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. We showed that Ras proteins are obligatory intermediates in the process of insulin-induced differentiation of 3T3 L1 cells into adipocytes. We demonstrated in this differentiation system 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. These 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) revealed the existence of multiple isoforms, 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. Recent work involves 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). Currently we are ascertaining the physiological role of each specific GEF in vivo, by analyzing the properties of genomic knock-out and transgenic mice strains for Sos1, GRF1 and H-ras recently generated in our laboratory. 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 RTK/Ras signaling pathways. The role of other regulators of Cdk activity and cell cycle progression, such as p27kip1, has also been characterized in the oocyte system.
