RasGEF1b: localização celular de seus domínios e papel regulador negativo sobre a ativação de NF-kappaB

Abstract

The guanine nucleotide exchange factor RasGEF1b is a protein encoded by a gene whose expression is strongly induced in response to stimuli with Toll-like receptor (TLR) agonists. RasGEF1b is found localized at the early endosomes associated with Ras, but no cellular function has yet been defined. Our goal was to assess the effect of the gain-offunction of this protein and its domains in the activation of NF-kB transcription factor induced in the inflammatory response, as well as to analyze its cellular distribution. To this end, luciferase reporter gene assays were carried out to investigate whether RasGEF1b displays any regulatory effect on the activation of NF-kB mediated by the signaling pathway of TLRs, Mal-Tirap, and TAK1/TAB1. In order to assess the effect of the amino and carboxiterminal domains of RasGEF1b in NF-kB activation, we cloned the coding regions corresponding to these domains in the eukaryotic expression vector pFLAG-CMV4. Transient transfection assays were initially carried out in HEK293A epithelial cell line. Reporter gene assays demonstrated that the amino and carboxi-terminal domains of RasGEF1b exhibit a negative regulatory effect in the activation of NF-kB mediated by the signaling pathway components of TLRs, Mal-Tirap, and TAK1/TAB1, but not by p65/Rel-A. However, no significant difference in the activation of NF-kB was observed between these two domains. Moreover, we observed that the transfection of either RAP2A or RasGEF1b, or both exhibit a negative regulatory effect on the activation of NF-kB mediated by TNF. To analyze the cellular distribution of the domains of RasGEF1b, the coding regions corresponding to the amino and carboxi-terminal domains of this GEF were cloned in the eukaryotic expression vector pEGFP-N1. The results of the fluorescence microscopy demonstrated that RasGEF1bamino- terminal is located in the cytoplasm, with a slightly increased fluorescence in regions associated with cell membranes. On the other hand, RasGEF1b-carboxi-terminal showed a distribution organized preferentially in the cell nucleus. Our results suggest that RasGEF1b has an important effect in the regulation of NF-kB and a potential role in the inflammatory response