCR signaling components by demonstrating that BCR-induced phosphorylation of RAB7A at S72 prevents its association with effector proteins and with endo-lysosomal compartments. In addition, we show that BCL10 is modified by LUBAC-mediated linear ubiquitylation, and demonstrate an important function of LUBAC in BCR-induced NF-κB signaling. Our results offer a global and integrated view of BCR signaling, and the provided datasets can serve as a valuable resource for further understanding BCR signaling networks.

Elucidation of tonic and activated B-cell receptor signaling in Burkitt’s lymphoma provides insights into regulation of cell survival

“…Recently, Satpathy et al (36) described phosphoproteomic changes that occurred upon stimulation of BCR in the murine B-cell line A20 (36). In addition to a common set of BCR signaling effectors that were identified in both studies, we identified more than 200 so far unreported BCR effectors.…”

Molecular Determinants of Scaffold-induced Linear Ubiquitinylation of B Cell Lymphoma/Leukemia 10 (Bcl10) during T Cell Receptor and Oncogenic Caspase Recruitment Domain-containing Protein 11 (CARD11) Signaling

“…Consistent with the latter, Sasaki et al (40) reported that the inducible deletion of the RBR domain of HOIP, which is required for catalytic activity, in murine B cells had little effect on BCR signaling to NF-B. However, in an unbiased survey of proteins ubiquitinylated during BCR signaling, Satpathy et al (41) discovered that Bcl10 is conjugated with linear ubiquitin chains in response to BCR engagement. Furthermore, Yang et al (42) also observed Bcl10 linear ubiquitinylation during the dysregulated chronic BCR signaling associated with ABC DLBCL, in a step proposed to lie downstream of the cIAP-mediated modification of Bcl10 with Lys-63-linked polyubiquitin chains.…”

Understanding Epstein-Barr Virus Life Cycle with Proteomics: A Temporal Analysis of Ubiquitination During Virus Reactivation

“…Because BCR activation triggers a dramatic downregulation in ubiquitination at later time points, it is not suitable to obtain a cutoff value using a normal distribution-based method for regulated ubiquitination. Therefore, we applied a commonly used cutoff value of twofold change for differentially regulated ubiquitination upon BCR activation (Satpathy et al, 2015;Tong et al, 2014). Peptides with ratios greater than twofold change were used for further analysis.…”

“…Previously, proteomic studies were performed to examine the ubiquitination events shortly after IgG cross-linking of mouse or human B cell lines ( Jayasundera et al, 2014;Satpathy et al, 2015). To obtain a global view of the long-term effects on protein ubiquitination, we utilized SILAC-based proteomics to survey the dynamic changes in ubiquitination in EBV + B cells upon IgG cross-linking (Harsha et al, 2008;Ong et al, 2002).…”

“…The cellular response to BCR activation involves the assembly of BCR signaling proteins that regulate a cascade of downstream signaling events, including protein phosphorylation and ubiquitination ( Jayasundera et al, 2014;Matsumoto et al, 2009;Satpathy et al, 2015). System-wide identification of downstream signaling events has revealed important roles for phosphorylation and ubiquitination within minutes of the BCR activation process.…”