Bosutinib, a Src/Abl Kinase Inhibitor Induces Apoptosis in CLL B Cells by Targeting Multiple Tyrosine Kinases and Overcomes Stroma Protection.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2368-2368
Author(s):  
Asish K Ghosh ◽  
Debabrata Mukhopadhyay ◽  
Neil E. Kay
Keyword(s):  
B Cells ◽  
B Cell ◽  
Tyrosine Kinases ◽  
Therapeutic Agent ◽  
Kinase Inhibitor ◽  
Src Kinase ◽  
Abl Kinase ◽  
Apoptotic Proteins ◽  
Constitutively Active

Abstract Abstract 2368 Poster Board II-345 Background: B-cell chronic lymphocytic leukemia (CLL) is characterized by the progressive accumulation of CD5+ B lymphocytes in the peripheral blood, lymphoid organs and bone marrow. Despite aggressive therapy, CLL is still incurable partly because of intrinsic defect in apoptosis induction. A novel therapeutic agent, Bosutinib, was initially developed as an inhibitor of Src and Abl kinases and is currently in phase II clinical trials for the treatment of several human malignancies. Recently, Bosutinib has been shown to inhibit phosphorylation of a novel receptor tyrosine kinase, Axl which has been reported to be overexpressed in several types of human cancers including colon, prostatic, thyroid, breast, gastric, renal and lung. Previously, Dasatinib, another Src/Abl kinase inhibitor, showed cytotoxic effects on CLL B cells by decreasing levels of activated, phosphorylated forms of Akt, Erk1/2 and p38 and reducing expression of anti-apoptotic proteins Mcl-1 and Bcl-xL in CLL B cells. Here, we wished to examine receptor or non-receptor tyrosine kinases active in primary CLL B-cells and determine their status after exposure to Bosutinib as well as the latter drug's effect on CLL B-cell viability. Methods: We used freshly isolated CLL B cells after obtaining written consent from patients. Bosutinib was used at various doses (2.5, 5.0, 10.0 and 20.0 μM) to treat CLL B cells in vitro for 24/48 hrs. Induction of apoptosis was assessed by annexin/propidium staining. To examine the impact of stroma on Bosutinib induced CLL B-cell death, primary CLL-bone marrow stromal cells (BMSC) were cocultured with CLL B cells at a cell density ratio of 1:20 and treated with various doses of Bosutinib for 24 hrs. Expression status of various kinases and downstream targets were analyzed in CLL B cell lysates with or without Bosutinib-treatment by Western blot using specific antibodies. Results: Treatment of CLL B-cells with Bosutinib induces a massive apoptotic cell death in a dose- and time-dependent manner (IC50 for 24 h; ∼10 μM and IC50 for 48 h: 5-10 μM) which involves PARP cleavage as demonstrated by Western blot analysis. Moreover, Bosutinib-treatment reduced expression of several key anti-apoptotic proteins, Mcl-1, XIAP and Bcl-2 reported to be overexpressed in CLL B cells. Interestingly, we detected that the majority of CLL B-cells express constitutively active Axl. Importantly, Bosutinib treatment inhibited phosphorylation of Axl in CLL B cells resulting in inhibition of AKT-activation, one of its downstream signaling pathways. Previous studies have suggested a possible physical association between Axl and Src kinase. We observed that expression of constitutively active Axl was associated with the presence of highly phosphorylated Src kinase when compared with that in CLL B-cells with low or unphosphorylated Axl. These observations suggest that phosphorylation of Axl may be an upstream event for Src activation in CLL. We found inhibition of constitutively active Axl resulted in subsequent inhibition of Src kinase activation in CLL B-cells following Bosutinib-treatment. We also detected inhibition of ZAP70/Syk-phosphorylation in CLL B cells upon Bosutinib-treatment. Finally, we found Bosutinib was able to overcome stomal protection of CLL B cells at a dose of 10 μM in an in vitro coculture system suggesting its potential as a therapeutic agent against CLL. Conclusion: Together, these observations suggest that Bosutinib induces apoptosis in CLL B-cells, even in the presence of stromal cells, in association with the down regulation of multiple kinases including the novel receptor-tyrosine kinase, Axl, and reduces expression of the anti-apoptotic proteins critical to CLL B-cell survival. In total, these findings for the first time indicate that Bosutinib has the potential to be a very potent therapeutic agent for CLL patients. Disclosures: Kay: Biogenc-Idec, Celgene, Genentech, genmab: Membership on an entity's Board of Directors or advisory committees; Genentech, Celgene, Hospira, Polyphenon Pharma, Sanofi-Aventis: Research Funding.

Association between B-lymphocyte membrane immunoglobulin and multiple members of the Src family of protein tyrosine kinases

1992 ◽  
Vol 12 (5) ◽  
pp. 2315-2321
Author(s):  
M A Campbell ◽  
B M Sefton
Keyword(s):  
B Cells ◽  
B Cell ◽  
Tyrosine Kinases ◽  
B Lymphocyte ◽  
Protein Tyrosine Kinases ◽  
Cell Type ◽  
Protein Tyrosine ◽  
Membrane Immunoglobulin ◽  
Family Protein

Treatment of B lymphocytes with antibodies to membrane immunoglobulin (Ig) stimulates protein tyrosine phosphorylation. We have examined the phosphorylation in vitro of proteins associated with membrane Ig. The Src family protein tyrosine kinases p53/56lyn, p59fyn, and p56lck are associated with membrane Ig in spleen B cells and B-cell lines and undergo phosphorylation in vitro. The pattern of expression of Src family protein tyrosine kinases in B cells varied. Our studies suggest that multiple kinases can potentially interact with membrane Ig and that within any one B-cell type, all of the Src family kinases expressed can be found in association with membrane Ig. We also observed that the Ig-associated Ig alpha protein, multiple forms of Ig beta, and proteins of 100 and 25 kDa were tyrosine phosphorylated in vitro. The 100- and 25-kDa proteins remain unidentified.

The Dual SRC/ABL Kinase Inhibitor BMS-354825 Potently Inhibits the Growth of Myeloid Leukemic Cells Characterized by FLT3-ITD Expression, GM-CSF Dependency, or G-CSF Responsiveness Via an ABL-Independent Mechanism.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4480-4480
Author(s):  
Jukka Kanerva ◽  
Ogonna Nwawka ◽  
Kevin Hwang ◽  
Francis Y. Lee ◽  
Seth J. Corey
Keyword(s):  
Growth Inhibition ◽  
Cell Lines ◽  
Western Blotting ◽  
Mtt Assay ◽  
Tyrosine Kinases ◽  
Kinase Inhibitor ◽  
Src Kinase ◽  
Myeloid Cell ◽  
Gm Csf ◽  
Abl Kinase

Abstract BMS-354825 is a dual SRC and ABL inhibitor, which has been shown effective in imatinib-resistant BCR-ABL+ cells, and it is in phase I trials for patients with imatinib-resistant leukemia. We conducted a study to evaluate growth inhibition and inhibition of Src v. Abl protein tyrosine kinases in human myeloid cell lines: MV4-11 expressing an internal tandem duplication of Flt3 (Flt3-ITD), the murine pro-B cell line Ba/F3 that expresses the Flt3-ITD, the GM-CSF dependent Mo7e, and the G-CSF-responsive BaF3-GR (Ba/F3 cells expressing the human G-CSF receptor). We compared BMS-354825 with PP1, a SRC kinase inhibitor with in vitro IC50 at sub-micromolar concentrations. We sought to correlate growth inhibition with SRC or ABL inhibition. In these myeloid cell lines, LYN is the predominant SRC kinase. Methods: Growth inhibition was assessed by Trypan blue exclusion and MTT assay using drug concentrations 0.1 uM – 10 uM. Drugs were added daily to the cell suspension during the 3-day experiment. After a 60 min incubation at concentrations 0.1 nM – 1 uM, SRC or ABL kinase inhibition was analyzed by blotting with a polyclonal phospho-SRC (Tyr416) antibody or a polyclonal phospho-ABL (Tyr245) antibody. Results: In MV4-11 cells BMS-354825 and PP1 caused similar growth inhibition IC50 at 5 uM. By western blotting, inhibition of phospho-Src 416 occurred at 1 nM concentrations of both compounds. Protein expression of ABL was not detected in MV4-11 cells. In Ba/F3-ITD cells, the IC50 for BMS-354825 was 1–10 uM (grown in IL-3) and 0.01 uM (without IL-3). The IC50 for PP1 was 1–10 uM (grown in IL-3) and 0.1 uM (without IL-3). Inhibition of phospho-SRC occurred at 10 nM. In Mo7e cells, grown in the presence of GM-CSF, the IC50 was 5 uM for BMS-354825 v. 10 uM for PP1 by MTT assay. By western blotting, inhibition of phospho-SRC 416 occurred at 1 nM for both BMS-354825 and PP1. To determine specific contribution of LYN to Mo7e growth, we treated Mo7e cells with LYN siRNA. With 70% knock-down of LYN, there was 50% growth inhibition. ABL was present in Mo7e cells, but no phosphoAbl was demonstrated (K562 cells served as positive control). In BaF3-GR cells grown in G-CSF, the IC50 was 5 uM for BMS-354825 vs. 10 uM for PP1 by MTT assay. In western blotting, inhibition of phospho-Src 416 was detected at 10 nM BMS-354825. ABL was present in Ba/F3GR cells, but no phospho-ABL was demonstrated (K562 cells served as positive control). Conclusions: BMS-354825 is more potent than PP1 in causing growth inhibition and SRC kinase inhibition in Mo7e and Ba/F3GR cells that serve as models for acute myeloid leukemia. It is unlikely that ABL is the drug target, because MV4-11 cells do not express ABL and phospho-ABL was not found in Mo7e or Ba/F3 cells. These results suggest that inhibition of SRC tyrosine kinases contributes predominantly to growth inhibition caused by the dual SRC/ABL kinase inhibitor BMS-354825 in myeloid cell lines expressing Flt3-ITD and cytokine-driven proliferation and survival via the IL-3/GM-CSF Receptor or G-CSF Receptor.

scholarly journals Association between B-lymphocyte membrane immunoglobulin and multiple members of the Src family of protein tyrosine kinases.

1992 ◽  
Vol 12 (5) ◽  
pp. 2315-2321 ◽  
Author(s):  
M A Campbell ◽  
B M Sefton
Keyword(s):  
B Cells ◽  
B Cell ◽  
Tyrosine Kinases ◽  
B Lymphocyte ◽  
Protein Tyrosine Kinases ◽  
Cell Type ◽  
Protein Tyrosine ◽  
Membrane Immunoglobulin ◽  
Family Protein

Treatment of B lymphocytes with antibodies to membrane immunoglobulin (Ig) stimulates protein tyrosine phosphorylation. We have examined the phosphorylation in vitro of proteins associated with membrane Ig. The Src family protein tyrosine kinases p53/56lyn, p59fyn, and p56lck are associated with membrane Ig in spleen B cells and B-cell lines and undergo phosphorylation in vitro. The pattern of expression of Src family protein tyrosine kinases in B cells varied. Our studies suggest that multiple kinases can potentially interact with membrane Ig and that within any one B-cell type, all of the Src family kinases expressed can be found in association with membrane Ig. We also observed that the Ig-associated Ig alpha protein, multiple forms of Ig beta, and proteins of 100 and 25 kDa were tyrosine phosphorylated in vitro. The 100- and 25-kDa proteins remain unidentified.

PP1 Inhibitors Depolarize HermissendaPhotoreceptors and Reduce K+ Currents

2001 ◽  
Vol 86 (3) ◽  
pp. 1297-1311 ◽  
Author(s):  
Haojiang Huang ◽  
Joseph Farley
Keyword(s):  
B Cells ◽  
B Cell ◽  
Kinase Inhibitor ◽  
Critical Role ◽  
Resting Membrane Potential ◽  
Type B ◽  
Phosphatase Inhibitors ◽  
Neural Excitability ◽  
Protein Phosphatase Inhibitors

Previous research indicates that activation of protein kinase C (PKC) plays a critical role in the induction and maintenance of memory-related changes in neural excitability of Type B photoreceptors in the eyes of nudibranch mollusk Hermissenda crassicornis (H.c.). The enhanced excitability of B cells is due in part to PKC-mediated reduction in somatic K+ currents. Here we examined the effects of protein phosphatase inhibitors on Type B photoreceptor excitability and K+ currents to determine the role(s) of protein phosphatases on memory formation in Hermissenda. Using electrophysiological and pharmacological methods, we found that the PP1 inhibitors calyculin A and inhibitor-2 depolarized Type B photoreceptors by 20–30 mV. A broad-spectrum kinase inhibitor, H7, blocked this effect. The depolarization induced by PP1 inhibition occluded that produced by an in vitro associative conditioning procedure. Calyculin and inhibitor-2 reduced the same B cell K+ currents ( I Aand I delayed) that are reduced by in vitro and behavioral conditioning. H7 blocked the reductions. Cantharidic acid (PP2A inhibitor) and cyclosporin (PP2B inhibitor) had negligible effects on B cell resting membrane potential, K+ currents, and in vitro conditioning-produced cumulative depolarization of B cells. These results suggest that the functional activity of K+ channels in B cells is sustained by basal activity of PP1. Inhibiting PP1 appears to allow one or more constitutively active kinase(s) to reduce K+ channel activity and thus mimic the effects of conditioning. Our results suggest that PP1 may oppose and/or constrain the extent of learning-produced changes in B cell excitability.

Cytokine-Dependent Imatinib Resistance in Mouse Bcr-Abl(+), Arf-Null Lymphoblastic Leukemia.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2632-2632
Author(s):  
Richard T. Williams ◽  
Willem den Besten ◽  
Nidal Boulos ◽  
Charles J. Sherr
Keyword(s):  
Stem Cells ◽  
Targeted Therapy ◽  
B Cells ◽  
B Cell ◽  
Kinase Inhibitor ◽  
Lymphoblastic Leukemia ◽  
Single Agent ◽  
Mouse Bone Marrow ◽  
Imatinib Resistance ◽  
Abl Kinase

Abstract Retroviral vector-mediated transduction of the BCR-ABL tyrosine kinase into Arf-deficient mouse bone marrow progenitors allows rapid ex vivo outgrowth of pure pre-B cell populations that induce a highly aggressive form of ALL when inoculated intravenously into healthy syngeneic mice. These leukemias resist therapy with the BCR-ABL kinase inhibitor imatinib, and surprisingly, drug resistance is non-tumor-cell autonomous. We now demonstrate that expression of the p185 BCR-ABL isoform (hereafter p185) in Arf-null pre-B cells is sufficient to generate polyclonal populations of leukemia-initiating cells (LICs), only 20 of which induce fatal, imatinib-resistant disease within one month after infusion. These p185+ Arf-null LICs are pre-B cells by immunophenotypic criteria, lack evidence of myeloid and stem cells markers, exhibit immunoglobulin heavy chain gene rearrangements and random proviral insertions, and are almost universally capable of initiating lethal, transplantable leukemias in healthy syngeneic recipient mice. Therefore, these LICs do not represent rare ‘cancer stem cells’. Resistance to targeted therapy with imatinib in this model system is not dependent upon mutations within the BCR-ABL kinase domain, but, rather, is mediated through interactions between the LICs and the host environment. While cytokines like IL-7 are dispensable for the efficient generation of p185+, Arf-null cells, these LICs remain IL-7-responsive and are significantly protected from imatinib-induced cytostasis by IL-7 in vitro. Introduction of p185 into Arf-null hematopoietic progenitors that also lack the common gamma chain (γc) for cytokine receptors bypasses their cytokine requirements and permits pre-B cell development. These p185+, Arf-null, γc-null LICs also initiate ALL but exhibit much greater sensitivity to imatinib in vivo, allowing a significant fraction of treated mice to sustain long term remissions even when therapy is discontinued. Therefore, salutary cytokines expressed in the hematopoietic microenvironment facilitate leukemic proliferation and confer resistance to targeted therapy. Not surprisingly, single agent therapy with dasatinib, a second generation BCR-ABL kinase inhibitor, is dramatically more efficacious in this pre-clinical Ph+ ALL model, although some recipient mice still succumb to disease despite continuous drug treatment. Using vectors co-expressing p185 and luciferase, we are now initiating dasatinib therapy when recipient animals have detectable disease burdens, thereby permitting a more dynamic assessment of therapeutic response and subsequent failure. We speculate that in human Ph+ ALL, the frequent deletion of the INK4A-ARF locus (∼50% of cases at diagnosis) might help to maintain LIC survival in the face of targeted therapy and so facilitate the emergence of drug-resistant BCR-ABL variants. As such, INK4A-ARF deletion might prove to be a poor prognostic indicator.

Proteinkinase C-β Inhibitors Mitigate Microenvironment-Mediated Survival Of CLL Cells and Sensitise Malignant B Cells To Cytotoxic Drugs

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 669-669
Author(s):  
Gloria Lutzny ◽  
Zhoulei Li ◽  
Madlen Oelsner ◽  
Jolanta Slawska ◽  
Thomas Kocher ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Stromal Cells ◽  
Cytotoxic Drugs ◽  
B Cell Lymphomas ◽  
Cytotoxic Effects ◽  
Pkc Β ◽  
Apoptotic Proteins

Abstract Heterotypic interactions between bone marrow stromal cells (BMSCs) and malignant B cells contribute to apoptosis-resistance of tumour cells, based on the provision of anti-apoptotic factors by stromal cells. For this reason, interference with the microenvironment may offer an alternative approach to chemotherapy to target B cell lymphomas/ leukaemias. However, the success of such treatments critically relies on specific targets expressed in the lymphoma microenvironment. We have previously reported that monoclonal B cells from patients with CLL, MCL und ALL activate and reprogram BMSCs. This activation of stromal cells is an essential prerequisite for microenvironment-mediated survival of malignant B cells and requires the induction and activation of protein-kinase C-β in stromal cells in vitro and in vivo. This is underscored by a complete resistance of PKC-β knockout mice to adoptively transferred B cell lymphomas from TCL1 transgenic mice (Lutzny et al. Cancer Cell. 2013 Jan 14; 23(1):77-92.). Analyses of primary CLL cells co-cultured on human or mouse BMSCs indicate that stromal cells protect malignant B cells from apoptosis primarily by enhancing the expression of the anti-apoptotic proteins Mcl1, XIAP, BclXL and Bcl2A1. Knockdown of Mcl1 expressed in CLL cells further demonstrates that its expression is crucial for stroma-mediated survival even in the presence of BMSCs. Since BMSC-mediated survival and propagation of CLL depends on the kinase activity of PKC-β, we hypothesized that pharmacological inhibition of the activation of stromal cells using small molecule inhibitors against PKC-β may display anti-leukemic effects. Here we report that the PKC-β inhibitor enzastaurin enhances the cytotoxic effects of standard chemotherapeutic drugs and of the BCL2-inhibitor ABT737. Dose-response analyses indicate that enzastaurin potentiates the cytotoxic effects of ABT737 in a synergistic manner, rapidly causing caspase-mediated apoptosis of malignant B cells. This drug-sensitising effect of enzastaurin is mediated by inhibition of PKC-β induced and expressed in activated stromal cells and not related to a direct cytotoxic effect on malignant B cells: PKC-β deficient BMSCs fail to maintain high expression levels of the anti-apoptotic proteins Mcl1, XIAP and Bcl2A1 in CLL cells, which show a significantly enhanced sensitivity to ABT737 compared to CLL cells cultured on PKC-β proficient stromal cells. These data provide evidence that PKC-β inhibitors can therapeutically be used to abolish microenvironment-mediated survival of malignant B cells. In order to assess whether drug combinations of enzastaurin and BCL2 inhibitors demonstrate synergistic anti-lymphoma effects in vivo, we adoptively transferred malignant B cells from diseased TCL1 mice into wild-type recipient mice. Tumour-bearing mice were treated with enzastaurin, ABT199 or a combination of both drugs. Response to treatment, assessed by PET-CT scan, indicates that the combination of enzastaurin and ABT199 is superior to single agent treatment. These data indicate that malignant B cells can be sensitized to cytotoxic drugs by inhibiting the tumour-promoting effects of the lymphoma microenvironment with PKC-β inhibitors. Our preclinical in vitro and in vivo experiments justify testing this drug combination as a new approach to leukaemia/ lymphoma therapy in a phase I/II clinical trial. Disclosures: No relevant conflicts of interest to declare.

Effectiveness of the dual specific Src/Abl kinase inhibitor AZD0530 in combination with tamoxifen in preventing acquired anti-estrogen resistance in breast cancer cells

2007 ◽  
Vol 25 (18_suppl) ◽  
pp. 14054-14054 ◽  
Author(s):  
S. Hiscox ◽  
T. P. Green ◽  
C. Smith ◽  
N. Jordan ◽  
M. James ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Cancer Cells ◽  
Breast Cancer Cells ◽  
Kinase Activity ◽  
Kinase Inhibitor ◽  
Acquired Resistance ◽  
Src Kinase ◽  
Abl Kinase ◽  
Invasive Capacity

14054 Background: AZD0530 is a novel, orally potent, once-daily, highly selective and dual-specific Src/Abl kinase inhibitor with potential for activity in a wide range of tumors. In the context of breast cancer, where tamoxifen resistance presents a major problem, Src inhibition may be a particularly valuable therapeutic strategy since we have previously observed that elevated Src kinase activity accompanies anti-estrogen resistance in vitro, promoting an aggressive cell phenotype. Here, we have explored the potential therapeutic effects of Src inhibition with AZD0530, alone and in combination with tamoxifen, on the acquisition of endocrine resistance in breast cancer cells. Methods: MCF7 and T47D breast cancer cells were exposed to tamoxifen (10–7 M), AZD0530 (1 μM), or both agents in combination for a minimum of 10 months with passaging as necessary, or until total cell death occurred. Cells were assayed at monthly intervals for intracellular signaling pathway activity (Western Blotting) and in vitro invasive capacity (Matrigel invasion assays). Apoptosis and proliferation were assessed by ELISA and Ki67 staining, respectively. Changes in c-Myc and cyclin-D1 were measured with RT-PCR. Results: Treatment of cells with tamoxifen alone ultimately resulted in acquired resistance, elevated Src kinase activity, and a Src- dependent increase in invasive capacity. Chronic exposure to AZD0530 alone resulted in outgrowth of AZD0530 resistant cells, in which Src kinase activity remained suppressed as did their in vitro invasiveness. Treatment of MCF7 and T47D cells with AZD0530 and tamoxifen combined resulted in a reduction of Src, FAK, and Akt activity, inhibition of c-Myc gene expression, and complete abrogation of their in vitro invasive behavior. Furthermore, combination treatment completely prevented cell proliferation and the subsequent emergence of a resistant phenotype, with a total loss of cells by 12 weeks. Conclusions: Inhibition of Src kinase with AZD0530, when used in conjunction with anti-estrogen therapies, effectively prevents acquired resistance in breast cancer cells in vitro suggesting a potential novel therapeutic benefit of Src kinase inhibitors clinically. No significant financial relationships to disclose.

scholarly journals In vivo and in vitro specificity of protein tyrosine kinases for immunoglobulin G receptor (FcgammaRII) phosphorylation.

1996 ◽  
Vol 16 (9) ◽  
pp. 4735-4743 ◽  
Author(s):  
N Bewarder ◽  
V Weinrich ◽  
P Budde ◽  
D Hartmann ◽  
H Flaswinkel ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Immunoglobulin G ◽  
Tyrosine Kinases ◽  
Cell Activation ◽  
Vitro Assay ◽  
Protein Tyrosine ◽  
Phosphorylation Pattern

Human B cells express four immunoglobulin G receptors, FcgammaRIIa, FcgammaRIIb1, FcgammaRIIb2, and FcgammaRIIc. Coligation of either FcgammaRII isoform with the B-cell antigen receptor (BCR) results in the abrogation of B-cell activation, but only the FcgammaRIIa/c and FcgammaIIb1 isoforms become phosphorylated. To identify the FcgammaRII-phosphorylating protein tyrosine kinase (PTK), we used the combination of an in vitro and an in vivo approach. In an in vitro assay using recombinant cytoplasmic tails of the different FcgammaRII isoforms as well as tyrosine exchange mutants, we show that each of the BCR-associated PTKs (Lyn, Blk, Fyn, and Syk) shows different phosphorylation patterns with regard to the different FcgammaR isoforms and point mutants. While each PTK phosphorylated FcgammaRIIa/c, FcgammaRIIb1 was phosphorylated by Lyn and Blk whereas FcgammaRIIb2 became phosphorylated only by Blk. Mutants lacking both tyrosine residues of the immune receptor tyrosine-based activation motif (ITAM) of FcgammaRIIa/c were not phosphorylated by Blk and Fyn, while Lyn-mediated phosphorylation was dependent on the presence of the C-terminal tyrosine of the ITAM. Results obtained in assays using an FcgammaR- B-cell line transfected with wild-type or mutated FcgammaRIIa demonstrated that exchange of the C-terminal tyrosine of the ITAM of FcgammaRIIa/c was sufficient to abolish FcgammaRIIa/c phosphorylation in B cells. Additionally, we could show that Lyn and Fyn bind to FcgammaRIIa/c, with the ITAM being necessary for association. Comparison of the phosphorylation pattern of each PTK observed in vitro with the phosphorylation pattern observed in vivo suggests that Lyn is the most likely candidate for FcgammaRIIa/c and FcgammaRIIb1 phosphorylation in vivo.

scholarly journals Protein Tyrosine Kinase Involvement in Learning-Produced Changes in Hermissenda Type B Photoreceptors

2009 ◽  
Vol 102 (6) ◽  
pp. 3573-3595 ◽  
Author(s):  
Iksung Jin ◽  
Haojiang Huang ◽  
Benjamin Smith ◽  
Joseph Farley
Keyword(s):  
B Cells ◽  
B Cell ◽  
Tyrosine Kinases ◽  
Protein Tyrosine Kinase ◽  
Protein Tyrosine Phosphatases ◽  
Inactivation Kinetics ◽  
Type B ◽  
Protein Tyrosine ◽  
Voltage Dependent

Learning-correlated changes in the excitability and photoresponses of Hermissenda 's ocular type B photoreceptors are mediated by reductions in two distinct K+ currents, IA and IK-Ca. The suppression of these K+ currents has been linked to conditioning-produced activation of protein kinase C (PKC). The question of whether PKC accounts completely for the changes in excitability and K+ currents or whether other kinase(s) are involved has received little attention. In the present experiments, we asked whether protein tyrosine kinases (PTKs) might also contribute to conditioning-produced alterations in B cells. We found that the PTK inhibitors genistein and lavendustin A greatly reduced cumulative depolarization of type B cells, a short-term correlate of associative learning. This disruption occurred even when PKC activation had been either occluded by preexposure of type B cells to a phorbol ester or otherwise prevented by the pseudosubstrate inhibitor peptide PKC[19–31]. PTK inhibitors also increased the amplitude of the transient ( IA) and delayed ( IDelayed) components of voltage-dependent K+ current that have previously been shown to be selectively reduced by conditioning and to contribute to cumulative depolarization. Genistein partially prevented the reduction of IA and IDelayed due to in vitro conditioning and blocked the changes in their voltage dependencies. Ionophoresis of pervanadate ion, a potent inhibitor of protein tyrosine phosphatases, depolarized type B photoreceptors and occluded conditioning-produced cumulative depolarization. Pervanadate also suppressed IA and IDelayed, reduced their voltage dependence, and altered inactivation kinetics for IA, mimicking conditioning. Western blot analysis using a phosphotyrosine antibody indicated that conditioning increased the phosphotyrosine content of many proteins within the Hermissenda CNS. Collectively, our results suggest that in addition to PKC, one or more PTKs play an important role in conditioning-produced changes in type B cell excitability. PTKs and PKCs converge to effect reductions in B cell K+ currents during conditioning, apparently through distinct biophysical mechanisms.

Ligation of tumour-produced mucins to CD22 dramatically impairs splenic marginal zone B-cells

2009 ◽  
Vol 417 (3) ◽  
pp. 673-683 ◽  
Author(s):  
Munetoyo Toda ◽  
Risa Hisano ◽  
Hajime Yurugi ◽  
Kaoru Akita ◽  
Kouji Maruyama ◽  
...  
Keyword(s):  
Sialic Acid ◽  
B Cells ◽  
B Cell ◽  
Marginal Zone ◽  
Cell Signalling ◽  
Negative Regulator ◽  
Mammary Adenocarcinoma ◽  
Marginal Zone B Cells ◽  
Tumour Bearing

CD22 [Siglec-2 (sialic acid-binding, immunoglobulin-like lectin-2)], a negative regulator of B-cell signalling, binds to α2,6- sialic acid-linked glycoconjugates, including a sialyl-Tn antigen that is one of the typical tumour-associated carbohydrate antigens expressed on various mucins. Many epithelial tumours secrete mucins into tissues and/or the bloodstream. Mouse mammary adenocarcinoma cells, TA3-Ha, produce a mucin named epiglycanin, but a subline of them, TA3-St, does not. Epiglycanin binds to CD22 and inhibits B-cell signalling in vitro. The in vivo effect of mucins in the tumour-bearing state was investigated using these cell lines. It should be noted that splenic MZ (marginal zone) B-cells were dramatically reduced in the mice bearing TA3-Ha cells but not in those bearing TA3-St cells, this being consistent with the finding that the thymus-independent response was reduced in these mice. When the mucins were administered to normal mice, a portion of them was detected in the splenic MZ associated with the MZ B-cells. Furthermore, administration of mucins to normal mice clearly reduced the splenic MZ B-cells, similar to tumour-bearing mice. These results indicate that mucins in the bloodstream interacted with CD22, which led to impairment of the splenic MZ B-cells in the tumour-bearing state.

Sign in / Sign up

Export Citation Format

Share Document