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Isoprenylation of RhoB Is Necessary for Its Degradation

2002; Elsevier BV; Volume: 277; Issue: 51 Linguagem: Inglês

10.1074/jbc.m204049200

1083-351X

Kostas Stamatakis, Eva Cernuda‐Morollón, Octavio Hernández‐Perera, Dolores Pérez‐Sala,

Metabolism, Diabetes, and Cancer

Statins improve vascular functions by mechanisms independent from their cholesterol-lowering effect. Rho GTPases are emerging as key targets for the vascular effects of statins. RhoB is a short-lived, early-response inducible protein involved in receptor endocytosis, apoptosis, and gene expression. Here we show that statins regulate RhoB expression by acting at multiple levels. Simvastatin increased RhoB protein levels by 8- to 10-fold. This effect was related to a depletion of isoprenoid intermediates, as deduced from the observation that several metabolites of the cholesterol biosynthetic pathway, namely, mevalonate and geranylgeranyl-pyrophosphate, attenuated simvastatin-induced RhoB up-regulation. Moreover, prenyltransferase inhibitors mimicked simvastatin effect. Cholesterol supplementation did not prevent simvastatin-elicited up-regulation but increased RhoB levels per se. Simvastatin moderately augmented RhoB transcript levels, but markedly impaired the degradation of RhoB protein, which accumulated in the cytosol in its non-isoprenylated form. Inhibition of RhoB isoprenylation was apparently required for simvastatin-induced up-regulation, because levels of an isoprenylation-deficient RhoB mutant were not affected by simvastatin. Moreover, this mutant was found to be markedly more stable than the wild-type protein. These results show that RhoB isoprenylation is necessary for rapid turnover of this protein and identify a novel link between the cholesterol biosynthetic pathway and the regulation of G-protein expression. Statins improve vascular functions by mechanisms independent from their cholesterol-lowering effect. Rho GTPases are emerging as key targets for the vascular effects of statins. RhoB is a short-lived, early-response inducible protein involved in receptor endocytosis, apoptosis, and gene expression. Here we show that statins regulate RhoB expression by acting at multiple levels. Simvastatin increased RhoB protein levels by 8- to 10-fold. This effect was related to a depletion of isoprenoid intermediates, as deduced from the observation that several metabolites of the cholesterol biosynthetic pathway, namely, mevalonate and geranylgeranyl-pyrophosphate, attenuated simvastatin-induced RhoB up-regulation. Moreover, prenyltransferase inhibitors mimicked simvastatin effect. Cholesterol supplementation did not prevent simvastatin-elicited up-regulation but increased RhoB levels per se. Simvastatin moderately augmented RhoB transcript levels, but markedly impaired the degradation of RhoB protein, which accumulated in the cytosol in its non-isoprenylated form. Inhibition of RhoB isoprenylation was apparently required for simvastatin-induced up-regulation, because levels of an isoprenylation-deficient RhoB mutant were not affected by simvastatin. Moreover, this mutant was found to be markedly more stable than the wild-type protein. These results show that RhoB isoprenylation is necessary for rapid turnover of this protein and identify a novel link between the cholesterol biosynthetic pathway and the regulation of G-protein expression. Statins are a family of drugs widely used in the treatment of hypercholesterolemia. They are competitive inhibitors of hydroxymethylglutaryl-CoA (HMG-CoA) 1The abbreviations used are: HMG-CoA, hydroxymethylglutaryl-CoA; BAEC, bovine aortic endothelial cells; GGPP, geranylgeranyl-pyrophosphate; GFP, green fluorescent protein; HA, hemagglutinin1The abbreviations used are: HMG-CoA, hydroxymethylglutaryl-CoA; BAEC, bovine aortic endothelial cells; GGPP, geranylgeranyl-pyrophosphate; GFP, green fluorescent protein; HA, hemagglutinin reductase (1Goldstein J.L. Brown M.S. Nature. 1990; 343: 425-430Crossref PubMed Scopus (4499) Google Scholar), the enzyme catalyzing the rate-limiting step in the synthesis of cholesterol, that is, the conversion of HMG-CoA to mevalonate (Scheme FS1). Mevalonate is also the precursor of farnesyl-pyrophosphate and geranylgeranyl-pyrophosphate, the isoprenoid lipids involved in the posttranslational modification of a number of proteins, including most G-proteins (2Zhang F.L. Casey P.J. Annu. Rev. Biochem. 1996; 65: 241-269Crossref PubMed Scopus (1725) Google Scholar, 3Rando R.R. Biochim. Biophys. Acta. 1996; 1300: 5-16Crossref PubMed Scopus (97) Google Scholar). For this reason, statins have also been used as pharmacological tools to assess the importance of protein isoprenylation in signal transduction (4Schafer W.R. Kim R. Sterne R. Thorner J. Kim S.-H. Rine J. Science. 1989; 245: 379-385Crossref PubMed Scopus (421) Google Scholar, 5Pérez-Sala D. Mollinedo F. Biochem. Biophys. Res. Commun. 1994; 199: 1209-1215Crossref PubMed Scopus (139) Google Scholar).The clinical use of statins has revealed that these drugs promote beneficial effects on cardiovascular functions that do not correlate with their ability to lower serum cholesterol levels (6Eichstädt H.W. Eskötter H. Hoffman I. Amthauer H.W. Weidinger G. Am. J. Cardiol. 1995; 76: 122A-125AAbstract Full Text PDF PubMed Scopus (128) Google Scholar, 7O'Driscoll G. Green D. Taylor R.R. Circulation. 1997; 95: 1126-1131Crossref PubMed Scopus (940) Google Scholar). Treatment of endothelial cells with statins increases or preserves the expression of endothelial nitric oxide synthase (8Laufs U. Liao J.K. J. Biol. Chem. 1998; 273: 24266-24271Abstract Full Text Full Text PDF PubMed Scopus (966) Google Scholar, 9Hernández-Perera O. Pérez-Sala D. Sánchez-Pascuala R. Navarro-Antolı́n J. Hernández G. Dı́az C. Lamas S. J. Clin. Invest. 1998; 101: 2711-2719Crossref PubMed Scopus (745) Google Scholar), while inhibiting the production of endothelin-1 (10Hernández-Perera O. Pérez-Sala D. Soria E. Lamas S. Circ. Res. 2000; 87: 616-622Crossref PubMed Scopus (173) Google Scholar). In addition, antiproliferative and anti-inflammatory effects of statins have been observed in several experimental systems (5Pérez-Sala D. Mollinedo F. Biochem. Biophys. Res. Commun. 1994; 199: 1209-1215Crossref PubMed Scopus (139) Google Scholar, 11Laufs U. Marra D. Node K. Liao J. J. Biol. Chem. 1999; 274: 21926-21931Abstract Full Text Full Text PDF PubMed Scopus (383) Google Scholar, 12Pahan K. Sheikh F.G. Namboodiri A.M. Singh I. J. Clin. Invest. 1997; 100: 2671-2679Crossref PubMed Scopus (507) Google Scholar, 13Bustos C. Hernández-Presa M.A. Ortego M. Tunon J. Ortega L. Pérez F. Dı́az C. Hernández G. Egido J. J. Am. Coll. Cardiol. 1998; 32: 2057-2064Crossref PubMed Scopus (413) Google Scholar) and in patients (14Weber C. Erl W. Weber K.S. Weber P.C. J. Am. Coll. Cardiol. 1997; 30: 1212-1217Crossref PubMed Scopus (392) Google Scholar, 15Romano M. Mezetti A. Marulli C. Ciabattoni G. Febo F. Ienno S.D. Roccaforte S. Vigneri S. Nubile G. Milani M. Davi G. J. Invest. Med. 2000; 48: 183-189PubMed Google Scholar). Elucidation of the mechanisms underlying these diverse effects has shown that small G-proteins of the Rho family are critical targets for statins in many systems. Rho GTPases participate in the regulation of numerous cellular functions, including cytoskeletal organization, cell adhesion, smooth muscle contraction, endocytosis, receptor signaling, cell cycle progression, and gene expression (16Kaibuchi K. Kuroda S. Amano M. Annu. Rev. Biochem. 1999; 68: 459-486Crossref PubMed Scopus (884) Google Scholar, 17Ellis S. Mellor H. Trends Cell Biol. 2000; 10: 85-88Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar, 18Mackay D.J. Hall A. J. Biol. Chem. 1998; 273: 20685-20688Abstract Full Text Full Text PDF PubMed Scopus (567) Google Scholar, 19Khosravi-Far R. Campbell S. Rossman K.L. Der C.J. Adv. Cancer Res. 1998; 72: 57-107Crossref PubMed Google Scholar). Rho proteins are posttranslationally modified by isoprenylation at a cysteine residue located near their C-terminal end (20Adamson P. Marshall C.J. Hall A. Tilbrook P.A. J. Biol. Chem. 1992; 267: 20033-20038Abstract Full Text PDF PubMed Google Scholar). Inhibition of Rho function by impairment of protein isoprenylation is at the basis of the reported effects of statins on smooth muscle cell proliferation (11Laufs U. Marra D. Node K. Liao J. J. Biol. Chem. 1999; 274: 21926-21931Abstract Full Text Full Text PDF PubMed Scopus (383) Google Scholar), endothelial nitric oxide synthase and inducible nitric oxide synthase expression (8Laufs U. Liao J.K. J. Biol. Chem. 1998; 273: 24266-24271Abstract Full Text Full Text PDF PubMed Scopus (966) Google Scholar, 21Hausding M. Witteck A. Rodrı́guez-Pascual F. Eichel-Streiber C.V. Fostermann U. Kleinert H. Br. J. Pharmacol. 2000; 131: 553-561Crossref PubMed Scopus (54) Google Scholar), pre-pro-endothelin-1 transcription (10Hernández-Perera O. Pérez-Sala D. Soria E. Lamas S. Circ. Res. 2000; 87: 616-622Crossref PubMed Scopus (173) Google Scholar), and protection against cerebral stroke (22Laufs U. Endres M. Stagliano N. Amin-Hanjani S. Chui D.-S. Yang S.-X. Simoncini T. Yamada M. Rabkin E. Allen P.G. Huang P.L. Böhm M. Schoen F.J. Moskowitz M.A. Liao J.K. J. Clin. Invest. 2000; 106: 15-24Crossref PubMed Scopus (250) Google Scholar). Evidence accumulating during the last decade indicates that, by limiting mevalonate availability, statins may influence not only the function but also the expression of several G-proteins, including Ras (23Dimster-Denk D. Schafer W.R. Rine J. Mol. Biol. Cell. 1995; 6: 59-70Crossref PubMed Scopus (18) Google Scholar), Rab (24Laezza C. Bucci C. Santillo M. Bruni C.B. Bifulco M. Biochem. Biophys. Res. Commun. 1998; 248: 469-472Crossref PubMed Scopus (19) Google Scholar), Rap (25Holstein S.A. Wohlford-Lenane C.L. Hohl R.J. J. Biol. Chem. 2002; 277: 10678-10682Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar), and Rho proteins (26Engel M.E. Datta P.K. Moses H.L. J. Biol. Chem. 1998; 273: 9921-9926Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar, 27Laufs U. Endress M. Custodis F. Gertz K. Nickenig G. Liao J.K. Böhm M. Circulation. 2000; 102: 3104-3110Crossref PubMed Scopus (255) Google Scholar, 28Pérez-Sala D. Cernuda-Morollón E. Hernández-Perera O. Eur. J. Biochem. 2001; 268: 143Google Scholar). However, the mechanisms responsible for these effects are not fully elucidated.RhoB is a short-lived protein, the levels of which can be regulated by growth factors and stress signals both transcriptionally and posttranslationally (26Engel M.E. Datta P.K. Moses H.L. J. Biol. Chem. 1998; 273: 9921-9926Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar, 29Fritz G. Kaina B. J. Biol. Chem. 1997; 272: 30637-30644Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). RhoB can be modified either by farnesylation or geranylgeranylation at its C terminus (20Adamson P. Marshall C.J. Hall A. Tilbrook P.A. J. Biol. Chem. 1992; 267: 20033-20038Abstract Full Text PDF PubMed Google Scholar), and it has been proposed that its function may depend on the presence and the nature of the isoprenoid moiety (30Lebowitz P.F. Casey P.J. Prendergast G.C. Thissen J.A. J. Biol. Chem. 1997; 272: 15591-15594Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar). Among the reported RhoB functions are the modulation of receptor endocytosis (31Gampel A. Parker P.J. Mellor H. Curr. Biol. 1999; 9: 955-958Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar), apoptosis (32Liu A.-X. Cerniglia G.J. Bernhard E.J. Prendergast G.C. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 6192-6197Crossref PubMed Scopus (131) Google Scholar), and vascular gene expression, including that of pre-pro-endothelin-1 (10Hernández-Perera O. Pérez-Sala D. Soria E. Lamas S. Circ. Res. 2000; 87: 616-622Crossref PubMed Scopus (173) Google Scholar) and inducible nitric oxide synthase (33Delarue F.L. Taylor B.S. Sebti S.M. Oncogene. 2001; 20: 6531-6537Crossref PubMed Scopus (31) Google Scholar). A detailed knowledge of the effect of statins on Rho protein biology is of primary importance in the context of the therapeutic strategies for hypercholesterolemia and atherosclerosis. In the present study we have explored the effect of statins on RhoB expression and processing in several cell types. Our results show that statins drastically increase the levels of cellular RhoB by mechanisms that involve the regulation of RhoB mRNA by the cholesterol biosynthetic pathway and the impairment of the proteolytic degradation of the non-isoprenylated RhoB protein. This latter observation provides the first evidence for the regulation of protein stability by isoprenylation.DISCUSSIONThe results presented in this work show that RhoB levels are modulated by the cholesterol biosynthetic pathway. Down-regulation of this pathway either by means of HMG-CoA reductase inhibitors or excess cholesterol is associated with an increase in the amount of RhoB. This regulation occurs in part at the mRNA level and largely at the protein level, because inhibition of RhoB isoprenylation either by pharmacological or genetic manipulation markedly reduces the degradation of this protein.The cholesterol biosynthetic pathway provides the isoprenoid intermediates needed for the posttranslational modification of G-proteins. Although protein isoprenylation is an irreversible modification, it is susceptible to a certain degree of regulation, because the activity of the prenyltransferases may be controlled by some growth factors and by the availability of isoprenoids. The cholesterol pathway is subjected to a complex feedback regulation (1Goldstein J.L. Brown M.S. Nature. 1990; 343: 425-430Crossref PubMed Scopus (4499) Google Scholar), and it can be modulated by stress (39Shack S. Gorospe M. Fawcett T.W. Hudgins W.R. Holbrook N.J. Oncogene. 1999; 18: 6021-6028Crossref PubMed Scopus (36) Google Scholar). In recent years it has become evident that the activity of this pathway may control the maturation of certain G-proteins such as Ras, thus resulting in the regulation of Ras-mediated cellular processes in response to cholesterol levels or cellular stress (39Shack S. Gorospe M. Fawcett T.W. Hudgins W.R. Holbrook N.J. Oncogene. 1999; 18: 6021-6028Crossref PubMed Scopus (36) Google Scholar, 40Gadbut A.P. Wu L. Tang D. Papageorge A. Watson J.A. Galper J.B. EMBO J. 1997; 16: 7250-7260Crossref PubMed Scopus (33) Google Scholar). In addition, several lines of evidence indicate that the mevalonate pathway may influence the expression of other members of the Ras superfamily, namely Ras1 and Ras2 in yeast (23Dimster-Denk D. Schafer W.R. Rine J. Mol. Biol. Cell. 1995; 6: 59-70Crossref PubMed Scopus (18) Google Scholar), and Rab5 and Rab7 in thyroid cells (24Laezza C. Bucci C. Santillo M. Bruni C.B. Bifulco M. Biochem. Biophys. Res. Commun. 1998; 248: 469-472Crossref PubMed Scopus (19) Google Scholar), through mechanisms not yet completely elucidated.The relationship between G-protein expression and cholesterol metabolism may be bidirectional. In a recent report, cdc42 proteins have been involved in the regulation of cholesterol efflux (41Hirano K.-I. Matsuura F. Tsukamoto K. Zhang Z. Matsuyama A. Takaishi K. Komuro R. Suehiro T. Yamashita S. Takai Y. Matsuzawa Y. FEBS Lett. 2000; 484: 275-279Crossref PubMed Scopus (47) Google Scholar). In addition, in fibroblasts from patients with the inherited disorder of lipid metabolism known as Tangier disease, characterized by cellular accumulation of cholesteryl esters, the expression of several Rho proteins, including RhoB, is increased (42Utech M. Hobbel G. Rust S. Reinecke H. Assmann G. Walter M. Biochem. Biophys. Res. Commun. 2001; 280: 229-236Crossref PubMed Scopus (31) Google Scholar). On this basis, a role for Rho proteins in cholesterol transport has been hypothesized. Our results raise the possibility that the observed increase in Rho proteins expression may be caused in part by the accumulation of cholesterol in cells.The mechanisms by which the mevalonate pathway regulates RhoB appear to be multiple. Recently, mevalonate depletion has been reported to result in an increase in RhoB mRNA (25Holstein S.A. Wohlford-Lenane C.L. Hohl R.J. J. Biol. Chem. 2002; 277: 10678-10682Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). However, the involvement of isoprenoid intermediates or isoprenylated proteins in this effect has not been explored. Here we have shown that inhibition of mevalonate biosynthesis with simvastatin moderately up-regulates RhoB mRNA levels and that this effect is prevented by GGPP. In addition, we have observed that inhibition of Rho signaling by means of bacterial toxins that target and inactivate Rho proteins (43Ridley A.J. Hall A. Cell. 1992; 70: 389-399Abstract Full Text PDF PubMed Scopus (3797) Google Scholar) or by a Rho kinase inhibitor, is also associated with a moderate increase in RhoB protein and mRNA levels. These observations suggest that geranylgeranylated proteins and, in particular, Rho proteins, exert a negative feedback on RhoB mRNA expression in endothelial cells. This interpretation is in accordance with previous studies that identified a negative role of RhoA and B on the activity of the mouse RhoB promoter (29Fritz G. Kaina B. J. Biol. Chem. 1997; 272: 30637-30644Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). Our results indicate that the main effect of simvastatin on RhoB expression takes place at a posttranslational level by reducing RhoB degradation. This could occur through several mechanisms. Simvastatin could affect the activity of proteolytic pathways within the cell. In fact, statins have been reported to modulate the activity of the proteasome (44Rao S. Porter D.C. Chen X. Herliczek T. Lowe M. Keyomarsi K. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 7797-7802Crossref PubMed Scopus (331) Google Scholar, 45Wojcik C. Bury M. Stoklosa T. Giermasz A. Feleszko W. Mlynarczuk I. Pleban E. Basak G. Omura S. Jakobisiak M. Int. J. Biochem. Cell Biol. 2000; 32: 957-965Crossref PubMed Scopus (39) Google Scholar), a protein-degradation pathway proposed to mediate RhoB turnover (26Engel M.E. Datta P.K. Moses H.L. J. Biol. Chem. 1998; 273: 9921-9926Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). However, the effect of simvastatin on RhoB levels and stability was completely prevented by co-treatment with cycloheximide, thus showing that de novo protein synthesis is required for its effect. Moreover, inhibition of protein isoprenylation by using peptidomimetic inhibitors of prenyltransferases reproduced the effect of simvastatin on RhoB levels. These observations suggest that the effect of simvastatin is related to the inhibition of protein isoprenylation rather than to a direct interference with cellular proteases. Because the increase in RhoB protein elicited by simvastatin occurs at the expense of non-prenylated cytosol-partitioned RhoB, it could be hypothesized that the immature cytosolic protein is a poorer substrate for proteases or it does not localize to the appropriate cellular compartment for degradation. This is supported by the fact that the Cys193Ser RhoB mutant, which lacks the isoprenylation site and is cytosolic, is markedly more stable than the wild-type protein. These observations indicate that isoprenylation of RhoB determines the half-life of the protein. Interestingly, carboxyl methylation of the isoprenylated cysteine in RhoA has been reported to reduce its turnover (46Backlund P.S. J. Biol. Chem. 1997; 272: 33175-33180Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar), thus providing another example of the regulation of Rho protein stability as a function of their posttranslational processing.Whether the increased stability of unprenylated RhoB is caused by changes in subcellular distribution, activity, or ability to interact with other cellular proteins will be the subject of further studies. Although inhibition of RhoB isoprenylation may abolish some of RhoB functions, like its cell transforming ability (47Lebowitz P.F. Du W. Prendergast G.C. J. Biol. Chem. 1997; 272: 16093-16095Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar) or the activation of the endothelin-1 promoter (10Hernández-Perera O. Pérez-Sala D. Soria E. Lamas S. Circ. Res. 2000; 87: 616-622Crossref PubMed Scopus (173) Google Scholar), other functions could be conserved, as it has been described for the activation of the serum-response element-dependent transcription in overexpression experiments (47Lebowitz P.F. Du W. Prendergast G.C. J. Biol. Chem. 1997; 272: 16093-16095Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). Future work will address whether unprenylated RhoB may have specific functions or whether it behaves as a dominant-negative RhoB protein.In conclusion, we have found that RhoB mRNA levels and protein stability can be regulated by the availability of isoprenoid intermediates needed for protein modification. These findings unveil the novel role of isoprenylation in the control of RhoB protein degradation and may contribute to the understanding of the complex structure-function relationships of RhoB posttranslational modifications. Statins are a family of drugs widely used in the treatment of hypercholesterolemia. They are competitive inhibitors of hydroxymethylglutaryl-CoA (HMG-CoA) 1The abbreviations used are: HMG-CoA, hydroxymethylglutaryl-CoA; BAEC, bovine aortic endothelial cells; GGPP, geranylgeranyl-pyrophosphate; GFP, green fluorescent protein; HA, hemagglutinin1The abbreviations used are: HMG-CoA, hydroxymethylglutaryl-CoA; BAEC, bovine aortic endothelial cells; GGPP, geranylgeranyl-pyrophosphate; GFP, green fluorescent protein; HA, hemagglutinin reductase (1Goldstein J.L. Brown M.S. Nature. 1990; 343: 425-430Crossref PubMed Scopus (4499) Google Scholar), the enzyme catalyzing the rate-limiting step in the synthesis of cholesterol, that is, the conversion of HMG-CoA to mevalonate (Scheme FS1). Mevalonate is also the precursor of farnesyl-pyrophosphate and geranylgeranyl-pyrophosphate, the isoprenoid lipids involved in the posttranslational modification of a number of proteins, including most G-proteins (2Zhang F.L. Casey P.J. Annu. Rev. Biochem. 1996; 65: 241-269Crossref PubMed Scopus (1725) Google Scholar, 3Rando R.R. Biochim. Biophys. Acta. 1996; 1300: 5-16Crossref PubMed Scopus (97) Google Scholar). For this reason, statins have also been used as pharmacological tools to assess the importance of protein isoprenylation in signal transduction (4Schafer W.R. Kim R. Sterne R. Thorner J. Kim S.-H. Rine J. Science. 1989; 245: 379-385Crossref PubMed Scopus (421) Google Scholar, 5Pérez-Sala D. Mollinedo F. Biochem. Biophys. Res. Commun. 1994; 199: 1209-1215Crossref PubMed Scopus (139) Google Scholar). The clinical use of statins has revealed that these drugs promote beneficial effects on cardiovascular functions that do not correlate with their ability to lower serum cholesterol levels (6Eichstädt H.W. Eskötter H. Hoffman I. Amthauer H.W. Weidinger G. Am. J. Cardiol. 1995; 76: 122A-125AAbstract Full Text PDF PubMed Scopus (128) Google Scholar, 7O'Driscoll G. Green D. Taylor R.R. Circulation. 1997; 95: 1126-1131Crossref PubMed Scopus (940) Google Scholar). Treatment of endothelial cells with statins increases or preserves the expression of endothelial nitric oxide synthase (8Laufs U. Liao J.K. J. Biol. Chem. 1998; 273: 24266-24271Abstract Full Text Full Text PDF PubMed Scopus (966) Google Scholar, 9Hernández-Perera O. Pérez-Sala D. Sánchez-Pascuala R. Navarro-Antolı́n J. Hernández G. Dı́az C. Lamas S. J. Clin. Invest. 1998; 101: 2711-2719Crossref PubMed Scopus (745) Google Scholar), while inhibiting the production of endothelin-1 (10Hernández-Perera O. Pérez-Sala D. Soria E. Lamas S. Circ. Res. 2000; 87: 616-622Crossref PubMed Scopus (173) Google Scholar). In addition, antiproliferative and anti-inflammatory effects of statins have been observed in several experimental systems (5Pérez-Sala D. Mollinedo F. Biochem. Biophys. Res. Commun. 1994; 199: 1209-1215Crossref PubMed Scopus (139) Google Scholar, 11Laufs U. Marra D. Node K. Liao J. J. Biol. Chem. 1999; 274: 21926-21931Abstract Full Text Full Text PDF PubMed Scopus (383) Google Scholar, 12Pahan K. Sheikh F.G. Namboodiri A.M. Singh I. J. Clin. Invest. 1997; 100: 2671-2679Crossref PubMed Scopus (507) Google Scholar, 13Bustos C. Hernández-Presa M.A. Ortego M. Tunon J. Ortega L. Pérez F. Dı́az C. Hernández G. Egido J. J. Am. Coll. Cardiol. 1998; 32: 2057-2064Crossref PubMed Scopus (413) Google Scholar) and in patients (14Weber C. Erl W. Weber K.S. Weber P.C. J. Am. Coll. Cardiol. 1997; 30: 1212-1217Crossref PubMed Scopus (392) Google Scholar, 15Romano M. Mezetti A. Marulli C. Ciabattoni G. Febo F. Ienno S.D. Roccaforte S. Vigneri S. Nubile G. Milani M. Davi G. J. Invest. Med. 2000; 48: 183-189PubMed Google Scholar). Elucidation of the mechanisms underlying these diverse effects has shown that small G-proteins of the Rho family are critical targets for statins in many systems. Rho GTPases participate in the regulation of numerous cellular functions, including cytoskeletal organization, cell adhesion, smooth muscle contraction, endocytosis, receptor signaling, cell cycle progression, and gene expression (16Kaibuchi K. Kuroda S. Amano M. Annu. Rev. Biochem. 1999; 68: 459-486Crossref PubMed Scopus (884) Google Scholar, 17Ellis S. Mellor H. Trends Cell Biol. 2000; 10: 85-88Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar, 18Mackay D.J. Hall A. J. Biol. Chem. 1998; 273: 20685-20688Abstract Full Text Full Text PDF PubMed Scopus (567) Google Scholar, 19Khosravi-Far R. Campbell S. Rossman K.L. Der C.J. Adv. Cancer Res. 1998; 72: 57-107Crossref PubMed Google Scholar). Rho proteins are posttranslationally modified by isoprenylation at a cysteine residue located near their C-terminal end (20Adamson P. Marshall C.J. Hall A. Tilbrook P.A. J. Biol. Chem. 1992; 267: 20033-20038Abstract Full Text PDF PubMed Google Scholar). Inhibition of Rho function by impairment of protein isoprenylation is at the basis of the reported effects of statins on smooth muscle cell proliferation (11Laufs U. Marra D. Node K. Liao J. J. Biol. Chem. 1999; 274: 21926-21931Abstract Full Text Full Text PDF PubMed Scopus (383) Google Scholar), endothelial nitric oxide synthase and inducible nitric oxide synthase expression (8Laufs U. Liao J.K. J. Biol. Chem. 1998; 273: 24266-24271Abstract Full Text Full Text PDF PubMed Scopus (966) Google Scholar, 21Hausding M. Witteck A. Rodrı́guez-Pascual F. Eichel-Streiber C.V. Fostermann U. Kleinert H. Br. J. Pharmacol. 2000; 131: 553-561Crossref PubMed Scopus (54) Google Scholar), pre-pro-endothelin-1 transcription (10Hernández-Perera O. Pérez-Sala D. Soria E. Lamas S. Circ. Res. 2000; 87: 616-622Crossref PubMed Scopus (173) Google Scholar), and protection against cerebral stroke (22Laufs U. Endres M. Stagliano N. Amin-Hanjani S. Chui D.-S. Yang S.-X. Simoncini T. Yamada M. Rabkin E. Allen P.G. Huang P.L. Böhm M. Schoen F.J. Moskowitz M.A. Liao J.K. J. Clin. Invest. 2000; 106: 15-24Crossref PubMed Scopus (250) Google Scholar). Evidence accumulating during the last decade indicates that, by limiting mevalonate availability, statins may influence not only the function but also the expression of several G-proteins, including Ras (23Dimster-Denk D. Schafer W.R. Rine J. Mol. Biol. Cell. 1995; 6: 59-70Crossref PubMed Scopus (18) Google Scholar), Rab (24Laezza C. Bucci C. Santillo M. Bruni C.B. Bifulco M. Biochem. Biophys. Res. Commun. 1998; 248: 469-472Crossref PubMed Scopus (19) Google Scholar), Rap (25Holstein S.A. Wohlford-Lenane C.L. Hohl R.J. J. Biol. Chem. 2002; 277: 10678-10682Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar), and Rho proteins (26Engel M.E. Datta P.K. Moses H.L. J. Biol. Chem. 1998; 273: 9921-9926Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar, 27Laufs U. Endress M. Custodis F. Gertz K. Nickenig G. Liao J.K. Böhm M. Circulation. 2000; 102: 3104-3110Crossref PubMed Scopus (255) Google Scholar, 28Pérez-Sala D. Cernuda-Morollón E. Hernández-Perera O. Eur. J. Biochem. 2001; 268: 143Google Scholar). However, the mechanisms responsible for these effects are not fully elucidated. RhoB is a short-lived protein, the levels of which can be regulated by growth factors and stress signals both transcriptionally and posttranslationally (26Engel M.E. Datta P.K. Moses H.L. J. Biol. Chem. 1998; 273: 9921-9926Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar, 29Fritz G. Kaina B. J. Biol. Chem. 1997; 272: 30637-30644Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). RhoB can be modified either by farnesylation or geranylgeranylation at its C terminus (20Adamson P. Marshall C.J. Hall A. Tilbrook P.A. J. Biol. Chem. 1992; 267: 20033-20038Abstract Full Text PDF PubMed Google Scholar), and it has been proposed that its function may depend on the presence and the nature of the isoprenoid moiety (30Lebowitz P.F. Casey P.J. Prendergast G.C. Thissen J.A. J. Biol. Chem. 1997; 272: 15591-15594Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar). Among the reported RhoB functions are the modulation of receptor endocytosis (31Gampel A. Parker P.J. Mellor H. Curr. Biol. 1999; 9: 955-958Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar), apoptosis (32Liu A.-X. Cerniglia G.J. Bernhard E.J. Prendergast G.C. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 6192-6197Crossref PubMed Scopus (131) Google Scholar), and vascular gene expression, including that of pre-pro-endothelin-1 (10Hernández-Perera O. Pérez-Sala D. Soria E. Lamas S. Circ. Res. 2000; 87: 616-622Crossref PubMed Scopus (173) Google Scholar) and inducible nitric oxide synthase (33Delarue F.L. Taylor B.S. Sebti S.M. Oncogene. 2001; 20: 6531-6537Crossref PubMed Scopus (31) Google Scholar). A detailed knowledge of the effect of statins on Rho protein biology is of primary importance in the context of the therapeutic strategies for hypercholesterolemia and atherosclerosis. In the present study we have explored the effect of statins on RhoB expression and processing in several cell types. Our results show that statins drastically increase the levels of cellular RhoB by mechanisms that involve the regulation of RhoB mRNA by the cholesterol biosynthetic pathway and the impairment of the proteolytic degradation of the non-isoprenylated RhoB protein. This latter observation provides the first evidence for the regulation of protein stability by isoprenylation. DISCUSSIONThe results presented in this work show that RhoB levels are modulated by the cholesterol biosynthetic pathway. Down-regulation of this pathway either by means of HMG-CoA reductase inhibitors or excess cholesterol is associated with an increase in the amount of RhoB. This regulation occurs in part at the mRNA level and largely at the protein level, because inhibition of RhoB isoprenylation either by pharmacological or genetic manipulation markedly reduces the degradation of this protein.The cholesterol biosynthetic pathway provides the isoprenoid intermediates needed for the posttranslational modification of G-proteins. Although protein isoprenylation is an irreversible modification, it is susceptible to a certain degree of regulation, because the activity of the prenyltransferases may be controlled by some growth factors and by the availability of isoprenoids. The cholesterol pathway is subjected to a complex feedback regulation (1Goldstein J.L. Brown M.S. Nature. 1990; 343: 425-430Crossref PubMed Scopus (4499) Google Scholar), and it can be modulated by stress (39Shack S. Gorospe M. Fawcett T.W. Hudgins W.R. Holbrook N.J. Oncogene. 1999; 18: 6021-6028Crossref PubMed Scopus (36) Google Scholar). In recent years it has become evident that the activity of this pathway may control the maturation of certain G-proteins such as Ras, thus resulting in the regulation of Ras-mediated cellular processes in response to cholesterol levels or cellular stress (39Shack S. Gorospe M. Fawcett T.W. Hudgins W.R. Holbrook N.J. Oncogene. 1999; 18: 6021-6028Crossref PubMed Scopus (36) Google Scholar, 40Gadbut A.P. Wu L. Tang D. Papageorge A. Watson J.A. Galper J.B. EMBO J. 1997; 16: 7250-7260Crossref PubMed Scopus (33) Google Scholar). In addition, several lines of evidence indicate that the mevalonate pathway may influence the expression of other members of the Ras superfamily, namely Ras1 and Ras2 in yeast (23Dimster-Denk D. Schafer W.R. Rine J. Mol. Biol. Cell. 1995; 6: 59-70Crossref PubMed Scopus (18) Google Scholar), and Rab5 and Rab7 in thyroid cells (24Laezza C. Bucci C. Santillo M. Bruni C.B. Bifulco M. Biochem. Biophys. Res. Commun. 1998; 248: 469-472Crossref PubMed Scopus (19) Google Scholar), through mechanisms not yet completely elucidated.The relationship between G-protein expression and cholesterol metabolism may be bidirectional. In a recent report, cdc42 proteins have been involved in the regulation of cholesterol efflux (41Hirano K.-I. Matsuura F. Tsukamoto K. Zhang Z. Matsuyama A. Takaishi K. Komuro R. Suehiro T. Yamashita S. Takai Y. Matsuzawa Y. FEBS Lett. 2000; 484: 275-279Crossref PubMed Scopus (47) Google Scholar). In addition, in fibroblasts from patients with the inherited disorder of lipid metabolism known as Tangier disease, characterized by cellular accumulation of cholesteryl esters, the expression of several Rho proteins, including RhoB, is increased (42Utech M. Hobbel G. Rust S. Reinecke H. Assmann G. Walter M. Biochem. Biophys. Res. Commun. 2001; 280: 229-236Crossref PubMed Scopus (31) Google Scholar). On this basis, a role for Rho proteins in cholesterol transport has been hypothesized. Our results raise the possibility that the observed increase in Rho proteins expression may be caused in part by the accumulation of cholesterol in cells.The mechanisms by which the mevalonate pathway regulates RhoB appear to be multiple. Recently, mevalonate depletion has been reported to result in an increase in RhoB mRNA (25Holstein S.A. Wohlford-Lenane C.L. Hohl R.J. J. Biol. Chem. 2002; 277: 10678-10682Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). However, the involvement of isoprenoid intermediates or isoprenylated proteins in this effect has not been explored. Here we have shown that inhibition of mevalonate biosynthesis with simvastatin moderately up-regulates RhoB mRNA levels and that this effect is prevented by GGPP. In addition, we have observed that inhibition of Rho signaling by means of bacterial toxins that target and inactivate Rho proteins (43Ridley A.J. Hall A. Cell. 1992; 70: 389-399Abstract Full Text PDF PubMed Scopus (3797) Google Scholar) or by a Rho kinase inhibitor, is also associated with a moderate increase in RhoB protein and mRNA levels. These observations suggest that geranylgeranylated proteins and, in particular, Rho proteins, exert a negative feedback on RhoB mRNA expression in endothelial cells. This interpretation is in accordance with previous studies that identified a negative role of RhoA and B on the activity of the mouse RhoB promoter (29Fritz G. Kaina B. J. Biol. Chem. 1997; 272: 30637-30644Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). Our results indicate that the main effect of simvastatin on RhoB expression takes place at a posttranslational level by reducing RhoB degradation. This could occur through several mechanisms. Simvastatin could affect the activity of proteolytic pathways within the cell. In fact, statins have been reported to modulate the activity of the proteasome (44Rao S. Porter D.C. Chen X. Herliczek T. Lowe M. Keyomarsi K. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 7797-7802Crossref PubMed Scopus (331) Google Scholar, 45Wojcik C. Bury M. Stoklosa T. Giermasz A. Feleszko W. Mlynarczuk I. Pleban E. Basak G. Omura S. Jakobisiak M. Int. J. Biochem. Cell Biol. 2000; 32: 957-965Crossref PubMed Scopus (39) Google Scholar), a protein-degradation pathway proposed to mediate RhoB turnover (26Engel M.E. Datta P.K. Moses H.L. J. Biol. Chem. 1998; 273: 9921-9926Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). However, the effect of simvastatin on RhoB levels and stability was completely prevented by co-treatment with cycloheximide, thus showing that de novo protein synthesis is required for its effect. Moreover, inhibition of protein isoprenylation by using peptidomimetic inhibitors of prenyltransferases reproduced the effect of simvastatin on RhoB levels. These observations suggest that the effect of simvastatin is related to the inhibition of protein isoprenylation rather than to a direct interference with cellular proteases. Because the increase in RhoB protein elicited by simvastatin occurs at the expense of non-prenylated cytosol-partitioned RhoB, it could be hypothesized that the immature cytosolic protein is a poorer substrate for proteases or it does not localize to the appropriate cellular compartment for degradation. This is supported by the fact that the Cys193Ser RhoB mutant, which lacks the isoprenylation site and is cytosolic, is markedly more stable than the wild-type protein. These observations indicate that isoprenylation of RhoB determines the half-life of the protein. Interestingly, carboxyl methylation of the isoprenylated cysteine in RhoA has been reported to reduce its turnover (46Backlund P.S. J. Biol. Chem. 1997; 272: 33175-33180Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar), thus providing another example of the regulation of Rho protein stability as a function of their posttranslational processing.Whether the increased stability of unprenylated RhoB is caused by changes in subcellular distribution, activity, or ability to interact with other cellular proteins will be the subject of further studies. Although inhibition of RhoB isoprenylation may abolish some of RhoB functions, like its cell transforming ability (47Lebowitz P.F. Du W. Prendergast G.C. J. Biol. Chem. 1997; 272: 16093-16095Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar) or the activation of the endothelin-1 promoter (10Hernández-Perera O. Pérez-Sala D. Soria E. Lamas S. Circ. Res. 2000; 87: 616-622Crossref PubMed Scopus (173) Google Scholar), other functions could be conserved, as it has been described for the activation of the serum-response element-dependent transcription in overexpression experiments (47Lebowitz P.F. Du W. Prendergast G.C. J. Biol. Chem. 1997; 272: 16093-16095Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). Future work will address whether unprenylated RhoB may have specific functions or whether it behaves as a dominant-negative RhoB protein.In conclusion, we have found that RhoB mRNA levels and protein stability can be regulated by the availability of isoprenoid intermediates needed for protein modification. These findings unveil the novel role of isoprenylation in the control of RhoB protein degradation and may contribute to the understanding of the complex structure-function relationships of RhoB posttranslational modifications. The results presented in this work show that RhoB levels are modulated by the cholesterol biosynthetic pathway. Down-regulation of this pathway either by means of HMG-CoA reductase inhibitors or excess cholesterol is associated with an increase in the amount of RhoB. This regulation occurs in part at the mRNA level and largely at the protein level, because inhibition of RhoB isoprenylation either by pharmacological or genetic manipulation markedly reduces the degradation of this protein. The cholesterol biosynthetic pathway provides the isoprenoid intermediates needed for the posttranslational modification of G-proteins. Although protein isoprenylation is an irreversible modification, it is susceptible to a certain degree of regulation, because the activity of the prenyltransferases may be controlled by some growth factors and by the availability of isoprenoids. The cholesterol pathway is subjected to a complex feedback regulation (1Goldstein J.L. Brown M.S. Nature. 1990; 343: 425-430Crossref PubMed Scopus (4499) Google Scholar), and it can be modulated by stress (39Shack S. Gorospe M. Fawcett T.W. Hudgins W.R. Holbrook N.J. Oncogene. 1999; 18: 6021-6028Crossref PubMed Scopus (36) Google Scholar). In recent years it has become evident that the activity of this pathway may control the maturation of certain G-proteins such as Ras, thus resulting in the regulation of Ras-mediated cellular processes in response to cholesterol levels or cellular stress (39Shack S. Gorospe M. Fawcett T.W. Hudgins W.R. Holbrook N.J. Oncogene. 1999; 18: 6021-6028Crossref PubMed Scopus (36) Google Scholar, 40Gadbut A.P. Wu L. Tang D. Papageorge A. Watson J.A. Galper J.B. EMBO J. 1997; 16: 7250-7260Crossref PubMed Scopus (33) Google Scholar). In addition, several lines of evidence indicate that the mevalonate pathway may influence the expression of other members of the Ras superfamily, namely Ras1 and Ras2 in yeast (23Dimster-Denk D. Schafer W.R. Rine J. Mol. Biol. Cell. 1995; 6: 59-70Crossref PubMed Scopus (18) Google Scholar), and Rab5 and Rab7 in thyroid cells (24Laezza C. Bucci C. Santillo M. Bruni C.B. Bifulco M. Biochem. Biophys. Res. Commun. 1998; 248: 469-472Crossref PubMed Scopus (19) Google Scholar), through mechanisms not yet completely elucidated. The relationship between G-protein expression and cholesterol metabolism may be bidirectional. In a recent report, cdc42 proteins have been involved in the regulation of cholesterol efflux (41Hirano K.-I. Matsuura F. Tsukamoto K. Zhang Z. Matsuyama A. Takaishi K. Komuro R. Suehiro T. Yamashita S. Takai Y. Matsuzawa Y. FEBS Lett. 2000; 484: 275-279Crossref PubMed Scopus (47) Google Scholar). In addition, in fibroblasts from patients with the inherited disorder of lipid metabolism known as Tangier disease, characterized by cellular accumulation of cholesteryl esters, the expression of several Rho proteins, including RhoB, is increased (42Utech M. Hobbel G. Rust S. Reinecke H. Assmann G. Walter M. Biochem. Biophys. Res. Commun. 2001; 280: 229-236Crossref PubMed Scopus (31) Google Scholar). On this basis, a role for Rho proteins in cholesterol transport has been hypothesized. Our results raise the possibility that the observed increase in Rho proteins expression may be caused in part by the accumulation of cholesterol in cells. The mechanisms by which the mevalonate pathway regulates RhoB appear to be multiple. Recently, mevalonate depletion has been reported to result in an increase in RhoB mRNA (25Holstein S.A. Wohlford-Lenane C.L. Hohl R.J. J. Biol. Chem. 2002; 277: 10678-10682Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). However, the involvement of isoprenoid intermediates or isoprenylated proteins in this effect has not been explored. Here we have shown that inhibition of mevalonate biosynthesis with simvastatin moderately up-regulates RhoB mRNA levels and that this effect is prevented by GGPP. In addition, we have observed that inhibition of Rho signaling by means of bacterial toxins that target and inactivate Rho proteins (43Ridley A.J. Hall A. Cell. 1992; 70: 389-399Abstract Full Text PDF PubMed Scopus (3797) Google Scholar) or by a Rho kinase inhibitor, is also associated with a moderate increase in RhoB protein and mRNA levels. These observations suggest that geranylgeranylated proteins and, in particular, Rho proteins, exert a negative feedback on RhoB mRNA expression in endothelial cells. This interpretation is in accordance with previous studies that identified a negative role of RhoA and B on the activity of the mouse RhoB promoter (29Fritz G. Kaina B. J. Biol. Chem. 1997; 272: 30637-30644Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). Our results indicate that the main effect of simvastatin on RhoB expression takes place at a posttranslational level by reducing RhoB degradation. This could occur through several mechanisms. Simvastatin could affect the activity of proteolytic pathways within the cell. In fact, statins have been reported to modulate the activity of the proteasome (44Rao S. Porter D.C. Chen X. Herliczek T. Lowe M. Keyomarsi K. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 7797-7802Crossref PubMed Scopus (331) Google Scholar, 45Wojcik C. Bury M. Stoklosa T. Giermasz A. Feleszko W. Mlynarczuk I. Pleban E. Basak G. Omura S. Jakobisiak M. Int. J. Biochem. Cell Biol. 2000; 32: 957-965Crossref PubMed Scopus (39) Google Scholar), a protein-degradation pathway proposed to mediate RhoB turnover (26Engel M.E. Datta P.K. Moses H.L. J. Biol. Chem. 1998; 273: 9921-9926Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). However, the effect of simvastatin on RhoB levels and stability was completely prevented by co-treatment with cycloheximide, thus showing that de novo protein synthesis is required for its effect. Moreover, inhibition of protein isoprenylation by using peptidomimetic inhibitors of prenyltransferases reproduced the effect of simvastatin on RhoB levels. These observations suggest that the effect of simvastatin is related to the inhibition of protein isoprenylation rather than to a direct interference with cellular proteases. Because the increase in RhoB protein elicited by simvastatin occurs at the expense of non-prenylated cytosol-partitioned RhoB, it could be hypothesized that the immature cytosolic protein is a poorer substrate for proteases or it does not localize to the appropriate cellular compartment for degradation. This is supported by the fact that the Cys193Ser RhoB mutant, which lacks the isoprenylation site and is cytosolic, is markedly more stable than the wild-type protein. These observations indicate that isoprenylation of RhoB determines the half-life of the protein. Interestingly, carboxyl methylation of the isoprenylated cysteine in RhoA has been reported to reduce its turnover (46Backlund P.S. J. Biol. Chem. 1997; 272: 33175-33180Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar), thus providing another example of the regulation of Rho protein stability as a function of their posttranslational processing. Whether the increased stability of unprenylated RhoB is caused by changes in subcellular distribution, activity, or ability to interact with other cellular proteins will be the subject of further studies. Although inhibition of RhoB isoprenylation may abolish some of RhoB functions, like its cell transforming ability (47Lebowitz P.F. Du W. Prendergast G.C. J. Biol. Chem. 1997; 272: 16093-16095Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar) or the activation of the endothelin-1 promoter (10Hernández-Perera O. Pérez-Sala D. Soria E. Lamas S. Circ. Res. 2000; 87: 616-622Crossref PubMed Scopus (173) Google Scholar), other functions could be conserved, as it has been described for the activation of the serum-response element-dependent transcription in overexpression experiments (47Lebowitz P.F. Du W. Prendergast G.C. J. Biol. Chem. 1997; 272: 16093-16095Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). Future work will address whether unprenylated RhoB may have specific functions or whether it behaves as a dominant-negative RhoB protein. In conclusion, we have found that RhoB mRNA levels and protein stability can be regulated by the availability of isoprenoid intermediates needed for protein modification. These findings unveil the novel role of isoprenylation in the control of RhoB protein degradation and may contribute to the understanding of the complex structure-function relationships of RhoB posttranslational modifications. We thank Dr. S. Lamas for helpful comments and discussion, Dr. M. Dı́az-Cazorla for culture of human umbilical vein endothelial cells and E. Soria and M. J. Carrasco for technical assistance.