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1.
Clin Transl Immunology ; 13(10): e70011, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-39364393

RESUMO

Objectives: Paediatric Burkitt's lymphoma (pBL) is the most common childhood non-Hodgkin B-cell lymphoma. Despite the encouraging survival rates for most children, treating cases with relapse/resistance to current therapies remains challenging. CD38 is a transmembrane protein highly expressed in pBL. This study investigates the effectiveness of CD38-targeting monoclonal antibodies (mAbs), daratumumab and isatuximab, in impairing crucial cellular processes and survival pathways in pBL malignant cells. Methods: In silico analyses of patient samples, combined with in vitro experiments using the Ramos cell line, were conducted to assess the impact of daratumumab and isatuximab on cellular proliferation, apoptosis and the phosphoinositide 3-kinase (PI3K) pathway. Results: Isatuximab was found to be more effective than daratumumab in disrupting B-cell receptor signalling, reducing cellular proliferation and inducing apoptosis. Additionally, isatuximab caused a significant impairment of the PI3K pathway and induced metabolic reprogramming in pBL cells. The study also revealed a correlation between CD38 and MYC expression levels in pBL patient samples, suggesting CD38 involvement in key oncogenic processes. Conclusion: The study emphasises the therapeutic potential of CD38-targeting mAbs, particularly isatuximab, in pBL.

2.
Front Immunol ; 15: 1432226, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-39139562

RESUMO

Introduction: The early transcription unit 3 (E3) of human adenoviruses (HAdVs) encodes several immunoevasins, including the E3/49K protein, which is unique for species D of HAdVs. It is expressed as surface transmembrane protein and shed. E3/49K of HAdV-D64 binds to the protein tyrosine phosphatase surface receptor CD45, thereby modulating activation of T and NK cells. Methods: Considering that E3/49K represents the most polymorphic viral protein among species D HAdVs, we demonstrate here that all tested E3/49K orthologs bind to the immunologically important regulator CD45. Thus, this feature is conserved regardless of the pathological associations of the respective HAdV types. Results: It appeared that modulation of CD45 is a unique property restricted to HAdVs of species D. Moreover, E3/49K treatment inhibited B cell receptor (BCR) signaling and impaired BCR signal phenotypes. The latter were highly comparable to B cells having defects in the expression of CD45, suggesting E3/49K as a potential tool to investigate CD45 specific functions. Conclusion: We identified B cells as new direct target of E3/49K-mediated immune modulation, representing a novel viral immunosubversive mechanism.


Assuntos
Proteínas E3 de Adenovirus , Adenovírus Humanos , Linfócitos B , Antígenos Comuns de Leucócito , Receptores de Antígenos de Linfócitos B , Transdução de Sinais , Humanos , Receptores de Antígenos de Linfócitos B/metabolismo , Receptores de Antígenos de Linfócitos B/imunologia , Transdução de Sinais/imunologia , Antígenos Comuns de Leucócito/metabolismo , Antígenos Comuns de Leucócito/imunologia , Adenovírus Humanos/imunologia , Proteínas E3 de Adenovirus/imunologia , Proteínas E3 de Adenovirus/metabolismo , Proteínas E3 de Adenovirus/genética , Linfócitos B/imunologia , Linfócitos B/metabolismo , Infecções por Adenovirus Humanos/imunologia , Infecções por Adenovirus Humanos/virologia , Infecções por Adenovirus Humanos/metabolismo , Células HEK293
3.
J Exp Med ; 219(9)2022 09 05.
Artigo em Inglês | MEDLINE | ID: mdl-35819358

RESUMO

CD38 is a multifunctional protein expressed on the surface of B cells in healthy individuals but also in B cell malignancies. Previous studies have suggested a connection between CD38 and components of the IgM class B cell antigen receptor (IgM-BCR) and its coreceptor complex. Here, we provide evidence that CD38 is closely associated with CD19 in resting B cells and with the IgM-BCR upon engagement. We show that targeting CD38 with an antibody, or removing this molecule with CRISPR/Cas9, inhibits the association of CD19 with the IgM-BCR, impairing BCR signaling in normal and malignant B cells. Together, our data suggest that CD38 is a new member of the BCR coreceptor complex, where it exerts a modulatory effect on B cell activation upon antigen recognition by regulating CD19. Our study also reveals a new mechanism where α-CD38 antibodies could be a valuable option in therapeutic approaches to B cell malignancies driven by aberrant BCR signaling.


Assuntos
ADP-Ribosil Ciclase 1/imunologia , Linfócitos B , Glicoproteínas de Membrana/imunologia , Receptores de Antígenos de Linfócitos B , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Antígenos CD19/metabolismo , Humanos , Imunoglobulina M , Ativação Linfocitária , Receptores de Antígenos de Linfócitos B/metabolismo
4.
Leukemia ; 36(3): 701-711, 2022 03.
Artigo em Inglês | MEDLINE | ID: mdl-34750506

RESUMO

FLT3-ITD is the most predominant mutation in AML being expressed in about one-third of AML patients and is associated with a poor prognosis. Efforts to better understand FLT3-ITD downstream signaling to possibly improve therapy response are needed. We have previously described FLT3-ITD-dependent phosphorylation of CSF2RB, the common receptor beta chain of IL-3, IL-5, and GM-CSF, and therefore examined its significance for FLT3-ITD-dependent oncogenic signaling and transformation. We discovered that FLT3-ITD directly binds to CSF2RB in AML cell lines and blasts isolated from AML patients. A knockdown of CSF2RB in FLT3-ITD positive AML cell lines as well as in a xenograft model decreased STAT5 phosphorylation, attenuated cell proliferation, and sensitized to FLT3 inhibition. Bone marrow from CSF2RB-deficient mice transfected with FLT3-ITD displayed decreased colony formation capacity and delayed disease onset together with increased survival upon transplantation into lethally irradiated mice. FLT3-ITD-dependent CSF2RB phosphorylation required phosphorylation of the FLT3 juxtamembrane domain at tyrosines 589 or 591, whereas the ITD insertion site and sequence were of no relevance. Our results demonstrate that CSF2RB participates in FLT3-ITD-dependent oncogenic signaling and transformation in vitro and in vivo. Thus, CSF2RB constitutes a rational treatment target in FLT3-ITD-positive AML.


Assuntos
Subunidade beta Comum dos Receptores de Citocinas/metabolismo , Leucemia Mieloide Aguda/metabolismo , Tirosina Quinase 3 Semelhante a fms/metabolismo , Animais , Linhagem Celular Tumoral , Subunidade beta Comum dos Receptores de Citocinas/genética , Técnicas de Silenciamento de Genes , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patologia , Camundongos , Camundongos Endogâmicos C57BL , Mutação , Fosforilação , Tirosina Quinase 3 Semelhante a fms/genética
5.
Life Sci Alliance ; 4(11)2021 11.
Artigo em Inglês | MEDLINE | ID: mdl-34526379

RESUMO

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.


Assuntos
Proteína Tirosina Fosfatase não Receptora Tipo 1/metabolismo , Receptores de Antígenos de Linfócitos B/metabolismo , Linfócitos B/metabolismo , Linhagem Celular , Proteína Adaptadora GRB2/metabolismo , Espectrometria de Massas/métodos , Fosforilação , Ligação Proteica , Proteína Tirosina Fosfatase não Receptora Tipo 1/genética , Proteína Tirosina Fosfatase não Receptora Tipo 1/fisiologia , Proteínas Tirosina Quinases/metabolismo , Proteômica/métodos , Proteínas Proto-Oncogênicas c-vav/metabolismo , Receptores de Antígenos de Linfócitos B/fisiologia , Lectina 2 Semelhante a Ig de Ligação ao Ácido Siálico/metabolismo , Transdução de Sinais/genética
6.
Proc Natl Acad Sci U S A ; 118(7)2021 02 16.
Artigo em Inglês | MEDLINE | ID: mdl-33563755

RESUMO

CD20 is a B cell-specific membrane protein and represents an attractive target for therapeutic antibodies. Despite widespread usage of anti-CD20 antibodies for B cell depletion therapies, the biological function of their target remains unclear. Here, we demonstrate that CD20 controls the nanoscale organization of receptors on the surface of resting B lymphocytes. CRISPR/Cas9-mediated ablation of CD20 in resting B cells resulted in relocalization and interaction of the IgM-class B cell antigen receptor with the coreceptor CD19. This receptor rearrangement led to a transient activation of B cells, accompanied by the internalization of many B cell surface marker proteins. Reexpression of CD20 restored the expression of the B cell surface proteins and the resting state of Ramos B cells. Similarly, treatment of Ramos or naive human B cells with the anti-CD20 antibody rituximab induced nanoscale receptor rearrangements and transient B cell activation in vitro and in vivo. A departure from the resting B cell state followed by the loss of B cell identity of CD20-deficient Ramos B cells was accompanied by a PAX5 to BLIMP-1 transcriptional switch, metabolic reprogramming toward oxidative phosphorylation, and a shift toward plasma cell development. Thus, anti-CD20 engagement or the loss of CD20 disrupts membrane organization, profoundly altering the fate of human B cells.


Assuntos
Antígenos CD20/metabolismo , Linfócitos B/imunologia , Antígenos CD19/metabolismo , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Células Cultivadas , Humanos , Ativação Linfocitária , Receptores de Antígenos de Linfócitos B/metabolismo
7.
Elife ; 82019 08 21.
Artigo em Inglês | MEDLINE | ID: mdl-31433296

RESUMO

In mice, neonatally-developing, self-reactive B-1 cells generate steady levels of natural antibodies throughout life. B-1 cells can, however, also rapidly respond to infections with increased local antibody production. The mechanisms regulating these two seemingly very distinct functions are poorly understood, but have been linked to expression of CD5, an inhibitor of BCR-signaling. Here we demonstrate that TLR-mediated activation of CD5+ B-1 cells induced the rapid reorganization of the IgM-BCR complex, leading to the eventual loss of CD5 expression, and a concomitant increase in BCR-downstream signaling, both in vitro and in vivo after infections of mice with influenza virus and Salmonella typhimurium. Both, initial CD5 expression and TLR-mediated stimulation, were required for the differentiation of B-1 cells to IgM-producing plasmablasts after infections. Thus, TLR-mediated signals support participation of B-1 cells in immune defense via BCR-complex reorganization.


Assuntos
Subpopulações de Linfócitos B/imunologia , Imunoglobulina M/metabolismo , Fatores Imunológicos/metabolismo , Orthomyxoviridae/imunologia , Proteínas Proto-Oncogênicas c-bcr/metabolismo , Salmonella typhimurium/imunologia , Receptores Toll-Like/metabolismo , Animais , Antígenos CD5/metabolismo , Modelos Animais de Doenças , Camundongos , Infecções por Orthomyxoviridae/imunologia , Salmonelose Animal/imunologia
8.
EMBO J ; 37(11)2018 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-29669863

RESUMO

Expression of the B-cell antigen receptor (BCR) is essential not only for the development but also for the maintenance of mature B cells. Similarly, many B-cell lymphomas, including Burkitt lymphoma (BL), require continuous BCR signaling for their tumor growth. This growth is driven by immunoreceptor tyrosine-based activation motif (ITAM) and PI3 kinase (PI3K) signaling. Here, we employ CRISPR/Cas9 to delete BCR and B-cell co-receptor genes in the human BL cell line Ramos. We find that Ramos B cells require the expression of the BCR signaling component Igß (CD79b), and the co-receptor CD19, for their fitness and competitive growth in culture. Furthermore, we show that in the absence of any other BCR component, Igß can be expressed on the B-cell surface, where it is found in close proximity to CD19 and signals in an ITAM-dependent manner. These data suggest that Igß and CD19 are part of an alternative B-cell signaling module that use continuous ITAM/PI3K signaling to promote the survival of B lymphoma and normal B cells.


Assuntos
Antígenos CD19/genética , Linfoma de Burkitt/genética , Antígenos CD79/genética , Aptidão Genética/genética , Linfócitos B/patologia , Linfoma de Burkitt/patologia , Sistemas CRISPR-Cas , Regulação Leucêmica da Expressão Gênica/genética , Humanos , Imunoglobulinas/genética , Motivo de Ativação do Imunorreceptor Baseado em Tirosina/genética , Fosfatidilinositol 3-Quinases/genética , Transdução de Sinais
9.
Methods Mol Biol ; 1707: 171-181, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29388107

RESUMO

The B cell antigen receptor (BCR) is found to be non-randomly organized at nano-scale distances on the B cell surface. Studying the organization and relocalization of the BCR is thus likely to provide new clues to understand the activation of the BCR. Indeed, with the in situ Fab proximity ligation assay (Fab-PLA), we now obtain proofs for the dissociation activation of BCRs and start to gain insight into how the relocalization of B cell surface signaling molecules could activate the cells. This chapter describes our methods to study the nano-scale organization of B cell surface receptors and co-receptors with Fab-PLA.


Assuntos
Linfócitos B/imunologia , Fragmentos Fab das Imunoglobulinas/química , Capeamento Imunológico , Receptores de Antígenos de Linfócitos B/imunologia , Transdução de Sinais/imunologia , Animais , Linfócitos B/citologia , Humanos
10.
Nat Immunol ; 18(10): 1150-1159, 2017 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-28805811

RESUMO

Caveolin-1 (Cav1) regulates the nanoscale organization and compartmentalization of the plasma membrane. Here we found that Cav1 controlled the distribution of nanoclusters of isotype-specific B cell antigen receptors (BCRs) on the surface of B cells. In mature B cells stimulated with antigen, the immunoglobulin M BCR (IgM-BCR) gained access to lipid domains enriched for GM1 glycolipids, by a process that was dependent on the phosphorylation of Cav1 by the Src family of kinases. Antigen-induced reorganization of nanoclusters of IgM-BCRs and IgD-BCRs regulated BCR signaling in vivo. In immature Cav1-deficient B cells, altered nanoscale organization of IgM-BCRs resulted in a failure of receptor editing and a skewed repertoire of B cells expressing immunoglobulin-µ heavy chains with hallmarks of poly- and auto-reactivity, which ultimately led to autoimmunity in mice. Thus, Cav1 emerges as a cell-intrinsic regulator that prevents B cell-induced autoimmunity by means of its role in plasma-membrane organization.


Assuntos
Linfócitos B/imunologia , Linfócitos B/metabolismo , Caveolina 1/metabolismo , Tolerância Imunológica , Receptores de Antígenos de Linfócitos B/metabolismo , Animais , Autoimunidade/genética , Autoimunidade/imunologia , Células da Medula Óssea/imunologia , Células da Medula Óssea/metabolismo , Caveolina 1/genética , Expressão Gênica , Tolerância Imunológica/genética , Imunoglobulina D/imunologia , Imunoglobulina D/metabolismo , Imunoglobulina M/imunologia , Imunoglobulina M/metabolismo , Ativação Linfocitária/genética , Ativação Linfocitária/imunologia , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Fosforilação , Ligação Proteica , Receptores de Antígenos de Linfócitos B/genética
11.
Nat Immunol ; 18(3): 321-333, 2017 03.
Artigo em Inglês | MEDLINE | ID: mdl-28135254

RESUMO

The FcµR receptor for the crystallizable fragment (Fc) of immunoglobulin M (IgM) can function as a cell-surface receptor for secreted IgM on a variety of cell types. We found here that FcµR was also expressed in the trans-Golgi network of developing B cells, where it constrained transport of the IgM-isotype BCR (IgM-BCR) but not of the IgD-isotype BCR (IgD-BCR). In the absence of FcµR, the surface expression of IgM-BCR was increased, which resulted in enhanced tonic BCR signaling. B-cell-specific deficiency in FcµR enhanced the spontaneous differentiation of B-1 cells, which resulted in increased serum concentrations of natural IgM and dysregulated homeostasis of B-2 cells; this caused the spontaneous formation of germinal centers, increased titers of serum autoantibodies and excessive accumulation of B cells. Thus, FcµR serves as a critical regulator of B cell biology by constraining the transport and cell-surface expression of IgM-BCR.


Assuntos
Linfócitos B/fisiologia , Imunoglobulina M/metabolismo , Células Precursoras de Linfócitos B/fisiologia , Receptores de Antígenos de Linfócitos B/metabolismo , Receptores Fc/metabolismo , Animais , Diferenciação Celular , Células Cultivadas , Citocinas/metabolismo , Feminino , Regulação da Expressão Gênica , Imunoglobulina M/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores de Antígenos de Linfócitos B/genética , Transdução de Sinais , Células Th1/imunologia , Células Th2/imunologia
12.
PLoS One ; 11(12): e0169159, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-28036404

RESUMO

The survival and proliferation of CLL cells depends on microenvironmental contacts in lymphoid organs. CD38 is a cell surface receptor that plays an important role in survival and proliferation signaling in CLL. In this study we demonstrate SYK's direct involvement in the CD38 signaling pathway in primary CLL samples. CD38 stimulation of CLL cells revealed SYK activation. SYK downstream target AKT was subsequently induced and MCL-1 expression was increased. Concomitant inhibition of SYK by the SYK inhibitor R406 resulted in reduced activation of AKT and prevented upregulation of MCL-1. Moreover, short-term CD38 stimulation enhanced BCR-signaling, as indicated by increased ERK phosphorylation. CXCL12-dependent migration was increased after CD38 stimulation. Treating CLL cells with R406 inhibited CD38-mediated migration. In addition, we observed marked downregulation of CD38 expression for CLL cells treated with R406 compared to vehicle control. Finally, we observed a clear correlation between CD38 expression on CLL cells and SYK-inhibitor efficacy. In conclusion, our study provides deeper mechanistic insight into the effect of SYK inhibition in CLL.


Assuntos
ADP-Ribosil Ciclase 1/metabolismo , Leucemia Linfocítica Crônica de Células B/patologia , Glicoproteínas de Membrana/metabolismo , Proteína de Sequência 1 de Leucemia de Células Mieloides/biossíntese , Proteínas Proto-Oncogênicas c-akt/metabolismo , Quinase Syk/antagonistas & inibidores , Quinase Syk/metabolismo , ADP-Ribosil Ciclase 1/biossíntese , ADP-Ribosil Ciclase 1/farmacologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Apoptose/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Sobrevivência Celular , Quimiocina CXCL12/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Feminino , Humanos , Masculino , Glicoproteínas de Membrana/biossíntese , Glicoproteínas de Membrana/farmacologia , Pessoa de Meia-Idade , Oxazinas/farmacologia , Fosforilação/efeitos dos fármacos , Piridinas/farmacologia , Células Tumorais Cultivadas
13.
J Immunol ; 195(4): 1548-63, 2015 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-26157172

RESUMO

The invariant chain (CD74), a chaperone in MHC class II-mediated Ag presentation, is sequentially processed by different endosomal proteases. We reported recently that clearance of the final membrane-bound N-terminal fragment (NTF) of CD74 is mediated by the intramembrane protease signal peptide peptidase-like (SPPL)2a, a process critical for B cell development. In mice, SPPL2a deficiency provokes the accumulation of this NTF in endocytic vesicles, which leads to a B cell maturation arrest at the transitional 1 stage. To define the underlying mechanism, we analyzed the impact of SPPL2a deficiency on signaling pathways involved in B cell homeostasis. We demonstrate that tonic as well as BCR-induced activation of the PI3K/Akt pathway is massively compromised in SPPL2a(-/-) B cells and identify this as major cause of the B cell maturation defect in these mice. Altered BCR trafficking induces a reduction of surface IgM in SPPL2a-deficient B cells, leading to a diminished signal transmission via the BCR and the tyrosine kinase Syk. We provide evidence that in SPPL2a(-/-) mice impaired BCR signaling is to a great extent provoked by the accumulating CD74 NTF, which can interact with the BCR and Syk, and that impaired PI3K/Akt signaling and reduced surface IgM are not directly linked processes. In line with disturbances in PI3K/Akt signaling, SPPL2a(-/-) B cells show a dysregulation of the transcription factor FOXO1, causing elevated transcription of proapoptotic genes. We conclude that SPPL2a-mediated processing of CD74 NTF is indispensable to maintain appropriate levels of tonic BCR signaling to promote B cell maturation.


Assuntos
Antígenos de Diferenciação de Linfócitos B/metabolismo , Ácido Aspártico Endopeptidases/metabolismo , Linfócitos B/metabolismo , Antígenos de Histocompatibilidade Classe II/metabolismo , Proteínas de Membrana/metabolismo , Receptores de Antígenos de Linfócitos B/metabolismo , Transdução de Sinais , Animais , Antígenos de Diferenciação de Linfócitos B/química , Apoptose/genética , Ácido Aspártico Endopeptidases/deficiência , Ácido Aspártico Endopeptidases/genética , Linfócitos B/citologia , Linfócitos B/imunologia , Diferenciação Celular , Membrana Celular/metabolismo , Endocitose/genética , Endocitose/imunologia , Proteína Forkhead Box O1 , Fatores de Transcrição Forkhead/metabolismo , Antígenos de Histocompatibilidade Classe II/química , Imunoglobulina M/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Ativação Linfocitária , Sistema de Sinalização das MAP Quinases , Proteínas de Membrana/deficiência , Proteínas de Membrana/genética , Camundongos , Camundongos Knockout , NF-kappa B/metabolismo , Subunidade p52 de NF-kappa B/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação , Células Precursoras de Linfócitos B/citologia , Células Precursoras de Linfócitos B/metabolismo , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Quinase Syk
14.
Dev Cell ; 32(5): 617-30, 2015 Mar 09.
Artigo em Inglês | MEDLINE | ID: mdl-25727005

RESUMO

The tuberous sclerosis proteins TSC1 and TSC2 are key integrators of growth factor signaling. They suppress cell growth and proliferation by acting in a heteromeric complex to inhibit the mammalian target of rapamycin complex 1 (mTORC1). In this study, we identify TSC1 as a component of the transforming growth factor ß (TGF-ß)-Smad2/3 pathway. Here, TSC1 functions independently of TSC2. TSC1 interacts with the TGF-ß receptor complex and Smad2/3 and is required for their association with one another. TSC1 regulates TGF-ß-induced Smad2/3 phosphorylation and target gene expression and controls TGF-ß-induced growth arrest and epithelial-to-mesenchymal transition (EMT). Hyperactive Akt specifically activates TSC1-dependent cytostatic Smad signaling to induce growth arrest. Thus, TSC1 couples Akt activity to TGF-ß-Smad2/3 signaling. This has implications for cancer treatments targeting phosphoinositide 3-kinases and Akt because they may impair tumor-suppressive cytostatic TGF-ß signaling by inhibiting Akt- and TSC1-dependent Smad activation.


Assuntos
Apoptose , Proliferação de Células , Transição Epitelial-Mesenquimal , Proteína Smad2/metabolismo , Proteína Smad3/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Western Blotting , Células Cultivadas , Citometria de Fluxo , Imunofluorescência , Humanos , Técnicas Imunoenzimáticas , Imunoprecipitação , Alvo Mecanístico do Complexo 1 de Rapamicina , Complexos Multiproteicos/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Transdução de Sinais , Serina-Treonina Quinases TOR/metabolismo , Proteína 1 do Complexo Esclerose Tuberosa , Proteína 2 do Complexo Esclerose Tuberosa
15.
J Immunol ; 193(1): 268-76, 2014 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-24899508

RESUMO

Signal transduction from the BCR is regulated by the equilibrium between kinases (e.g., spleen tyrosine kinase [Syk]) and phosphatases (e.g., Shp-1). Previous studies showed that Syk-deficient B cells have a developmental block at the pro/pre-B cell stage, whereas a B cell-specific Shp-1 deficiency promoted B-1a cell development and led to autoimmunity. We generated B cell-specific Shp-1 and Syk double-knockout (DKO) mice and compared them to the single-knockout mice deficient for either Syk or Shp-1. Unlike Syk-deficient mice, the DKO mice can generate mature B cells, albeit at >20-fold reduced B cell numbers. The DKO B-2 cells are all Syk-negative, whereas the peritoneal B1 cells of the DKO mice still express Syk, indicating that they require this kinase for their proper development. The DKO B-2 cells cannot be stimulated via the BCR, whereas they are efficiently activated via TLR or CD40. We also found that in DKO pre-B cells, the kinase Zap70 is associated with the pre-BCR, suggesting that Zap70 is important to promote B cell maturation in the absence of Syk and SHP-1. Together, our data show that a properly balanced kinase/phosphatase equilibrium is crucial for normal B cell development and function.


Assuntos
Subpopulações de Linfócitos B/imunologia , Peptídeos e Proteínas de Sinalização Intracelular/imunologia , Proteína Tirosina Fosfatase não Receptora Tipo 6/imunologia , Proteínas Tirosina Quinases/imunologia , Transdução de Sinais/imunologia , Animais , Subpopulações de Linfócitos B/citologia , Peptídeos e Proteínas de Sinalização Intracelular/genética , Camundongos , Camundongos Knockout , Proteína Tirosina Fosfatase não Receptora Tipo 6/genética , Proteínas Tirosina Quinases/genética , Transdução de Sinais/genética , Quinase Syk , Proteína-Tirosina Quinase ZAP-70/genética , Proteína-Tirosina Quinase ZAP-70/imunologia
16.
Elife ; 3: e02069, 2014 Jun 24.
Artigo em Inglês | MEDLINE | ID: mdl-24963139

RESUMO

Binding of antigen to the B cell antigen receptor (BCR) initiates a multitude of events resulting in B cell activation. How the BCR becomes signaling-competent upon antigen binding is still a matter of controversy. Using a high-resolution proximity ligation assay (PLA) to monitor the conformation of the BCR and its interactions with co-receptors at a 10-20 nm resolution, we provide direct evidence for the opening of BCR dimers during B cell activation. We also show that upon binding Syk opens the receptor by an inside-out signaling mechanism that amplifies BCR signaling. Furthermore, we found that on resting B cells, the coreceptor CD19 is in close proximity with the IgD-BCR and on activated B cells with the IgM-BCR, indicating nanoscale reorganization of receptor clusters during B cell activation.DOI: http://dx.doi.org/10.7554/eLife.02069.001.


Assuntos
Linfócitos B/metabolismo , Ativação Linfocitária , Receptores de Antígenos de Linfócitos B/metabolismo , Transdução de Sinais , Animais , Linhagem Celular , Dimerização , Humanos , Camundongos
17.
J Immunol ; 192(11): 5406-14, 2014 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-24790146

RESUMO

Siglec-G is an inhibitory receptor on B1 cells. Siglec-G-deficient mice show a large B1 cell expansion, owing to higher BCR-induced Ca(2+) signaling and enhanced cellular survival. It was unknown why Siglec-G shows a B1 cell-restricted inhibitory function. With a new mAb we could show a comparable Siglec-G expression on B1 cells and conventional B2 cells. However, Siglec-G has a different ligand sialic acid-binding pattern on peritoneal B1 cells than on splenic B cells, and its sialic acid ligands are expressed differentially on these two B cell populations, suggesting that cis-ligand binding plays a crucial role on B1 cells. This observation was further studied by generation of Siglec-G knockin mice with a mutated ligand-binding domain. These mice show increased B1 cell numbers, increased B1 cell Ca(2+) signaling, better B1 cell survival, and changes in the B1 cell Ig repertoire. These phenotypes are very similar to Siglec-G-deficient mice. The mutation of the ligand-binding domain of Siglec-G strongly reduces the Siglec-G-IgM association on the B cell surface. Thus, Siglec-G sialic acid-dependent binding to the BCR is crucial for the B1 cell-restricted inhibitory function of Siglec-G and is regulated in an opposite way to that of the related protein CD22 (Siglec-2) on B cells.


Assuntos
Subpopulações de Linfócitos B/imunologia , Sinalização do Cálcio/imunologia , Lectinas/imunologia , Receptores de Antígenos de Linfócitos B/imunologia , Animais , Subpopulações de Linfócitos B/citologia , Imunoglobulina M/imunologia , Lectinas/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Mutantes , Especificidade de Órgãos/genética , Especificidade de Órgãos/imunologia , Estrutura Terciária de Proteína , Receptores de Antígenos de Linfócitos B/genética , Lectinas Semelhantes a Imunoglobulina de Ligação ao Ácido Siálico
18.
Cell ; 154(4): 859-74, 2013 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-23953116

RESUMO

Mammalian target of rapamycin complex 1 (mTORC1) controls growth and survival in response to metabolic cues. Oxidative stress affects mTORC1 via inhibitory and stimulatory inputs. Whereas downregulation of TSC1-TSC2 activates mTORC1 upon oxidative stress, the molecular mechanism of mTORC1 inhibition remains unknown. Here, we identify astrin as an essential negative mTORC1 regulator in the cellular stress response. Upon stress, astrin inhibits mTORC1 association and recruits the mTORC1 component raptor to stress granules (SGs), thereby preventing mTORC1-hyperactivation-induced apoptosis. In turn, balanced mTORC1 activity enables expression of stress factors. By identifying astrin as a direct molecular link between mTORC1, SG assembly, and the stress response, we establish a unifying model of mTORC1 inhibition and activation upon stress. Importantly, we show that in cancer cells, apoptosis suppression during stress depends on astrin. Being frequently upregulated in tumors, astrin is a potential clinically relevant target to sensitize tumors to apoptosis.


Assuntos
Apoptose , Neoplasias da Mama/metabolismo , Proteínas de Ciclo Celular/metabolismo , Complexos Multiproteicos/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Grânulos Citoplasmáticos/metabolismo , Humanos , Alvo Mecanístico do Complexo 1 de Rapamicina , Estresse Oxidativo , Proteína Regulatória Associada a mTOR
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