RESUMEN
In hippocampus endocannabinoids modulate synaptic function and plasticity and increase tyrosine phosphorylation of several proteins, including focal adhesion kinase (FAK). Autophosphorylation of FAK on Tyr-397 is generally a critical step for its activation, allowing the recruitment of Src family kinases, and phosphorylation of FAK and associated proteins. We have examined the mechanisms of the regulation of FAK by cannabinoids in rat and mouse hippocampal slices. Anandamide and 2-arachidonoylglycerol, two endocannabinoids, and Delta9-tetrahydrocannabinol, stimulated tyrosine phosphorylation of FAK+6,7, a neuronal splice isoform of FAK, on several residues including Tyr-397. Cannabinoids increased phosphorylation of p130-Cas, a protein associated with FAK, but had no effect on PYK2, a tyrosine kinase related to FAK and enriched in hippocampus. Pharmacological experiments and the use of knockout mice demonstrated that the effects of cannabinoids were mediated through CB1 receptors. These effects were sensitive to manipulation of cAMP-dependent protein kinase, suggesting that they were mediated by inhibition of a cAMP pathway. PP2, an Src family kinase inhibitor, prevented the effects of cannabinoids on p130-Cas and on FAK+6,7 tyrosines 577 and 925, but not 397, indicating that FAK autophosphorylation was upstream of Src family kinases in response to CB1-R stimulation. Endocannabinoids increased the association of Fyn, but not Src, with FAK+6,7. In hippocampal slices from Fyn -/- mice, the levels of p130-Cas were increased, and the effects of endocannabinoids on tyrosine phosphorylation, including of Tyr-397, were completely abolished. These results demonstrate the specific functional association of Fyn with FAK+6,7 in a pathway regulated by endocannabinoids, in which Fyn may play roles dependent and independent of its catalytic activity.
Asunto(s)
Cannabinoides/farmacología , Hipocampo/efectos de los fármacos , Proteínas Tirosina Quinasas/fisiología , Proteínas , Proteínas Proto-Oncogénicas/fisiología , Animales , Moduladores de Receptores de Cannabinoides , Proteína Sustrato Asociada a CrK , Activación Enzimática , Quinasa 1 de Adhesión Focal , Proteína-Tirosina Quinasas de Adhesión Focal , Hipocampo/enzimología , Técnicas In Vitro , Ratones , Ratones Noqueados , Fosfoproteínas/fisiología , Fosforilación , Proteínas Tirosina Quinasas/genética , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas c-fyn , Ratas , Receptores de Cannabinoides , Receptores de Droga/fisiología , Proteína p130 Similar a la del Retinoblastoma , Transducción de Señal , Tirosina/metabolismoRESUMEN
Ezrin, a membrane-cytoskeleton linker, is required for cell morphogenesis, motility, and survival through molecular mechanisms that remain to be elucidated. Using the N-terminal domain of ezrin as a bait, we found that p125 focal adhesion kinase (FAK) interacts with ezrin. We show that the two proteins coimmunoprecipitate from cultured cell lysates. However, FAK does not interact with full-length ezrin in vitro, indicating that the FAK binding site on ezrin is cryptic. Mapping experiments showed that the entire N-terminal domain of FAK (amino acids 1-376) is required for optimal ezrin binding. While investigating the role of the ezrin-FAK interaction, we observed that, in suspended kidney-derived epithelial LLC-PK1 cells, overproduction of ezrin promoted phosphorylation of FAK Tyr-397, the major autophosphorylation site, creating a docking site for FAK signaling partners. Treatment of the cells with a Src family kinase inhibitor reduced the phosphorylation of Tyr-577 but not that of Tyr-397, indicating that ezrin-mediated FAK activation does not require the activity of Src kinases. Altogether, these observations indicate that ezrin is able to trigger FAK activation in signaling events that are not elicited by cell-matrix adhesion.
Asunto(s)
Uniones Célula-Matriz/metabolismo , Fosfoproteínas/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Animales , Proteínas del Citoesqueleto , Quinasa 1 de Adhesión Focal , Proteína-Tirosina Quinasas de Adhesión Focal , Humanos , Fosforilación , Pruebas de Precipitina , Estructura Terciaria de Proteína , Porcinos , Células Tumorales Cultivadas , Tirosina/metabolismo , Familia-src Quinasas/metabolismoRESUMEN
The 206-kDa protein of turnip yellow mosaic virus belongs to an expanding group of proteins containing a domain which includes the consensus nucleotide binding site GxxxxGKS/T. A portion of this protein (amino acids [aa] 916 to 1259) was expressed in Escherichia coli and purified by affinity chromatography to near homogeneity. In the absence of any other viral factors, it exhibited ATPase and GTPase activities in vitro. A mutant protein with a single amino acid substitution in the consensus nucleotide binding site (Lys-982 to Ser) exhibited only low levels of both activities, implying that Lys-982 is important for nucleoside triphosphatase activity. The protein also possessed nonspecific RNA binding capacity. Deletion mutants revealed that an N-terminal domain (aa 916 to 1061) and a C-terminal domain (aa 1182 to 1259) participate in RNA binding. The results presented here provide the first experimental evidence that turnip yellow mosaic virus encodes nucleoside triphosphatase and RNA binding activities.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , GTP Fosfohidrolasas/metabolismo , Lisina , Proteínas de Unión al ARN/metabolismo , Tymovirus/metabolismo , Proteínas Virales/metabolismo , Adenosina Trifosfatasas/genética , Adenosina Trifosfatasas/aislamiento & purificación , Clonación Molecular , Secuencia Conservada , GTP Fosfohidrolasas/genética , GTP Fosfohidrolasas/aislamiento & purificación , Expresión Génica , Mutagénesis Sitio-Dirigida , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/aislamiento & purificación , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/metabolismo , Serina , Tymovirus/genética , Proteínas Virales/genética , Proteínas Virales/aislamiento & purificaciónRESUMEN
The large non-structural polyprotein (206 kDa) of turnip yellow mosaic tymovirus (TYMV) undergoes auto-cleavage, producing N- and C-terminal proteins. Here we show that the viral proteinase responsible for this event is active when produced in Escherichia coli, as monitored in Western blots by examining the generation of the C-terminal cleavage product after induction by IPTG. The outer boundaries and critical amino acids of the proteinase domain were characterized by deletion analysis and site-directed mutagenesis. A miniproteinase of 273 residues resulting from combined N- and C-terminal deletions still performed efficient cleavage. Sequence analysis of the bacterially-purified C-terminal cleavage product indicated that cleavage occurs between Ala1259 and Thr1260 of the non-structural protein.
Asunto(s)
Endopeptidasas/metabolismo , Tymovirus/enzimología , Proteínas no Estructurales Virales/metabolismo , Secuencia de Aminoácidos , Aminoácidos/análisis , Secuencia de Bases , Sitios de Unión , Endopeptidasas/química , Endopeptidasas/genética , Escherichia coli , Datos de Secuencia Molecular , Proteínas Recombinantes/metabolismo , Homología de Secuencia de Aminoácido , Proteínas no Estructurales Virales/químicaRESUMEN
Total nucleotide sequencing of the RNA genome of various tymoviruses has demonstrated that the overall genome organization of these viruses is identical. Furthermore, the strategies of expression of the turnip yellow mosaic virus (TYMV) genome have been established by in vitro translation studies; these include the synthesis of a subgenomic RNA, the utilization of overlapping open reading frames (ORFs) and maturation of a polyprotein. In the experiments described here, the strategies of expression of other tymovirus (eggplant mosaic virus, ononis yellow mosaic virus, belladonna mottle virus and physalis mottle virus) genomes have been compared to those used by the TYMV genome, in particular to determine whether these tymoviruses also resort to the expression of overlapping ORFs and maturation of a polyprotein.