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1.
Sci Transl Med ; 15(726): eadf9561, 2023 12 13.
Artículo en Inglés | MEDLINE | ID: mdl-38091405

RESUMEN

Immunoglobulin E (IgE) is a key driver of type 1 hypersensitivity reactions and allergic disorders, which are globally increasing in number and severity. Although eliminating pathogenic IgE may be a powerful way to treat allergy, no therapeutic strategy reported to date can fully ablate IgE production. Interleukin-4 receptor α (IL-4Rα) signaling is required for IgE class switching, and IL-4Rα blockade gradually reduces, but does not eliminate, IgE. The persistence of IgE after IL-4Rα blockade may be due to long-lived IgE+ plasma cells that maintain serological memory to allergens and thus may be susceptible to plasma cell-targeted therapeutics. We demonstrate that transient administration of a B cell maturation antigen x CD3 (BCMAxCD3) bispecific antibody markedly depletes IgE, as well as other immunoglobulins, by ablating long-lived plasma cells, although IgE and other immunoglobulins rapidly rebound after treatment. Concomitant IL-4Rα blockade specifically and durably prevents the reemergence of IgE by blocking IgE class switching while allowing the restoration of other immunoglobulins. Moreover, this combination treatment prevented anaphylaxis in mice. Together with additional cynomolgus monkey and human data, our studies demonstrate that allergic memory is primarily maintained by both non-IgE+ memory B cells that require class switching and long-lived IgE+ plasma cells. Our combination approach to durably eliminate pathogenic IgE has potential to benefit allergy in humans while preserving antibody-mediated immunity.


Asunto(s)
Anafilaxia , Inmunoglobulina E , Ratones , Humanos , Animales , Macaca fascicularis , Células Plasmáticas , Alérgenos
2.
Cell Rep Methods ; 3(7): 100522, 2023 07 24.
Artículo en Inglés | MEDLINE | ID: mdl-37533642

RESUMEN

Following activation by cognate antigen, B cells undergo fine-tuning of their antigen receptors and may ultimately differentiate into antibody-secreting cells (ASCs). While antigen-specific B cells that express surface receptors (B cell receptors [BCRs]) can be readily cloned and sequenced following flow sorting, antigen-specific ASCs that lack surface BCRs cannot be easily profiled. Here, we report an approach, TRAPnSeq (antigen specificity mapping through immunoglobulin [Ig] secretion TRAP and Sequencing), that allows capture of secreted antibodies on the surface of ASCs, which in turn enables high-throughput screening of single ASCs against large antigen panels. This approach incorporates flow cytometry, standard microfluidic platforms, and DNA-barcoding technologies to characterize antigen-specific ASCs through single-cell V(D)J, RNA, and antigen barcode sequencing. We show the utility of TRAPnSeq by profiling antigen-specific IgG and IgE ASCs from both mice and humans and highlight its capacity to accelerate therapeutic antibody discovery from ASCs.


Asunto(s)
Células Productoras de Anticuerpos , Antígenos , Humanos , Animales , Ratones , Linfocitos B , Anticuerpos/genética , Receptores de Antígenos de Linfocitos B/genética
3.
J Vis Exp ; (167)2021 01 22.
Artículo en Inglés | MEDLINE | ID: mdl-33554965

RESUMEN

Extensive studies have characterized the development and differentiation of murine B cells in secondary lymphoid organs. Antibodies secreted by B cells have been isolated and developed into well-established therapeutics. Validation of murine B cell development, in the context of autoimmune prone mice, or in mice with modified immune systems, is a crucial component of developing or testing therapeutic agents in mice and is an appropriate use of flow cytometry. Well established B cell flow cytometric parameters can be used to evaluate B cell development in the murine peritoneum, bone marrow, and spleen, but a number of best practices must be adhered to. In addition, flow cytometric analysis of B cell compartments should also complement additional readouts of B cell development. Data generated using this technique can further our understanding of wild type, autoimmune prone mouse models as well as humanized mice that can be used to generate antibody or antibody-like molecules as therapeutics.


Asunto(s)
Linfocitos B/citología , Citometría de Flujo/métodos , Animales , Linfocitos B/inmunología , Células de la Médula Ósea/citología , Recuento de Células , Diferenciación Celular , Separación Celular , Análisis de Datos , Femenino , Cadenas lambda de Inmunoglobulina/metabolismo , Inmunoglobulinas/metabolismo , Activación de Linfocitos , Subgrupos Linfocitarios/citología , Ratones Endogámicos C57BL , Peritoneo/citología , Bazo/citología , Coloración y Etiquetado
4.
Sci Immunol ; 5(43)2020 01 10.
Artículo en Inglés | MEDLINE | ID: mdl-31924685

RESUMEN

Immunoglobulin E (IgE) plays an important role in allergic diseases. Nevertheless, the source of IgE serological memory remains controversial. We reexamined the mechanism of serological memory in allergy using a dual reporter system to track IgE+ plasma cells in mice. Short-term allergen exposure resulted in the generation of IgE+ plasma cells that resided mainly in secondary lymphoid organs and produced IgE that was unable to degranulate mast cells. In contrast, chronic allergen exposure led to the generation of long-lived IgE+ plasma cells that were primarily derived from sequential class switching of IgG1, accumulated in the bone marrow, and produced IgE capable of inducing anaphylaxis. IgE+ plasma cells were found in the bone marrow of human allergic, but not nonallergic donors, and allergen-specific IgE produced by these cells was able to induce mast cell degranulation when transferred to mice. These data demonstrate that long-lived IgE+ bone marrow plasma cells arise during chronic allergen exposure and establish serological memory in both mice and humans.


Asunto(s)
Alérgenos/inmunología , Inmunoglobulina E/sangre , Memoria Inmunológica , Células Plasmáticas/inmunología , Pyroglyphidae/inmunología , Anafilaxia/inmunología , Animales , Células de la Médula Ósea/inmunología , Exposición a Riesgos Ambientales , Humanos , Mastocitos/inmunología , Ratones
6.
Nature ; 521(7552): 357-61, 2015 May 21.
Artículo en Inglés | MEDLINE | ID: mdl-25799995

RESUMEN

B cells are selected for an intermediate level of B-cell antigen receptor (BCR) signalling strength: attenuation below minimum (for example, non-functional BCR) or hyperactivation above maximum (for example, self-reactive BCR) thresholds of signalling strength causes negative selection. In ∼25% of cases, acute lymphoblastic leukaemia (ALL) cells carry the oncogenic BCR-ABL1 tyrosine kinase (Philadelphia chromosome positive), which mimics constitutively active pre-BCR signalling. Current therapeutic approaches are largely focused on the development of more potent tyrosine kinase inhibitors to suppress oncogenic signalling below a minimum threshold for survival. We tested the hypothesis that targeted hyperactivation--above a maximum threshold--will engage a deletional checkpoint for removal of self-reactive B cells and selectively kill ALL cells. Here we find, by testing various components of proximal pre-BCR signalling in mouse BCR-ABL1 cells, that an incremental increase of Syk tyrosine kinase activity was required and sufficient to induce cell death. Hyperactive Syk was functionally equivalent to acute activation of a self-reactive BCR on ALL cells. Despite oncogenic transformation, this basic mechanism of negative selection was still functional in ALL cells. Unlike normal pre-B cells, patient-derived ALL cells express the inhibitory receptors PECAM1, CD300A and LAIR1 at high levels. Genetic studies revealed that Pecam1, Cd300a and Lair1 are critical to calibrate oncogenic signalling strength through recruitment of the inhibitory phosphatases Ptpn6 (ref. 7) and Inpp5d (ref. 8). Using a novel small-molecule inhibitor of INPP5D (also known as SHIP1), we demonstrated that pharmacological hyperactivation of SYK and engagement of negative B-cell selection represents a promising new strategy to overcome drug resistance in human ALL.


Asunto(s)
Linfocitos B/metabolismo , Linfocitos B/patología , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/patología , Transducción de Señal , Secuencias de Aminoácidos/genética , Animales , Antígenos CD/metabolismo , Linfocitos B/efectos de los fármacos , Muerte Celular/efectos de los fármacos , Línea Celular Tumoral , Transformación Celular Neoplásica , Modelos Animales de Enfermedad , Resistencia a Antineoplásicos/efectos de los fármacos , Activación Enzimática/efectos de los fármacos , Femenino , Proteínas de Fusión bcr-abl/genética , Eliminación de Gen , Humanos , Inositol Polifosfato 5-Fosfatasas , Péptidos y Proteínas de Señalización Intracelular/agonistas , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Ratones , Ratones Endogámicos NOD , Ratones SCID , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatasas , Monoéster Fosfórico Hidrolasas/antagonistas & inhibidores , Monoéster Fosfórico Hidrolasas/metabolismo , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamiento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Células Precursoras de Linfocitos B/efectos de los fármacos , Células Precursoras de Linfocitos B/metabolismo , Células Precursoras de Linfocitos B/patología , Proteína Tirosina Fosfatasa no Receptora Tipo 6/deficiencia , Proteína Tirosina Fosfatasa no Receptora Tipo 6/genética , Proteína Tirosina Fosfatasa no Receptora Tipo 6/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Receptores de Antígenos de Linfocitos B/deficiencia , Receptores de Antígenos de Linfocitos B/genética , Receptores de Antígenos de Linfocitos B/metabolismo , Receptores Inmunológicos/genética , Receptores Inmunológicos/metabolismo , Transducción de Señal/efectos de los fármacos , Quinasa Syk , Tirosina/metabolismo , Ensayos Antitumor por Modelo de Xenoinjerto
7.
Mol Cell Biol ; 34(8): 1474-85, 2014 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-24515435

RESUMEN

Protein kinase Cδ (PKCδ) deficiency causes autoimmune pathology in humans and mice and is crucial for the maintenance of B cell homeostasis. However, the mechanisms underlying autoimmune disease in PKCδ deficiency remain poorly defined. Here, we address the antigen-dependent and -independent roles of PKCδ in B cell development, repertoire selection, and antigen responsiveness. We demonstrate that PKCδ is rapidly phosphorylated downstream of both the B cell receptor (BCR) and the B cell-activating factor (BAFF) receptor. We found that PKCδ is essential for antigen-dependent negative selection of splenic transitional B cells and is required for activation of the proapoptotic Ca(2+)-Erk pathway that is selectively activated during B cell-negative selection. Unexpectedly, we also identified a previously unrecognized role for PKCδ as a proximal negative regulator of BCR signaling that substantially impacts survival and proliferation of mature follicular B cells. As a consequence of these distinct roles, PKCδ deficiency leads to the survival and development of a B cell repertoire that is not only aberrantly autoreactive but also hyperresponsive to antigen stimulation.


Asunto(s)
Linfocitos B/inmunología , Tolerancia Inmunológica/inmunología , Células Precursoras de Linfocitos B/citología , Proteína Quinasa C-delta/metabolismo , Receptores de Antígenos de Linfocitos B/metabolismo , Transducción de Señal , Animales , Factor Activador de Células B/inmunología , Factor Activador de Células B/metabolismo , Linfocitos B/citología , Linfocitos B/metabolismo , Diferenciación Celular/inmunología , Activación de Linfocitos/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Fosforilación/fisiología , Células Precursoras de Linfocitos B/inmunología , Proteína Quinasa C-delta/deficiencia , Proteína Quinasa C-delta/inmunología , Receptores de Antígenos de Linfocitos B/inmunología , Transducción de Señal/inmunología
8.
Elife ; 2: e01020, 2013 Dec 12.
Artículo en Inglés | MEDLINE | ID: mdl-24336796

RESUMEN

Missense variants are a major source of human genetic variation. Here we analyze a new mouse missense variant, Rasgrp1(Anaef), with an ENU-mutated EF hand in the Rasgrp1 Ras guanine nucleotide exchange factor. Rasgrp1(Anaef) mice exhibit anti-nuclear autoantibodies and gradually accumulate a CD44(hi) Helios(+) PD-1(+) CD4(+) T cell population that is dependent on B cells. Despite reduced Rasgrp1-Ras-ERK activation in vitro, thymocyte selection in Rasgrp1(Anaef) is mostly normal in vivo, although CD44 is overexpressed on naïve thymocytes and T cells in a T-cell-autonomous manner. We identify CD44 expression as a sensitive reporter of tonic mTOR-S6 kinase signaling through a novel mouse strain, chino, with a reduction-of-function mutation in Mtor. Elevated tonic mTOR-S6 signaling occurs in Rasgrp1(Anaef) naïve CD4(+) T cells. CD44 expression, CD4(+) T cell subset ratios and serum autoantibodies all returned to normal in Rasgrp1(Anaef)Mtor(chino) double-mutant mice, demonstrating that increased mTOR activity is essential for the Rasgrp1(Anaef) T cell dysregulation. DOI: http://dx.doi.org/10.7554/eLife.01020.001.


Asunto(s)
Autoanticuerpos/inmunología , Factores de Intercambio de Guanina Nucleótido/fisiología , Receptores de Hialuranos/inmunología , Mutación , Linfocitos T/inmunología , Serina-Treonina Quinasas TOR/fisiología , Animales , Motivos EF Hand , Factores de Intercambio de Guanina Nucleótido/genética , Ratones
9.
Elife ; 2: e00813, 2013 Jul 30.
Artículo en Inglés | MEDLINE | ID: mdl-23908768

RESUMEN

RasGRP1 and SOS are Ras-specific nucleotide exchange factors that have distinct roles in lymphocyte development. RasGRP1 is important in some cancers and autoimmune diseases but, in contrast to SOS, its regulatory mechanisms are poorly understood. Activating signals lead to the membrane recruitment of RasGRP1 and Ras engagement, but it is unclear how interactions between RasGRP1 and Ras are suppressed in the absence of such signals. We present a crystal structure of a fragment of RasGRP1 in which the Ras-binding site is blocked by an interdomain linker and the membrane-interaction surface of RasGRP1 is hidden within a dimerization interface that may be stabilized by the C-terminal oligomerization domain. NMR data demonstrate that calcium binding to the regulatory module generates substantial conformational changes that are incompatible with the inactive assembly. These features allow RasGRP1 to be maintained in an inactive state that is poised for activation by calcium and membrane-localization signals. DOI:http://dx.doi.org/10.7554/eLife.00813.001.


Asunto(s)
Proteínas de Unión al ADN/metabolismo , Factores de Intercambio de Guanina Nucleótido/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Calcio/metabolismo , Cristalografía por Rayos X , Proteínas de Unión al ADN/antagonistas & inhibidores , Proteínas de Unión al ADN/química , Factores de Intercambio de Guanina Nucleótido/antagonistas & inhibidores , Factores de Intercambio de Guanina Nucleótido/química , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Resonancia Magnética Nuclear Biomolecular , Conformación Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Homología de Secuencia de Aminoácido
10.
Front Biol (Beijing) ; 8(5): 508-532, 2013 Oct 01.
Artículo en Inglés | MEDLINE | ID: mdl-24744772

RESUMEN

RasGRP proteins are activators of Ras and other related small GTPases by the virtue of functioning as guanine nucleotide exchange factors (GEFs). In vertebrates, four RasGRP family members have been described. RasGRP-1 through -4 share many structural domains but there are also subtle differences between each of the different family members. Whereas SOS RasGEFs are ubiquitously expressed, RasGRP proteins are expressed in distinct patterns, such as in different cells of the hematopoietic system and in the brain. Most studies have concentrated on the role of RasGRP proteins in the development and function of immune cell types because of the predominant RasGRP expression profiles in these cells and the immune phenotypes of mice deficient for Rasgrp genes. However, more recent studies demonstrate that RasGRPs also play an important role in tumorigenesis. Examples are skin- and hematological-cancers but also solid malignancies such as melanoma or prostate cancer. These novel studies bring up many new and unanswered questions related to the molecular mechanism of RasGRP-driven oncogenesis, such as new receptor systems that RasGRP appears to respond to as well as regulatory mechanism for RasGRP expression that appear to be perturbed in these cancers. Here we will review some of the known aspects of RasGRP biology in lymphocytes and will discuss the exciting new notion that RasGRP Ras exchange factors play a role in oncogenesis downstream of various growth factor receptors.

11.
Nat Immunol ; 12(5): 425-33, 2011 May.
Artículo en Inglés | MEDLINE | ID: mdl-21441934

RESUMEN

Clonal deletion of autoreactive B cells is crucial for the prevention of autoimmunity, but the signaling mechanisms that regulate this checkpoint remain undefined. Here we characterize a previously unrecognized Ca(2+)-driven pathway for activation of the kinase Erk, which was proapoptotic and biochemically distinct from Erk activation induced by diacylglycerol (DAG). This pathway required protein kinase C-δ (PKC-δ) and the guanine nucleotide-exchange factor RasGRP and depended on the concentration of the Ca(2+) sensor STIM1, which controls the magnitude of Ca(2+) entry. Developmental regulation of these proteins was associated with selective activation of the pathway in B cells prone to negative selection. This checkpoint was impaired in PKC-δ-deficient mice, which developed B cell autoimmunity. Conversely, overexpression of STIM1 conferred a competitive disadvantage to developing B cells. Our findings establish Ca(2+)-dependent Erk signaling as a critical proapoptotic pathway that mediates the negative selection of B cells.


Asunto(s)
Linfocitos B/inmunología , Quinasas MAP Reguladas por Señal Extracelular/inmunología , Factores de Intercambio de Guanina Nucleótido/inmunología , Glicoproteínas de Membrana/inmunología , Proteína Quinasa C-delta/inmunología , Animales , Apoptosis/inmunología , Linfocitos B/enzimología , Canales de Calcio , Línea Celular , Activación Enzimática/inmunología , Citometría de Flujo , Regulación del Desarrollo de la Expresión Génica , Immunoblotting , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Fosforilación/inmunología , Transducción de Señal , Molécula de Interacción Estromal 1
12.
Small GTPases ; 2(5): 282-288, 2011 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-22292132

RESUMEN

Signaling via the Ras/Erk pathway has long been recognized to be critical in lymphocyte development and function, yet the mechanisms that control the distinct functional outputs of this pathway in different cellular contexts remain poorly understood. Our recent results have demonstrated unexpected involvement of Ras/Erk signaling in the sensitization of B cells to apoptosis in order to eliminate autoreactive cells. Increases in cytosolic Ca(2+) are necessary and sufficient to induce activation of this Ras/Erk pathway, and the biochemical events involved in its activation are different from the ones involved in diacylglycerol (DAG)-mediated Ras/Erk activation. Developmental regulation of upstream mediators of these distinct pathways contributes to their predominant activation at different stages of B cell development. These findings have revealed a mechanism by which antigen stimulation can activate distinct Ras/Erk pathways at different developmental stages to mediate appropriate functional outputs that control the selection, development and activation of B cells. Despite these recent findings, however, much remains to be learned about the molecular mechanisms that confer functional specificity to common Ras/Erk signaling modules.

13.
Blood ; 111(3): 1677-85, 2008 Feb 01.
Artículo en Inglés | MEDLINE | ID: mdl-18042805

RESUMEN

The precise mechanisms by which Abl oncogenes transform hematopoietic cells are unknown. We have examined the role of Pim kinases in v-Abl-mediated transformation. In v-Abl transformants, expression of Pim-1 and Pim-2, but not Pim-3, is dependent on Abl kinase activity. Transformation assays demonstrate that v-Abl cannot efficiently transform bone marrow cells derived from Pim-1(-/-)/Pim-2(-/-) mice. Ectopic expression of either Pim-1 or Pim-2 in Pim-1(-/-)/Pim-2(-/-) cells restores transformation by v-Abl, strongly suggesting that either Pim-1 or Pim-2 is required for v-Abl-mediated tumorigenesis. Interestingly, the combined deficiency of Pim-1, Pim-2, and Suppressor of Cytokine Signalling (SOCS)-1 resulted in partial restoration of v-Abl transformation efficiency. In addition, Pim kinases are involved in modification of SOCS-1 and in regulating SOCS-1 protein levels in v-Abl-transformed cells. Furthermore, Pim kinases regulate the proapoptotic proteins Bcl-XS and BAD. Pim kinases inhibit the expression of Bcl-XS. Pim deficiency decreases the phosphorylation levels of BAD, whereas ectopic expression of Pim-1 increases the amount of phospho-BAD. This correlates with an increased protection from apoptosis in Abl transformants expressing Pim kinases. Together, these data suggest that Pim kinases play a key role in the v-Abl transformation, possibly via participating in modulation of SOCS-1 and via regulating the apoptotic signaling.


Asunto(s)
Linfocitos B/citología , Linfocitos B/metabolismo , Transformación Celular Neoplásica , Proteínas Oncogénicas v-abl/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-pim-1/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Animales , Apoptosis/efectos de los fármacos , Benzamidas , Células Cultivadas , Mesilato de Imatinib , Hígado/citología , Hígado/metabolismo , Ratones , Ratones Noqueados , Proteínas Oncogénicas v-abl/genética , Fosforilación , Piperazinas/farmacología , Proteínas Serina-Treonina Quinasas/deficiencia , Proteínas Serina-Treonina Quinasas/genética , Proteínas Proto-Oncogénicas/deficiencia , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas c-pim-1/deficiencia , Proteínas Proto-Oncogénicas c-pim-1/genética , Pirimidinas/farmacología , Proteína 1 Supresora de la Señalización de Citocinas , Proteínas Supresoras de la Señalización de Citocinas/deficiencia , Proteínas Supresoras de la Señalización de Citocinas/genética , Proteínas Supresoras de la Señalización de Citocinas/metabolismo , Proteína Letal Asociada a bcl/metabolismo
14.
J Cell Biol ; 177(2): 317-28, 2007 Apr 23.
Artículo en Inglés | MEDLINE | ID: mdl-17452533

RESUMEN

Activation of the B cell receptor complex in B lymphocytes causes Ca(2+) release from intracellular stores, which, in turn, activates ion channels known as Icrac. We investigated the mechanisms that link Ca(2+) store release to channel gating in DT40 B lymphocyte cell lines genetically manipulated to suppress the expression of several tyrosine kinases: Btk, Lyn, Syk, and the Blnk adaptor molecule. The simultaneous but not the independent suppression of Lyn and Syk expression prevents the activation of Icrac without interfering with thapsigargin-sensitive Ca(2+) store release. Icrac activation by Ca(2+) is reversed in mutant cells by the homologous expression of the missing kinases. Pharmacological inhibition of kinase activity by LavendustinA and PP2 cause the same functional deficit as the genetic suppression of enzyme expression. Biochemical assays demonstrate that kinase activity is required as a tonic signal: targets must be phosphorylated to link Ca(2+) store release to Icrac gating. The action of kinases on Icrac activation does not arise from control of the expression level of the stromal interaction molecule 1 and Orai1 proteins.


Asunto(s)
Linfocitos B/metabolismo , Canales de Calcio/metabolismo , Calcio/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Familia-src Quinasas/metabolismo , Animales , Línea Celular Tumoral , Pollos , Electrofisiología , Péptidos y Proteínas de Señalización Intracelular/genética , Activación del Canal Iónico , Activación de Linfocitos , Proteínas de la Membrana/metabolismo , Mutación , Fenoles/metabolismo , Fosforilación , Proteínas Tirosina Quinasas/genética , ARN Mensajero , Quinasa Syk , Tapsigargina/farmacología , Familia-src Quinasas/genética
15.
Cell Cycle ; 4(2): 310-4, 2005 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-15655368

RESUMEN

The v-Abl tyrosine kinase activates several signaling pathways during transformation of bone marrow cells in mice. Because the SH2-containing inositol 5'-phosphatase (SHIP) and Downstream of tyrosine kinase 1 (Dok1) have been shown to interact with Abl, the effect of SHIP and Dok1 deficiency on v-Abl transformation was investigated. Bone marrow cells from either Dok1- or SHIP-deficient mice are more susceptible to transformation by v-Abl. v-Abl-transformed preB cells from these knockout mice show Abl kinase-dependent hyperproliferation and moderate resistance to apoptosis. Elevated activation of Ras, Raf-1, and Erk, but not of Akt, was observed in either SHIP(-/-) or Dok1(-/-) v-Abl-transformed cells. This activation is sensitive to treatment with STI571. Furthermore, treatment of these cells with either a farnesyltransferase inhibitor or a MEK1/2 inhibitor abrogates the increased proliferation of SHIP(-/-) or Dok1(-/-) cells in a dose-dependent manner. Complementation of SHIP(-/-) or Dok1(-/-) cells abrogates their hyperproliferation and intracellular Erk activation. These data indicate that both SHIP and Dok1 functionally regulate the activation of Ras-Erk pathway by v-Abl and affect the mitogenic activity of v-Abl transformed bone marrow cells.


Asunto(s)
Transformación Celular Neoplásica/genética , Proteínas de Unión al ADN/fisiología , Proteínas Oncogénicas v-abl/fisiología , Fosfoproteínas/fisiología , Monoéster Fosfórico Hidrolasas/fisiología , Leucemia-Linfoma Linfoblástico de Células Precursoras B/fisiopatología , Proteínas de Unión al ARN/fisiología , Proteínas ras/metabolismo , Animales , Apoptosis/genética , Benzamidas , Células de la Médula Ósea/efectos de los fármacos , Células de la Médula Ósea/patología , Células de la Médula Ósea/fisiología , Línea Celular , Proliferación Celular , Transformación Celular Neoplásica/patología , Proteínas de Unión al ADN/deficiencia , Proteínas de Unión al ADN/genética , Relación Dosis-Respuesta a Droga , Activación Enzimática/genética , Quinasas MAP Reguladas por Señal Extracelular/genética , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Farnesiltransferasa/antagonistas & inhibidores , Regulación Leucémica de la Expresión Génica , Mesilato de Imatinib , MAP Quinasa Quinasa 1/antagonistas & inhibidores , MAP Quinasa Quinasa 2/antagonistas & inhibidores , Metionina/análogos & derivados , Metionina/farmacología , Ratones , Proteínas Oncogénicas v-abl/genética , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatasas , Fosfoproteínas/deficiencia , Fosfoproteínas/genética , Monoéster Fosfórico Hidrolasas/deficiencia , Monoéster Fosfórico Hidrolasas/genética , Piperazinas , Leucemia-Linfoma Linfoblástico de Células Precursoras B/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras B/patología , Pirimidinas/farmacología , Proteínas de Unión al ARN/genética , Transducción de Señal/genética , Proteínas ras/genética
16.
Cell Cycle ; 3(12): 1486-8, 2004 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-15611644

RESUMEN

In normal cells, the strength and duration of proliferative signaling pathways are tightly regulated. In oncogenic settings, negative regulation is often bypassed to allow constitutive activation of these pathways. In our recent manuscript, we identify a mechanism that allows the v-Abl oncogene to bypass negative regulation by SOCS-1 to constitutively activate Jak-Stat signaling. The mechanism involves post-translational modifications of SOCS-1 that disrupt its interaction with the proteasome, thereby preventing it from targeting activated Jak kinases for degradation. In this review, we discuss the implications of these findings for our understanding of v-Abl oncogenesis and the regulation of SOCS protein function.


Asunto(s)
Proteínas Oncogénicas v-abl/genética , Proteínas Oncogénicas v-abl/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Factor de Transcripción STAT1/metabolismo , Transducción de Señal , Animales , Regulación de la Expresión Génica , Janus Quinasa 1 , Proteínas Supresoras de la Señalización de Citocinas/metabolismo
17.
Mol Cell Biol ; 24(20): 9092-101, 2004 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-15456882

RESUMEN

The regulation of cytokine signaling is critical for controlling cellular proliferation and activation during an immune response. SOCS-1 is a potent inhibitor of Jak kinase activity and of signaling initiated by several cytokines. SOCS-1 protein levels are tightly regulated, and recent data suggest that SOCS-1 may regulate the protein levels of some signaling proteins by the ubiquitin proteasome pathway; however, the cellular mechanism by which SOCS-1 directs proteins for degradation is unknown. In this report, SOCS-1 is found to colocalize and biochemically copurify with the microtubule organizing complex (MTOC) and its associated 20S proteasome. The SOCS-1 SH2 domain is required for the localization of SOCS-1 to the MTOC. Overexpression of SOCS-1 targets Jak1 in an SH2-dependent manner to a perinuclear distribution resembling the MTOC-associated 20S proteasome. Analysis of MTOCs fractionated from SOCS-1-deficient cells demonstrates that SOCS-1 may function redundantly to regulate the localization of Jak1 to the MTOC. Nocodazole inhibits the protein turnover of SOCS-1, demonstrating that the minus-end transport of SOCS-1 to the MTOC-associated 20S proteasome is required to regulate SOCS-1 protein levels. These data link SOCS-1 directly with the proteasome pathway and suggest another function for the SH2 domain of SOCS-1 in the regulation of Jak/STAT signaling.


Asunto(s)
Péptidos y Proteínas de Señalización Intracelular/metabolismo , Centro Organizador de los Microtúbulos/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteínas Represoras/metabolismo , Animales , Fraccionamiento Celular , Línea Celular , Centrosoma/química , Centrosoma/metabolismo , Chlorocebus aethiops , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Janus Quinasa 1 , Microtúbulos/metabolismo , Estructura Terciaria de Proteína , Proteínas Tirosina Quinasas/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Represoras/genética , Fracciones Subcelulares/metabolismo , Proteína 1 Supresora de la Señalización de Citocinas , Proteínas Supresoras de la Señalización de Citocinas
18.
Mol Cell ; 15(3): 329-41, 2004 Aug 13.
Artículo en Inglés | MEDLINE | ID: mdl-15304214

RESUMEN

The v-Abl oncogene activates Jak-Stat signaling during transformation of pre-B cells in mice. Disrupting Jak activation by deleting the Jak binding domain of v-Abl or by expressing a dominant-negative Jak1 decreases v-Abl transformation efficiency. As SOCS-1 is a known potent inhibitor of Jak kinases, the mechanism by which v-Abl bypasses SOCS-1 regulation to constitutively activate Jak kinases was investigated. SOCS-1 is expressed in v-Abl-transformed cells but is unable to inhibit v-Abl-mediated Jak-Stat signaling. In v-Abl transformants, SOCS-1 can inhibit cytokine signals, but it is more efficient at doing so when the cells are treated with STI571, an Abl kinase inhibitor. Downstream effects of v-Abl signaling include phosphorylation of SOCS-1 on nontyrosine residues, disruption of the interaction between SOCS-1 and the Elongin BC complex, and inhibition of SOCS-1-mediated proteasomal targeting of activated Jaks. These findings reveal a mechanism by which Jak-dependent oncogenes may bypass SOCS-1 inhibition.


Asunto(s)
Linfocitos B/metabolismo , Proteínas Portadoras/metabolismo , Transformación Celular Neoplásica/metabolismo , Péptidos y Proteínas de Señalización Intracelular , Proteínas Oncogénicas v-abl/metabolismo , Proteínas Represoras/metabolismo , Transducción de Señal/fisiología , Animales , Células de la Médula Ósea/metabolismo , Células de la Médula Ósea/patología , Transformación Celular Neoplásica/patología , Humanos , Ratones , Mutación , Proteínas Oncogénicas v-abl/genética , Proteínas Tirosina Quinasas/metabolismo , Proteína 1 Supresora de la Señalización de Citocinas , Proteínas Supresoras de la Señalización de Citocinas
19.
Blood ; 100(3): 966-73, 2002 Aug 01.
Artículo en Inglés | MEDLINE | ID: mdl-12130510

RESUMEN

Activation of intracellular signaling pathways is important for cellular transformation and tumorigenesis. The nonreceptor tyrosine kinases Jak1 and Jak3, which bind to the v-Abl oncoprotein, are constitutively activated in cells transformed with the Abelson murine leukemia virus. A mutant of p160 v-Abl lacking the Jak1-binding region (v-Abl Delta858-1080) has a significant defect in Jak/STAT (signal transducers and activators of transcription) activation, cytokine-independent cell growth/survival, and tumorigenesis. To identify the pathways downstream of Jak kinases in v-Abl-mediated signaling, we examined the activation of several signaling molecules by p160 v-Abl or the v-Abl Delta858-1080 mutant. We demonstrate that, in addition to the decreased Ras activation, signaling through phosphatidylinositol-3 kinase and Akt are impaired in cells expressing mutant v-Abl. The proliferative defect of v-Abl Delta858-1080 was rescued by activated v-Akt and was also moderately rescued by activated v-H-Ras. However, constitutive active phosphatidylinositol-3 kinase (p110CAAX) did not complement this effect. Cells expressing v-Abl Delta858-1080 demonstrated reduced tumor formation in nude mice. In contrast, cells coexpressing v-Akt with v-Abl Delta858-1080 demonstrated reduced latency and increased frequency of tumor formation in nude nice compared with cells expressing v-Abl Delta858-1080 alone, whereas v-H-Ras or p110CAAX had minimum effects on tumor formation. These results suggest that Jak1-dependent Akt activation is important in v-Abl-mediated transformation.


Asunto(s)
Células Madre Hematopoyéticas/metabolismo , Activación de Linfocitos/fisiología , Proteínas Oncogénicas v-abl/farmacología , Proteínas Serina-Treonina Quinasas , Proteínas Tirosina Quinasas/fisiología , Proteínas Proto-Oncogénicas/fisiología , Transducción de Señal , Animales , Apoptosis , Sitios de Unión , Línea Celular , Transformación Celular Neoplásica/efectos de los fármacos , Células Madre Hematopoyéticas/efectos de los fármacos , Células Madre Hematopoyéticas/enzimología , Janus Quinasa 1 , Activación de Linfocitos/efectos de los fármacos , Ratones , Ratones Desnudos , Proteínas Oncogénicas v-abl/administración & dosificación , Proteínas Oncogénicas v-abl/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-akt , Receptor Cross-Talk , Eliminación de Secuencia , Proteínas ras/metabolismo
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