RESUMEN

Mutation of KIT receptor tyrosine kinase at residue D816 results in ligand-independent constitutive kinase activity. This mutation occurs in most patients with mastocytosis, a myeloproliferative neoplasm, and is detected at lower frequencies in acute myeloid leukemia and in germ cell tumors. Other KIT mutations occur in gastrointestinal stromal tumors (GIST) and mucosal melanoma. KIT is considered as a bona fide therapeutic target as c-kit mutations are driving oncogenes in these pathologies. However, several evidences suggest that KIT-D816V mutant is not as aggressive as other KIT mutants. Here, we show that an intracellular docking site in the juxtamembrane region of KIT maintains a negative regulation on KIT-D816V transforming potential. Sixteen signaling proteins were shown to interact with this motif. We further demonstrate that mutation of this site results in signaling modifications, altered gene expression profile and increased transforming activity of KIT-D816V mutant. This result was unexpected as mutations of the homologous sites on wild-type (WT) KIT, or on the related oncogenic FLT3-ITD receptor, impair their function. Our results support the hypothesis that, KIT-D816V mutation is a mild oncogenic event that is sufficient to confer partial transforming properties, but requires additional mutations to acquire its full transforming potential.

Asunto(s)

Mutación Missense , Proteínas Proto-Oncogénicas c-kit/genética , Secuencia de Aminoácidos , Animales , Sitios de Unión , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Transformación Celular Neoplásica/genética , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Ratones , Ratones Desnudos , Datos de Secuencia Molecular , Trasplante de Neoplasias , Proteínas Proto-Oncogénicas c-kit/química , Transducción de Señal , Transcriptoma

RESUMEN

The closely related non-receptor tyrosine kinases FEline Sarcoma (FES) and FEs Related (FER) are activated by cell surface receptors in hematopoietic cells. Despite the early description of oncogenic viral forms of fes, v-fes, and v-fps, the implication of FES and FER in human pathology is not known. We have recently shown that FES but not FER is necessary for oncogenic KIT receptor signaling. Here, we report that both FES and FER kinases are activated in primary acute myeloid leukemia (AML) blasts and in AML cell lines. FES and FER activation is dependent on FLT3 in cell lines harboring constitutively active FLT3 mutants. Moreover, both FES and FER proteins are critical for FLT3-internal tandem duplication (ITD) signaling and for cell proliferation in relevant AML cell lines. FER is required for cell cycle transitions, whereas FES seems necessary for cell survival. We concluded that FES and FER kinases mediate essential non-redundant functions downstream of FLT3-ITD.

Asunto(s)

Secuencias Invertidas Repetidas/genética , Leucemia Mieloide Aguda/metabolismo , Proteínas Proto-Oncogénicas c-fes/metabolismo , Transducción de Señal , Tirosina Quinasa 3 Similar a fms/metabolismo , Western Blotting , Ciclo Celular , Proliferación Celular , Técnica del Anticuerpo Fluorescente , Humanos , Técnicas para Inmunoenzimas , Inmunoprecipitación , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patología , Mutación/genética , Fosforilación , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Proteínas Tirosina Quinasas/genética , Proteínas Tirosina Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-fes/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-fes/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Interferente Pequeño/farmacología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células Tumorales Cultivadas , Tirosina Quinasa 3 Similar a fms/antagonistas & inhibidores , Tirosina Quinasa 3 Similar a fms/genética

RESUMEN

Cytokines play an essential role during early T-cell development. However, the mechanisms controlling cytokine signaling in developing thymocytes have not been elucidated. Cytokine receptor signaling can be modulated by suppressor of cytokine signaling-1 (SOCS-1), which acts as a negative regulator of Janus kinases. SOCS-1 is normally expressed throughout thymocyte development; however, retroviral-mediated overexpression of SOCS-1 in fetal liver-derived hematopoietic progenitors prevented their progression beyond the earliest stage of T-cell development. Further analysis revealed that SOCS-1 expression is transiently suppressed following pre-T-cell receptor (TCR) signaling. Moreover, constitutive expression of SOCS-1 abrogated pre-TCR- mediated expansion of immature thymocytes but did not interfere with differentiation. These findings reveal that SOCS-1 serves to regulate cytokine signaling at critical checkpoints during early T-cell development.

Asunto(s)

Proteínas Portadoras/fisiología , Proteínas Represoras , Transducción de Señal , Subgrupos de Linfocitos T/metabolismo , Animales , Proteínas Portadoras/biosíntesis , Proteínas Portadoras/genética , Diferenciación Celular , División Celular , Linaje de la Célula , Proteínas de Unión al ADN/deficiencia , Proteínas de Unión al ADN/genética , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/metabolismo , Subunidad gamma Común de Receptores de Interleucina , Interleucina-7/fisiología , Hígado/embriología , Activación de Linfocitos , Glicoproteínas de Membrana/fisiología , Ratones , Ratones Noqueados , Técnicas de Cultivo de Órganos , Estructura Terciaria de Proteína , Receptores de Antígenos de Linfocitos T alfa-beta , Receptores de Interleucina-7/fisiología , Proteínas Recombinantes de Fusión/fisiología , Proteína 1 Supresora de la Señalización de Citocinas , Proteínas Supresoras de la Señalización de Citocinas , Subgrupos de Linfocitos T/citología , Timo/citología , Timo/embriología

RESUMEN

Macrophage colony-stimulating factor receptor (M-CSF-R) is a tyrosine kinase that regulates proliferation, differentiation, and cell survival during monocytic lineage development. Upon activation, M-CSF-R dimerizes and autophosphorylates on specific tyrosines, creating binding sites for several cytoplasmic SH2-containing signaling molecules that relay and modulate the M-CSF signal. Here we show that M-CSF-R interacts with suppressor of cytokine signaling 1 (Socs1), a negative regulator of various cytokine and growth factor signaling pathways. Using the yeast two-hybrid system, in vitro glutathione S-transferase-M-CSF-R pull-down, and in vivo coimmunoprecipitation experiments, we demonstrated a direct interaction between the SH2 domain of Socs1 and phosphorylated tyrosines 697 or 721 of the M-CSF-R kinase insert region. Moreover, Socs1 is tyrosine-phosphorylated in response to M-CSF. Ectopic expression of Socs1 in FDC-P1/MAC and EML hematopoietic cell lines decreased their growth rates in the presence of limiting concentrations of M-CSF. However, Socs1 expression did not totally suppress long term cell growth in the presence of saturating M-CSF concentrations, in contrast to other cytokines such as stem cell factor and interleukin 3. Taken together, these results suggest that Socs1 is an M-CSF-R-binding partner involved in negative regulation of proliferation signaling and that it differentially affects cytokine receptor signals.

Asunto(s)

Proteínas Portadoras/metabolismo , Receptor de Factor Estimulante de Colonias de Macrófagos/metabolismo , Proteínas Represoras , Transducción de Señal , Animales , División Celular , Línea Celular , Macrófagos/citología , Ratones , Fosforilación , Fosfotirosina/metabolismo , Unión Proteica , Receptor de Factor Estimulante de Colonias de Macrófagos/química , Proteínas Recombinantes/metabolismo , Spodoptera , Proteína 1 Supresora de la Señalización de Citocinas , Proteínas Supresoras de la Señalización de Citocinas , Técnicas del Sistema de Dos Híbridos

RESUMEN

Suppressor of cytokine signaling-1 (SOCS1) is an inducible Src homology 2 (SH2)-containing protein that negatively regulates cytokine and growth factor signaling required during thymic development. Recent evidence indicates that SOCS1 interacts with elongins B and C, which are components of a ubiquitin ligase complex, VCB (VHL/elonginC/B), based on the VHL (von Hippel Lindau) tumor suppressor protein. SOCS1 has previously been shown to operate as an inhibitor of Janus kinases. Here we show that SOCS1 has the distinct function of targeting the hematopoietic specific guanine nucleotide exchange factor, VAV, for ubiquitin-mediated protein degradation. VAV and SOCS1 form a protein complex through interactions between the VAV NH(2)-terminal regulatory region and the SH2 domain of SOCS1 in a phosphotyrosine-independent manner. SOCS1 decreases the steady state levels of cotransfected VAV and onco-VAV and reduces the focus forming activity of onco-VAV. SOCS1 stimulates the polyubiquitination of VAV proteins in vivo, which was stabilized by proteasomal inhibitors. These results suggest that SOCS1 programs VAV degradation by acting as a substrate-specific recognition component of a VCB-like ubiquitin ligase complex.

Asunto(s)

Proteínas Portadoras/metabolismo , Péptidos y Proteínas de Señalización Intracelular , Proteínas Oncogénicas/antagonistas & inhibidores , Proteínas Represoras , Transducción de Señal , Animales , Línea Celular , Humanos , Hidrólisis , Proteínas Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-vav , Proteína 1 Supresora de la Señalización de Citocinas , Proteínas Supresoras de la Señalización de Citocinas , Ubiquitinas/metabolismo

RESUMEN

We have identified Socs1 as a downstream component of the Kit receptor tyrosine kinase signalling pathway. We show that the expression of Socs1 mRNA is rapidly increased in primary bone marrow-derived mast cells following exposure to Steel factor, and Socs1 inducibly binds to the Kit receptor tyrosine kinase via its Src homology 2 (SH2) domain. Previous studies have shown that Socs1 suppresses cytokine-mediated differentiation in M1 cells inhibiting Janus family kinases. In contrast, constitutive expression of Socs1 suppresses the mitogenic potential of Kit while maintaining Steel factor-dependent cell survival signals. Unlike Janus kinases, Socs1 does not inhibit the catalytic activity of the Kit tyrosine kinase. In order to define the mechanism by which Socs1-mediated suppression of Kit-dependent mitogenesis occurs, we demonstrate that Socs1 binds to the signalling proteins Grb-2 and the Rho-family guanine nucleotide exchange factors Vav. We show that Grb2 binds Socs1 via its SH3 domains to putative diproline determinants located in the N-terminus of Socs1, and Socs1 binds to the N-terminal regulatory region of Vav. These data suggest that Socs1 is an inducible switch which modulates proliferative signals in favour of cell survival signals and functions as an adaptor protein in receptor tyrosine kinase signalling pathways.

Asunto(s)

Proteínas Adaptadoras Transductoras de Señales , Proteínas Portadoras/genética , Transducción de Señal , Factor de Células Madre/metabolismo , Proteínas Portadoras/metabolismo , División Celular , Supervivencia Celular , Citocinas/farmacología , Proteína Adaptadora GRB2 , Regulación de la Expresión Génica/genética , Mastocitos , Mitógenos/metabolismo , Proteínas Oncogénicas/metabolismo , Unión Proteica , Proteínas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-kit/metabolismo , Proteínas Proto-Oncogénicas c-vav , ARN Mensajero/metabolismo , Proteínas Tirosina Quinasas Receptoras/metabolismo , Receptor 1 de Factores de Crecimiento Endotelial Vascular , Dominios Homologos src

RESUMEN

Transgenic mice carrying either a 1.008 or a 4.225 kb of the mouse c-kit 5'-flanking sequences linked to the oncogenic large T antigen (TAg) region of the simian virus 40 (SV40) genome were generated to test if the c-kit promoter could be used to develop useful mouse models. Both constructs promote tumourigenesis in the pituitary and the thyroid with high efficiency. The cell types from which each of these tumours derives were identified. Tumours of the pituitary derive from alpha-MSH-expressing cells located in the intermediate lobe. Transformed cells of the thyroid were calcitonin-positive, implying that the tumours derive from C cells or their precursors. Chromogranin A and neuron-specific enolase, general neuroendocrine cell markers, were expressed in both tumour types. Furthermore a variety of tumours appeared in the transgenic mice. Several of them stained positively for chromogranin A and/or neuron-specific enolase. This suggests a previously unsuspected tissue-specificity of the c-kit 5' flanking sequences for neuroendocrine cells. The Kit-TAg transgenic mouse lines may represent a valuable model for the study of the development and the biology of neuroendocrine tumours.

Asunto(s)

Antígenos Transformadores de Poliomavirus/genética , Neoplasia Endocrina Múltiple/genética , Proteínas Proto-Oncogénicas c-kit/genética , Virus 40 de los Simios/inmunología , Animales , Clonación Molecular , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos CBA , Ratones Transgénicos

RESUMEN

In the mouse, the Kit receptor and its ligand, the stem cell factor (SCF), are encoded at the W/Kit and Steel loci, respectively. The Kit/SCF transduction pathway is involved in promoting cellular migration, proliferation and/or survival of melanoblasts, hematopoietic progenitors and primordial germ cells. Furthermore, a functional Kit/SCF pathway is required for the development of interstitial cells of Cajal (ICC) in the small intestine. Whereas all c-kit-expressing cells in embryogenesis were not identified, previous studies clearly demonstrated that the c-kit expression pattern extends well beyond cells known to be affected by W mutations. To investigate further Kit function, we specifically marked the c-kit-expressing cells and followed their fate during embryogenesis. A mutation was introduced by gene targeting at the W/Kit locus in mouse embryonic stem cells. The lacZ reporter gene was inserted into the first exon of c-kit, thus creating a null allele, called WlacZ. The lacZ expression reflects normal expression of the c-kit gene in WlacZ/+ embryos. The comparison of the patterns of lacZ-expressing cells between WlacZ/+ and WlacZ/WlacZ embryos allowed us to detect where and when melanoblasts, primordial germ cells and hematopoietic progenitors failed to survive in the absence of Kit. We also observed that ICC express c-kit during embryogenesis. ICC are found identically in WlacZ/+ and WlacZ/WlacZ embryos. Therefore, ICC do not depend on Kit expression during embryogenesis. These results indicate that the function of the c-kit gene is only required for the postnatal development of the ICC. Unexpected sites of c-kit expression were uncovered in embryos, including endothelial, epithelial and endocrine cells. None of these cells are dependent on Kit expression for their migration, proliferation and/or survival during embryogenesis. Nevertheless, we assume that the Kit/SCF pathway could be involved in the growth of transformed endothelial, epithelial and endocrine cells.

Asunto(s)

Expresión Génica , Operón Lac , Proteínas Proto-Oncogénicas c-kit/genética , Animales , Células Cultivadas , Femenino , Hematopoyesis , Masculino , Ratones , Ratones Endogámicos C57BL , Mutagénesis

RESUMEN

The mouse W locus encodes Kit, the receptor tyrosine kinase for stem cell factor (SCF). Kit is required for several developmental processes, including the proliferation and survival of melanoblasts. Because of the nearly complete failure of Wrio/+ melanoblasts to colonize the skin, the costs of Wrio/+ mice are characterized by a majority of white hairs interspersed among pigmented hairs, giving a roan effect. However, 3.6% of Wrio/+ mice exhibit phenotypic reversions, i.e., spots of wild-type color on their coats with an otherwise mutant phenotype. Melanocyte cell lines were derived from each of six independent reversion spots on the skin of (C57BL/6 x DBA/2)F1 Wrio/+ mice. All six melanocyte cell lines exhibited the general characteristics common to normal, nonimmortal mouse melanocytes. Of these, three revertant cell lines had lost the dominant-negative Wrio allele following mitotic recombination between the centromere and the W locus. One of the cell lines remained Wrio/+ but showed (i) stimulation in response to SCF and (ii) increased Kit expression, suggesting that the Wrio mutation can be rescued by increased endogenous expression of the c-kit proto-oncogene. Finally, two cell lines showed no detectable genetic change at the W/Kit locus and failed to respond to SCF stimulation in vitro. These results demonstrate that mitotic recombination can create large patches of wild-type hair on the coats of Wrio/+ mutant mice. This shows that mitotic recombination occurs spontaneously in normal healthy tissue in vivo. Moreover, these experiments confirm that other mechanisms, not associated with loss of heterozygosity, may account for the coat color reversion phenotype.

Asunto(s)

Mitosis , Proteínas Proto-Oncogénicas c-kit/genética , Proto-Oncogenes , Recombinación Genética , Alelos , Animales , Secuencia de Bases , División Celular , Células Cultivadas , Mapeo Cromosómico , Genes , Melanocitos/citología , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Ratones Mutantes , Datos de Secuencia Molecular , Fenotipo , Pigmentación , Polimorfismo Genético , Eliminación de Secuencia

RESUMEN

We have cloned and sequenced 4.2 kb of the murine c-kit promoter region. Analysis of transgenic embryos for a kit/lacZ fusion gene showed (i) an unexpected expression in the neural retina, and (ii) an unusual sensitivity to position effects. This indicates that major c-kit tissue specific regulating elements lie outside the proximal promoter. We took advantage to the intrinsic properties of the kit/lacZ transgene to investigated the potential of scaffold attachment regions (SARs) to confer position-independence on gene regulation in an in vivo context. No SAR-dependent isolation of the kit/lacZ transgene was found in the transgenic embryos, indicating a limited capacity of SAR sequences to buffer the effect of flanking sequences on transgene expression.

Asunto(s)

Embriología , Regulación de la Expresión Génica , Interferón beta/genética , Proteínas Proto-Oncogénicas/genética , Proteínas Tirosina Quinasas Receptoras/genética , Receptores del Factor Estimulante de Colonias/genética , Secuencias Reguladoras de Ácidos Nucleicos/genética , Animales , Genes Reporteros , Operón Lac/genética , Ratones , Ratones Transgénicos , Regiones Promotoras Genéticas/genética , Proteínas Proto-Oncogénicas/biosíntesis , Proteínas Proto-Oncogénicas c-kit , Proteínas Tirosina Quinasas Receptoras/biosíntesis , Receptores del Factor Estimulante de Colonias/biosíntesis , Proteínas Recombinantes de Fusión/biosíntesis , Retina/embriología , Distribución Tisular

RESUMEN

Mutations at the mouse W/c-kit locus have pleiotropic defects including impaired development of the melanocyte lineage. We have characterized the molecular basis of the Wei mutation. We show here that Wei is the result of a missense mutation in the ATP binding site domain of c-kit proto-oncogene which affects the tyrosine kinase function of the receptor. As a result, few melanoblasts survive during embryogenesis in heterozygous Wei/+ foetuses. Therefore the adult skin is partly devoid of differentiated pigmented cells giving rise to a mottled coat colour phenotype. However, three per cent of Wei/+ mice exhibit spots of wild-type pigmentation on the coat which is otherwise of mutant phenotype. Such areas are known as phenotypic reversions. To dissect the molecular events responsible for the phenotypic instability of the Wei mutation, we have isolated pure cultures of continuously proliferating melanocytes from two independent reversion spots. These melanocyte lines, designated Wei-R1 and Wei-R2, were shown to exhibit none of the characteristics associated with transformed melanocytes. We have used a polymorphic restriction site generated by the Wei mutation to show that both melanocyte lines are still heterozygous at the W focus. Furthermore, Wei-R1 and Wei-R2 melanocytes express both the mutated and the wild-type c-kit RNA. These results indicate that the somatic mutation events responsible for reversion spots are not necessarily associated with loss of heterozygosity at the W/c-kit locus. Together with previous data, this points to the fact that several mechanisms account for the coat colour reversion phenotype.

Asunto(s)

Melanocitos/metabolismo , Ratones/genética , Mutación , Proteínas Proto-Oncogénicas/genética , Proteínas Tirosina Quinasas Receptoras/genética , Receptores del Factor Estimulante de Colonias/genética , Alelos , Animales , Línea Celular , Deleción Cromosómica , Mapeo Cromosómico , Color del Cabello , Ratones Endogámicos C57BL , Proteínas Proto-Oncogénicas c-kit