Adenovirus-mediated gene transfer of dominant negative ras(asn17) in 3T3L1 adipocytes does not alter insulin-stimulated P13-kinase activity or glucose transport.
Recent studies suggest that the ras-map kinase and PI3-kinase cascades converge. We sought to determine whether PI3-kinase is downstream of ras in insulin signaling in a classic insulin target cell. We generated a recombinant adenovirus encoding dominant negative ras by cloning the human H-ras cDNA with a ser to asn substitution at amino acid 17 (ras(asn17)) into the pACCMVpLpA vector and cotransfecting 293 cells with the pJM17 plasmid containing the adenoviral genome. Efficiency of gene transfer was assessed by infecting fully differentiated 3T3L1 adipocytes with a recombinant adenovirus expressing beta-galactosidase (beta-gal); greater than 70% of cells were infected. Infection of adipocytes with ras(asn17) resulted in 10-fold greater expression than endogenous ras. This high efficiency gene transfer allowed biochemical assays. Insulin stimulation of ras-GTP formation was inhibited in ras(asn17)-expressing cells. Map kinase gel mobility shift revealed that insulin (1 UM) or epidermal growth factor (100 ng/ml) resulted in the appearance of a hyperphosphorylated species of p42 map kinase in uninfected cells and those expressing beta-gal but not in cells expressing ras(asn17). In contrast, insulin increased IRS-1-associated PI3-kinase activity approximately 10-fold in control cells and high level overexpression of ras(asn17) did not impair this effect. Similarly, insulin and epidermal growth factor activation of total (no immunoprecipitation) PI3-kinase activity in both cytosol and total cellular membranes and insulin stimulation of glucose transport were not affected by expression of dominant negative ras. Thus, adenovirus-mediated gene transfer is effective for studying insulin signaling in fully differentiated insulin target cells. Inhibition of ras activation abolishes insulin-stimulated phosphorylation of map kinase but does not affect insulin stimulation of PI3-kinase activity. In normal cell physiology, PI3-kinase does not appear to be downstream of ras in mediating the actions of insulin.
Gnudi
L
L
Division of Endocrinology, Dana Farber Cancer Institute, Boston, Massachusetts 02215, USA.
Frevert
E U
EU
Houseknecht
K L
KL
Erhardt
P
P
Kahn
B B
BB
D015056:1-Methyl-3-isobutylxanthine / Q000494:pharmacology; D016475:3T3 Cells / Q000187:drug effects* / Q000378:metabolism; D000256:Adenoviridae / Q000235:genetics*; D000273:Adipose Tissue / Q000166:cytology*; D000818:Animals; D001692:Biological Transport; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism; D002454:Cell Differentiation / Q000187:drug effects; D003907:Dexamethasone / Q000494:pharmacology; D004815:Epidermal Growth Factor / Q000494:pharmacology; D005799:Genes, Dominant; D017930:Genes, Reporter; D011905:Genes, ras; D005822:Genetic Vectors; D005947:Glucose / Q000378:metabolism*; D051272:Glucose Transporter Type 1; D051275:Glucose Transporter Type 4; D006160:Guanosine Triphosphate / Q000378:metabolism; D006801:Humans; D007328:Insulin / Q000494:pharmacology*; D051379:Mice; D009004:Monosaccharide Transport Proteins / Q000096:biosynthesis / Q000235:genetics; D009124:Muscle Proteins; D019869:Phosphatidylinositol 3-Kinases; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000096:biosynthesis* / Q000235:genetics; D017354:Point Mutation; D016283:Proto-Oncogene Proteins p21(ras) / Q000235:genetics / Q000502:physiology*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D015398:Signal Transduction / Q000187:drug effects / Q000235:genetics*; D001616:beta-Galactosidase / Q000096:biosynthesis / Q000235:genetics
D051272:Glucose Transporter Type 1; D051275:Glucose Transporter Type 4; D007328:Insulin; D009004:Monosaccharide Transport Proteins; D009124:Muscle Proteins; D011993:Recombinant Fusion Proteins; C495677:SLC2A1 protein, human; C495692:SLC2A4 protein, human; C495693:Slc2a4 protein, mouse; D004815:Epidermal Growth Factor; D003907:Dexamethasone; D006160:Guanosine Triphosphate; D019869:Phosphatidylinositol 3-Kinases; D017853:Phosphotransferases (Alcohol Group Acceptor); D017871:Calcium-Calmodulin-Dependent Protein Kinases; D001616:beta-Galactosidase; C501009:HRAS protein, human; D016283:Proto-Oncogene Proteins p21(ras); D005947:Glucose; D015056:1-Methyl-3-isobutylxanthine