RhoA Prenylation Is Required for Promotion of Cell Growth and Transformation and Cytoskeleton Organization but Not for Induction of Serum Response Element Transcription

Abstract

The importance of post-translational geranylgeranylation of the GTPase RhoA for its ability to induce cellular proliferation and malignant transformation is not well understood. In this manuscript we demonstrate that geranylgeranylation is required for the proper cellular localization of V14RhoA and for its ability to induce actin stress fiber and focal adhesion formation. Furthermore, V14RhoA geranylgeranylation was also required for suppressing p21 WAF transcription, promoting cell cycle progression and cellular proliferation. The ability of V14RhoA to induce focus formation and enhance plating efficiency and oncogenic Ras anchoragedependent growth was also dependent on its geranylgeranylation. The only biological activity of V14RhoA that was not dependent on its prenylation was its ability to induce serum response element transcriptional activity. Furthermore, we demonstrate that a farnesylated form of V14RhoA was also able to bind RhoGDI-1, was able to induce cytoskeleton organization, proliferation, and transformation, and was just as potent as geranylgeranylated V14RhoA at suppressing p21 WAF transcriptional activity. These results demonstrate that RhoA geranylgeranylation is required for its biological activity and that the nature of the lipid modification is not critical.Small G proteins of the Ras superfamily are regulatory proteins whose activity is controlled by a GDP/GTP cycle. Several members of the Ras superfamily are regulators of signaling pathways that control cell growth, differentiation, and oncogenic transformation as well as actin cytoskeletal organization (1). The Rho protein branch of this superfamily includes at least eight distinct Rho families (RhoA, B, C, D, and G, Rac1 and 2, TC10, Cdc42, and Rnd1, 2, and 3) (2) that are regulated by Rho-GTPase activating proteins and a large family of guanine nucleotide exchange factors of the Dbl family proteins. Moreover Rho guanine nucleotide dissociation inhibitors (RhoGDIs) 1 stabilize the inactive GDP-bound form of the Rho proteins. Rho proteins notably regulate signal transduction from cell surface receptors to intracellular molecules and are involved in a variety of cellular processes including cell morphology (3), motility (4), cytokinesis (5, 6), cell proliferation (7, 8), and tumor progression (9 -11).Ras and Rho proteins are post-translationally modified by the isoprenoid lipids, farnesyl, and geranylgeranyl (12). Two prenyltransferases, farnesyltransferase (FTase) and geranylgeranyltransferase I (GGTase I), catalyze the covalent attachment of the farnesyl and geranylgeranyl groups, respectively, to the carboxyl-terminal cysteine of proteins ending in a CAAX motif (C is a cysteine, A usually aliphatic amino acid, and X any amino acid). FTase prefers CAAX sequences where X is a serine, methionine, cysteine, alanine, or glutamine, as in Ras or in nuclear lamins (13-15). When X is a leucine or isoleucine the protein, as in the Rho/Rac family of proteins, is geranylgeranylated by GGTase I (16, 17). Protein prenylation is important in target...

High Affinity for Farnesyltransferase and Alternative Prenylation Contribute Individually to K-Ras4B Resistance to Farnesyltransferase Inhibitors

“…We observed no significant differences at the mRNA level between the set of genes expressed in either cell line (data not shown), suggesting that signaling by K-Ras is independent of the identity of the prenyl group. This is consistent with reports in which RhoA signaling was shown to be independent of the identity of the prenyl group (41,42). We conclude that K-Ras-F and K-Ras-GG are functionally equivalent and that the effect of FTIs on K-Ras-transformed cells probably results from inhibition of other farnesylated proteins.…”

Effects of lovastatin on Rho isoform expression, activity, and association with guanine nucleotide dissociation inhibitors

“…This allows separating extracts into an aqueous and detergent phase, and previous work showed that unprenylated Ras and Rho proteins partition into the aqueous phase, while prenylated proteins partition into the detergent phase [7,20]. In the absence of lovastatin, RhoA was largely in the detergent phase (Fig.…”

“…Prenylation of Rho proteins is essential for proper membrane localization, and appears to be required for most, but not all, known Rho functions [5][6][7][8]. HMG-CoA reductase (EC1.1.1.88) inhibitors (statins) reduce the synthesis of mevalonate, the precursor of cholesterol, and of farnesyl and geranyl-geranyl isoprenoids [9].…”

Preclinical and Clinical Evidence that Deoxy-2-[18F]fluoro-D-glucose Positron Emission Tomography with Computed Tomography Is a Reliable Tool for the Detection of Early Molecular Responses to Erlotinib in Head and Neck Cancer

“…Immunofluorescence histology was done as described elsewhere (9). Cells were seeded on glass coverslips in six-well plates and were treated with either vehicle or erlotinib at 5 μmol/L for 48 hours.…”