Caracterização parcial e localização subcelular de um fator de troca de nucleotídeos guanina associado à proteína Ras (RasGEF1b) induzido por agonistas de receptores do tipo Toll

Abstract

The guanine nucleotide exchange factors of the Ras protein super family (RasGEFs) are essential cellular components in the process of Ras activation in response to diverse extra cellular stimuli. The rasGEF1b is a highly conserved guanine exchange factor (GEF). In macrophagesexpression of rasGEF1b mRNA is induced by different TLRs agonists, such as LPS (TLR4), GPImucin (TLR2) and Poli I: C (TLR3). First, by using bioinformatics tolls we analyzed the probable RasGEF1b subcellular localization. The pFLAGCMV2 encoding the recombinant protein FLAGRasGEF1b was used to transfect HEK 293T cells and protein expression evaluated by Western Blot using an anti-FLAG mAb, it showed a protein with apparent molecular weight of 56kDa. By using differential centrifugation, HEK 293T cells were fractioned and the different fractions were recognized using monoclonal antibodies specific to protein of them, using this methodology, we showed that the protein FLAGRasGEF1b was present mostly in the nuclear and heavy membrane fractions. The FLAGRasGEF1b was inserted into a plasmid in fusion with mRFP or YFP fluorescent proteins, and by using confocal microscopy we showed that the proteins were present at early endosome and endolysosome. By using RNA interference (RNAi) we inhibit the expression of FLAGRasGEF1b-YFP, which was capable to activate Ras in HEK 293T cells in vitro.