Functional and mechanistic aspects of Ras participation in signaling pathways initiated by receptor tyrosine kinases were studied in eukaryotic models of Ras-mediated cell proliferation and differentiation including NIH 3T3 fibroblasts, Xenopus oocytes and 3T3 L1 preadipocytes. We showed that Ras proteins are obligatory signaling intermediates in the process of insulin-induced differentiation of 3T3 L1 cells into adipocytes. We demonstrated also in this differentiation system that Ras proteins are necessary and sufficient for insulin-induced activation of cytosolic kinases including Raf-1, MAPK and RSK. However, whereas Raf-1 kinase activation mediates insulin/Ras-induced adipocytic differentiation, MAP kinase activation appears not to be required for the differentition process. In addition, and in contrast to proliferating cells, Raf-1 kinase activation is completely dissociated from activation of the MAP kinases by insulin in these differentiating cells, indicating that at least two separate, parallel signals emerge from Ras after insulin stimulation. These novel observations indicate that Ras proteins elicit different signaling networks activating downstream kinases depending on whether they are in a differentiating or proliferating cellular context. Analysis of the expression patterns of the Ras guanine nucleotide exchangers (GEF) hGRF and and hSOS1 revealed the existence of multiple isoforms that are expressed differentially in various human fetal and adult tissues or in different tumor cell lines analyzed. Comparison of two hSos1 isoforms expressed, respectively, in fetal brain and adult skeletal muscle demonstrated significant functional differences in vivo and in vitro. Alternatively spliced, functionally different GEF isoforms, with differential tissue expression and distribution, may play important regulatory roles in the fine control of Ras activation in different tissues or at different developmental stages. Injection of purified peptides into Xenopus oocytes was used to ascertain the functional contribution of specific modular domains, including the Sos1 PH domains and the N- and C-terminal SH2 domains of p85 PI 3-Kinase in Tyrosine kinase(TK)/Ras signaling pathways.
