Early endosome localization and activity of RasGEF1b, a toll-like receptor-inducible Ras guanine-nucleotide exchange factor.

Guanine-nucleotide exchange factors (GEFs) stimulate the intrinsic GDP/GTP exchange activity of Ras and promote the formation of active Ras-GTP, which in turn controls diverse signalling networks important for the regulation of cell proliferation, survival, differentiation, vesicular trafficking, and gene expression. RasGEF1b is a GEF, whose expression is induced in macrophages on stimulation with toll-like receptor (TLR) agonists. Here, we showed that in vitro RasGEF1b expression by macrophages is mostly induced by TLR3 (poly I:C) and TLR4 (lipopolysaccharyde) through the MyD88-independent pathway. In vivo infection with the protozoan parasites Trypanosoma cruzi and Plasmodium chabaudi induced RasGEF1b in an MyD88-, TRIF-, and IFN-gamma-dependent manner. Ectopically expressed RasGEF1b was found, mostly, in the heavy membrane fraction of HEK 293T, and by confocal microscopy, it was found to be located at early endosomes. Computational modelling of the RasGEF1b-Ras interaction revealed that RasGEF1b interacts with the binding domain site of Ras, a critical region for interacting with GEFs involved in the activation of Ras-Raf-MEK-ERK pathway. More important, RasGEF1b was found to be closely associated with Ras in live cells and to trigger Ras activity. Altogether, these results indicate that on TLR activation, RasGEF1b may trigger Ras-like proteins and regulate specific biological activities described for this subtype of GTPases.

Relationship between protein kinase C and derepression of different enzymes.

The PKC1 gene in the yeast Saccharomyces cerevisiae encodes for protein kinase C which is known to control a MAP kinase cascade consisting of different kinases: Bck1, Mkk1 and Mkk2, and Mpk1. This cascade affects the cell wall integrity but the phenotype of pkc1Delta mutants suggests additional targets that have not yet been identified [Heinisch et al., Mol. Microbiol. 32 (1999) 671-680]. The pkc1Delta mutant, as opposed to other mutants in the MAP kinase cascade, displays defects in the control of carbon metabolism. One of them occurs in the derepression of SUC2 gene after exhaustion of glucose from the medium, suggesting an involvement of Pkc1p in the derepression process that is not shared by the downstream MAP kinase cascade. In this work, we demonstrate that Pkc1p is required for the increase of the activity of enzymatic systems during the derepression process. We observed that Pkc1p is involved in the derepression of invertase and alcohol dehydrogenase activities. On the other hand, it seems not to be necessary for the derepression of the enzymes of the GAL system. Our results suggest that Pkc1p is acting through the main glucose repression pathway, since introduction of an additional mutation in the PKC1 gene in yeast strains already presenting mutations in the HXKII or MIG1 genes does not interfere with the typical derepressed phenotype observed in these single mutants. Moreover, our data indicate that Pkc1p participates in this process through the control of the cellular localization of the Mig1 transcriptional factor.