Dissociation between activation of Raf-1 kinase and the 42-kDa mitogen-activated protein kinase/90-kDa S6 kinase (MAPK/RSK) cascade in the insulin/Ras pathway of adipocytic differentiation of 3T3 L1 cells.

Insulin treatment of untransfected 3T3 L1 cells quickly induced activation of a cytosolic 42-kDa mitogen-activated protein kinase (MAPK) and a 90-kDa S6 kinase (RSK). The activation of these cytosolic kinases was also mimicked by Ras expression (in the absence of insulin) in the same cells transfected with inducible ras oncogenes. Furthermore, insulin-induced activation of MAPK and RSK could be blocked by expression of a transfected inducible dominant negative Ras mutant (Asn-17). These results indicate that Ras proteins are obligatory intermediates in the activation of the cytosolic MAPK/RSK cascade by insulin. Insulin treatment of 3T3 L1 cells or expression of transfected ras oncogenes resulted also in hyperphosphorylation of cellular Raf-1. Insulin-induced Raf hyperphosphorylation was inhibited by expression of an inducible dominant negative Ras mutant (Asn-17). We also showed that expression of transfected raf oncogenes induces adipocytic differentiation, as detected by expression of the specific adipocytic marker aP2. In addition, insulin-induced differentiation was significantly blocked by expression of a dominant negative raf mutant. Interestingly, however, the expression of transfected raf oncogenes did not induce MAPK or RSK activation, and the insulin-induced activation of these kinases was not blocked by expression of transfected dominant negative raf mutants. These results are consistent with Raf kinases acting downstream of Ras, but not upstream of MAPK and RSK in insulin-signaling pathways leading to 3T3 L1 differentiation.