Rho Family GTPase Modification and Dependence on CAAX Motif-signaled Posttranslational Modification (original) (raw)

2008, Journal of Biological Chemistry

Rho GTPases (20 human members) comprise a major branch of the Ras superfamily of small GTPases, and aberrant Rho GTPase function has been implicated in oncogenesis and other human diseases. Although many of our current concepts of Rho GTPases are based on the three classical members (RhoA, Rac1, and Cdc42), recent studies have revealed the diversity of biological functions mediated by other family members. A key basis for the functional diversity of Rho GTPases is their association with distinct subcellular compartments, which is dictated in part by three posttranslational modifications signaled by their carboxyl-terminal CAAX (where C represents cysteine, A is an aliphatic amino acid, and X is a terminal amino acid) tetrapeptide motifs. CAAX motifs are substrates for the prenyltransferase-catalyzed addition of either farnesyl or geranylgeranyl isoprenoid lipids, Rce1-catalyzed endoproteolytic cleavage of the AAX amino acids, and Icmt-catalyzed carboxyl methylation of the isoprenylcysteine. We utilized pharmacologic, biochemical, and genetic approaches to determine the sequence requirements and roles of CAAX signal modifications in dictating the subcellular locations and functions of the Rho GTPase family. Although the classical Rho GTPases are modified by geranylgeranylation, we found that a majority of the other Rho GTPases are substrates for farnesyltransferase. We found that the membrane association and/or function of Rho GTPases are differentially dependent on Rce1-and Icmt-mediated modifications. Our results further delineate the sequence requirements for prenyltransferase specificity and functional roles for protein prenylation in Rho GTPase function. We conclude that a majority of Rho GTPases are targets for pharmacologic inhibitors of farnesyltransferase, Rce1, and Icmt. Rho proteins are members of the Ras superfamily of small GTPases and function as GDP/GTP-regulated switches (1, 2). Much of our current understanding of the biochemistry and biology of the Rho family has come from the extensive evaluation of three classical members, RhoA, Rac1, and Cdc42 (3). Similar to Ras, Rho GDP/GTP cycling is regulated by guanine nucleotide exchange factors that promote the formation of the active GTP-bound form (4) and GTPase-activating proteins that catalyze the intrinsic GTPase activity and promote the formation of inactive GDP-bound Rho (5). Active, GTP-bound Rho GTPases bind preferentially to downstream effectors, stimulating diverse cytoplasmic signaling cascades that control actin reorganization and regulate cell shape, polarity, motility, adhesion, and membrane trafficking (6). As such, it is thought that activated Rho proteins contribute to cancer progression by influencing the ability of cells to migrate and thus to invade and metastasize. In addition to these alterations in cellular function, aberrant activation of Rho proteins has also been shown to contribute to other cancer phenotypes by promoting cell growth, proliferation, survival, and angiogenesis (7). Therefore, defining pharmacologic approaches for inhibition of Rho GTPase function represents an important direction for target-based anti-cancer drug discovery. Similar to Ras, the majority of Rho family GTPases are known or anticipated to undergo a series of posttranslational modifications that promote proper subcellular localization to the plasma membrane and/or endomembranes, which is required for biological activity. This series of modifications is initiated by the recognition of a carboxyl-terminal CAAX tetrapeptide motif (where C represents cysteine, A is an aliphatic amino acid, and X is any amino acid), which is found on 16 of 20 Rho GTPases (Table 1; canonical CAAX motifs are not present in the Wrch-1, Chp/Wrch-2, RhoBTB1, or RhoBTB2). The first step, mediated by farnesyltransferase (FTase) 2 and/or geranylgeranyltransferase type I (GGTase-I), results in the covalent addition of a farnesyl or geranylgeranyl isoprenoid lipid, respectively, to the cysteine residue of the CAAX sequence. Next, the-AAX peptide is cleaved from the carboxyl terminus by the Rce1 (Ras-converting enzyme 1) endoprotease. Finally, isoprenylcysteine-O-carboxyl methyltransferase (Icmt) catalyzes the addition of a methyl group to the prenylated cysteine residue (8). Together, these modifications increase protein hydrophobicity and facilitate membrane association. Where studied, mutation * This work was supported, in whole or in part, by National Institutes of Health Grants CA063071, CA67771, and CA92240 (to C. J. D.) and CA063071, CA67771, and CA109550 (to A. D. C.