Early Events in B Cell Activation

Harwood, Naomi E.; Batista, Facundo D.

doi:10.1146/annurev-immunol-030409-101216

Cited by 337 publications

(355 citation statements)

References 152 publications

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“…3A). Because Syk is activated in B cells following aggregation of the BCR, we treated DG75 cells with antibodies against surface IgM (25). We detected the receptor-stimulated phosphorylation of HCA59, CLNS1A, Nek9, and BOB1, indicating that these substrates may be involved in signaling pathways downstream of the BCR.…”

Section: Resultsmentioning

confidence: 96%

Sensitive kinase assay linked with phosphoproteomics for identifying direct kinase substrates

Xue¹,

Wang²,

Iliuk³

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

119

138

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Our understanding of the molecular control of many disease pathologies requires the identification of direct substrates targeted by specific protein kinases. Here we describe an integrated proteomic strategy, termed kinase assay linked with phosphoproteomics, which combines a sensitive kinase reaction with endogenous kinase-dependent phosphoproteomics to identify direct substrates of protein kinases. The unique in vitro kinase reaction is carried out in a highly efficient manner using a pool of peptides derived directly from cellular kinase substrates and then dephosphorylated as substrate candidates. The resulting newly phosphorylated peptides are then isolated and identified by mass spectrometry. A further comparison of these in vitro phosphorylated peptides with phosphopeptides derived from endogenous proteins isolated from cells in which the kinase is either active or inhibited reveals new candidate protein substrates. The kinase assay linked with phosphoproteomics strategy was applied to identify unique substrates of spleen tyrosine kinase (Syk), a protein-tyrosine kinase with duel properties of an oncogene and a tumor suppressor in distinctive cell types. We identified 64 and 23 direct substrates of Syk specific to B cells and breast cancer cells, respectively. Both known and unique substrates, including multiple centrosomal substrates for Syk, were identified, supporting a unique mechanism that Syk negatively affects cell division through its centrosomal kinase activity.P rotein kinases and their substrates represent the largest signaling network that regulates protein-protein interactions, subcellular localization, and ultimately cellular functions (1, 2). Deregulation of the signaling network often leads to disease states such as human malignancies, diabetes, and immune disorders. Although many kinases are excellent therapeutic targets, the precise connection between protein kinases and their direct substrates has not been fully elucidated for a majority of protein kinases. Besides classical genetic and biochemical methods, there have been a number of high throughput approaches for the identification of potential kinase substrates. Common methods include in vitro kinase assays using libraries of synthetic peptides (3), phase expression libraries (4), protein/peptide arrays (5-7), or cell extracts (8, 9), but these methods can often be misleading and provide many false positive results. The discovery of physiological substrates for specific protein kinases has remained challenging, even with recent advances in mass spectrometry.Mass spectrometry-based proteomics has become a powerful tool and been applied to map protein interaction networks, including kinase/phosphatase-substrate networks (10). Large-scale phosphoproteomics, however, does not typically reveal precise connections between protein kinases and their direct substrates (11,12). In recent years, there have been increasing attempts to develop mass spectrometry-based proteomic strategies for the identification of elusive kinase substrates (7,13,1...

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Section: Resultsmentioning

confidence: 96%

Sensitive kinase assay linked with phosphoproteomics for identifying direct kinase substrates

Xue¹,

Wang²,

Iliuk³

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

119

138

View full text Add to dashboard Cite

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“…This will finally lead to the transformation into plasma cell and the antibody secretion. 89 B cells can, however, also present antigen and produce cytokines. Little is known about the participation of B cells in pregnancy, and their study has been mostly concentrated on the production of autoantibodies during pathologic pregnancies.…”

Section: The Final Players: B Cells Directing the Immune Response Tomentioning

confidence: 99%

Adaptive Immune Responses During Pregnancy

Zenclussen

2013

American J Rep Immunol

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It has long been believed that there is no immune interaction between mother and conceptus during pregnancy. This concept changed after evidence was provided that the maternal immune system is aware of the semiallogeneic conceptus and develops strategies to tolerate it. Since then, finely regulated mechanisms of active tolerance toward the fetus have been described. This Special Issue of the American Journal of Reproductive Immunology deals with these mechanisms. It begins with the description of minor histocompatibility antigens in the placenta; it further goes through adaptive immune responses toward paternal fetal antigens, mostly concentrating on regulatory T cells and molecules modulating the Th1/Th2 balance. The participation of antibody‐producing B cells in normal and pathological pregnancies is also discussed. This introductory chapter resumes the concepts presented throughout the Issue and discusses the clinical applications raised from these concepts.

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“…Mature B cells are maintained through tonic BCR, CD40, and B-cell-activating factor receptor (BAFFR) signaling (4). Activation by cross-linking the BCR leads to rapid proliferation, somatic hypermutation, and further differentiation of B cells (5,6). The expansion of activated B-cell compartment is subsequently downmodulated through activation-induced cell death (AICD) (7,8).…”

mentioning

confidence: 99%

Endoplasmic reticulum-resident E3 ubiquitin ligase Hrd1 controls B-cell immunity through degradation of the death receptor CD95/Fas

Kong¹,

Yang²,

Xu³

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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Humoral immunity involves multiple checkpoints during B-cell development, maturation, and activation. The cell death receptor CD95/Fas-mediated apoptosis plays a critical role in eliminating the unwanted activation of B cells by self-reactive antigens and in maintaining B-cell homeostasis through activation-induced B-cell death (AICD). The molecular mechanisms controlling AICD remain largely undefined. Herein, we show that the E3 ubiquitin ligase Hrd1 protected B cells from activation-induced cell death by degrading the death receptor Fas. Hrd1-null B cells exhibited high Fas expression during activation and rapidly underwent Fas-mediated apoptosis, which could be largely inhibited by FasL neutralization. Fas mutation in Hrd1 KO mice abrogated the increase in B-cell AICD. We identified Hrd1 as the first E3 ubiquitin ligase of the death receptor Fas and Hrd1-mediated Fas destruction as a molecular mechanism in regulating B-cell immunity.Hrd1 | Fas | B cells | ubiquitination B -cell immunity involves several checkpoints, which are important to orchestrate and balance survival and apoptotic signals and control the quality and size of the B-cell compartment (1, 2). The earliest steps in B-cell development occur in the bone marrow, where assembly of the pre-B-cell receptor (pre-BCR) followed by the mature BCR occurs in distinct stages. Once the mature BCR is expressed, B lymphocytes then progress into the immature B-cell stage, where further selection checkpoints occur before the immature B cells transition to the periphery and mature into circulating B cells (2, 3). Mature B cells are maintained through tonic BCR, CD40, and B-cell-activating factor receptor (BAFFR) signaling (4). Activation by cross-linking the BCR leads to rapid proliferation, somatic hypermutation, and further differentiation of B cells (5, 6). The expansion of activated B-cell compartment is subsequently downmodulated through activation-induced cell death (AICD) (7,8).The death receptor Fas has been identified as a key regulator of AICD of B cells (9-11). Fas is highly expressed on activated B cells, particularly in the germinal center, where it mediates the deletion of autoreactive or unproductive somatic hypermutated B cells (12)(13)(14). Fas, as well as its ligand FasL, play critical roles in B-cell apoptosis. The selective removal of Fas from activated B cells results in lupus-like disease and autoantibody production (15-17). It has been a widely accepted view that the Fas/FasL mutations result in, at least in part, a failure to eliminate self-reactive GC B cells in autoimmune patients and animal models (12, 18). However, more recent studies show that FAS is in fact not required for the elimination of self-reactive GC B cells. Instead, Fas inactivation led to accumulation of a population of unconventional GC B cells that underwent somatic hypermutation and survived despite losing antigen reactivity (8). Nevertheless, it is clear that Fasmediated B-cell death is critically involved in B-cell-mediated autoimmune diseases. However, the molecular...

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Early Events in B Cell Activation

Cited by 337 publications

References 152 publications

Sensitive kinase assay linked with phosphoproteomics for identifying direct kinase substrates

Sensitive kinase assay linked with phosphoproteomics for identifying direct kinase substrates

Adaptive Immune Responses During Pregnancy

Endoplasmic reticulum-resident E3 ubiquitin ligase Hrd1 controls B-cell immunity through degradation of the death receptor CD95/Fas

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