Editors' ChoiceProteomics

B cell receptor signaling dynamics

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Sci. Signal.  07 Jul 2015:
Vol. 8, Issue 384, pp. ec186
DOI: 10.1126/scisignal.aac9222

Antigen binding to B cell receptors (BCRs) leads to the assembly of BCR signaling complexes (signalosomes), which initiate downstream signaling through the phosphorylation and ubiquitylation of cellular proteins. Satpathy et al. used stable isotope labeling by amino acids (SILAC)–based quantitative mass spectrometry to investigate BCR signaling in A20 cells, a murine B cell lymphoma cell line that expresses IgG2a-type BCRs. To investigate the dynamics of BCR signalosome assembly, signaling was activated by binding BCRs with biotin-labeled anti-IgG F(ab′)2 and terminated at 5 and 15 min by lysing the cells. Mass spectrometric analysis of streptavidin-purified complexes revealed that 154 proteins were present in the signalosomes and that the number and identity of the recruited proteins changed over time. Some of these proteins, including KLHL6, SPRED1, and ANKRD13A, were not previously thought to form BCR signalosomes. To analyze the dynamics of the phosphorylation and ubiquitylation of downstream signaling components, signaling was initiated by crosslinking BCRs with the F(ab′)2 fragment of rabbit anti-mouse IgG. Phosphorylated peptides in the cell lysates were enriched for both serine/threonine and tyrosine-containing peptides and then were analyzed by mass spectrometry. BCR stimulation resulted in the phosphorylation of 1829 serine and threonine sites and 124 tyrosine sites. Immunofluorescence analysis of cells expressing wild-type or mutant forms of a regulator of the early-to-late maturation of endosomes, RAB7A, which was phosphorylated at Ser72 in response to BCR stimulation, confirmed that its association with endolysosomal compartments was blocked by phosphorylation. Analysis of purified ubiquitylated peptides indicated the presence of ~300 ubiquitylation sites after BCR stimulation. Further analysis revealed that BCL10, which is required to activate nuclear factor κB (NF-κB) signaling, underwent linear ubiquitylation by LUBAC in response to BCR stimulation. Approximately 65 proteins were regulated by both phosphorylation and ubiquitylation. In characterizing the dynamic nature of BCR signalosomes and the complex regulation of proteins by phosphorylation and ubiquitylation in response to BCR stimulation, this study should serve as a resource for the analysis of B cell signaling.

S. Satpathy, S. A. Wagner, P. Beli, R. Gupta, T. A. Kristiansen, D. Malinova, C. Francavilla, P. Tolar, G. A. Bishop, B. S. Hostager, C. Choudhary, Systems-wide analysis of BCR signalosomes and downstream phosphorylation and ubiquitylation. Mol. Syst. Biol. 11, 810 (2015). [PubMed]