Abstract
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.
Highlights
Mitogen-activated Protein Kinase/SO-kDSa6 Kinase (WK/RSK) Cascade in the InsulidRasPathway of Adipocytic Differentiation of 3T3 L1 Cells*
3T3 L1 cells lecular mechanism whereby Ras activation by upstream tyroquickly induced activation of a cytosolic 42-kDa mito- sine kinasesis mediated by the action of guanosine nucleotide gen-activated protein kinase (MAPK) and a 90-kDa 56 exchange factors that are brought into theproximity of memkinase (RSK).The activation of these cytosolic kinases brane Ras proteins through formation of a complex with the was mimicked byRas expression (inthe absence of tyrosine phosphorylated receptor in thseame cells transfected with inducibrlues insulin receptor substrate-1)through SH2lSH3“connector”moloncogenes
Insulin-induced activation of MAPK and RSK could be blocked by expressionof a transfectedinducibledominantnegative Ras mutant (Asn-17).These results indicate that Ras proteins are obligatory intermediates in the activation of the cytosolic MAP-K cascade by insulin
Summary
LCMB, NCI, NIH, Bldg. 37, Rm. 1D28, Bethesda, MD 20892.Tel.: 301-496-1070;Fax: 301-496-8479. The insulin-induced activation ofMAPK and RSK could not be inhibited by transfected dominant negative raf mutants, and the expression of transfected raf oncogenes was unable to induce the activation of these kinases. These mitted to electrophoresis,blotted, and immunodetectedby using a polyclonal anti-mouse rskl S6 kinase antibody (UBI 06-185). To demonstrate the specificity activity of growth factors or of expression of the transfected genes was of the 74-kDa band, immunoprecipitations were done using anti-Raf studied, cells were serum-starved overnight before being submitted to antibody preincubated with a peptide that competes with Raf for the the specific treatments as described. Vector pMAMneo (Clontech)was used as a negative control in
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