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

Abstract

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-g-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.

Coiled-Coil Domain Containing Protein 124 Is a Novel Centrosome and Midbody Protein That Interacts with the Ras-Guanine Nucleotide Exchange Factor 1B and Is Involved in Cytokinesis

“…S3). Then, by using this antibody, we observed that even though in HeLa cells at interphase and prophase it displays characteristics of previously suggested cytoplasmic/early endosome localization [29], in metaphase cells RasGEF1B was located at a pericentrosomal/centrosomal position, as assessed by its co-localization with γ-tubulin, a subcellular localization similar to that Ccdc124 at metaphase cells (Fig. 6A, compare with Fig.…”

“…In parallel to functional studies to establish cellular roles of Ccdc124 and RasGEF1B, we sought to determine whether the subcellular localizations of these two proteins were comparable throughout the cell cycle. In fact, in a previous study, RasGEF1B was shown to localize in endosomal vesicles when fluorescent protein-fused versions (YFP-RasGEF1B or mRFP-RasGEF1B) were ectopically expressed in asynchronous CHO cells [29]. However, the subcellular localization of RasGEF1B was not previously addressed in cell cycle-synchronized cells.…”

“…Furthermore, by using in vitro assay systems [24] we previously characterized RasGEF1B as an exchange factor exclusively activating the small G protein Rap2. RasGEF1B was also identified in murine macrophages as a toll-like receptor inducible protein with a subcellular localization in early endosomal vesicles [29].…”

Inducible RasGEF1B circular RNA is a positive regulator of ICAM-1 in the TLR4/LPS pathway

“…25,26 Physiologically, ICAM-1 is expressed at a low basal level, 27 however during inflammatory and immune responses, ICAM-1 level increased substantially and aberrantly at sites of inflammation contributing to a number of inflammation-related diseases and injuries such as endotoxin-induced airway disease 28,29 and asthma, 27,30 arthritis, 31 ulcerative colitis 32 and chronic cholangiopathies. 21 In cancer, ICAM-1 has been mainly implicated in local inflammatory tumor microenvironment, 33 tumor progression and metastasis. 34 The molecular mechanisms underlying the transcriptional regulation of ICAM-1 gene has an important implication in term of inflammatoryrelated diseases.…”

“…21 In addition, flagellin stimulates TLR5 while imiquimod (R837) can be used to specifically activate TLR7. 22 To test if mcircRasGEF1B is regulated by TLRs other than TLR4, we treated RAW264.7 cells with PAM3CSK4, ODN1826, LPS, FLA-ST (flagellin from S. typhimurium), poly I:C and R837.…”