RESUMEN
Mutations of the reelin gene cause severe defects in cerebral cortex development and profound intellectual impairment. While many aspects of the reelin signaling pathway have been identified, the molecular and ultimate cellular consequences of reelin signaling remain unknown. Specifically, it is unclear if termination of reelin signaling is as important for normal cortical neuron migration as activation of reelin signaling. Using mice that are single or double deficient, we discovered that combined loss of the suppressors of cytokine signaling, SOCS6 and SOCS7, recapitulated the cortical layer inversion seen in mice lacking reelin and led to a dramatic increase in the reelin signaling molecule disabled (DAB1) in the cortex. The SRC homology domains of SOCS6 and SOCS7 bound DAB1 ex vivo. Mutation of DAB1 greatly diminished binding and protected from degradation by SOCS6. Phosphorylated DAB1 was elevated in cortical neurons in the absence of SOCS6 and SOCS7. Thus, constitutive activation of reelin signaling was observed to be equally detrimental as lack of activation. We hypothesize that, by terminating reelin signaling, SOCS6 and SOCS7 may allow new cycles of reelin signaling to occur and that these may be essential for cortical neuron migration.
Asunto(s)
Moléculas de Adhesión Celular Neuronal/metabolismo , Corteza Cerebral/embriología , Corteza Cerebral/metabolismo , Proteínas de la Matriz Extracelular/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Serina Endopeptidasas/metabolismo , Proteínas Supresoras de la Señalización de Citocinas/deficiencia , Animales , Moléculas de Adhesión Celular Neuronal/genética , Movimiento Celular/fisiología , Corteza Cerebral/patología , Proteínas de la Matriz Extracelular/genética , Células HEK293 , Humanos , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas del Tejido Nervioso/genética , Neuronas/metabolismo , Fosforilación , Proteína Reelina , Serina Endopeptidasas/genética , Proteínas Supresoras de la Señalización de Citocinas/genéticaRESUMEN
We present the results of in vitro DNA-binding assays for a mutant protein (Q44K) of the E. coli methionine repressor, MetJ, as well as the crystal structure at 2.2 A resolution of the apo-mutant bound to a 10-mer oligonucleotide encompassing an 8 bp met-box sequence. The wild-type protein binds natural operators co-operatively with respect to protein concentration forming at least a dimer of repressor dimers along operator DNAs. The minimum operator length is thus 16 bp, each MetJ dimer interacting with a single met-box site. In contrast, the Q44K mutant protein can also bind stably as a single dimer to 8 bp target sites, in part due to additional contacts made to the phosphodiester backbone outside the 8 bp target via the K44 side-chains. Protein-protein co-operativity in the mutant is reduced relative to the wild-type allowing the properties of an intermediate on the pathway to operator site saturation to be examined for the first time. The crystal structure of the decamer complex shows a unique conformation for the protein bound to the single met-box site, possibly explaining the reduced protein-protein co-operativity. In both the extended and minimal DNA complexes formed, the mutant protein makes slightly different contacts to the edges of DNA base-pairs than the wild-type, even though the site of amino acid substitution is distal from the DNA-binding motif. Quantitative binding assays suggest that this is not due to artefacts caused by the crystallisation conditions but is most likely due to the relatively small contribution of such direct contacts to the overall binding energy of DNA-protein complex formation, which is dominated by sequence-dependent distortions of the DNA duplex and by the protein-protein contact between dimers.
Asunto(s)
Proteínas Bacterianas/química , Escherichia coli/metabolismo , Regiones Operadoras Genéticas , Proteínas Represoras/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Emparejamiento Base , Cristalografía por Rayos X , ADN Bacteriano/genética , ADN Bacteriano/metabolismo , Escherichia coli/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Oligonucleótidos/química , Oligonucleótidos/metabolismo , Unión Proteica , Estructura Cuaternaria de Proteína , Proteínas Represoras/genética , Proteínas Represoras/metabolismoRESUMEN
MetJ is a member of the ribbon-helix-helix class of DNA-binding proteins whose affinity for operators is apparently controlled by an unprecedented long-range electrostatic effect from the tertiary sulphur atom of its co-repressor, S-adenosyl methionine. We report here the results of kinetic assays of DNA binding with MetJ mutant proteins having altered net charges. The results (a) suggest that MetJ locates its operators via a sliding mechanism, (b) support the idea that electrostatic steering is important in the initial DNA binding event and (c) highlight the sensitivity of this system to electrostatic effects.
Asunto(s)
Proteínas Bacterianas/química , Regiones Operadoras Genéticas , Proteínas Represoras/química , Proteínas Bacterianas/genética , Técnicas Biosensibles , Proteínas de Escherichia coli/genética , Cinética , Mutagénesis Sitio-Dirigida , Mutación , Unión Proteica , Proteínas Represoras/genética , Sales (Química)/farmacología , Electricidad Estática , Resonancia por Plasmón de SuperficieRESUMEN
Eph receptor tyrosine kinases and ephrins regulate morphogenesis in the developing embryo where they effect adhesion and motility of interacting cells. Although scarcely expressed in adult tissues, Eph receptors and ephrins are overexpressed in a range of tumours. In malignant melanoma, increased Eph and ephrin expression levels correlate with metastatic progression. We have examined cellular and biochemical responses of EphA3-expressing melanoma cell lines and human epithelial kidney 293T cells to stimulation with polymeric ephrin-A5 in solution and with surfaces of defined ephrin-A5 densities. Within minutes, rapid reorganisation of the actin and myosin cytoskeleton occurs through activation of RhoA, leading to the retraction of cellular protrusions, membrane blebbing and detachment, but not apoptosis. These responses are inhibited by monomeric ephrin-A5, showing that receptor clustering is required for this EphA3 response. Furthermore, the adapter CrkII, which associates with tyrosine-phosphorylated EphA3 in vitro, is recruited in vivo to ephrin-A5-stimulated EphA3. Expression of an SH3-domain mutated CrkII ablates cell rounding, blebbing and detachment. Our results suggest that recruitment of CrkII and activation of Rho signalling are responsible for EphA3-mediated cell rounding, blebbing and de-adhesion, and that ephrin-A5-mediated receptor clustering and EphA3 tyrosine kinase activity are essential for this response.