RESUMEN
During the development of the nervous system, the migration of many cells and axons is guided by extracellular molecules. These molecules bind to receptors at the tips of the growth cones of migrating axons and trigger intracellular signaling to steer the axons along the correct trajectories. We have identified a novel mutant, enu-3 (enhancer of Unc), that enhances the motor neuron axon outgrowth defects observed in strains of Caenorhabditis elegans that lack either the UNC-5 receptor or its ligand UNC-6/Netrin. Specifically, the double-mutant strains have enhanced axonal outgrowth defects mainly in DB4, DB5 and DB6 motor neurons. enu-3 single mutants have weak motor neuron axon migration defects. Both outgrowth defects of double mutants and axon migration defects of enu-3 mutants were rescued by expression of the H04D03.1 gene product. ENU-3/H04D03.1 encodes a novel predicted putative trans-membrane protein of 204 amino acids. It is a member of a family of highly homologous proteins of previously unknown function in the C. elegans genome. ENU-3 is expressed in the PVT interneuron and is weakly expressed in many cell bodies along the ventral cord, including those of the DA and DB motor neurons. We conclude that ENU-3 is a novel C. elegans protein that affects both motor axon outgrowth and guidance.
Asunto(s)
Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/fisiología , Caenorhabditis elegans/crecimiento & desarrollo , Caenorhabditis elegans/fisiología , Proteínas de la Membrana/genética , Proteínas de la Membrana/fisiología , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/fisiología , Secuencia de Aminoácidos , Animales , Axones/fisiología , Secuencia de Bases , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/química , Mapeo Cromosómico , Cartilla de ADN/genética , Regulación del Desarrollo de la Expresión Génica , Proteínas de la Membrana/química , Datos de Secuencia Molecular , Neuronas Motoras/fisiología , Mutación , Proteínas del Tejido Nervioso/química , Neurogénesis/genética , Neurogénesis/fisiología , Homología de Secuencia de AminoácidoRESUMEN
One of the challenges to understanding nervous system development has been to establish how a fairly limited number of axon guidance cues can set up the patterning of very complex nervous systems. Studies on organisms with relatively simple nervous systems such as Drosophila melanogaster and C. elegans have provided many insights into axon guidance mechanisms. The axons of many neurons migrate along both the dorsal-ventral (DV) and the anterior-posterior (AP) axes at different phases of development, and in addition they may also cross the midline. Axon migration in the dorsal-ventral (DV) direction is mainly controlled by Netrins with their receptors; UNC-40/DCC and UNC-5, and the Slits with their receptors; Robo/SAX-3. Axon guidance in the anterior-posterior (AP) axis is mainly controlled by Wnts with their receptors; the Frizzleds/Fz. An individual axon may be subjected to opposing attractive and repulsive forces coming from opposite sides in the same axis but there may also be opposing cues in the other axis of migration. All the information from the cues has to be integrated within the growth cone at the leading edge of the migrating axon to elicit a response. Recent studies have provided insight into how this is achieved. Evidence suggests that the axis of axon migration is determined by the manner in which Netrin, Slit and Wnt receptors are polarized (localized) within the neuron prior to axon outgrowth. The same molecules are involved in both axon outgrowth and axon guidance, for at least some neurons in C. elegans, whether the cue is the attractive cue UNC-6/Netrin working though UNC-40/DCC or the repulsive cue SLT-1/Slit working though the receptor SAX-3/Robo (Adler et al., 2006, Chang et al., 2006, Quinn et al., 2006, 2008). The molecules involved in cell signaling in this case are polarized within the cell body of the neuron before process outgrowth and direct the axon outgrowth. Expression of the Netrin receptor UNC-40/DCC or the Slit receptor SAX-3/Robo in axons that normally migrate in the AP direction causes neuronal polarity reversal in a Netrin and Slit independent manner (Levy-Strumpf and Culotti 2007, Watari-Goshima et al., 2007). Localization of the receptors in this case is caused by the kinesin-related VAB-8L which appears to govern the site of axon outgrowth in these neurons by causing receptor localization. Therefore, asymmetric localization of axon guidance receptors is followed by axon outgrowth in vivo using the receptor's normal cue, either attractive, repulsive or unknown cues.
Asunto(s)
Axones/metabolismo , Movimiento Celular , Glicoproteínas/metabolismo , Factores de Crecimiento Nervioso/metabolismo , Receptores de Superficie Celular/metabolismo , Proteínas Wnt/metabolismo , AnimalesRESUMEN
Juvenile myelomonocytic leukemia (JMML) is an aggressive myeloproliferative neoplasm in children characterized by the overproduction of monocytic cells that infiltrate the spleen, lung, and liver. JMML remains a disease for which few curative therapies are available other than myeloablative hematopoietic stem cell transplant (HSCT); however, relapse remains a major cause of treatment failure and the long-term morbidities of HSCT for survivors are substantial. A hallmark feature of JMML is acquired hypersensitivity by clonal myeloid progenitor cells to granulocyte macrophage-colony stimulating factor (GM-CSF) via a largely unknown mechanism. Here, we identify c-Cbl (henceforth referred to as Cbl) as a GM-CSF receptor (GMR) adaptor protein that targets Src for ubiquitin-mediated destruction upon GM-CSF stimulation and show that a loss of negative regulation of Src is pivotal in the hyperactivation of GMR signaling in Cbl-mutated JMML cells. Notably, dasatinib, an U.S. Food and Drug Administration-approved multikinase inhibitor that also targets Src family, dramatically attenuated the spontaneous and GM-CSF-induced hypersensitive growth phenotype of mononuclear cells from peripheral blood and bone marrow collected from JMML patients harboring Cbl or other known JMML-associated mutations. These findings reveal Src kinase as a critical oncogenic driver underlying JMML.
Asunto(s)
Resistencia a Antineoplásicos , Factor Estimulante de Colonias de Granulocitos y Macrófagos/farmacología , Leucemia Mielomonocítica Juvenil/metabolismo , Familia-src Quinasas/antagonistas & inhibidores , Antineoplásicos/farmacología , Línea Celular Tumoral , Subunidad beta Común de los Receptores de Citocinas/genética , Subunidad beta Común de los Receptores de Citocinas/metabolismo , Dasatinib , Células HEK293 , Humanos , Leucemia Mielomonocítica Juvenil/genética , Leucemia Mielomonocítica Juvenil/terapia , Fosforilación/efectos de los fármacos , Unión Proteica , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas c-cbl/genética , Proteínas Proto-Oncogénicas c-cbl/metabolismo , Pirimidinas/farmacología , Tiazoles/farmacología , Ubiquitinación/efectos de los fármacos , Familia-src Quinasas/metabolismoRESUMEN
Chuvash polycythemia is a rare congenital form of polycythemia caused by homozygous R200W and H191D mutations in the VHL (von Hippel-Lindau) gene, whose gene product is the principal negative regulator of hypoxia-inducible factor. However, the molecular mechanisms underlying some of the hallmark abnormalities of Chuvash polycythemia, such as hypersensitivity to erythropoietin, are unclear. Here we show that VHL directly binds suppressor of cytokine signaling 1 (SOCS1) to form a heterodimeric E3 ligase that targets phosphorylated JAK2 (pJAK2) for ubiquitin-mediated destruction. In contrast, Chuvash polycythemia-associated VHL mutants have altered affinity for SOCS1 and do not engage with and degrade pJAK2. Systemic administration of a highly selective JAK2 inhibitor, TG101209, reversed the disease phenotype in Vhl(R200W/R200W) knock-in mice, an experimental model that recapitulates human Chuvash polycythemia. These results show that VHL is a SOCS1-cooperative negative regulator of JAK2 and provide biochemical and preclinical support for JAK2-targeted therapy in individuals with Chuvash polycythemia.
Asunto(s)
Janus Quinasa 2/fisiología , Policitemia/etiología , Proteínas Supresoras de la Señalización de Citocinas/genética , Ubiquitina-Proteína Ligasas/genética , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/genética , Animales , Modelos Animales de Enfermedad , Humanos , Janus Quinasa 2/antagonistas & inhibidores , Ratones , Mutación/genética , Policitemia/genética , Multimerización de Proteína/genética , Pirimidinas/farmacología , Sulfonamidas/farmacología , Proteína 1 Supresora de la Señalización de Citocinas , Proteínas Supresoras de la Señalización de Citocinas/fisiología , Ubiquitina-Proteína Ligasas/fisiología , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/fisiologíaRESUMEN
CBL encodes a member of the Cbl family of proteins, which functions as an E3 ubiquitin ligase. We describe a dominant developmental disorder resulting from germline missense CBL mutations, which is characterized by impaired growth, developmental delay, cryptorchidism and a predisposition to juvenile myelomonocytic leukemia (JMML). Some individuals experienced spontaneous regression of their JMML but developed vasculitis later in life. Importantly, JMML specimens from affected children show loss of the normal CBL allele through acquired isodisomy. Consistent with these genetic data, the common p.371Y>H altered Cbl protein induces cytokine-independent growth and constitutive phosphorylation of ERK, AKT and S6 only in hematopoietic cells in which normal Cbl expression is reduced by RNA interference. We conclude that germline CBL mutations have developmental, tumorigenic and functional consequences that resemble disorders that are caused by hyperactive Ras/Raf/MEK/ERK signaling and include neurofibromatosis type 1, Noonan syndrome, Costello syndrome, cardiofaciocutaneous syndrome and Legius syndrome.