Btk regulation in human and mouse B cells via protein kinase C phosphorylation of IBtkγ

Blood ◽  
2011 ◽  
Vol 117 (24) ◽  
pp. 6520-6531 ◽  
Author(s):  
Elzbieta Janda ◽  
Camillo Palmieri ◽  
Antonio Pisano ◽  
Marilena Pontoriero ◽  
Enrico Iaccino ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
B Cells ◽  
Protein Kinase ◽  
Tyrosine Kinase ◽  
B Cell ◽  
Serine Phosphorylation ◽  
Negative Regulator ◽  
B Cell Differentiation ◽  
Kinase C ◽  
Bruton Tyrosine Kinase

Abstract The inhibitor of Bruton tyrosine kinase γ (IBtkγ) is a negative regulator of the Bruton tyrosine kinase (Btk), which plays a major role in B-cell differentiation; however, the mechanisms of IBtkγ-mediated regulation of Btk are unknown. Here we report that B-cell receptor (BCR) triggering caused serine-phosphorylation of IBtkγ at protein kinase C consensus sites and dissociation from Btk. By liquid chromatography and mass-mass spectrometry and functional analysis, we identified IBtkγ-S87 and -S90 as the critical amino acid residues that regulate the IBtkγ binding affinity to Btk. Consistently, the mutants IBtkγ carrying S87A and S90A mutations bound constitutively to Btk and down-regulated Ca2+ fluxes and NF-κB activation on BCR triggering. Accordingly, spleen B cells from Ibtkγ−/− mice showed an increased activation of Btk, as evaluated by Y551-phosphorylation and sustained Ca2+ mobilization on BCR engagement. These findings identify a novel pathway of Btk regulation via protein kinase C phosphorylation of IBtkγ.

scholarly journals Protein Kinase C β Controls Nuclear Factor κB Activation in B Cells Through Selective Regulation of the IκB Kinase α

2002 ◽  
Vol 195 (12) ◽  
pp. 1647-1652 ◽  
Author(s):  
Kaoru Saijo ◽  
Ingrid Mecklenbräuker ◽  
Angela Santana ◽  
Michael Leitger ◽  
Christian Schmedt ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
B Cells ◽  
Protein Kinase ◽  
Tyrosine Kinase ◽  
B Cell ◽  
B Cell Antigen Receptor ◽  
Nuclear Factor ◽  
Cell Antigen ◽  
Kinase C ◽  
Iκb Kinase

Activation of the nuclear factor (NF)-κB transcription complex by signals derived from the surface expressed B cell antigen receptor controls B cell development, survival, and antigenic responses. Activation of NF-κB is critically dependent on serine phosphorylation of the IκB protein by the multi-component IκB kinase (IKK) containing two catalytic subunits (IKKα and IKKβ) and one regulatory subunit (IKKγ). Using mice deficient for protein kinase C β (PKCβ) we show an essential role of PKCβ in the phosphorylation of IKKα and the subsequent activation of NF-κB in B cells. Defective IKKα phosphorylation correlates with impaired B cell antigen receptor–mediated induction of the pro-survival protein Bcl-xL. Lack of IKKα phosphorylation and defective NF-κB induction in the absence of PKCβ explains the similarity in immunodeficiencies caused by PKCβ or IKKα ablation in B cells. Furthermore, the well established functional cooperation between the protein tyrosine kinase Bruton's tyrosine kinase (Btk), which regulates the activity of NF-κB and PKCβ, suggests PKCβ as a likely serine/threonine kinase component of the Btk-dependent NF-κB activating signal transduction chain downstream of the BCR.

scholarly journals B Cell Antigen Receptor Engagement Inhibits Stromal Cell–derived Factor (SDF)-1α Chemotaxis and Promotes Protein Kinase C (PKC)-induced Internalization of CXCR4

1999 ◽  
Vol 189 (9) ◽  
pp. 1461-1466 ◽  
Author(s):  
Rodolphe Guinamard ◽  
Nathalie Signoret ◽  
Masamichi Ishiai ◽  
Mark Marsh ◽  
Tomohiro Kurosaki ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
B Cells ◽  
Protein Kinase ◽  
B Cell ◽  
Stromal Cell ◽  
B Cell Antigen Receptor ◽  
Cell Antigen ◽  
Kinase C ◽  
Stromal Cell Derived Factor ◽  
Secondary Lymphoid Organs

The entry of B lymphocytes into secondary lymphoid organs is a critical step in the development of an immune response, providing a site for repertoire shaping, antigen-induced activation and selection. These events are controlled by signals generated through the B cell antigen receptor (BCR) and are associated with changes in the migration properties of B cells in response to chemokine gradients. The chemokine stromal cell–derived factor (SDF)-1α is thought to be one of the driving forces during those processes, as it is produced inside secondary lymphoid organs and induces B lymphocyte migration that arrests upon BCR engagement. The signaling pathway that mediates this arrest was genetically dissected using B cells deficient in specific BCR-coupled signaling components. BCR-induced inhibition of SDF-1α chemotaxis was dependent on Syk, BLNK, Btk, and phospholipase C (Plc)γ2 but independent of Ca2+ mobilization, suggesting that the target of BCR stimulation was a protein kinase C (PKC)-dependent substrate. This target was identified as the SDF-1α receptor, CXCR4, which undergoes PKC- dependent internalization upon BCR stimulation. Mutation of the internalization motif SSXXIL in the COOH terminus of CXCR4 resulted in B cells that constitutively expressed this receptor upon BCR engagement. These studies suggest that one pathway by which BCR stimulation results in inhibition of SDF-1α migration is through PKC-dependent downregulation of CXCR4.

scholarly journals Induction of bcl-2 expression by phosphorylated CREB proteins during B-cell activation and rescue from apoptosis.

1996 ◽  
Vol 16 (10) ◽  
pp. 5546-5556 ◽  
Author(s):  
B E Wilson ◽  
E Mochon ◽  
L M Boxer
Keyword(s):  
Protein Kinase C ◽  
B Cells ◽  
Protein Kinase ◽  
B Cell ◽  
Regulatory Element ◽  
Regulatory Region ◽  
Cross Linking ◽  
Surface Immunoglobulin ◽  
Kinase C ◽  
Upstream Regulatory Element

Engagement of surface immunoglobulin on mature B cells leads to rescue from apoptosis and to proliferation. Levels of bcl-2 mRNA and protein increase with cross-linking of surface immunoglobulin. We have located the major positive regulatory region for control of bcl-2 expression in B cells in the 5'-flanking region. The positive region can be divided into an upstream and a downstream regulatory region. The downstream regulatory region contains a cyclic AMP-responsive element (CRE). We show by antibody supershift experiments and UV cross-linking followed by denaturing polyacrylamide gel electrophoresis that both CREB and ATF family members bind to this region in vitro. Mutations of the CRE site that result in loss of CREB binding also lead to loss of functional activity of the bcl-2 promoter in transient-transfection assays. The presence of an active CRE site in the bcl-2 promoter implies that the regulation of bcl-2 expression is linked to a signal transduction pathway in B cells. Treatment of the mature B-cell line BAL-17 with either anti-immunoglobulin M or phorbol 12-myristate 13-acetate leads to an increase in bcl-2 expression that is mediated by the CRE site. Treatment of the more immature B-cell line, Ramos, with phorbol esters rescues the cells from calcium-dependent apoptosis. bcl-2 expression is increased following phorbol ester treatment, and the increased expression is dependent on the CRE site. These stimuli result in phosphorylation of CREB at serine 133. The phosphorylation of CREB that results in activation is mediated by protein kinase C rather than by protein kinase A. Although the CRE site is necessary, optimal induction of bcl-2 expression requires participation of the upstream regulatory element, suggesting that phosphorylation of CREB alters its interaction with the upstream regulatory element. The CRE site in the bcl-2 promoter appears to play a major role in the induction of bcl-2 expression during the activation of mature B cells and during the rescue of immature B cells from apoptosis. It is possible that the CRE site is responsible for induction of bcl-2 expression in other cell types, particularly those in which protein kinase C is involved.

scholarly journals Analysis of signal transduction in B chronic lymphocytic leukemia cells

Blood ◽  
1988 ◽  
Vol 71 (5) ◽  
pp. 1461-1469
Author(s):  
HG Drexler ◽  
MK Brenner ◽  
E Coustan-Smith ◽  
SM Gignac ◽  
AV Hoffbrand
Keyword(s):  
Signal Transduction ◽  
Protein Kinase C ◽  
B Cells ◽  
Protein Kinase ◽  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
B Lymphocytes ◽  
Lymphocytic Leukemia ◽  
Receptor Expression ◽  
Kinase C

We report here experiments on the analysis of cellular signal transduction in a series of patients with chronic B cell disorders (B cell chronic lymphocytic leukemia [B-CLL] and prolymphocytic leukemia). We compared the response of the leukemic cells with primary external signals (interleukin 2 [IL-2] or B cell differentiation factors [BCDF or IL-6]) with their response to secondary inducers (the phorbol ester (12–O-tetradecanoylphorbol-13-acetate [TPA] or the calcium ionophore A23187) that circumvent the first part of the signal transduction pathway by directly activating the key enzyme protein kinase C. One BCDF was synthesized by mitogen-activated peripheral blood B lymphocytes; a second BCDF was constitutively produced by the human bladder carcinoma cell line T24. Changes in morphology, Tac (IL-2 receptor) expression, RNA synthesis measured by 3H-uridine uptake, and immunoglobulin production tested by enzyme-linked immunosorbent assay were used as parameters of successful signal transduction. TPA alone and TPA plus A23187 (synergistically) effectively initiated differentiation in all the leukemia cases. Neither IL-2 nor BCDF (singly or in combinations) caused equivalent responses. On the other hand, IL-2 and BCDF produced a substantial differentiation effect on normal B lymphocytes. Our data suggest that (a) B-CLL cells are able to respond to direct stimulation of the second messenger pathway (through protein kinase C) but not to the physiological stimuli IL-2 or BCDF; (b) the defect in signal transduction appears to be located upstream of protein kinase C (a possible candidate is a G protein); (c) malignant B cells may spontaneously or after treatment with inducers express the IL- 2 receptor (Tac antigen) in the absence of a functional differentiating response to IL-2; and (d) signs of proliferation/differentiation in B- CLL samples after incubation with IL-2 or BCDF might be due to contamination of the cell populations with residual normal B cells.

scholarly journals Transcriptional Regulatory Elements Within the First Intron of Bruton's Tyrosine Kinase

Blood ◽  
1998 ◽  
Vol 91 (1) ◽  
pp. 214-221
Author(s):  
Jurg Rohrer ◽  
Mary Ellen Conley
Keyword(s):  
B Cells ◽  
Tyrosine Kinase ◽  
B Cell ◽  
Regulatory Elements ◽  
Negative Regulator ◽  
Bruton’S Tyrosine Kinase ◽  
B Cell Differentiation ◽  
Bruton's Tyrosine Kinase ◽  
First Intron ◽  
Transcriptional Regulatory

Defects in the gene for Bruton's tyrosine kinase (Btk) result in the disorder X-linked agammaglobulinemia (XLA). Whereas XLA is characterized by a profound defect in B-cell development, Btk is expressed in both the B lymphocyte and myeloid cell lineages. We evaluated a patient with XLA who had reduced amounts of Btk transcript but no abnormalities in his coding sequence. A single base-pair substitution in the first intron of Btk was identified in this patient, suggesting that this region may contain regulatory elements. Using reporter constructs we identified two transcriptional control elements in the first 500 bp of intron 1. A strong positive regulator, active in both pre-B cells and B cells, was identified within the first 43 bp of the intron. Gel-shift assays identified two Sp1 binding sites within this element. The patient's mutation results in an altered binding specificity of the proximal Sp1 binding site. A negative regulator, active in pre-B cells only, was located between base pairs 281 and 491 of the intron. These findings indicate that regulation of Btk transcription is complex and may involve several transcriptional regulatory factors at the different stages of B-cell differentiation.

scholarly journals Mitogenic Response of Murine B Lymphocytes to Salmonella typhimurium Lipopolysaccharide Requires Protein Kinase C-Dependent Late Tyrosine Phosphorylations

1998 ◽  
Vol 66 (6) ◽  
pp. 2547-2552 ◽  
Author(s):  
Anne Mey ◽  
Jean-Pierre Revillard
Keyword(s):  
Protein Synthesis ◽  
Protein Kinase C ◽  
B Cells ◽  
Protein Kinase ◽  
Tyrosine Kinase ◽  
Kinase Inhibitors ◽  
Cross Linking ◽  
Kinase Activation ◽  
Herbimycin A ◽  
Kinase C

ABSTRACT Unlike the cross-linking of membrane immunoglobulins, the activation of B cells by lipopolysaccharide (LPS) does not involve the phosphoinositol turnover and the initial activation of tyrosine kinases. However, LPS-induced B-cell proliferation was inhibited by the tyrosine kinase inhibitors genistein and herbimycin A even when added 48 h after the beginning of the culture. Tyrosyl-phosphorylated proteins were detected by Western blotting after 24 h of culture with LPS, reaching a maximum concentration after 72 h. Late tyrosine phosphorylations were also detected in B cells activated for 72 h with anti-immunoglobulin M antibody and were abrogated by the protein synthesis inhibitor cycloheximide, the tyrosine kinase inhibitors genistein and herbimycin A, and the protein kinase C inhibitor chelerythrine. The role of protein kinase C in late tyrosine kinase activation is independent of Ca2+mobilization and was confirmed by detection of a comparable but restricted pattern of tyrosine-phosphorylated substrates in B cells treated with phorbol myristate acetate alone or in association with ionomycin. Tyrosine kinase activation was dependent on de novo protein synthesis. However, culture supernatants of LPS-activated B cells were devoid of mitogenic activity and induced a phosphorylation pattern more restricted than that achieved by LPS. Altogether these data indicate that proliferation signals induced by LPS or by the cross-linking of membrane immunoglobulins are controlled by late tyrosine phosphorylations occurring throughout the first 3 days of culture, controlled in part by protein kinase C activation, and dependent on the synthesis of an intermediate protein(s) either not secreted in the culture supernatant or present but biologically inactive in naive B cells.

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