We used eukaryotic models of Ras-mediated cell proliferation and differentiation, including NIH 3T3 fibroblasts, Xenopus oocytes and 3T3 L1 preadipocytes, to functionally analyze mechanistic aspects of Ras participation in receptor tyrosine kinase (RTK) signaling pathways.Ras proteins are obligatory intermediates in insulin-induced processes of adipocytic differentiation of 3T3 L1 cells and meiotic maturation of Xenopus oocytes. We demonstrated in 3T3 L1 preadipocytes that Ras proteins are necessary and sufficient for insulin-induced activation of cytosolic kinases including Raf-1, MAPK and RSK. Surprisingly, and in contrast to proliferating cells, Raf-1 kinase activation was dissociated from activation of the MAPK/RSK cascade in insulin/Ras signaling pathways of 3T3 L1 cells, indicating that at least two separate, parallel signals emerge from Ras after insulin stimulation. We also demonstrated that one of these branched signals, Raf-1 kinase activation, mediates insulin/Ras-induced adipocytic differentiation, whereas the other, MAP kinase activation, is not required for the differentiation process, and in fact opposses it. Using specific inhibitors we have also recently shown that PI3K acts upstream of Ras in insulin-induced signaling pathways triggering differentiation of 3T3 L1 cells and downstream of Ras in insulin-induced pathways leading to G2/M transition of Xenopus oocytes. These observations indicate that Ras proteins elicit different signaling networks depending on whether they are in a differentiating or proliferating cellular context.We also analyzed the functional contribution of guanine nucleotide exchange factors (GEF) to the activation of Ras family proteins by upstream signals. We documented the existence of multiple isoforms of the human Ras exchange factors Sos1, GRF1 and C3G in different tissues or at different stages of development. We also demonstrated that two specific Sos1 isoforms, expressed exclusively in human fetal brain or adult skeletal muscle, exhibit very different Grb2 binding affinity and biological activity. These results indicate that functionally different GEF isoforms, with differential tissue expression and distribution, play a regulatory role in the mechanisms controlling the intensity of Ras activation in different tissues and/or developmental stages. Since quantitative differences in Ras-mediated MAP kinase activation appear to be responsible for proliferative or differentiative decisions made by specific cell types, these observations may provide a starting point for understanding how such different biological responses as proliferation, differentiation or even cell death, may be mediated by the same Ras proteins in different biological contexts. Recent work involved characterization of newly identified GEFs, such as C3G, and structure/function analysis of modular domains (DH, PH, REM, CD25-H) present in various GEF proteins (Grf1, Sos1, C3G). Current emphasis is on ascertaining the physiological role of each specific GEF in vivo, by analyzing the properties of genomic knock-out and transgenic mice strains for Sos1, Sos2 GRF1, GRF2 and H-ras that have been developed in the laboratory. Whereas Sos1 -/- animals died in utero due to placental malformation, homozygous disruption of the other GEF loci gave rise to viable animals whose phenotypes could be analyzed. Thus, GRF1 -/- animals exhibited significant reduction in body weight and in the level of circulating insulin present in serum. Biochemical analysis of cell lines generated from Sos1 -/- embryos allowed us to conclude that Sos1, but not Sos2, is required for sustained long-term activation of the Ras/MAPK pathway. - Cancer cell growth regulation, cell proliferation, Cell signaling, Differentiation, Oncogenes, Transformation, guanine nucleotide exchange factor, Ras, Xenopus, Insulin,
