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Endocrinology Vol. 138, No. 12 5119-5124
Copyright © 1997 by The Endocrine Society

ARTICLES

Insulin Stimulates the Phosphorylation and Activity of Farnesyltransferase via the Ras-Mitogen-Activated Protein Kinase Pathway1

Marc Goalstone, Kirstin Carel, J. Wayne Leitner and Boris Draznin

Medical Research Service and the Department of Medicine, Veterans Affairs Medical Center and the University of Colorado Health Sciences Center, Denver, Colorado 80220

Address all correspondence and requests for reprints to: Dr. Boris Draznin, Veterans Affairs Medical Center, Chief, Section of Endocrinology (111H), 1055 Clermont Street, Denver, Colorado 80220. E-mail: bdraznin{at}sembilan.uchsc.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Farnesylation of p21Ras by farnesyltransferase (FTase) is obligatory for anchoring p21Ras to the plasma membrane, where it can be activated by growth factors. Insulin significantly stimulates the phosphorylation of the {alpha}-subunit of FTase (4-fold) and the enzymatic activity of FTase in 3T3-L1 fibroblasts and adipocytes. FTase activity was assessed by the amount of [3H] mevalonate (a precursor of farnesyl) incorporated into p21Ras in vivo and by quantitating the amount of farnesylated p21Ras before and after insulin administration. Insulin-stimulated phosphorylation of the {alpha}-subunit of FTase in 3T3-L1 fibroblasts and adipocytes was blocked by the mitogen-activated protein/extracellular-signal regulated kinase-kinase inhibitor, PD98059, but not by wortmannin or bisindolylmaleimide. Additionally, PD98059 blocked insulin-stimulated [3H]mevalonic incorporation and farnesylation of unprocessed p21Ras in both cell lines. Furthermore, expression of the dominant negative mutant of p21Ras precluded insulin-stimulated phosphorylation of the FTase {alpha}-subunit and activation of its enzymatic activity. In contrast, 3T3-L1 fibroblasts, expressing the constitutively active Raf-1, exhibited enhanced phosphorylation of the FTase {alpha}-subunit. It seems that insulin’s effect on the phosphorylation and activation of FTase in both fibroblasts and adipocytes is mediated via the Ras pathway, resulting in a positive feedback augmentation of the cellular pool of farnesylated p21Ras.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
FARNESYLTRANSFERASE (FTase), a ubiquitous protein prenyltransferase enzyme, promotes prenylation of p21Ras by catalyzing the attachment of the cholesterol intermediate, farnesyl, to cysteine 186 (a part of the CAAX motif) at the carboxyl terminus of the p21Ras protein (1, 2, 3). Farnesylation of p21Ras is an obligatory step for subsequent translocation of p21Ras to the plasma membrane, where it exists in either the GDP (inactive) or GTP (active) conformation.

Our recent observations have demonstrated that insulin, in a time- and dose-dependent manner, stimulates FTase activity and phosphorylation of the FTase {alpha}-subunit (4). The intracellular signaling mechanism leading to the phosphorylation of the {alpha}-subunit of FTase and the relationship between the phosphorylation of this enzyme and its activity remain unknown at this time.

Insulin signaling involves a rapid activation of p21Ras via stimulation of the guanine nucleotide exchange factor (Sos), which promotes an exchange of GTP for GDP on the membrane-associated p21Ras (5, 6). The insulin signal then travels from the active (GTP-loaded) p21Ras to Raf-1 and subsequently to mitogen-activated protein (MAP) kinase kinase (MEK) and MAP kinase (7, 8). In this study, we examined whether the Ras-MAP kinase pathway is involved in the mechanism of insulin effect on FTase in 3T3-L1 fibroblasts and adipocytes. It seems that in both cell types, insulin promotes phosphorylation and activation of FTase via the Ras-MAP kinase pathway.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Tissue culture media, gentamicin, methotrexate, and phosphate-free DMEM were from Life Technologies, Inc. (Gaithersburg, MD). FCS was from Gemini Bio-Products, Inc. (Calabasas, CA). BSA and other biochemicals were from Sigma (St. Louis, MO). The anti-p21Ras rat monoclonal antibody (Y13–259) and Protein G-PLUS/Protein A-agarose immunoprecipitation reagents were from Oncogene Science, Inc. (Uniondale, NY). [32P]Orthophosphate and [3H]mevalonolactone were from Du Pont New England Nuclear (Boston, MA). The FTase {alpha}-subunit antibody was from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). SDS-PAGE supplies and reagents were from Bio-Rad (Hercules, CA); lovastatin was from Merck and Company (West Point, PA); and the enhanced chemiluminescence kit was a product of Amersham (Arlington Heights. IL). Bisindolylmaleimide and wortmannin were from Calbiochem (San Diego, CA). The LacSwitch Inducible Mammalian Expression System was from Stratagene (La Jolla, CA). The BxBRaf gene was a kind gift from Dr. Paul Wojtaszek (University of Colorado Health Sciences), and PD98059 was obtained from Dr. Alan Saltiel (Park-Davis, Ann Arbor, MI). The Wizard Mega-Prep DNA Purification System kit was from Promega (Madison, WI). Hygromycin B, ampicillin, and Geneticin (G418) were from Boehringer Mannheim (Indianapolis, IN). Rat-1 fibroblasts that expressed N17 dominant negative mutant of p21Ras were a gift from Dr. Jerrold Olefsky (University of California, San Diego).

Cell culture and differentiation
3T3-L1 fibroblasts were grown to confluence in fibroblast growth medium (DMEM containing 5.5 mM glucose, 10% FCS, 50 µg/ml gentamicin, and 0.5 mM glutamine). Ten days after confluence, fibroblasts were fed differentiation medium (DMEM containing 25 mM glucose, 10% FCS, 50 µg/ml gentamicin, 0.5 mM glutamine) plus differentiation mix (2.5 ml of 10x PBS, 55 mg of 3-isobutyl-1-methylxanthine, 20 ml of deionized water, 250 µl of 49 µM dexamethasone, 2.5 mg of insulin).

Separation of farnesylated and unfarnesylated p21Ras
Confluent cells were serum-starved overnight; preincubated in the presence or absence of 20 µM PD98059 (1 h), 100 nM wortmannin (30 min), or 100 nM bisindolylmaleimide (30 min); and then incubated with or without 100 nM insulin for 1 h. Cells were lysed in 1 ml lysis buffer (150 mM NaCl, 5 mM MgCl2, 1 mM phenylmethyl-sulfonyl fluoride, 1 mM dithiothreitol, 1 mM sodium vanadate, 1 mM sodium phosphate, 1% Triton X-100, 0.05% SDS, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 50 mM HEPES, pH 7.5). Crude lysates were sonicated and centrifuged at 10,000 rpm. Total protein was determined by the bicinchoninic acid protein assay (Pierce, Rockford, IL) and diluted to 0.2 mg/ml per sample. Equal volumes of lysate and 2% Triton X-114 (see Ref.11) were combined in a borosilicate glass tube (12 x 75 mm), vortexed, and incubated at 37 C for 3 min. Solutions were kept at room temperature until aqueous and detergent phases had separated. Equal samples from each phase were placed in separate 1.5-ml Eppendorf tubes, and p21Ras was immunoprecipitated using the monoclonal antibody, Y13–259. Relative amounts of p21Ras were determined by Western blotting, followed by densitometry.

In vivo [3H]mevalonic acid incorporation
Confluent 3T3-L1 fibroblasts or 10-day-old 3T3-L1 adipocytes were placed in serum-free medium and incubated at 37 C for 3 h with 2 µg/ml lovastatin. Cells were then labeled overnight with 25 µCi of [3H]mevalonic acid (33 Ci/mmol) in the presence of lovastatin. The following day, cells in medium containing lovastatin and [3H]mevalonic acid were incubated at 37 C for 60 min with or without 100 nM insulin. Lysates were centrifuged and normalized to 0.2 mg/ml and a monoclonal antibody (Y13–259) was used to immunoprecipitate p21Ras. [3H]Mevalonic acid that was incorporated into p21Ras was quantified by liquid scintillation.

32P-phosphorylation of FTase {alpha}-subunit
3T3-L1 fibroblasts or adipocytes were serum- and phosphate-starved for 6 h, then incubated at 37 C overnight with 250 µCi [32P]orthophosphate (10 mCi/mmol). Cells were then preincubated in the presence or absence of 20 µM PD98059 (1 h), 100 nM wortmannin (30 min), or 100 nM bisindolylmaleimide (30 min), and then incubated for 1 h with or without 100 nM insulin. Lysates were sonicated, centrifuged, and protein concentrations diluted to 0.5 mg/ml. FTase {alpha}-subunit was immunoprecipitated with antiserum to the {alpha}-subunit, analyzed by 12% SDS-PAGE, and visualized by autoradiography. Relative intensity of the signal was quantified by densitometry.

Induction of the BxBRaf gene
3T3-L1 fibroblasts were stably transfected with the BxBRaf gene using the commercially available LacSwitch System from Stratagene. Clones, containing both the Lac repressor and operator, were selected for by using Geneticin (G148) and hyrgromycin B-containing medium. The BxBRaf gene coupled to the Lac operator was induced by incubating the cells for 12 h with 5 mM isopropyl-ß-thiogalactopyranoside (IPTG). Induction of the BxBRaf gene resulted in a 3- to 5-fold increase in the MAP kinase activity (not shown).

Statistical analysis
Statistics were analyzed by Student’s t test or paired t test, with P < 0.05 considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Intracellular insulin signaling represents a complex network of numerous signaling intermediates that are believed to be responsible for various branches of pleiotropic action of insulin. Although the precise contribution of each of these intermediates to the specific aspects of insulin action has not been firmly established, it seems that the Ras-Raf-1-MEK-MAP kinase pathway is involved in the mitogenic effects of insulin, and the phosphatidylinositol-3 kinase (PI-3 kinase) contributes to both mitogenic and metabolic effects of insulin (9, 10). In our initial experiments, we determined whether wortmannin (an inhibitor of PI-3 kinase), PD98059 (MEK inhibitor), and bisindolylmaleimide (inhibitor of protein kinase C) interfered with the ability of insulin to promote the phosphorylation of the {alpha}-subunit of FTase (Fig. 1Go). Exposure of cells to insulin (100 nM for 60 min) resulted in a dramatic increase in the phosphorylation of the {alpha}-subunit of FTase. Insulin had no effect on the amount of the {alpha}-subunit protein, as assessed by Western blotting (not shown). In both 3T3-L1 fibroblasts (Fig. 1AGo) and 3T3-L1 adipocytes (Fig. 1BGo), only the MEK inhibitor blocked the effect of insulin on the phosphorylation of FTase {alpha}-subunit. Wortmannin and bisindolylmaleimide did not interfere with the insulin-induced phosphorylation of the FTase {alpha}-subunit.



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  		Figure 1. Effect of MEK inhibitor (PD98059), wortmannin, and bisindolylmaleimide on insulin-stimulated phosphorylation of the FTase {alpha}-subunit in 3T3-L1 fibroblasts and adipocytes. Confluent fibroblasts (A) and adipocytes (B) were incubated in serum- and phosphate-free medium for 6 h, followed by overnight incubation with 250 µCi of [32P]orthophosphate. Before incubation in the presence or absence of 100 nM insulin, cells were preincubated without (lanes 1 and 2) or with 20 µM PD98059 (lane 3), 100 nM wortmannin (lane 4), or 100 nM bisindolylmaleimide (lane 5), as described in Materials and Methods. Lysates were immunoprecipitated with the FTase {alpha}-subunit antibody, analyzed by SDS-PAGE, and visualized by autoradiography.

 
Because PD98059 was the only inhibitor of the three to block the effect of insulin, we were interested in determining what concentration of MEK inhibitor would be necessary to effect this repression. Thus, we preincubated cells with the indicated concentrations of PD98059 (0.1–100 µM) and challenged the cells with 100 nM insulin (Fig. 2Go). The MEK inhibitor blocked the insulin-stimulated phosphorylation of the FTase {alpha}-subunit in a dose-response manner with a half maximal effect (IC50) of 3 µM PD98059.



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  		Figure 2. Dose-response of insulin-stimulated phosphorylation of the FTase {alpha}-subunit to increasing concentrations of PD98059. Confluent 3T3-L1 fibroblasts were incubated in serum- and phosphate-free medium for 6 h, followed by overnight incubation with 250 µCi of [32P]orthophosphate. Cells were preincubated for 1 h with the indicated concentrations of PD98059, followed by a 1-h incubation with or without 100 nM insulin. Lysates were immunoprecipitated with the FTase {alpha}-subunit antibody, analyzed by SDS-PAGE, and visualized by autoradiography.

 
Inhibition of phosphorylation of FTase was accompanied by a decrease in FTase activity. Because FTase catalyzes the attachment of farnesyl to p21Ras, its enzymatic activity can be assessed by the amount of [3H]mevalonate (an immediate precursor of farnesyl) incorporated into endogenous p21Ras after insulin challenge (Fig. 3Go). Insulin significantly stimulated the incorporation of [3H]mevalonate into p21Ras in 3T3-L1 fibroblasts (P < 0.01) (Fig. 3AGo) and adipocytes (P < 0.05) (Fig. 3BGo), thus reflecting an increase in p21Ras farnesylation. In contrast, in the presence of the MEK inhibitor, PD98059, but not wortmannin or bisindolylmaleimide, the effect of insulin was abrogated in both cell lines.



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  		Figure 3. Effect of MEK inhibitor (PD98059), wortmannin, and bisindolylmaleimide on insulin-stimulated incorporation of [3H]mevalonic acid in p21Ras in 3T3-L1 fibroblasts and adipocytes. Confluent fibroblasts (A) and adipocytes (B) were labeled overnight with 25 µCi of [3H]mevalonic acid (33 Ci/mmol) in the presence of 2 µg/ml lovastatin (see Materials and Methods). The following day, cells were preincubated with the indicated inhibitors and then incubated at 37 C for 60 min with 100 nM insulin. Cell lysates were immunoprecipitated with the anti-p21Ras monoclonal antibody (Y13–259), and incorporation of [3H]mevalonic acid into p21Ras was quantified by liquid scintillation. Results represent mean ± SEM of five experiments performed in triplicate. *, P < 0.01; **, P < 0.05 vs. controls; #, P < 0.05 vs. insulin-stimulated.

 
Activation of FTase by insulin should also result in an increase in the amount of cellular farnesylated p21Ras. Consequently, changes in the amount of farnesylated p21Ras were directly measured by separating farnesylated p21Ras from unfarnesylated proteins by extraction with Triton X-114 (4, 11). The relative amounts of farnesylated (detergent-extracted) and unfarnesylated (aqueous) p21Ras were then quantified by Western blotting, followed by densitometry (Fig. 4Go). In unstimulated 3T3-L1 fibroblasts (Fig. 4AGo) and adipocytes (Fig. 4BGo), approximately 35–45% of the total cellular p21Ras existed in the farnesylated form. In contrast, the percentage of farnesylated p21Ras increased to 60–70% at 60 min of incubation with insulin. This rise was completely prevented by the presence of PD98059, but not by wortmannin or bisindolylmaleimide, in fibroblasts and adipocytes, confirming the important role of MEK in the phosphorylation and activation of FTase by insulin. In these experiments, insulin did not influence the amounts of the total cellular Ras, as determined by Western blotting.



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  		Figure 4. Effect of MEK inhibitor (PD98059), wortmannin, and bisindolylmaleimide on the amount of farnesylated p21Ras in 3T3-L1 fibroblasts and adipocytes. Confluent 3T3-L1 fibroblasts (A) or adipocytes (B) were serum-starved overnight, preincubated in the presence of the indicated kinase inhibitor, and then incubated with 100 nM insulin for 1 h. Equal volumes of cell lysate and 2% Triton X-114 were combined, as described in Materials and Methods and separated into aqueous and detergent phases. Equal samples from each phase were immunoprecipitated with the p21Ras monoclonal antibody, Y13–259. Relative amounts of p21Ras, immunoprecipitated from each phase, were determined by Western blotting and densitometry. Results represent the percent of farnesylated p21Ras to total cellular p21Ras and are expressed as the means ± SEM of five experiments. *, P < 0.05 vs. controls; **, P < 0.05 vs. insulin-stimulated.

 
To further examine the role of the Ras pathway in mediating the effect of insulin on FTase, we looked at the effect of insulin on the phosphorylation of FTase after insulin challenge in fibroblasts transfected with a dominant negative mutant of Ras (Fig. 5AGo) or with the inducible constitutively active mutant of Raf-1 (LacSwitch System) (Fig. 5BGo). Insulin failed to stimulate the phosphorylation of FTase in the Rat-1 fibroblasts stably transfected with the dominant negative (N17) mutant of p21Ras (Fig. 5AGo). In these cells, insulin also failed to increase the amount of farnesylated p21Ras (not shown). In contrast, 3T3-L1 fibroblasts, which were induced with 5 mM IPTG to express a constitutively active Raf-1 (BxBRaf), demonstrated a significantly enhanced activity of MAP Kinase (not shown) and an increased phosphorylation of FTase, even in the absence of insulin (Fig. 5BGo). Enhanced phosphorylation of the FTase {alpha}-subunit was accompanied by increased amounts of farnesylated p21Ras in these cells (Fig 6Go), supporting the notion that phosphorylation of FTase results in the augmentation of its activity.



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  		Figure 5. Phosphorylation of the FTase {alpha}-subunit in Rat-1 fibroblasts expressing N17 p21Ras and in 3T3-L1 fibroblasts expressing constitutively active Raf-1 (BxBRaf). Cells were serum- and phosphate-starved for 6 h, then incubated at 37 C overnight with 250 µCi [32P]ortho-phosphate (10 mCi/mmol) before a 1 h exposure to insulin (100 nM) or 12 h exposure to IPTG (5 mM). (A) Lysates from wild-type Rat-1 fibroblasts (lanes 1 and 2) and Rat-1 fibroblasts transfected with the N17 p21Ras (lanes 3 and 4) were immunoprecipitated with antiserum to the FTase {alpha}-subunit. The FTase {alpha}-subunit was analyzed by SDS-PAGE and visualized by autoradiography. (B) Wild-type 3T3-L1 fibroblasts (lanes 1–3) were incubated with or without 100 nM insulin. 3T3-L1 fibroblasts, transfected with the inducible constitutively active Raf-1 (lanes 4–6), were incubated with or without 5 mM IPTG for 12 h. The presence of 5 mM IPTG for 12 h induced the BxBRaf-1 gene (LacSwitch System). Cell lysates were immunoprecipitated with the {alpha}-subunit antibody. Immunoprecipitates were analyzed by SDS-PAGE and visualized by autoradiography.

 


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  		Figure 6. Effect of constitutively active Raf-1 (BxBRaf) on the amount of farnesylated p21Ras in 3T3-L1 fibroblasts. The expression of BxBRaf is described in Fig. 4BGo. Lysates of control and experimental cells were combined with 2% Triton X-114 and separated into aqueous and detergent phases, as described in Materials and Methods. Relative amounts of p21Ras immunoprecipitated from each phase were determined by Western blotting and densitometry. Results represent the percent of farnesylated p21Ras to total cellular p21Ras and are expressed as the mean ± SEM of two experiments conducted in duplicate. *, P < 0.05 vs. control.

 
The {alpha}-subunit of FTase is shared with geranylgeranyl-transferase I (GGTase I), another cellular prenyltransferase, which is responsible for the prenylation of Rho, Rac, and Rap proteins (reviewed in Ref.12). In spite of strong substrate specificity, GGTase I has been shown to farnesylate Rho-ß, and possibly, geranylgeranylate K-Ras (13, 14). Thus, if insulin also activates GGTase I, this might contribute to insulin’s effect on the farnesylation of endogenous p21Ras. To evaluate a potential contribution of GGTase activity to the effect of insulin on the farnesylation of p21Ras, we performed the following experiments. Lysates from control and insulin-treated cells (source of either FTase or GGTase I) were incubated with either [3H]farnesyl-pyrophosphate ([3H]FPP) or [3H]geranylgeranylpyrophosphate ([3H)GGPP) and bacterially expressed Ras. Incorporation of FPP or GGPP into Ras reflected either FTase or GGTase I activity, respectively (Fig. 7Go). Insulin significantly increased FTase activity and did not affect GGTase activity. Both the inhibitor of FTase, {alpha}-hydroxyfarnesylphosphonic acid ({alpha}-HFPA) and the removal of Mg2+ (an obligatory element for FTase, but not GGTase, activity) completely blocked FTase activity. These experiments indicate that even if insulin stimulated GGTase I activity, this would not contribute to the farnesylation of p21Ras.



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  		Figure 7. Effect of insulin on FTase and GGTase I activities. Lysates from 3T3-L1 fibroblasts, preincubated with or without insulin (100 nM for 60 min), were incubated with either [3H]FPP or [3]GGPP and bacterially expressed H-Ras (100-nM) for 30 min at 37 C. Measurements of incorporation of either labeled product into Ras proteins were performed as described in our previous publication (4). The effect of insulin on FTase activity also was assessed in the presence of {alpha}-HFPA (300 nM) or Mg2+ chelator, 10 mM EDTA. Results are expressed as the mean ± SEM of two experiments performed in triplicate. *, P < 0.05 vs. control.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Farnesylation and subsequent carboxymethylation of p21Ras are mandatory steps for anchoring p21Ras proteins to the plasma membrane (1, 2, 3), where they are activated (GTP loaded) by growth factors (15). Attachment of farnesyl to cysteine residue 186 of p21Ras is catalyzed by FTase, a heterodimeric enzyme that consists of an {alpha}- and ß-subunits (16). The ß-subunit seems to bind p21Ras (16, 17), and although the role of the {alpha}-subunit remains unclear, it has been suggested to bind the prenyl group and stabilize the {alpha}/ß heterodimer (18, 19, 20).

Although the process of farnesylation of p21Ras has been well recognized, the regulation of FTase activity has not been examined in detail. Several studies have shown that the {alpha}-subunit of FTase interacts with, and is subsequently phosphorylated by, the active transforming growth factor ß receptor-1 (TBR-1) (18, 21, 22). The data from the Massagué lab have suggested, however, the lack of association between the phosphorylation of FTase and its activity (21). Because TBR-1 is a serine-threonine kinase and the antiphosphotyrosine antibody does not interact with the phosphorylated FTase (Goalstone and Draznin, unpublished observation), one would assume that the FTase {alpha}-subunit is phosphorylated on serine-threonine residue(s). This assumption is supported by the present findings that the {alpha}-subunit of FTase is phosphorylated in the cells transfected with the constitutively active Raf-1 kinase (Fig. 5Go). Further tests, using peptide mapping, should reveal the phosphorylation sites on the {alpha}-subunit of FTase in response to insulin and TBR-1.

Our previous experiments have demonstrated that insulin stimulates both phosphorylation of the {alpha}-subunit and activity of FTase in a dose- and time-dependent manner (4). In the present study, we have examined the mechanism of the insulin-stimulated phosphorylation of FTase and the relationship between its phosphorylation and activity. Our present observations indicate that activation of the Ras pathway by insulin is necessary to phosphorylate and stimulate FTase. Inhibition of this pathway, either at the level of p21Ras itself (N17, dominant negative mutant of Ras) or at the level of MEK (MEK inhibitor PD98059), completely eliminated the ability of insulin to phosphorylate FTase and stimulate its activity ( Figs. 1–3GoGoGo). In contrast, wortmannin (an inhibitor of PI-3 kinase) and bisindolylmaleimide (an inhibitor of protein kinase C) were without effect. Because the inhibition of insulin-induced phosphorylation of the FTase {alpha}-subunit was accompanied by the abrogation of FTase activity, we postulate that phosphorylation of the {alpha}-subunit of this enzyme results in its activation.

Furthermore, it seems that there exists a positive feedback relationship between an activation of the Ras pathway and stimulation of FTase activity (Fig. 8Go). Insulin activates p21Ras (via phosphorylation of IRS-1 and Shc, and activation of the guanine nucleotide exchange activity of Sos), thereby initiating a phosphorylation cascade, leading to activation of MAP kinase (7). MAP kinase phosphorylates and therefore activates FTase, resulting in a significant increase in the pool of farnesylated p21Ras available for subsequent activation. Conversely, inhibition of FTase activity would predictably decrease the rate of farnesylation of unprocessed p21Ras, eventually diminishing the amount of p21Ras anchored to the plasma membrane and available for GTP loading.



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  		Figure 8. Proposed positive feedback relationship between the Ras pathway and activation of FTase by insulin (see Discussion for details).

 
Finally, because FTase and GGTase I share the same {alpha}-subunit (a subject of phosphorylation in response to insulin), insulin might increase GGTase activity, as well. GGTase I promotes the geranylgeranylation of Rho, Rac, and Rap proteins (12). To evaluate the potential influence of insulin on this process would be of great importance. Because these proteins are involved in intracellular trafficking, it is conceivable that insulin action on protein redistribution might be, at least in part, related to insulin’s effect on GGTase I. In the case of farnesylation of p21Ras, examined in the present investigation, it seems that insu-lin’s effect is mediated by FTase. Both an inhibitor of FTase, {alpha}-HFPA, and the absence of Mg2+ completely blocked insulin’s effect (Fig. 7Go), thus ruling out an involvement of GGTase.

In summary, insulin’s effect on the phosphorylation and activation of FTase is mediated via the Ras-MAP kinase pathway. Activation of p21Ras and MAP kinase by insulin results in a positive-feedback fashion in augmentation of the cellular pool of farnesylated p21Ras that is available for subsequent activation by growth factors.


   Acknowledgments
 
We thank Ms. Gloria Smith for her help in preparing this manuscript.


   Footnotes
 
1 This work was supported by the Medical Research service of the Department of Veterans Affairs, Colorado Diabetes Research Foundation; and by the Foundation for Biomedical Education and Research. Back

Received May 27, 1997.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Casey PJ, Solski PA, Der CJ, Buss JE 1989 p21Ras is modified by a farnesyl isoprenoid. Proc Natl Acad Sci USA 86:8323–8327[Abstract/Free Full Text]
  2. Manne V, Roberts D, Tobin A, O’Rourke E, DeVirgilia M, Meyers C, Ahemed N, Kurz B, Resh M, Kung H, Barbicid M 1990 Identification and preliminary characterization of protein-cysteine farnesyltransferase. Proc Natl Acad Sci USA 87:7541–7545[Abstract/Free Full Text]
  3. Schaber MD, O’Hara MB, Garsky VD, Mosser SD, Bergstrom JD, Moores, SL, Marshall MS, Friedman PA, Dixon RAF, Gibbs JB 1990 Polyisoprenylation of ras in vitro by a farnesyl-protein transferase. J Biol Chem 265:14701–14704[Abstract/Free Full Text]
  4. Goalstone M, Draznin B 1996 Effect of insulin on farnesyltransferase activity in 3T3–L1 adipocytes. J Biol Chem 271:27585–27589[Abstract/Free Full Text]
  5. Medema RH, De Vries-Smits AMM, Van Der Zon GCM, Maassen JA, Bos JL 1993 Ras activation by insulin and epidermal growth factor through enhanced exchange of guanine nucleotides on p21ras. Mol Cell Biol 13:155–162[Abstract/Free Full Text]
  6. Draznin B, Chang L, Leitner JW, Takata Y, Olefsky JM 1993 Insulin activates p21Ras and guanine nucleotide releasing factor in cells expressing wild type and mutant insulin receptors. J Biol Chem 68:19998–20001
  7. Cheatham B, Kahn CR 1995 Insulin action and the insulin signaling network. Endocr Rev 16:117–142[CrossRef][Medline]
  8. Cobb MH, Goldsmith EJ 1995 How MAP kinases are regulated. J Biol Chem 270:14843–14846[Free Full Text]
  9. Reusch JE-B, Bhuripanyo P, Carel K, Leitner JW, Hsieh P, DePaolo D, Draznin B 1995 Differential requirement for p21Ras activation in the metabolic signaling by insulin. J Biol Chem 270:2036–2040[Abstract/Free Full Text]
  10. Sun XJ, Miralpeix M, Myers Jr MG, Glashen EM, Backer JM, Kahn CR, White MF 1992 Expression and function of IRS-1 in insulin signal transmission. J Biol Chem 267:22662–22672[Abstract/Free Full Text]
  11. Bordier C 1981 Phase separation of integral membrane proteins in Triton-X 114 solution. J Biol Chem 256:1604–1607[Abstract/Free Full Text]
  12. Zhang FL, Casey PJ 1996 Protein prenylation: molecular mechanisms and functional consequences. Annu Rev Biochem 65:241–269[CrossRef][Medline]
  13. James GL, Goldstein JL, Brown MS 1995 Polylysine and CVIM sequences of K-RasB dictate specificity of prenylation and confer resistance to benzodiazepine peptidomimetic in vitro. J Biol Chem 270:6221–6226[Abstract/Free Full Text]
  14. Armstrong SA, Hannah VC, Goldstein JL, Brown MS 1995 CAAX geranylgeranyl transferase transfers farnesyl as efficiently as geranylgeranyl to RhoB. J Biol Chem 270:7864–7868[Abstract/Free Full Text]
  15. Jhun BH, Meinkoth JL, Leitner JW, Draznin B, Olefsky JM 1994 Insulin and insulin-like growth factor-I signal transduction requires p21Ras. J Biol Chem 269:5699–5704[Abstract/Free Full Text]
  16. Reiss Y, Seabra MC, Armstrong SA, Slaughter CA, Goldstein J, Brown MS 1991 Nonidentical subunits of p21H-ras farnesyltransferase. Peptide binding and farnesyl pyrophosphate carrier functions. J Cell Biol 266:10672–10677
  17. Moores SL, Schaber MD, Mosser SD, Rands E, O’Hara MB, Garsky VM, Marshall MS, Pompliano DL, Gibbs J 1991 Sequence dependence of protein isoprenylation. J Biol Chem 2661:14603–14610
  18. Kawabata M, Imamura T, Miyazono K, Engel ME, Moses HL 1995 Interaction of the transforming growth factor-beta type I receptor with farnesyl-protein transferase-alpha. J Biol Chem 270:29628–29631[Abstract/Free Full Text]
  19. Chen W-J, Moomaw HF, Overton L, Kost TA, Casey PJ 1993 High level expression of mammalian protein farnesyltransferase in a baculovirus system. The purified protein contains zinc. J Biol Chem 268:9675–9680[Abstract/Free Full Text]
  20. Andres DA, Goldstein JL, Ho YK, Brown MS 1993 Mutational analysis of alpha-subunit of protein farnesyltransferase. Evidence for a catalytic role. J Biol Chem 268:1383–1390[Abstract/Free Full Text]
  21. Wang T, Danielson PD, Li B-Y, Shah PC, Kim SD, Donahoe PK 1996 The p21Ras farnesyltransferase alpha-subunit in TGF-beta and activin signaling. Science 271:1120–1122[Abstract]
  22. Ventura F, Lie F, Doody J, Massagué J 1996 Interaction of transforming growth factor-beta receptor I with farnesyl-protein transferase-alpha in yeast and mammalian cells. J Biol Chem 271:13931–13934[Abstract/Free Full Text]



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