Abstract
Activation of p21(ras) by GTP loading is a critical step in a cascade of intracellular insulin signaling. Farnesylation of p21(ras) protein is an obligatory event that facilitates Ras migration to the plasma membrane and subsequent activation. Farnesyltransferase (FTase) is a ubiquitous enzyme that catalyzes the lipid modification of p21(ras) by the addition of farnesyl to the C-terminal "CAAX" motif. In vitro and in vivo FTase activities were studied in 3T3-L1 adipocytes in response to insulin challenge. Insulin exerted a biphasic stimulatory effect on FTase activity measured in vitro with a 31% increase at 5 min and a 130% increase at 60 min. Insulin-stimulated farnesylation of p21(ras) pools in vivo correlated with FTase activity seen in vitro by displaying an increase in farnesylated p21(ras) from 40% of total cellular Ras in control cells to 63% by 5 min and 80% by 60 min (p < 0.05) in insulin-treated cells. Insulin challenge of 3T3-L1 adipocytes increased incorporation of tritiated mevalonic acid in p21(ras) in a dose-dependent manner and stimulated a 2-fold increase in phosphorylation of the alpha-subunit of FTase at 5 min and a 4-fold increase at 60 min.
Highlights
Membrane association of p21ras is promoted by lipid modification of the C terminus of p21ras by the protein prenyltransferase enzyme, farnesyltransferase (FTase)1 [5,6,7]
The influence of insulin on adipocyte FTase activity was assessed by the ability of cell lysates to stimulate the transfer of labeled farnesyl from tritiated farnesyl pyrophosphate to p21ras in vitro
Two min after insulin challenge, the percentage of lipid modified p21ras increased to 50 Ϯ 0.5% of the total cellular p21ras, and by 5 min, the percentage increased to 63 Ϯ 6% (p Ͻ 0.05) (Fig. 2B)
Summary
Vol 271, No 44, Issue of November 1, pp. 27585–27589, 1996 Printed in U.S.A. From the Medical Research Service and the Department of Medicine, Veterans Affairs Medical Center and the University of Colorado Health Sciences Center, Denver, Colorado 80220. Farnesylation of p21ras protein is an obligatory event that facilitates Ras migration to the plasma membrane and subsequent activation. Insulin challenge of 3T3-L1 adipocytes increased incorporation of tritiated mevalonic acid in p21ras in a dose-dependent manner and stimulated a 2-fold increase in phosphorylation of the ␣-subunit of FTase at 5 min and a 4-fold increase at 60 min. Membrane association of p21ras is promoted by lipid modification of the C terminus of p21ras by the protein prenyltransferase enzyme, farnesyltransferase (FTase)1 [5,6,7]. We show that insulin promotes the phosphorylation of the ␣-subunit of FTase and stimulates the activity of FTase in a time- and dose-dependent manner in 3T3-L1 adipocytes. As a result of the activation of FTase by insulin, the cellular pool of farnesylated p21ras doubled after 1 h of cell exposure to insulin
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