Cbl-b Negatively Regulates B Cell Antigen Receptor Signaling in Mature B Cells through Ubiquitination of the Tyrosine Kinase Syk

Members of the Cbl family of molecular adaptors play key roles in regulating tyrosine kinase-dependent signaling in a variety of cellular systems. Here we provide evidence that in B cells Cbl-b functions as a negative regulator of B cell antigen receptor (BCR) signaling during the normal course of a response. In B cells from Cbl-b–deficient mice cross-linking the BCRs resulted in sustained phosphorylation of Igα, Syk, and phospholipase C (PLC)-γ2, leading to prolonged Ca2+ mobilization, and increases in extracellular signal–regulated kinase (ERK) and c-Jun NH2-terminal protein kinase (JNK) phosphorylation and surface expression of the activation marker, CD69. Image analysis following BCR cross-linking showed sustained polarization of the BCRs into large signaling-active caps associated with phosphorylated Syk in Cbl-b–deficient B cells in contrast to the BCRs in Cbl-b–expressing B cells that rapidly proceeded to form small, condensed, signaling inactive caps. Significantly, prolonged phosphorylation of Syk correlated with reduced ubiquitination of Syk indicating that Cbl-b negatively regulates BCR signaling by targeting Syk for ubiquitination.

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Identification of the SH2 Domain Binding Protein of Bruton’s Tyrosine Kinase as BLNK—Functional Significance of Btk-SH2 Domain in B-Cell Antigen Receptor-Coupled Calcium Signaling

Blood ◽

10.1182/blood.v94.7.2357.419k40_2357_2364 ◽

1999 ◽

Vol 94 (7) ◽

pp. 2357-2364 ◽

Cited By ~ 143

Author(s):

Shoji Hashimoto ◽

Akihiro Iwamatsu ◽

Masamichi Ishiai ◽

Katsuya Okawa ◽

Tomoki Yamadori ◽

...

Keyword(s):

B Cells ◽

Tyrosine Kinase ◽

Calcium Signaling ◽

B Cell ◽

Functional Significance ◽

Antigen Receptor ◽

Sh2 Domain ◽

B Cell Antigen Receptor ◽

Cell Antigen Receptor ◽

Cell Antigen

Bruton’s tyrosine kinase (Btk) is a critical component in the B-cell antigen receptor (BCR)-coupled signaling pathway. Its deficiency in B cells leads to loss or marked reduction in the BCR-induced calcium signaling. It is known that this BCR-induced calcium signaling depends on the activation of phospholipase Cγ (PLCγ), which is mediated by Btk and another tyrosine kinase Syk and that the SH2 and pleckstrin homology (PH) domains of Btk play important roles in this activation process. Although the importance of the PH domain of Btk has been explained by its role in the membrane targeting of Btk, the functional significance of the SH2 domain in the calcium signaling has remained merely a matter of speculation. In this report, we identify that one of the major Btk-SH2 domain-binding proteins in B cells is BLNK (B-cell linker protein) and present evidences that the interaction of BLNK and the SH2 domain of Btk contributes to the complete tyrosine phosphorylation of PLCγ.

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Quantitative Proteomics Identifies PTP1B as Modulator of B Cell Antigen Receptor Signaling

10.1101/2021.03.30.437652 ◽

2021 ◽

Author(s):

Jennifer J. Schwarz ◽

Lorenz Grundmann ◽

Thomas Kokot ◽

Kathrin Kläsener ◽

Sandra Fotteler ◽

...

Keyword(s):

B Cells ◽

B Cell ◽

Protein Tyrosine Phosphatase ◽

Tyrosine Phosphatase ◽

Antigen Receptor ◽

B Cell Antigen Receptor ◽

Cell Antigen Receptor ◽

Cell Antigen ◽

Bcr Signaling ◽

Signaling Components

ABSTRACTB cell antigen receptor (BCR) signaling is initiated by protein kinases and limited by counteracting phosphatases that currently are less well studied in their regulation of BCR signaling. We here used the B cell line Ramos to identify and quantify human B cell signaling components. Specifically, a protein tyrosine phosphatase profiling revealed a high expression of the protein tyrosine phosphatase 1B (PTP1B) in Ramos and human naïve B cells. The loss of PTP1B leads to increased B cell activation. Through substrate trapping in combination with quantitative mass spectrometry, we identified 22 putative substrates or interactors of PTP1B. We validated Igα, CD22, PLCγ1/2, CBL, BCAP and APLP2 as specific substrates of PTP1B in Ramos B cells. The tyrosine kinase BTK and the two adaptor proteins GRB2 and VAV1 were identified as direct binding partners and potential substrates of PTP1B. We showed that PTP1B dephosphorylates the inhibitory receptor protein CD22 at phosphotyrosine 807. We conclude that PTP1B negatively modulates BCR signaling by dephosphorylating distinct phosphotyrosines in B cell specific receptor proteins and various downstream signaling components.

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Quantitative proteomics identifies PTP1B as modulator of B cell antigen receptor signaling

Life Science Alliance ◽

10.26508/lsa.202101084 ◽

2021 ◽

Vol 4 (11) ◽

pp. e202101084

Author(s):

Jennifer J Schwarz ◽

Lorenz Grundmann ◽

Thomas Kokot ◽

Kathrin Kläsener ◽

Sandra Fotteler ◽

...

Keyword(s):

B Cells ◽

B Cell ◽

Protein Tyrosine Phosphatase ◽

Tyrosine Phosphatase ◽

Antigen Receptor ◽

B Cell Antigen Receptor ◽

Cell Antigen Receptor ◽

Cell Antigen ◽

Bcr Signaling ◽

Signaling Components

B cell antigen receptor (BCR) signaling is initiated by protein kinases and limited by counteracting phosphatases that currently are less well studied in their regulation of BCR signaling. Here, we used the B cell line Ramos to identify and quantify human B cell signaling components. Specifically, a protein tyrosine phosphatase profiling revealed a high expression of the protein tyrosine phosphatase 1B (PTP1B) in Ramos and human naïve B cells. The loss of PTP1B leads to increased B cell activation. Through substrate trapping in combination with quantitative mass spectrometry, we identified 22 putative substrates or interactors of PTP1B. We validated Igα, CD22, PLCγ1/2, CBL, BCAP, and APLP2 as specific substrates of PTP1B in Ramos B cells. The tyrosine kinase BTK and the two adaptor proteins GRB2 and VAV1 were identified as direct binding partners and potential substrates of PTP1B. We showed that PTP1B dephosphorylates the inhibitory receptor protein CD22 at phosphotyrosine 807. We conclude that PTP1B negatively modulates BCR signaling by dephosphorylating distinct phosphotyrosines in B cell-specific receptor proteins and various downstream signaling components.

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A role for Bruton's tyrosine kinase in B cell antigen receptor-mediated activation of phospholipase C-gamma 2.

Journal of Experimental Medicine ◽

10.1084/jem.184.1.31 ◽

1996 ◽

Vol 184 (1) ◽

pp. 31-40 ◽

Cited By ~ 336

Author(s):

M Takata ◽

T Kurosaki

Keyword(s):

B Cells ◽

Tyrosine Kinase ◽

B Cell ◽

Phospholipase C ◽

Antigen Receptor ◽

Bruton’S Tyrosine Kinase ◽

B Cell Antigen Receptor ◽

Bruton's Tyrosine Kinase ◽

Cell Antigen Receptor ◽

Cell Antigen

Defects in the gene encoding Bruton's tyrosine kinase (Btk) result in a disease called X-linked agammaglobulinemia, in which there is a profound decrease of mature B cells due to a block in B cell development. Recent studies have shown that Btk is tyrosine phosphorylated and activated upon B cell antigen receptor (BCR) stimulation. To elucidate the functions of this kinase, we examined BCR signaling of DT40 B cells deficient in Btk. Tyrosine phosphorylation of phospholipase C (PLC)-gamma 2 upon receptor stimulation was significantly reduced in the mutant cells, leading to the loss of both BCR-coupled phosphatidylinositol hydrolysis and calcium mobilization. Pleckstrin homology and Src-homology 2 domains of Btk were required for PLC-gamma 2 activation. Since Syk is also required for the BCR-induced PLC-gamma 2 activation, our findings indicate that PLC-gamma 2 activation is regulated by Btk and Syk through their concerted actions.

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