In 3T3-L1 fibroblasts, Ras proteins mediate both insulin-induced differentiation to adipocytes and its activation of cytosolic serine/threonine kinases, including Raf-1 kinase, mitogen-activated protein kinase (MAPK), and Rsk. Here, we report that insulin- and Ras-induced activation of MAPK is not required for the differentiation process and in fact antagonizes it. The treatment of 3T3-L1 preadipocytes with MEK-specific inhibitor PD98059 blocked insulin- and Ras-induced MAPK activation but had no effect on or slightly enhanced adipocytic differentiation. Tumor necrosis factor alpha (TNF-alpha), an inhibitor of insulin-stimulated adipogenesis, activated MAPK in 3T3-L1 cells. PD98059 treatment blocked MAPK activation by TNF-alpha and reversed the blockade of adipogenesis mediated by low (1 ng/ml) TNF-alpha concentrations. 3T3-L1 transfectants containing hyperactivated MEK1 or overexpressed MAPK displayed impaired adipocytic differentiation. PD98059 treatment also reversed the blockade of differentiation in MEK1 transfectants. These results indicate that MAPK does not promote but can contribute to inhibition of the process of adipocytic differentiation of 3T3-L1 cells.

A number of reports have identified phosphatidylinositol 3‐kinase as a downstream effector of Ras in various cellular settings, in contrast to others supporting the notion that phosphatidylinositol 3‐kinase acts upstream of Ras. Here, we used Xenopus oocytes, a model of Ras‐mediated cell cycle progression (G2/M transition) to analyze the contribution of phosphatidylinositol 3‐kinase to insulin/Ras‐dependent signaling pathways leading to germinal vesicle breakdown and to ascertain whether phosphatidylinositol 3‐kinase acts upstream or downstream of Ras in those signaling pathways. We analyzed the process of meiotic maturation induced by progesterone, insulin or micro‐injected oncogenic Ras (Lys12) proteins in the presence and absence of specific inhibitors of phosphatidylinositol 3‐kinase activity. As expected, the progesterone‐induced maturation was independent of phosphatidylinositol 3‐kinase since similar rates of germinal vesicle breakdown were produced by the hormone in the presence and absence of wortmannin and LY294002. In contrast, insulin‐induced germinal vesicle breakdown was completely blocked by pre‐incubation with the inhibitors prior to insulin treatment. Interestingly, similar rates of germinal vesicle breakdown were obtained in Ras (Lys12)‐injected oocytes, independently of whether or not they had been pre‐treated with phosphatidylinositol 3‐kinase inhibitors. The effect of wortmannin or LY294002 on MAPK and Akt activation by progesterone, insulin or Ras was also analyzed. Whereas insulin activated those kinases in a phosphatidylinositol 3‐kinase‐dependent manner, progesterone and Ras were able to activate those kinases in the absence of phosphatidylinositol 3‐kinase activity. Since Ras is a necessary and sufficient downstream component of insulin signaling pathways leading to germinal vesicle breakdown, these observations demonstrate that phosphatidylinositol 3‐kinase is not a downstream effector of Ras in insulin/Ras‐dependent signaling pathways leading to entry into the M phase in Xenopus oocytes.

Purified, bacterially expressed PH domains of Sos1, IRS-1, βARK, and PLCδ₁ were analyzed functionally by means of microinjection into full grown, stage VI Xenopus laevis oocytes. Whereas the PH domains from IRS-1, βARK, or PLCδ₁ did not show any effect in the oocytes, injection of the purified Sos1 PH domain resulted in induction of significant rates of germinal vesicle breakdown and meiotic maturation. Furthermore, the Sos1 PH domain exhibited also significant synergy with insulin or coinjected normal Ras protein in induction of germinal vesicle breakdown, although it did not affect the rate of progesterone-induced maturation. These results suggest that purified, isolated PH domains retain, at least in part, their functional specificity and that Xenopus oocytes may constitute a useful biological system to analyze the functional role of the Sos1 PH domain in Ras signaling pathways.