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Characterization of Hydrophobic Prenyl Groups of Isoprenylated Proteins in Human Cancer Cells
Biochemical and Biophysical Research Communications 288, 736 –741 (2001) doi:10.1006/bbrc.2001.5830, available online at http://www.idealibrary.com on
Characterization of Hydrophobic Prenyl Groups of Isoprenylated Proteins in Human Cancer Cells Magnus Hjertman, Johan Wejde, and Olle Larsson 1 Department of Oncology and Pathology, CCK R8:04, Karolinska Hospital, SE-171 76 Stockholm, Sweden
Received September 18, 2001
Extensive protease digestion of delipidated [ 3H]mevalonate (MVA)-labeled proteins, followed by HPLC separation of the products, is one approach to identify and study prenyl cysteines. Using this methodology three major [ 3H]MVA-labeled peaks appeared. Two of them represent farnesyl cysteine (FC) and geranylgeranyl cysteine (GGC). The third peak represents unknown products that are considerably more hydrophobic than FC and GGC, here designated HPC. Previously, we provided evidence that cysteine residues may also be modified by dolichyl groups. Dolichyl cysteines (DolC) belong to HPC. However, as shown in the present study, DolC only represents a minor portion of HPC. Data obtained from different sets of experiments, including [ 3H]GGOH-labeling and use of prenyl transferase inhibitors, suggest that HPC mainly involves CXC or CC residues with double-linked GG groups. In turn this points to the possibility that proteins modified by double GG groups are quite common, and may probably involve other proteins than the rab family of GTPases. © 2001 Academic Press Key Words: mevalonate; geranylgeraniol; protein isoprenylation; prenyl cysteine; prenyl transferase inhibitors.
Inhibition of MVA synthesis blocks DNA replication and cell cycle progression (1–3). Among the different compounds derived from MVA, the substrates for prenylation, farnesyl pyrophosphate (FPP) and geranylgeranyl pyrophosphate (GGPP) have attracted particular attention as candidates for MVA-dependent cell growth. Three distinct enzymes are responsible for the posttranslational modification with isoprenoids. Farnesyl transferase (FTase) and the closely related geranylgeranyl transferase I (GGTase I) transfer the activated lipid substrate to a C-terminal CAAX motif, where the X amino acid mainly determines the choice of isoprene (4 – 6). A second mechanism for prenylation To whom correspondence should be addressed. Fax. ⫹46 8 7588397. E-mail: [email protected]. 1
0006-291X/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.
exists for low molecular GTPases of the Rab family, containing CC, CXC or CCXX motifs. These proteins, involved in targeting and fusion of transport vesicles, are geranylgeranylated at two adjacent cysteine residues and the enzyme responsible for this reaction is the cytosolic GGTase II (7). The prenyl modification functionally activates the proteins and enhances the propensity for membrane association. The farnesylation of p21 Ras has been a subject of intense research, since this reaction is known to be a necessity for its malignant transforming activity (8). Most proteins modified with prenyl groups are members of the Ras super family of GTPases, key players in signal transduction, regulating processes such as cell proliferation (9). A number of studies have recently shown that geranylgeranyl rather than farnesyl is the isoprenoid of importance for induction of DNA synthesis in cells growth-arrested by 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (10, 11). This indicates important roles of geranylgeranylated proteins in cell proliferation (12). Inhibitors of the enzymes responsible for the modification and processing of prenylated proteins have been developed and these substances have shown promising results as antitumor agents (13, 14). Because of the regulatory importance of prenylated proteins, a method to determine the expression level and distribution of the different prenyl substitutents would provide valuable information. In a previous work we demonstrated that reversed phase HPLC analysis of Pronase E digested [ 3H]MVA/[ 35S]cysteine-labeled proteins resulted in three distinct radioactive peaks (15). Two of them corresponded to farnesyl cysteine (FC) and geranylgeranyl cysteine (GGC) standards. The third peak (HPC), representing much more hydrophobic compounds, contained digest products labeled with [ 3H]Dolichol (Dol) and [ 35S]cysteine. However, by measuring the amount of [ 3H]MVA-labeled isoprenes released from proteins by sulfonium salt cleavage reaction, we found that dolichol only accounted for 2% (compared to 20% for HPC) of totally released [ 3H]MVA-labeled isoprenes
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3
[ H]MVA-Labeled Products Released by Sulfonium Salt Cleavage or Pronase E Digestion of Delipidated WiDr Proteins
Cleavage method
Short-chained isoprenoids F ⫹ GG (% of total)
Long-chained/ hydrophobic isoprenoids Dol, HPC (% of total)
Sulfonium salt (CH 3I) Pronase E
98 80
2 20
Note. Free isoprenoids and prenyl-modified amino acids were analyzed on C18 or C4 reversed phase HPLC, respectively.
(see Table 1). This suggests that other prenyl groups than dolichyl modified residues predominate in the HPC fraction. In the present study we extended the characterization of the prenyl cysteine expression with a main focus on the hydrophobic prenyl-containing peak. Our data strongly suggest that the HPC mainly consists of CC, CXC or CCXX fragments modified with double GG groups.
Differential MVA labeling. MDA-231 cells were grown in 144-mm dishes to near confluence. Fresh medium containing 25 M lovastatin was added. After a 2 h incubation period, 10 Ci [ 3H]MVA/ml together with unlabeled MVA (0 to 770 M) was added and the incubation was continued for 22 h. Proteins were isolated as described. Delipidation. The delipidation procedure was performed by washing the protein precipitate three times each with acetone, chloroform/methanol, 2:1 (v/v) and absolute ethanol, as described (16 –18). To remove any residual dolichyl pyrophosphoryl oligosaccharides the precipitate was finally washed five times with chloroform/methanol/water, 10:10:3 (by vol.) (19). Proteolytic cleavage of isoprene labeled proteins. Delipidated protein residues were suspended in 0.3 ml 50 mM Hepes-buffer, pH 7.4 containing 10 mM calcium acetate. Proteins were digested with protease type XIV from Streptomyces griseus (Pronase E) at 37°C for 20 h (20). Prenyl containing products were extracted into water saturated butanol and evaporated under a stream of nitrogen. HPLC. Butanol-extractable products, redissolved in methanol were purified on a reversed-phase HPLC system consisting of a Hypersil WP butyl (C4) column (150 ⫻ 4.6 mm). The elution was achieved with a linear gradient starting with 95% solvent A (methanol/water, 1/1 by volume containing 10 mM ammonium acetate) reaching 50% solvent B (isopropanol, 10 mM ammonium acetate) after 60 min. The flow rate was 0.8 ml/min. The absorbance was monitored at 210 nm. 1.6-ml fractions were collected and samples from each fraction were taken for scintillation counting.
RESULTS
MATERIALS AND METHODS Chemicals. , t, t, t-[1- 3H]geranylgeraniol (20 Ci/mmol), , t, t-[1- 3H]farnesol (60 Ci/mmol), Farnesyl-cysteine (FC) and geranylgeranyl cysteine (GGC) were purchased from American Radio labeled Chemicals, Inc. (St. Louis, MO). RS-[5- 3H]mevalonolactone (24 Ci/mmol) was from NEN DuPont. Lovastatin was obtained from Merck, Sharp and Dohme and was converted to its sodium salt before experimental use. (S)-(⫺)-perillyl alcohol (POH) was from Aldrich. The farnesyl transferase inhibitor FTI-277 and the geranylgeranyl transferase I inhibitor GGTI-286 were from Calbiochem. All other chemicals, if not otherwise specified, were from Sigma (St Louis, MO). Cell culture. The breast cancer cell line MDA-231, the colonic cancer cell line WiDr and the Ewing’s sarcoma cell line RD-ES, purchased from American Tissue Culture Collection (Rockville, MD), were grown in monolayers in tissue culture flasks maintained in a 95% air/5% CO 2 atmosphere at 37°C in a humidified incubator. RD-ES cells were cultured in RPMI 40 medium supplemented 15% (v/v) fetal calf serum (FCS). MDA-231 cells were grown in Dulbecco’s Modified Eagle Medium with 10% (v/v) FCS. Metabolic radiolabeling. Semiconfluent RD-ES cells, grown in 100-mm dishes were labeled with 10 Ci/ml [ 3H]F-OH, 10 Ci/ml [ 3H]GG-OH or [ 3H]-mevalonolactone (25 Ci/ml). The medium was removed after 24 h, the dishes carefully rinsed with PBS, after which the cells were harvested. After brief centrifugation the cell pellet was re-suspended in distilled water. A small sample was taken for determination of protein content. Total cellular proteins were precipitated over night with 10 volumes of ice-cold acetone. Protein prenyl transferase inhibition experiments. Exponentially growing subconfluent RD-ES cells cultured in 100-mm dishes were shifted to fresh medium containing 25 Ci [ 3H]MVA/ml, 10 M lovastatin, FTI-277 or GGTI-286 (1 or 10 M) or vehicle (10 mM dithiothreitol in dimethyl sulfoxide). Perillyl alcohol was added from stock solutions in ethanol to final concentrations of 100, 200 or 500 M. Proteins were isolated after 24 h incubation and precipitated with 10 volumes of acetone.
Extensive protease digestion of [ 3H]MVA-labeled proteins generates in addition to free amino acids, prenyl-modified cysteines which can be resolved and identified by reversed phase HPLC. Three major peaks can thus be detected, two of them co-elutes with standards of farnesyl-cysteine (FC) and geranylgeranyl cysteine (GGC) respectively. The third peak (HPC) represents [ 3H]MVA labeled products that are much more hydrophobic (Fig. 1). First we investigated the saturation concentration of MVA (0 –770 M), using nonradioactive MVA, on
FIG. 1. Rechromatography of HPC by RP-HPLC. Total cellular proteins were isolated from WiDr cells labeled with 25 Ci [ 3H]MVA/ ml in the presence of 10 M lovastatin. Butanol extractable products, derived after pronase E digestion of protein precipitates, were loaded on a C18 SEP-PAK cartridge. The methanol eluate was dried with nitrogen and subjected to C4 reversed phase HPLC. Fractions between 48 to 64 min were pooled, dried and analyzed on the same HPLC system. The mobility of farnesyl-cysteine and geranylgeranyl cysteine was measured by UV-detection at 210 nm.
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FIG. 2. MDA cells were grown in 150-mm dishes to near confluence. New fresh medium containing 25 M lovastatin was added. After a 3 h incubation period, 10 Ci [ 3H]MVA/ml and 0 to 770 M MVA, as indicated, was added and the incubation continued for 22 h. Isolation and analysis of prenyl-cysteines was performed as described.
[ 3H]MVA incorporation of FC, GGC and HPC. The prenyl-cysteine distribution pattern was examined in the breast cancer cell line MDA-231, which was also incubated by 25 M lovastatin to completely block the HMG-CoA reductase activity. At the lowest concentrations of MVA tested (0 or 7.7 M) the [ 3H]MVA incorporation into FC and GGC was strongly favored (Fig. 2). A dramatic change in the incorporation distribution occurred at higher concentrations of MVA in the medium (77 and 770 M). Two thirds of the radioactivity were now found in the HPC fraction and not more than 15% of the [ 3H]MVA labeling was in the fraction corresponding to FC. There was no notable difference in the distribution of the [ 3H]MVA incorporation into the prenyl groups at 77 and 770 M (Fig. 2), suggesting that all three pathways are saturated at 77 M MVA. To achieve saturation and simultaneously as high radioactive labeling as possible, we found that 50 Ci [ 3H]MVA/ml in the presence of the 50 M lovastatin was an optimal and reproducible condition [12], and therefore applied in the following experiments. To further characterize the HPC peak, the Ewing sarcoma cell line RD-ES was labeled with [ 3H]F-OH, [ 3H]GG-OH and [ 3H]Dol-OH. Expectedly, [ 3H]F-OH was found to be incorporated in all peaks with approximately 50% of the radiolabeling in the FC fraction (Fig. 3A). Surprisingly, over 90% of the total [ 3H]GGOH labeling was found in the HPC fraction, and the remaining labeling in the GGC fraction (Fig. 3B). The labeled compounds in HPC cannot represent dolichol since all-trans GG-OH, used in our experiments, is not utilized as a precursor in dolichol synthesis [21]. In a separate experiment we could also confirm that alltrans GG-OH is not incorporated into dolichol (data not shown). This result emphasizes the existence of mammalian GG-OH kinases, which might be activated un-
der limiting substrate conditions for prenylation. As expected no [ 3H]GG-OH was detected in the FC peak. All incorporated [ 3H]Dol-OH was found in the HPC peak (Fig. 3C). A control sample of [ 3H]MVA-labeled products is shown in Fig. 3D. In the next experiment RD-ES cells were incubated with 1 or 10 M of non-radioactive farnesol, geranylgeraniol or dolichol (⫺17, ⫺19, ⫺20 mixture) (Fig. 4). The addition of GG-OH resulted in a decreased [ 3H]MVA incorporation in the HPC fraction, 27 and 86%, respectively. The corresponding decreases in [ 3H]MVA incorporation in the GGC fraction accounted for 14 and 95%. A marked increased [ 3H]MVA labeling was observed in the FC fraction in cell cultures incubated with GG-OH. A similar increase was seen in the GGC and HPC fractions when F-OH was added to the medium (Figs. 4A and 4B). Incubation of Dol did not change the prenyl-cysteine distribution compared to the control (Fig. 4C). The effects of three different prenylation inhibitors on the [ 3H]MVA incorporation into the 3 prenyl cysteines were now investigated in RD-ES cells (Fig. 5). The peptidomimetic inhibitor FTI-277, specific for farnesyl transferase (22), led to an 80% reduction of FC synthesis at the highest concentration (10 M). The corresponding GGC labeling decreased with 18%. The GGTase I inhibitor GGTI-286 was less specific and showed the same inhibition profile at both 1 and 10 M (Fig. 5). Neither FTI-277 nor GGTI-286 affected the
FIG. 3. Distribution of radio labeled peaks on RP-HPLC. RD-ES cells were metabolically labeled with [ 3H]F-OH (A), [ 3H]GG-OH (B) or [ 3H]MVA (D). Protein fractions were isolated, delipidated and digested with Pronase E. Products extracted into butanol were analyzed on C4 reversed phase HPLC. A culture of WiDr cells were incubated with [ 3H]Dol/[ 35S]cysteine (C). Isolation and analysis of hydrophobic proteolytic products was as described. Eluates in fractions between 48 – 64 min were pooled and rechromatographed.
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DISCUSSION In this study we showed that [ 3H]GG-OH is heavily incorporated into products that are significantly more hydrophobic than FC and GGC. Specific inhibitors of FTase or GGTase I did not affect the expression of HPC. However the dietary monoterpene perillyl alcohol, currently being under investigation as an antitumor agent (24 –26), caused a dose dependent inhibition of [ 3H]MVA incorporation in this fraction. Our findings that farnesol and geranylgeraniol can serve as substrates for protein prenylation are supported by other studies (10, 11). The isoprenols are easily taken up by several mammalian cell lines and are probably then converted to the corresponding allylic pyrophosphates prior to incorporation into proteins (27). Enzyme systems catalyzing the phosphorylation reaction have been demonstrated in mammalians and lower organisms (28, 29). Several other studies have, using different proteolytic methods and subsequent analysis on chromatographic systems, also observed a fraction of MVAlabeled products that are more hydrophobic than GGC (10, 30, 31). It has been suggested that these products might consist of incompletely degraded isoprenylated peptides or di-geranylgeranylated CC or CXC sequences (10, 27). However, to our knowledge they have still not been further characterized. In a previous study
FIG. 4. Incorporation of [ 3H]MVA into prenyl-cysteines at incubations with cold F-OH (A), GG-OH (B) or Dol (C). Semiconfluent RD-ES cell cultures were labeled with 25 Ci [ 3H] MVA, 10 mM lovastatin and one of the indicated prenyl alcohol at two different concentrations (1 or 10 M). Prenyl-cysteines were isolated and analyzed as described under Materials and Methods.
incorporation of [ 3H]MVA into HPC. The monoterpene perillyl alcohol (POH), recently found to inhibit GGTase II [23], was tested at three different concentrations (100, 200 and 500M). This drug caused a dose dependent decrease in HPC labeling, the magnitude of which was 80% at the highest concentration (500 M). A small decrease of GGC synthesis was seen, while the [ 3H]MVA incorporation into FC increased with 34% compared to the control.
FIG. 5. The effect of isoprenylation inhibitors on the prenylcysteine distribution pattern. Semiconfluent RD-ES cultures were metabolically labeled with 25 Ci [ 3H]MVA/ml, 10 M lovastatin and indicated concentration of FTI-277, GGTI-286 (A) or POH (B). Prenyl-cysteines were isolated and analyzed as described under Materials and Methods.
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we found that digest products of [ 3H]dolichol-labeled proteins eluted in this chromatographic fraction (15). However, when we now compared the pronase digestion method with the sulfonium salt cleavage reaction, regarding released [ 3H]MVA-labeled products in the HPC versus free-dolichol fraction, 10 times more labeling was found in the aforementioned fraction. Based on this fact we concluded that HPC mainly consists of other MVA-derived products than dolichols. The high labeling with [ 3H]GG-OH suggests that GG is the main isoprene component of HPC. In addition, after co-incubation with [ 3H]MVA and non-radioactive GG-OH this [ 3H]MVA incorporation was drastically decreased. However, our data suggest that the geranylgeranylated HPC compounds are not products synthesized by GGTase I. Firstly, the incorporation of [ 3H]MVA into GGC and HPC was saturated at different MVA concentrations. This indicates that different enzymes are involved in the synthesis GGC and HPC. Secondly, inhibition of GGTase I using GGTI-286 did not affect the [ 3H]MVA incorporation into HPC, which would be expected if HPC would consist of partly degraded peptides modified by single GG groups. It was confirmed that this inhibitor decreased GGC synthesis adequately. Thirdly, the monoterpene perillyl alcohol, which is a semiselective inhibitor of GGTase II activity (23, 32), resulted in a dose-dependent decrease in HPC synthesis. There was also a decrease, but much smaller, in GGC labeling following treatment with this inhibitor. Taken together, our data strongly suggest that [ 3H]MVA incorporation into HPC involves a reaction that is distinct from carboxy terminal CAAX prenylation. Rather the hydrophobic prenyl cysteine products, assayed by reversed phase HPLC, could be composed of doubly geranylgeranylated CC or CXC products. Unpublished data from our laboratory showed that neither the chromatographic behavior nor the amount of [ 3H]MVA labeling of the HPC fraction changed at different times of protease incubation or after inclusion of carboxypeptidase Y. An intriguing question is why the CC and CXC sequences are resistant to the enzymatic proteolytic procedure. A reasonable explanation might be that adjacent bound prenyl groups protect the peptide bonds from cleavage. The large incorporation of [ 3H]MVA into HPC suggest that proteins modified by double GG groups may be relatively common in human cells. So far only Rab proteins have been found to be prenylated in this manner. However, the relative abundance of [ 3H]MVA incorporation into HPC may indicate that other proteins, still unknown, are di-geranylgeranylated. Since all experiments in this study was performed on malignant cells it will also be interesting to investigate whether this type of modification is associated with cancer.
ACKNOWLEDGMENTS This project was supported by grants from the Swedish Cancer Society, the Cancer Society in Stockholm, and the Karolinska Institute.
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26. Hardcastle, I. R., Rowlands, M. G., Barber, A. M., Grimshaw, R. M., Mohan, M. K., Nutley, B. P., and Jarman, M. (1999) Inhibition of protein prenylation by metabolites of limonene. Biochem. Pharmacol. 57, 801– 809. 27. Andres, D. A., Crick, D. C., Finlin, B. S., and Waechter, C. J. (1999) Rapid identification of cysteine-linked isoprenyl groups by metabolic labeling with [ 3H]farnesol and [ 3H]geranylgeraniol. Methods Mol. Biol. 116, 107–123. 28. Shin-ichi, O., Watanabe, M., and Nishino, T. (1996) Identification and characterization of geranylgeraniol kinase and geranylgeranylphosphate kinase from the Archebacterium Sulfolobus acidocaldarius. J. Biochem. 119, 541–547. 29. Westfall, D., Aboushadi, N., Shackelford, J. E., and Krisans, S. (1997) Metabolism of farnesol: Phosphorylation of farnesol by rat liver microsomial and peroxisomal fractions. Biochem. Biophys. Res. Commun. 230, 562–568. 30. Epstein, W. W., Lever, D. C., and Rilling, H. C. (1990) Prenylated proteins: Synthesis of geranylgeranylcysteine and identification of this thioether amino acid as a component of proteins in CHO cells. Proc. Natl. Acad. Sci. USA 87, 7352–7354. 31. Leining, L. M., Epstein, W. W., and Rilling H. C. (1994) Thioprenols as hydrazinolysis products of prenylated proteins: Dependence upon methylation of the prenylcysteine. Arch. Biochem. Biophys. 311, 199 –204. 32. Goalstone, M. L., Leitner, J. W., Golovchenko, I., Stjernholm, M. R., Cormon, M., Le Marchand-Brustel, Y., and Draznin, B. (1999) Insulin promotes phosphorylation and activation of geranylgeranyltransferase II. J. Biol. Chem. 274, 2880 –2884.
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Characterization of Hydrophobic Prenyl Groups of Isoprenylated Proteins in Human Cancer Cells Magnus Hjertman, Johan Wejde, and Olle Larsson 1 Department of Oncology and Pathology, CCK R8:04, Karolinska Hospital, SE-171 76 Stockholm, Sweden
Received September 18, 2001
Extensive protease digestion of delipidated [ 3H]mevalonate (MVA)-labeled proteins, followed by HPLC separation of the products, is one approach to identify and study prenyl cysteines. Using this methodology three major [ 3H]MVA-labeled peaks appeared. Two of them represent farnesyl cysteine (FC) and geranylgeranyl cysteine (GGC). The third peak represents unknown products that are considerably more hydrophobic than FC and GGC, here designated HPC. Previously, we provided evidence that cysteine residues may also be modified by dolichyl groups. Dolichyl cysteines (DolC) belong to HPC. However, as shown in the present study, DolC only represents a minor portion of HPC. Data obtained from different sets of experiments, including [ 3H]GGOH-labeling and use of prenyl transferase inhibitors, suggest that HPC mainly involves CXC or CC residues with double-linked GG groups. In turn this points to the possibility that proteins modified by double GG groups are quite common, and may probably involve other proteins than the rab family of GTPases. © 2001 Academic Press Key Words: mevalonate; geranylgeraniol; protein isoprenylation; prenyl cysteine; prenyl transferase inhibitors.
Inhibition of MVA synthesis blocks DNA replication and cell cycle progression (1–3). Among the different compounds derived from MVA, the substrates for prenylation, farnesyl pyrophosphate (FPP) and geranylgeranyl pyrophosphate (GGPP) have attracted particular attention as candidates for MVA-dependent cell growth. Three distinct enzymes are responsible for the posttranslational modification with isoprenoids. Farnesyl transferase (FTase) and the closely related geranylgeranyl transferase I (GGTase I) transfer the activated lipid substrate to a C-terminal CAAX motif, where the X amino acid mainly determines the choice of isoprene (4 – 6). A second mechanism for prenylation To whom correspondence should be addressed. Fax. ⫹46 8 7588397. E-mail: [email protected]. 1
0006-291X/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.
exists for low molecular GTPases of the Rab family, containing CC, CXC or CCXX motifs. These proteins, involved in targeting and fusion of transport vesicles, are geranylgeranylated at two adjacent cysteine residues and the enzyme responsible for this reaction is the cytosolic GGTase II (7). The prenyl modification functionally activates the proteins and enhances the propensity for membrane association. The farnesylation of p21 Ras has been a subject of intense research, since this reaction is known to be a necessity for its malignant transforming activity (8). Most proteins modified with prenyl groups are members of the Ras super family of GTPases, key players in signal transduction, regulating processes such as cell proliferation (9). A number of studies have recently shown that geranylgeranyl rather than farnesyl is the isoprenoid of importance for induction of DNA synthesis in cells growth-arrested by 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (10, 11). This indicates important roles of geranylgeranylated proteins in cell proliferation (12). Inhibitors of the enzymes responsible for the modification and processing of prenylated proteins have been developed and these substances have shown promising results as antitumor agents (13, 14). Because of the regulatory importance of prenylated proteins, a method to determine the expression level and distribution of the different prenyl substitutents would provide valuable information. In a previous work we demonstrated that reversed phase HPLC analysis of Pronase E digested [ 3H]MVA/[ 35S]cysteine-labeled proteins resulted in three distinct radioactive peaks (15). Two of them corresponded to farnesyl cysteine (FC) and geranylgeranyl cysteine (GGC) standards. The third peak (HPC), representing much more hydrophobic compounds, contained digest products labeled with [ 3H]Dolichol (Dol) and [ 35S]cysteine. However, by measuring the amount of [ 3H]MVA-labeled isoprenes released from proteins by sulfonium salt cleavage reaction, we found that dolichol only accounted for 2% (compared to 20% for HPC) of totally released [ 3H]MVA-labeled isoprenes
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3
[ H]MVA-Labeled Products Released by Sulfonium Salt Cleavage or Pronase E Digestion of Delipidated WiDr Proteins
Cleavage method
Short-chained isoprenoids F ⫹ GG (% of total)
Long-chained/ hydrophobic isoprenoids Dol, HPC (% of total)
Sulfonium salt (CH 3I) Pronase E
98 80
2 20
Note. Free isoprenoids and prenyl-modified amino acids were analyzed on C18 or C4 reversed phase HPLC, respectively.
(see Table 1). This suggests that other prenyl groups than dolichyl modified residues predominate in the HPC fraction. In the present study we extended the characterization of the prenyl cysteine expression with a main focus on the hydrophobic prenyl-containing peak. Our data strongly suggest that the HPC mainly consists of CC, CXC or CCXX fragments modified with double GG groups.
Differential MVA labeling. MDA-231 cells were grown in 144-mm dishes to near confluence. Fresh medium containing 25 M lovastatin was added. After a 2 h incubation period, 10 Ci [ 3H]MVA/ml together with unlabeled MVA (0 to 770 M) was added and the incubation was continued for 22 h. Proteins were isolated as described. Delipidation. The delipidation procedure was performed by washing the protein precipitate three times each with acetone, chloroform/methanol, 2:1 (v/v) and absolute ethanol, as described (16 –18). To remove any residual dolichyl pyrophosphoryl oligosaccharides the precipitate was finally washed five times with chloroform/methanol/water, 10:10:3 (by vol.) (19). Proteolytic cleavage of isoprene labeled proteins. Delipidated protein residues were suspended in 0.3 ml 50 mM Hepes-buffer, pH 7.4 containing 10 mM calcium acetate. Proteins were digested with protease type XIV from Streptomyces griseus (Pronase E) at 37°C for 20 h (20). Prenyl containing products were extracted into water saturated butanol and evaporated under a stream of nitrogen. HPLC. Butanol-extractable products, redissolved in methanol were purified on a reversed-phase HPLC system consisting of a Hypersil WP butyl (C4) column (150 ⫻ 4.6 mm). The elution was achieved with a linear gradient starting with 95% solvent A (methanol/water, 1/1 by volume containing 10 mM ammonium acetate) reaching 50% solvent B (isopropanol, 10 mM ammonium acetate) after 60 min. The flow rate was 0.8 ml/min. The absorbance was monitored at 210 nm. 1.6-ml fractions were collected and samples from each fraction were taken for scintillation counting.
RESULTS
MATERIALS AND METHODS Chemicals. , t, t, t-[1- 3H]geranylgeraniol (20 Ci/mmol), , t, t-[1- 3H]farnesol (60 Ci/mmol), Farnesyl-cysteine (FC) and geranylgeranyl cysteine (GGC) were purchased from American Radio labeled Chemicals, Inc. (St. Louis, MO). RS-[5- 3H]mevalonolactone (24 Ci/mmol) was from NEN DuPont. Lovastatin was obtained from Merck, Sharp and Dohme and was converted to its sodium salt before experimental use. (S)-(⫺)-perillyl alcohol (POH) was from Aldrich. The farnesyl transferase inhibitor FTI-277 and the geranylgeranyl transferase I inhibitor GGTI-286 were from Calbiochem. All other chemicals, if not otherwise specified, were from Sigma (St Louis, MO). Cell culture. The breast cancer cell line MDA-231, the colonic cancer cell line WiDr and the Ewing’s sarcoma cell line RD-ES, purchased from American Tissue Culture Collection (Rockville, MD), were grown in monolayers in tissue culture flasks maintained in a 95% air/5% CO 2 atmosphere at 37°C in a humidified incubator. RD-ES cells were cultured in RPMI 40 medium supplemented 15% (v/v) fetal calf serum (FCS). MDA-231 cells were grown in Dulbecco’s Modified Eagle Medium with 10% (v/v) FCS. Metabolic radiolabeling. Semiconfluent RD-ES cells, grown in 100-mm dishes were labeled with 10 Ci/ml [ 3H]F-OH, 10 Ci/ml [ 3H]GG-OH or [ 3H]-mevalonolactone (25 Ci/ml). The medium was removed after 24 h, the dishes carefully rinsed with PBS, after which the cells were harvested. After brief centrifugation the cell pellet was re-suspended in distilled water. A small sample was taken for determination of protein content. Total cellular proteins were precipitated over night with 10 volumes of ice-cold acetone. Protein prenyl transferase inhibition experiments. Exponentially growing subconfluent RD-ES cells cultured in 100-mm dishes were shifted to fresh medium containing 25 Ci [ 3H]MVA/ml, 10 M lovastatin, FTI-277 or GGTI-286 (1 or 10 M) or vehicle (10 mM dithiothreitol in dimethyl sulfoxide). Perillyl alcohol was added from stock solutions in ethanol to final concentrations of 100, 200 or 500 M. Proteins were isolated after 24 h incubation and precipitated with 10 volumes of acetone.
Extensive protease digestion of [ 3H]MVA-labeled proteins generates in addition to free amino acids, prenyl-modified cysteines which can be resolved and identified by reversed phase HPLC. Three major peaks can thus be detected, two of them co-elutes with standards of farnesyl-cysteine (FC) and geranylgeranyl cysteine (GGC) respectively. The third peak (HPC) represents [ 3H]MVA labeled products that are much more hydrophobic (Fig. 1). First we investigated the saturation concentration of MVA (0 –770 M), using nonradioactive MVA, on
FIG. 1. Rechromatography of HPC by RP-HPLC. Total cellular proteins were isolated from WiDr cells labeled with 25 Ci [ 3H]MVA/ ml in the presence of 10 M lovastatin. Butanol extractable products, derived after pronase E digestion of protein precipitates, were loaded on a C18 SEP-PAK cartridge. The methanol eluate was dried with nitrogen and subjected to C4 reversed phase HPLC. Fractions between 48 to 64 min were pooled, dried and analyzed on the same HPLC system. The mobility of farnesyl-cysteine and geranylgeranyl cysteine was measured by UV-detection at 210 nm.
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FIG. 2. MDA cells were grown in 150-mm dishes to near confluence. New fresh medium containing 25 M lovastatin was added. After a 3 h incubation period, 10 Ci [ 3H]MVA/ml and 0 to 770 M MVA, as indicated, was added and the incubation continued for 22 h. Isolation and analysis of prenyl-cysteines was performed as described.
[ 3H]MVA incorporation of FC, GGC and HPC. The prenyl-cysteine distribution pattern was examined in the breast cancer cell line MDA-231, which was also incubated by 25 M lovastatin to completely block the HMG-CoA reductase activity. At the lowest concentrations of MVA tested (0 or 7.7 M) the [ 3H]MVA incorporation into FC and GGC was strongly favored (Fig. 2). A dramatic change in the incorporation distribution occurred at higher concentrations of MVA in the medium (77 and 770 M). Two thirds of the radioactivity were now found in the HPC fraction and not more than 15% of the [ 3H]MVA labeling was in the fraction corresponding to FC. There was no notable difference in the distribution of the [ 3H]MVA incorporation into the prenyl groups at 77 and 770 M (Fig. 2), suggesting that all three pathways are saturated at 77 M MVA. To achieve saturation and simultaneously as high radioactive labeling as possible, we found that 50 Ci [ 3H]MVA/ml in the presence of the 50 M lovastatin was an optimal and reproducible condition [12], and therefore applied in the following experiments. To further characterize the HPC peak, the Ewing sarcoma cell line RD-ES was labeled with [ 3H]F-OH, [ 3H]GG-OH and [ 3H]Dol-OH. Expectedly, [ 3H]F-OH was found to be incorporated in all peaks with approximately 50% of the radiolabeling in the FC fraction (Fig. 3A). Surprisingly, over 90% of the total [ 3H]GGOH labeling was found in the HPC fraction, and the remaining labeling in the GGC fraction (Fig. 3B). The labeled compounds in HPC cannot represent dolichol since all-trans GG-OH, used in our experiments, is not utilized as a precursor in dolichol synthesis [21]. In a separate experiment we could also confirm that alltrans GG-OH is not incorporated into dolichol (data not shown). This result emphasizes the existence of mammalian GG-OH kinases, which might be activated un-
der limiting substrate conditions for prenylation. As expected no [ 3H]GG-OH was detected in the FC peak. All incorporated [ 3H]Dol-OH was found in the HPC peak (Fig. 3C). A control sample of [ 3H]MVA-labeled products is shown in Fig. 3D. In the next experiment RD-ES cells were incubated with 1 or 10 M of non-radioactive farnesol, geranylgeraniol or dolichol (⫺17, ⫺19, ⫺20 mixture) (Fig. 4). The addition of GG-OH resulted in a decreased [ 3H]MVA incorporation in the HPC fraction, 27 and 86%, respectively. The corresponding decreases in [ 3H]MVA incorporation in the GGC fraction accounted for 14 and 95%. A marked increased [ 3H]MVA labeling was observed in the FC fraction in cell cultures incubated with GG-OH. A similar increase was seen in the GGC and HPC fractions when F-OH was added to the medium (Figs. 4A and 4B). Incubation of Dol did not change the prenyl-cysteine distribution compared to the control (Fig. 4C). The effects of three different prenylation inhibitors on the [ 3H]MVA incorporation into the 3 prenyl cysteines were now investigated in RD-ES cells (Fig. 5). The peptidomimetic inhibitor FTI-277, specific for farnesyl transferase (22), led to an 80% reduction of FC synthesis at the highest concentration (10 M). The corresponding GGC labeling decreased with 18%. The GGTase I inhibitor GGTI-286 was less specific and showed the same inhibition profile at both 1 and 10 M (Fig. 5). Neither FTI-277 nor GGTI-286 affected the
FIG. 3. Distribution of radio labeled peaks on RP-HPLC. RD-ES cells were metabolically labeled with [ 3H]F-OH (A), [ 3H]GG-OH (B) or [ 3H]MVA (D). Protein fractions were isolated, delipidated and digested with Pronase E. Products extracted into butanol were analyzed on C4 reversed phase HPLC. A culture of WiDr cells were incubated with [ 3H]Dol/[ 35S]cysteine (C). Isolation and analysis of hydrophobic proteolytic products was as described. Eluates in fractions between 48 – 64 min were pooled and rechromatographed.
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DISCUSSION In this study we showed that [ 3H]GG-OH is heavily incorporated into products that are significantly more hydrophobic than FC and GGC. Specific inhibitors of FTase or GGTase I did not affect the expression of HPC. However the dietary monoterpene perillyl alcohol, currently being under investigation as an antitumor agent (24 –26), caused a dose dependent inhibition of [ 3H]MVA incorporation in this fraction. Our findings that farnesol and geranylgeraniol can serve as substrates for protein prenylation are supported by other studies (10, 11). The isoprenols are easily taken up by several mammalian cell lines and are probably then converted to the corresponding allylic pyrophosphates prior to incorporation into proteins (27). Enzyme systems catalyzing the phosphorylation reaction have been demonstrated in mammalians and lower organisms (28, 29). Several other studies have, using different proteolytic methods and subsequent analysis on chromatographic systems, also observed a fraction of MVAlabeled products that are more hydrophobic than GGC (10, 30, 31). It has been suggested that these products might consist of incompletely degraded isoprenylated peptides or di-geranylgeranylated CC or CXC sequences (10, 27). However, to our knowledge they have still not been further characterized. In a previous study
FIG. 4. Incorporation of [ 3H]MVA into prenyl-cysteines at incubations with cold F-OH (A), GG-OH (B) or Dol (C). Semiconfluent RD-ES cell cultures were labeled with 25 Ci [ 3H] MVA, 10 mM lovastatin and one of the indicated prenyl alcohol at two different concentrations (1 or 10 M). Prenyl-cysteines were isolated and analyzed as described under Materials and Methods.
incorporation of [ 3H]MVA into HPC. The monoterpene perillyl alcohol (POH), recently found to inhibit GGTase II [23], was tested at three different concentrations (100, 200 and 500M). This drug caused a dose dependent decrease in HPC labeling, the magnitude of which was 80% at the highest concentration (500 M). A small decrease of GGC synthesis was seen, while the [ 3H]MVA incorporation into FC increased with 34% compared to the control.
FIG. 5. The effect of isoprenylation inhibitors on the prenylcysteine distribution pattern. Semiconfluent RD-ES cultures were metabolically labeled with 25 Ci [ 3H]MVA/ml, 10 M lovastatin and indicated concentration of FTI-277, GGTI-286 (A) or POH (B). Prenyl-cysteines were isolated and analyzed as described under Materials and Methods.
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we found that digest products of [ 3H]dolichol-labeled proteins eluted in this chromatographic fraction (15). However, when we now compared the pronase digestion method with the sulfonium salt cleavage reaction, regarding released [ 3H]MVA-labeled products in the HPC versus free-dolichol fraction, 10 times more labeling was found in the aforementioned fraction. Based on this fact we concluded that HPC mainly consists of other MVA-derived products than dolichols. The high labeling with [ 3H]GG-OH suggests that GG is the main isoprene component of HPC. In addition, after co-incubation with [ 3H]MVA and non-radioactive GG-OH this [ 3H]MVA incorporation was drastically decreased. However, our data suggest that the geranylgeranylated HPC compounds are not products synthesized by GGTase I. Firstly, the incorporation of [ 3H]MVA into GGC and HPC was saturated at different MVA concentrations. This indicates that different enzymes are involved in the synthesis GGC and HPC. Secondly, inhibition of GGTase I using GGTI-286 did not affect the [ 3H]MVA incorporation into HPC, which would be expected if HPC would consist of partly degraded peptides modified by single GG groups. It was confirmed that this inhibitor decreased GGC synthesis adequately. Thirdly, the monoterpene perillyl alcohol, which is a semiselective inhibitor of GGTase II activity (23, 32), resulted in a dose-dependent decrease in HPC synthesis. There was also a decrease, but much smaller, in GGC labeling following treatment with this inhibitor. Taken together, our data strongly suggest that [ 3H]MVA incorporation into HPC involves a reaction that is distinct from carboxy terminal CAAX prenylation. Rather the hydrophobic prenyl cysteine products, assayed by reversed phase HPLC, could be composed of doubly geranylgeranylated CC or CXC products. Unpublished data from our laboratory showed that neither the chromatographic behavior nor the amount of [ 3H]MVA labeling of the HPC fraction changed at different times of protease incubation or after inclusion of carboxypeptidase Y. An intriguing question is why the CC and CXC sequences are resistant to the enzymatic proteolytic procedure. A reasonable explanation might be that adjacent bound prenyl groups protect the peptide bonds from cleavage. The large incorporation of [ 3H]MVA into HPC suggest that proteins modified by double GG groups may be relatively common in human cells. So far only Rab proteins have been found to be prenylated in this manner. However, the relative abundance of [ 3H]MVA incorporation into HPC may indicate that other proteins, still unknown, are di-geranylgeranylated. Since all experiments in this study was performed on malignant cells it will also be interesting to investigate whether this type of modification is associated with cancer.
ACKNOWLEDGMENTS This project was supported by grants from the Swedish Cancer Society, the Cancer Society in Stockholm, and the Karolinska Institute.
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