RESUMEN
Recognition that Ral guanine nucleotide exchange factors (RalGEFs) are direct Ras effectors and that Ral G-protein activation is a direct consequence of Ras activation has spurred focused efforts to establish the contribution of RalGEF/Ral signaling to oncogenic transformation. Here, we provide a broad-strokes overview of the mechanistic organization of the RalGEF/Ral signaling network, evaluate the evidence for participation of this network in tumorigenic regulatory milieus, consider targeting strategies, and discuss the challenges to and opportunities for clinical development of these targeting strategies.
Asunto(s)
Antineoplásicos/farmacología , Neoplasias/tratamiento farmacológico , Transducción de Señal/efectos de los fármacos , Proteínas de Unión al GTP ral/antagonistas & inhibidores , Factor de Intercambio de Guanina Nucleótido ral/antagonistas & inhibidores , Animales , HumanosRESUMEN
The small GTP-binding protein Ral has been implicated in regulated exocytosis via its interaction with the mammalian exocyst complex. We have previously demonstrated that Ral is involved in exocytosis of Weibel-Palade bodies (WPBs). Little is known about intracellular signaling pathways that promote activation of Ral in response to ligand binding of G protein-coupled receptors. Here we show that RNAi-mediated knockdown of RalGDS, an exchange factor for Ral, results in inhibition of thrombin- and epinephrine-induced exocytosis of WPBs, while overexpression of RalGDS promotes exocytosis of WPBs. A RalGDS variant lacking its exchange domain behaves in a dominant negative manner by blocking release of WPBs. We also provide evidence that RalGDS binds calmodulin (CaM) via an amino-terminal CaM-binding domain. RalGDS association to CaM is required for Ral activation because a cell-permeable peptide comprising this RalGDS CaM-binding domain inhibits Ral activation and WPB exocytosis. Together our findings suggest that RalGDS plays a vital role in the regulation of Ral-dependent WPB exocytosis after stimulation with Ca(2+)- or cAMP-raising agonists.
Asunto(s)
Exocitosis/fisiología , Cuerpos de Weibel-Palade/fisiología , Factor de Intercambio de Guanina Nucleótido ral/fisiología , Secuencia de Aminoácidos , Sitios de Unión/genética , Calmodulina/metabolismo , Línea Celular , Células Endoteliales/efectos de los fármacos , Células Endoteliales/fisiología , Células Endoteliales/ultraestructura , Epinefrina/farmacología , Exocitosis/efectos de los fármacos , Variación Genética , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Interferencia de ARN , ARN Interferente Pequeño/genética , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Transducción de Señal , Trombina/farmacología , Transfección , Proteínas de Unión al GTP ral/metabolismo , Factor de Intercambio de Guanina Nucleótido ral/antagonistas & inhibidores , Factor de Intercambio de Guanina Nucleótido ral/química , Factor de Intercambio de Guanina Nucleótido ral/genéticaRESUMEN
Neurofibromatosis type 2 (NF2) is the most commonly mutated gene in benign tumors of the human nervous system such as schwannomas and meningiomas. The NF2 gene encodes a protein called schwannomin or merlin, which is involved in regulating cell growth and proliferation through protein-protein interactions with various cellular proteins. In order to better understand the mechanism by which merlin exerts its function, yeast two-hybrid screening was performed and Ral guanine nucleotide dissociation stimulator (RalGDS), a downstream molecule of Ras, was identified as a merlin-binding protein. The direct interaction between merlin and RalGDS was confirmed both in vitro and in the NIH3T3 cells. The domain analyses revealed that the broad C-terminal region of merlin (aa 141-595) is necessary for the interaction with the C-terminal Ras-binding domain (RBD) of RalGDS. Functional studies showed that merlin inhibits the RalGDS-induced RalA activation, the colony formation and the cell migration in mammalian cells. These results suggest that merlin can function as a tumor suppressor by inhibiting the RalGDS-mediated oncogenic signals.