Isoprenoid Pyrophosphate Analogues Regulate Expression of Ras-Related Proteins ² Sarah A. Holstein, ‡ Christine L. Wohlford-Lenane, § David F. Wiemer, | and Raymond J. Hohl* ,‡,§ Departments of Pharmacology, Internal Medicine, and Chemistry, UniVersity of Iowa, Iowa City, Iowa 52242 ReceiVed NoVember 25, 2002; ReVised Manuscript ReceiVed February 20, 2003 ABSTRACT: The isoprenoids farnesyl pyrophosphate (FPP) and geranylgeranyl pyrophosphate (GGPP) are synthetic precursors for numerous molecules essential for cellular function as well as substrates in isoprenylation reactions. We have previously demonstrated that depletion of mevalonate results in the upregulation of Ras-related proteins which can be prevented by FPP or GGPP, independent of restoration of protein isoprenylation. To better define the regulatory properties of isoprenoid pyrophosphates, we have investigated the abilities of isoprenoid analogues to regulate the expression of the Ras-related proteins. Farnesyl phosphonic acids potentiate the upregulation of these proteins induced by mevalonate depletion independent of inhibitory activity against farnesyl protein transferase, geranylgeranyl protein transferase I, FPP synthase, or GGPP synthase. The potentiation of RhoB upregulation is at both the mRNA and protein level. The ability of these analogues to serve as functional antagonists of the isoprenoid pyrophosphates is dependent on the nature of the functional group at the head of the molecule, the charge of the molecule, and the length of the isoprenoid chain. Metabolites and additional analogues of isoprenoid pyrophosphates were found to possess agonist properties relative to FPP and GGPP. Interestingly, the structurally related retinoids all-trans-retinoic acid and 9-cis-retinoic acid also display slight agonist properties. These studies provide evidence for direct roles of FPP and GGPP in regulating transcriptional and post-transcriptional events. Ras, Rap1a, RhoA, and RhoB belong to the Ras super- family of small GTPases. These proteins play important roles in regulating diverse cellular functions, including cell survival, proliferation, differentiation, and cytoskeletal or- ganization (1, 2). Out of necessity, these roles require protein expression, proper intracellular localization, and interaction with downstream signal transducing elements. For example, central to the function of these proteins is their association with cellular membranes which is achieved, in part, by isoprenylation (3, 4). Isoprenylation involves the addition of either a 15-carbon farnesyl chain or a 20-carbon gera- nylgeranyl chain to a cysteine sulfhydryl group near the carboxy terminus. The lipid donors in these reactions, FPP 1 and GGPP, are derived from mevalonate and are intermedi- ates in the isoprenoid biosynthetic pathway. Critically important, but less well characterized, are the factors that regulate the expression of these small GTPases. We have previously reported that mevalonate depletion, due to HMG-CoA reductase inhibition, results in the upregulation of Ras, Rap1a, RhoA, and RhoB (5). This upregulation is mediated transcriptionally for RhoB and translationally/post- translationally for Ras, RhoA, and Rap1a (5). Analysis of this upregulation has revealed that it is a consequence of depletion of specific isoprenoid species rather than a diminu- tion of protein isoprenylation (6). Specifically, FPP prevents the upregulation of Ras and RhoB induced by mevalonate depletion, while GGPP prevents the upregulation of RhoA, RhoB, and Rap1a (6). Thus far, other isoprenoids have not been evaluated as regulators of the transcription and transla- tion of these small GTPases. There is precedence in the literature for the regulatory roles of farnesyl derivatives. The degradation of HMG-CoA reductase is regulated by farnesol (7-9). The farnesoid X receptor (FXR), a member of the nuclear receptor super- family, has been shown to be activated by farnesol and juvenile hormone III (10). The mechanisms underlying the isoprenoid pyrophosphate-mediated regulation of the small GTPases are unknown. On the basis of our prior results (5, 6), we reasoned that since FPP itself demonstrates biochemi- cal activities, the use of FPP analogues would allow definition of the structural basis for these activities. We found that the structure-function relationship depends on charge, isoprene chain length, and differences in functional groups. Furthermore, comparisons to members of the retinoid family revealed interesting similarities to select isoprenoid ana- logues. EXPERIMENTAL PROCEDURES Cell Cultures and Reagents. The K562 cell line was purchased from the American Type Culture Collection ² This project was supported by the Leukemia and Lymphoma Society in the form of a Translational Research Grant, the Roy J. Carver Charitable Trust, and the Roland W. Holden Family Program for Experimental Cancer Therapeutics. * To whom correspondence should be addressed: Department of Internal Medicine, C32 GH, University of Iowa, Iowa City, IA 52242. Telephone: (319) 356-8110. Fax: (319) 353-8383. E-mail: raymond- hohl@uiowa.edu. ‡ Department of Pharmacology. § Department of Internal Medicine. | Department of Chemistry. 1 Abbreviations: HMG-CoA, hydroxymethylglutaryl coenzyme A; PBS, phosphate-buffered saline; SDS, sodium dodecyl sulfate; PMSF, phenylmethanesulfonyl fluoride; FPTase, farnesyl protein transferase; GGPTase, geranylgeranyltransferase; IPP, isopentenyl pyrophosphate; GPP, geranyl pyrophosphate; FPP, farnesyl pyrophosphate; GGPP, geranylgeranyl pyrophosphate; cmpd, compound. 4384 Biochemistry 2003, 42, 4384-4391 10.1021/bi027227m CCC: $25.00 © 2003 American Chemical Society Published on Web 03/29/2003