Insulin Promotes Phosphorylation and Activation of Geranylgeranyltransferase II
STUDIES WITH GERANYLGERANYLATION OF Rab-3 AND Rab-4*

Marc L. GoalstoneDagger §, J. Wayne LeitnerDagger §, Inga GolovchenkoDagger §, M. Richard StjernholmDagger §, Mireille Cormont, Yannick Le Marchand-Brustel, and Boris DrazninDagger §parallel

From the Dagger  Research Service, Veterans Affairs Medical Center, Denver, Colorado 80220, the § Department of Medicine, University of Colorado Health Sciences Center, Denver, Colorado 80220, and  INSERM U145, Faculty of Medicine, 06107 Nice, France

    ABSTRACT
Top
Abstract
Introduction
References

Rab proteins play a crucial role in the trafficking of intracellular vesicles. Rab proteins are GTPases that cycle between an inactive GDP-bound form and an active GTP-bound conformation. A prerequisite to Rab activation by GTP loading is its post-translational modification by the addition of geranylgeranyl moieties to highly conserved C-terminal cysteine residues. We examined the effect of insulin on the activity of geranylgeranyltransferase II (GGTase II) in 3T3-L1 fibroblasts and adipocytes. In fibroblasts, insulin increased the enzymatic activity of GGTase II 2.5-fold after 1 h of incubation, an effect that is blocked by perillyl alcohol, an inhibitor of prenyltransferases, but not by the geranylgeranyltransferase I inhibitor, GGTI-298, or the farnesyltransferase inhibitor, alpha -hydroxyfarnesylphosphonic acid. Concomitantly, insulin stimulated the phosphorylation of the GGTase II alpha -subunit without any effect on the GGTase II beta -subunit. At the same time, insulin also increased the amounts of geranylgeranylated Rab-3 in 3T3-L1 fibroblasts from 44 ± 1.2% in control cells to 63 ± 3.8 and 64 ± 6.1% after 1 and 24 h of incubation, respectively. In adipocytes, insulin increased the amounts of geranylgeranylated Rab-4 from 38 ± 0.6% in control cells to 56 ± 1.7 and 60 ± 2.6% after 1 and 24 h of incubation, respectively. In both fibroblasts and adipocytes, the presence of perillyl alcohol blocked the ability of insulin to increase geranylgeranylation of Rab-4, whereas GGTI-298 and alpha -hydroxyfarnesylphosphonic acid were without effect, indicating that insulin activates GGTase II. In summary, insulin promotes phosphorylation and activation of GGTase II in both 3T3 L1 fibroblasts and adipocytes and increases the amounts of geranylgeranylated Rab-3 and Rab-4 proteins.

    INTRODUCTION
Top
Abstract
Introduction
References

The family of Rab proteins is believed to play a crucial role in the intracellular trafficking of vesicles along the endocytic and exocytic pathways (1, 2). To date, over 30 members of this family have been identified (3, 4). Although the exact role and mechanism of action of Rab proteins remain to be elucidated, it appears that Rab proteins are involved in the process of vesicular transport and precise targeting of membranous vesicles to their docking and/or fusion sites (5, 6). Rab proteins are GTPases that cycle between an inactive GDP-bound form, cystosolic Rab, and an active GTP-bound conformation, membrane-associated Rab, under the influence of Rab-associated guanine nucleotide exchange factors and guanosine triphosphatase-activating proteins (1-7). Although Rab protein activity is based on GTP loading, a prerequisite to Rab activation is its post-translational modification (4-6, 8, 9).

Post-translational modification of small molecular mass GTP-binding proteins is accomplished by the isoprenylation of conserved cysteine residues found on their C termini (10, 11). Ras and Rho proteins are prenylated on a single cysteine residue of the CAAX box (where C is cysteine, A is the aliphatic residue, and X is methionine, serine, or glutamine for Ras and leucine for Rho proteins) by farnesyltransferase (FTase)1 and geranylgeranyltransferase I (GGTase I), respectively. In contrast, Rab proteins, which terminate with C-terminal cysteine motifs of CC or CXC, are double prenylated by geranylgeranyltransferase II (GGTase II) (10-13), an enzyme that catalyzes the attachment of a geranylgeranyl moiety (four isoprenes, 20 carbons) to each C-terminal cysteine residue. GGTase II, which is also known as Rab GGTase, consists of a 60-kDa alpha -subunit and a 38-kDa beta -subunit heterodimer that are closely associated with a 95-kDa protein called the Rab escort protein (10-13). The Rab escort protein binds together with the alpha /beta dimer of GGTase II as a holoenzyme complex and functions to present Rab proteins to GGTase II for prenylation.

We have recently demonstrated that insulin promotes the phosphorylation and activation of FTase, a ubiquitous enzyme responsible for the attachment of farnesyl (3 isoprenes, 15 carbons) to p21ras proteins (14, 15). Additionally, we found that insulin, in a time-dependent fashion, stimulated the phosphorylation of the FTase alpha -subunit (16), an event that correlated well with insulin-stimulated FTase activity (16). Because FTase and GGTase I share the same alpha -subunit (17), one could assume that the enzymatic activity of GGTase I might also be regulated by insulin. However, the potential influence of insulin on GGTase II, which has approximately 20-30% homology with FTase and GGTase I, has not been verified experimentally.

In the present study, we utilized Rab-3 and Rab-4, two substrates of GGTase II, to evaluate the effect of insulin on GGTase II activity in 3T3-L1 fibroblasts and adipocytes. We observed that insulin promoted the phosphorylation of the alpha -subunit of GGTase II and an increase in GGTase II activity. Consequently, we found insulin increased amounts of geranylgeranylated Rab-3 and Rab-4 in the insulin-challenged cells.

    EXPERIMENTAL PROCUDURES

Materials-- Cell culture media and supplies were from Life Technologies, Inc. and Gemini Biological Products (Calabasas, CA). Radioisotopes were from NEN Life Science Products, and all standard chemicals were from Sigma. Anti-Rab-3A monoclonal antibodies were from Transduction Laboratories (Lexington, KY); polyclonal antibodies H-492 to Rab geranylgeranyltransferase II were a gift from Dr. Miguel Seabra (Imperial College School of Medicine, Norfolk, United Kingdom), and protein G-PLUS/protein A-agarose was from Oncogene Research Products, Inc. (Cambridge, MA). Rab-4 antibodies (produced against the protein) were developed in the laboratory of Dr. Yannick Le Marchand-Brustel (INSERM, Nice, France) (18), and the farnesyltransferase inhibitor, alpha -hydroxyfarnesylphosphonic acid (alpha -HFPA), was from Biomol (Plymouth Meeting, PA). (S)-(-)-perillyl alcohol (POH) was from Aldrich; the GGTase I inhibitor GGTI-298 was a gift from Dr. Saïd Sebti (University of South Florida), and lovastatin was from Merck. All supplies and reagents for SDS-PAGE were from Bio-Rad, and the enhanced chemiluminescence kit (ECL) was from Amersham Pharmacia Biotech.

Cell Culture-- 3T3-L1 fibroblasts were grown to confluence in fibroblast growth medium (Dulbecco's modified Eagle's medium containing 5.5 mM glucose, 10% fetal calf serum, 50 µg/ml gentamicin, 0.5 mM glutamine, 0.5 µg/ml Fungizone). Two days after confluence, fibroblasts were fed differentiation medium (Dulbecco's modified Eagle's medium containing 25 mM glucose, 10% fetal calf serum, 50 µg/ml gentamicin, 0.5 mM glutamine) plus differentiation mix (2.5 ml of 10× phosphate-buffered saline, 55 mg of 3-isobutyl-1-methylxanthine, 20 ml of deionized water, 250 µl of 49 mM dexamethasone, 2.5 mg of insulin). On day 4, adipocytes were fed adipocyte growth medium (Dulbecco's modified Eagle's medium with 25 mM glucose, 10% fetal calf serum, 50 µg/ml gentamicin, 0.5 mM glutamine) plus 100 nM insulin. Cells were refed every 2 days with adipocyte growth medium and used on days 10-12.

Measurements of Geranylgeranylated Rab Protein-- Confluent cells were serum-starved for 24 h, incubated with or without insulin (100 nM), and then lysed in 500 µl of buffer A (150 mM NaCl, 5 mM MgCl2, 1 mM phenylmethylsulfonyl 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 from the resultant supernatant was determined by the bicinchoninic acid assay (Pierce) and diluted to 1 mg/ml. Equal volumes of lysate and 4% Triton X-114 were combined in a borosilicate glass tube, vortexed, and incubated at 37 °C for 3 min. Solutions were kept at room temperature until phases had separated. Equal volumes from each phase were placed into separate 1.5-ml Eppendorf tubes, and Rab-3 or Rab-4 was immunoprecipitated using antibodies to Rab-3A or Rab-4. Relative amounts of Rab proteins were determined by Western blotting followed by densitometry.

32P-Labeled Phosphorylation of the alpha - and beta -Subunits of GGTase II-- Cultured cells were serum- and phosphate-starved for 6 h and then incubated at 37 °C overnight with 250 µCi of [32P]orthophosphate (10 mCi/mmol). Cells were then incubated for various times with or without insulin (100 nM). Lysates (in buffer A) were sonicated and centrifuged. Protein concentrations were diluted to 1 mg/ml. GGTase II alpha - and beta -subunits were immunoprecipitated with GGTase alpha - or beta -subunit rabbit antiserum, analyzed by SDS-PAGE, and visualized by autoradiography.

In Vitro Assay of GGTase II Activity-- GGTase II activity was assayed in vitro using a modified method of Moores et al. (19). In brief, cells were serum-starved for 24 h, incubated for designated times with insulin (100 nM) in the presence or absence of inhibitors, and lysed in 500 µl of buffer A. The in vitro enzymatic reaction was initiated by adding a 5-µl aliquot of diluted extract (0.5 mg/ml) to 45 µl of reaction assay solution (5 mM MgCl2, 5 mM dithiothreitol, 100 nM Rab-3 protein, 100 nM tritiated geranylgeranyl pyrophosphate ([3H]GGPP) (15 mCi/mmol), 50 mM HEPES, pH 7.5) and incubated at 37 °C. After 30 min the assay was stopped with 1 ml of ice-cold 1 M HCl in ethanol, and the samples were placed on ice for 15 min. Solutions were filtered through Whatman GF/C glass-fiber filters. Each filter was air-dried, placed in a scintillation vial with 3 ml of scintillation fluid, and quantified by liquid scintillation spectrometry. The in vitro GGTase II assay was linear with respect to time and extract protein.

In Vivo Assay of Labeled Rab-3 Using [3H]Mevalonic Acid-- Confluent 3T3-L1 fibroblasts were placed in serum-free medium containing lovastatin (2 µg/ml) and [3H]mevalonolactone (10 µCi) for 24 h. Cells were then incubated for 1 h with or without insulin (100 nM), lysed, and normalized for protein. Rab-3 proteins were immunoprecipitated from the aqueous and detergent fractions and analyzed by SDS-PAGE. Proteins and labeled product were determined by Western blotting and autoradiography, respectively, and quantified by densitometry.

Triton X-114 Extraction of in Vitro Labeled Rab-3 Using [3H]GGPP-- Confluent cells were serum-starved for 24 h and then incubated with or without insulin (100 nM) for 1 h, lysed, and normalized for protein. The in vitro reaction was initiated by adding a 5-µl aliquot of diluted extract (0.5 mg/ml) to 45 µl of reaction assay solution (5 mM MgCl2, 5 mM dithiothreitol, 100 nM Rab-3 protein, 100 nM tritiated geranylgeranyl pyrophosphate ([3H]GGPP) (15 mCi/mmol), 50 mM HEPES, pH 7.5) and incubated at 37 °C. After 30 min the assay was stopped with 50 µl of ice-cold M HCl in ethanol, and the samples were placed on ice for 15 min. Samples were diluted to 800 µl with 1× phosphate-buffered saline and incubated with an equal volume of 4% Triton X-114 for 3 min. Rab-3 proteins were then immunoprecipitated from the aqueous and detergent fractions and analyzed by SDS-PAGE. Proteins and labeled product were determined by Western blotting and autoradiography, respectively.

Statistical Analysis-- Data were analyzed by Student's paired or unpaired t test with a p value of <0.05 considered significant.

    RESULTS

Insulin, a potent modulator of FTase activity in various tissues, increases the amounts of cellular farnesylated p21ras (14-16, 20). To determine whether insulin can also stimulate other prenyltransferases, we examined its effect on GGTase II activity. Initially, we performed an in vitro assay of GGTase II activity in control and insulin-treated 3T3-L1 fibroblasts. The cells were exposed to insulin for up to 2 h, and their lysates were then used as a source of enzyme (GGTase II) as described under "Experimental Procedures." Enzymatic activity of GGTase II is expressed as the amount of [3H]GGPP (counts/min) attached to the recombinant Rab-3 in vitro. Insulin significantly increased the enzymatic activity of GGTase II 1.5-fold (p < 0.05) after 45 min of incubation and 2.5-fold (p < 0.01) after 1 h of incubation (Fig. 1). This effect of insulin was blocked by 1 mM POH, an inhibitor of prenyltransferases, but not by GGTI-298, an inhibitor of GGTase I, or alpha -HFPA, an inhibitor of FTase.


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Fig. 1.   Effect of insulin and POH on the activity of GGTase II. Confluent 3T3-L1 cells were serum-starved for 24 h, incubated with insulin (100 nM) in the absence (solid line) or presence (dotted line) of POH (1 mM) for indicated times, lysed, and normalized for protein. A 5-µl sample was incubated at 37 °C with 45 µl of buffer containing [3H]GGPP and Rab-3 protein for 30 min as described under "Experimental Procedures." The reaction was stopped and the solution was filtered through Whatman GF/C glass-fiber filters. Filters were placed in 3 ml of scintillation fluid, and labeled Rab-3 was quantified by spectrometry. Results represent mean ± S.E. of three independent experiments performed in duplicate. *, p < 0.05; **, p < 0.01.

Because insulin-stimulated activation of FTase has been shown to be related to the phosphorylation of its alpha -subunit (16), we examined whether or not insulin could also promote the phosphorylation of GGTase II. Insulin appears to induce a rapid phosphorylation of the GGTase II alpha -subunit, without any effect on its beta -subunit (Fig. 2). Insulin-stimulated phosphorylation of the alpha -subunit was detectable by 10 min and clearly evident by 1 h. Taken together, these data indicated that, as with FTase, insulin-stimulated activation of GGTase II correlated with its phosphorylation.


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Fig. 2.   Effect of insulin on the phosphorylation of alpha - and beta -subunits of GGTase II. Cultured cells were serum- and phosphate-starved for 6 h and then incubated at 37 °C overnight with [32P]orthophosphate. Cells were then incubated with or without insulin (100 nM) for 1 h, and cell lysates were diluted to 1 mg/ml protein. GGTase II alpha - and beta -subunits were immunoprecipitated with GGTase alpha - or beta -subunit rabbit antiserum, analyzed by SDS-PAGE, and visualized by autoradiography.

Because we previously showed that the activation of FTase by insulin resulted in significant increases in the amount of farnesylated p21ras (14-16), we asked if insulin-stimulated augmentation in GGTase II activity might result in increases in the amount of geranylgeranylated Rab proteins. To address this possibility, we examined the effect of insulin on the geranylgeranylation of Rab-3 in 3T3-L1 fibroblasts and PC-12 cells, using the method of Triton X-114 extraction (21).

To validate this technique, we needed to establish that only prenylated Rab-3 protein was extracted into the detergent phase. To this end, we extracted the lysis buffer A containing unprocessed (bacterially expressed) Rab-3 protein with Triton X-114 and determined that unprocessed (nonprenylated) Rab-3 was detected only in the aqueous phase (Fig. 3A). Conversely, endogenous geranylgeranylated Rab-3 was detected only in the detergent phase (Fig. 3, B and D). In these experiments, the cells were incubated with lovastatin (2 µg/ml) to inhibit isoprenoid synthesis and then with 10 µCi of [3H]mevalonate, a precursor of geranylgeranyl moiety that enters the synthetic pathway downstream of the lovastatin block. The cells were then lysed and extracted with Triton X-114. Newly labeled geranylgeranylated Rab-3 was detected only in the detergent phase (Fig. 3D), whereas Rab-3 detected in the aqueous phase by Western blotting (Fig. 3B) was not prenylated. Finally, we determined that the recombinant Rab-3 that was prenylated in vitro with labeled geranylgeranyl pyrophosphate was recovered only in the detergent phase (Fig. 3, C and E).


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Fig. 3.   Triton X-114 extraction of geranylgeranylated Rab-3. A, lysis buffer containing unprocessed Rab-3 was extracted with Triton X-114 as described under "Experimental Procedures." Rab-3 was immunoprecipitated from aqueous (a) (lane 1) and detergent (d) (lane 2) phases and determined by Western blotting. B and D, cells were incubated in lovastatin (2 µg/ml) and [3H]mevalonolactone as described under "Experimental Procedures." After 1 h of incubation with insulin (100 nM) lysates were normalized for protein and extracted with Triton X-114. Aqueous (a) (lane 3) and detergent (d) (lane 4) phases were immunoprecipitated with anti-Rab-3 antibodies, analyzed by SDS-PAGE, and determined by either Western blotting (B) for protein or autoradiography (D) for labeled product. C and E, lysates of the insulin-treated cells were used as a source of GGTase II to geranylgeranylated Rab-3 in vitro as described under "Experimental Procedures." The reaction mixture was then incubated with Triton X-114. Protein and labeled product were determined by Western blotting (C) and autoradiography (D), respectively, in aqueous (a) (lane 5) and detergent (d) (lane 6) phases.

Subsequently, using the Triton X-114 extraction method we found that geranylgeranylation of Rab-3 was significantly enhanced by insulin in both 3T3-L1 fibroblasts (Fig. 4) and PC-12 cells (not shown). Insulin significantly increased the amounts of geranylgeranylated Rab-3 in 3T3-L1 fibroblasts from 44 ± 1.2% in control cells to 63 ± 3.8% (p < 0.01) after 1 h of incubation and to 64 ± 6.1% (p < 0.01) after 24 h of incubation. POH but not GGTI-298 or alpha -HFPA blocked the effects of insulin on geranylgeranylation of Rab-3. Taken together, these results suggest that geranylgeranylation of Rab-3 is mediated by the activity of GGTase II and augmented by insulin action.


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Fig. 4.   Effect of insulin on geranylgeranylation of Rab-3. Serum-starved 3T3-L1 fibroblasts were challenged with insulin (INS) (100 nM) in the absence or presence (for the same length of time) of POH (1 mM), GGTI-298 (3 µM), or alpha -HFPA (1 µM). Following Triton X-114 extraction, Rab-3 was immunoprecipitated from aqueous and detergent phases, determined by Western blotting, and quantified by densitometry. A, a representative experiment where Rab-3 was immunoprecipitated from aqueous (a) and detergent (d) phases from control cells (lanes 1 and 2) and cells treated with insulin for 1 h (lanes 3 and 4), insulin for 24 h (lanes 5 and 6), insulin and POH for 24 h (lanes 7 and 8), insulin and GGTI-298 for 24 h (lanes 9 and 10), and insulin and alpha -HFPA for 24 h (lanes 11 and 12). B, summary of five independent experiments (mean ± S.E. of detergent-extracted Rab-3 as a percentage of total cellular Rab-3). *, p < 0.05 versus control; #, p < 0.05 versus insulin alone.

Because Rab-4 has been implicated in the mechanism of the insulin-stimulated glucose uptake (22, 23), we were also interested in the effect of insulin on Rab-4 prenylation. In the next series of experiments we examined the effect of insulin on the geranylgeranylation of Rab-4 in 3T3-L1 adipocytes. Insulin significantly increased the amounts of geranylgeranylated Rab-4 from 38 ± 0.6% in control cells to 56 ± 1.7% (p < 0.01) after 1 h of incubation and to 60 ± 2.6% (p < 0.01) after 24 h of incubation (Fig. 5). Similarly to the experiments with Rab-3, the presence of POH (1 mM) blocked the ability of insulin to increase geranylgeranylation of Rab-4, whereas GGTI-298 and alpha -HFPA were without effect. Interestingly, the ability of insulin to increase the amounts of geranylgeranylated Rab-4 was also inhibited by a mitogen-activated protein kinase kinase inhibitor, PD 98056, but not by wortmannin, an inhibitor of phosphatidylinositol 3-kinase (Fig. 6).


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Fig. 5.   Effect of insulin on geranylgeranylation of Rab-4. Serum-starved 3T3-L1 adipocytes were incubated for 1 and 24 h without or with insulin (INS) (100 nM) in the presence of the indicated inhibitor as described in Fig. 4. Rab-4 was immunoprecipitated from equal volumes of aqueous and detergent phases as described under "Experimental Procedures." Amounts of protein were analyzed by SDS-PAGE, determined by Western blotting, and quantified by densitometry. A, representative experiment where Rab-4 protein was immunoprecipitated from aqueous (a) and detergent (d) phases from cells treated with control (lanes 1 and 2), insulin for 1 h (lanes 3 and 4), insulin for 24 h (lanes 5 and 6), insulin and POH for 24 h (lanes 7 and 8), insulin and GGTI-298 for 24 h (lanes 9 and 10), and insulin and alpha -HFPA for 24 h (lanes 11 and 12). B, summary of four independent experiments (mean ± S.E. of detergent-extracted Rab-4 as a percentage of total cellular Rab-4). *, p < 0.05 versus control; #, p < 0.05 versus insulin alone.


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Fig. 6.   Effect of PD98056 and wortmannin on insulin-stimulated geranylgeranylation of Rab-4. 3T3-L1 adipocytes were serum-starved for 24 h and then incubated in the absence (white bar, control) or presence (black bar) of insulin (INS) (100 nM) and with or without the designated inhibitor for 24 h. Prior to the insulin challenge, designated groups were preincubated with the mitogen-activated protein kinase kinase inhibitor (hatched bar), PD98059 (20 µM), for 1 h or the phosphatidylinositol 3-kinase inhibitor (cross-hatched bar), wortmannin (100 nM), for 30 min. Rab-4 was immunoprecipitated from aqueous and detergent fractions, analyzed by SDS-PAGE, determined by Western blotting, and quantified by densitometry. Results represent the percentage of geranylgeranylated Rab-4 to total cellular Rab-4 and are expressed as the mean ± S.E. of five experiments. *, p < 0.05 versus control; #, p < 0.05 versus insulin for 24 h.


    DISCUSSION

The present experiments demonstrate that insulin-stimulated phosphorylation and activation of GGTase II are accompanied by increases in the amounts of geranylgeranylated Rab proteins in fibroblasts, adipocytes, and PC-12 cells. This aspect of insulin action appears to be similar to its ability to phosphorylate and activate FTase and to increase the amounts of farnesylated p21ras (14-16). We have previously demonstrated that insulin promotes the phosphorylation of the alpha -subunit of the FTase in association with augmented FTase activity (16). Because the same alpha -subunit belongs to GGTase I as well (17), one could predict that insulin would also activate GGTase I. Our ongoing studies support this prediction.2 Thus, based on the current findings (activation of GGTase II) and previous observations (activation of FTase), we conclude that insulin is a major regulator of isoprenylation of the small molecular mass GTP-binding proteins. To further strengthen this conclusion, the following questions must be addressed. What is the precise mechanism of the effect of insulin on prenyltransferases? What is the physiological significance of this aspect of insulin action?

Our previous work has addressed certain elements of both questions. We have determined that the effect of insulin on FTase is specific. Other growth factors such as insulin-like growth factor 1, epidermal growth factor, and platelet-derived growth factor failed to promote FTase phosphorylation and stimulate its activity (15). Furthermore, insulin appears to promote the phosphorylation of the FTase alpha -subunit in a positive feedback fashion (16). We found that the ability of insulin to activate the Ras-mitogen-activated protein kinase pathway was critical for the subsequent phosphorylation and activation of FTase. The latter, in turn, increased the amounts of farnesylated p21ras proteins making them available for activation by GTP loading (15, 20). It appears that the effect of insulin on GGTase II also involves phosphorylation of the alpha -subunit of this enzyme (Fig. 2). Moreover, the ability of insulin to increase the amounts of geranylgeranylated Rab-4 was blocked by the inhibitor of the mitogen-activated protein kinase kinase pathway (Fig. 6), emphasizing a similarity to the mechanism of the effect of insulin on FTase and farnesylation of Ras (16).

The physiological and/or pathophysiological role of the insulin-induced modulation of isoprenylation of small molecular mass GTPases constitutes the second major question in this field. At present, we can only state that insulin, via its ability to regulate the amounts of prenylated Ras and Rab, creates a certain background that is responsible for the magnitude of activation of these GTPases by their appropriate stimuli. Interestingly, cells that are derived from insulin receptor-deficient mice exhibited normal basal levels of farnesylated p21ras, although they failed to respond to an insulin challenge (15). Moreover, even using potent inhibitors of FTase (15, 20) and GGTase I and II (present study) we were unable to deplete the cells of the prenylated Ras or Rab within a time frame of our experiments (24 h). These observations suggest that insulin is only responsible for augmenting and modulating prenyltransferase activity over the basal rates of their activity. Clearly, additional experiments are needed to gain more insight into the mechanism of the insulin action on FTase and GGTase I and II.

Geranylgeranylation of Rab proteins is believed to be the first step in their post-translational modification, allowing them to interact with cellular membranes (8-11). Reversible phosphorylation-dephosphorylation of Rab-4 and Rab-1 appears to add an extra layer of control that determines cellular localization of these proteins during the cell cycle (interphase versus mitosis) (24, 25). Prenylated Rab-4 and Rab-3 have been found in both soluble and membranous cellular fractions (26), suggesting that prenylation itself is not solely responsible for the membrane association. Moreover, van der Sluijs et al. (25) have demonstrated that 82% of the newly geranylgeranylated wild-type Rab-4 was found in the cytosol of the mitotic Chinese hamster ovary cells. Mutation of Ser-196 to glutamine or aspartic acid completely prevented Rab-4 phosphorylation in mitotic cells and blocked its appearance in the cytosol (25). Association of Rab proteins with the GDP-dissociation inhibitor protein represents an additional mechanism that regulates the interaction of Rab proteins with cellular membranes (26-29). Chinni et al. (30) have demonstrated that even though the release of GDP-dissociation inhibitor from the intracellular membranes did not affect the cellular distribution of Rab-4 (only Rab-5 was affected) it was associated with the insulin-stimulated glucose transporter (GLUT)-4 membrane trafficking.

Whether or not geranylgeranylation plays a significant physiological role in the mechanism of Rab-4 involvement in the insulin-stimulated glucose uptake remains unknown. Our previous experiments with inhibition of prenylation by lovastatin (31) suggest that insulin essentially stimulates glucose transport normally in cells pretreated with lovastatin for up to 24 h. However, within the time frame of the experiments (24-48 h) inhibitors do not deplete the pool of prenylated Rab-4 below the basal levels that may be sufficient to assure the normal function of Rab-4 in response to insulin. Longer term experiments in the future, both with insulin and the GGTase II inhibitors, are needed to examine this relationship.

Although the physiological and pathophysiological significance of the ability of insulin to increase the amounts of geranylgeranylated Rab-3 and Rab-4 remains unknown, new data presented here indicate that insulin is an important regulator of the cellular pool of prenylated Rab proteins available for GTP loading and carrying out their specific biological functions.

    FOOTNOTES

* This work was supported by the Veterans Affairs Medical Center Research Service, the Foundation for Biomedical Education and Research, the American Heart Association (fellowship to M. L. G.), and the Institut National de la Recherche Médicale APEX 97-01 (to Y. L. M. B.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

parallel To whom correspondence should be addressed: Research Service (151), Veterans Affairs Medical Center, 1055 Clermont St., Denver, CO 80220. Tel.: 303-393-4619; Fax: 303-377-5686; E-mail: DRAZNINB{at}den-res.org.

The abbreviations used are: FTase, farnesyltransferase; GGTase, geranylgeranyltransferase; alpha -HFPA, alpha -hydroxyfarnesylphosphonic acid; POH, (S)-(-)-perillyl alcohol; GGPP, geranylgeranyl pyrophosphate; PAGE, polyacrylamide gel electrophoresis.

2 M. L. Goalstone, M. R. Stjernholm, J. W. Leitner, and B. Draznin, unpublished observations.

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