Rab GTPases containing a CAAX motif are processed post-geranylgeranylation by proteolysis and methylation

Post-translational modification by protein prenylation is required for membrane targeting and biological function of monomeric GTPases. Ras and Rho proteins possess a C-terminal CAAX motif (C is cysteine, A is usually an aliphatic residue, and X is any amino acid), in which the cysteine is prenylated, followed by proteolytic cleavage of the AAX peptide and carboxyl methylation by the Rce1 CAAX protease and Icmt methyltransferase, respectively. Rab GTPases usually undergo double geranylgeranylation within CC or CXC motifs. However, very little is known about processing and membrane targeting of Rabs that naturally contain a CAAX motif. We show here that a variety of Rab-CAAX proteins undergo carboxyl methylation, both in vitro and in vivo, with one exception. Rab38(CAKS) is not methylated in vivo, presumably because of the inhibitory action of the lysine residue within the AAX motif for cleavage by Rce1. Unlike farnesylated Ras proteins, we observed no targeting defects of overexpressed Rab-CAAX proteins in cells deficient in Rce1 or Icmt, as reported for geranylgeranylated Rho proteins. However, endogenous geranylgeranylated non-methylated Rab-CAAX and Rab-CXC proteins were significantly redistributed to the cytosol at steady-state levels and redistribution correlates with higher affinity of RabGDI for non-methylated Rabs in Icmt-deficient cells. Our data suggest a role for methylation in Rab function by regulating the cycle of Rab membrane recruitment and retrieval. Our findings also imply that those Rabs that undergo post-prenylation processing follow an indirect targeting pathway requiring initial endoplasmic reticulum membrane association prior to specific organelle targeting.     

Rho Family GTPase modification and dependence on CAAX motif-signaled posttranslational modification

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.     

Isoprenylcysteine carboxyl methyltransferase deficiency in mice

After isoprenylation, Ras and other CAAX proteins undergo endoproteolytic processing by Rce1 and methylation of the isoprenylcysteine by Icmt (isoprenylcysteine carboxyl methyltransferase). We reported previously that Rce1-deficient mice died during late gestation or soon after birth. We hypothesized that Icmt deficiency might cause a milder phenotype, in part because of reports suggesting the existence of more than one activity for methylating isoprenylated proteins. To address this hypothesis and also to address the issue of other methyltransferase activities, we generated Icmt-deficient mice. Contrary to our expectation, Icmt deficiency caused a more severe phenotype than Rce1 deficiency, with virtually all of the knockout embryos (Icmt-/-) dying by mid-gestation. An analysis of chimeric mice produced from Icmt-/- embryonic stem cells showed that the Icmt-/- cells retained the capacity to contribute to some tissues (e.g. skeletal muscle) but not to others (e.g. brain). Lysates from Icmt-/- embryos lacked the ability to methylate either recombinant K-Ras or small molecule substrates (e.g. N-acetyl-S-geranylgeranyl-l-cysteine). In addition, Icmt-/- cells lacked the ability to methylate Rab proteins. Thus, Icmt appears to be the only enzyme participating in the carboxyl methylation of isoprenylated proteins.     

Absence of the CAAX Endoprotease Rce1: Effects on Cell Growth and Transformation

After isoprenylation, the Ras proteins and other CAAX proteins undergo two additional enzymatic modifications-endoproteolytic release of the last three amino acids of the protein by the protease Rce1 and methylation of the carboxyl-terminal isoprenylcysteine by the methyltransferase Icmt. This postisoprenylation processing is thought to be important for the association of Ras proteins with membranes. Blocking postisoprenylation processing, by inhibiting Rce1, has been suggested as a potential approach for retarding cell growth and blocking cellular transformation. The objective of this study was to develop a cell culture system for addressing these issues. We generated mice with a conditional Rce1 allele (Rce1(flox)) and produced Rce1(flox/flox) fibroblasts. Cre-mediated excision of Rce1 (thereby producing Rce1(Delta/Delta) fibroblasts) eliminated Ras endoproteolytic processing and methylation and caused a partial mislocalization of truncated K-Ras and H-Ras fusion proteins within cells. Rce1(Delta/Delta) fibroblasts grew more slowly than Rce1(flox/flox) fibroblasts. The excision of Rce1 also reduced Ras-induced transformation, as judged by the growth of colonies in soft agar. The excision of Rce1 from a Rce1(flox/flox) skin carcinoma cell line also significantly retarded the growth of cells, and this effect was exaggerated by cotreatment of the cells with a farnesyltransferase inhibitor. These studies support the idea that interference with postisoprenylation processing retards cell growth, limits Ras-induced transformation, and sensitizes tumor cells to a farnesyltransferase inhibitor.     

Disruption of the mouse Rce1 gene results in defective Ras processing and mislocalization of Ras within cells

Little is known about the enzyme(s) required for the endoproteolytic processing of mammalian Ras proteins. We identified a mouse gene (designated Rce1) that shares sequence homology with a yeast gene (RCE1) implicated in the proteolytic processing of Ras2p. To define the role of Rce1 in mammalian Ras processing, we generated and analyzed Rce1-deficient mice. Rce1 deficiency was lethal late in embryonic development (after embryonic day 15.5). Multiple lines of evidence revealed that Rce1-deficient embryos and cells lacked the ability to endoproteolytically process Ras proteins. First, Ras proteins from Rce1-deficient cells migrated more slowly on SDS-polyacrylamide gels than Ras proteins from wild-type embryos and fibroblasts. Second, metabolic labeling of Rce1-deficient cells revealed that the Ras proteins were not carboxymethylated. Finally, membranes from Rce1-deficient fibroblasts lacked the capacity to proteolytically process farnesylated Ha-Ras, N-Ras, and Ki-Ras or geranylgeranylated Ki-Ras. The processing of two other prenylated proteins, the farnesylated Ggamma1 subunit of transducin and geranylgeranylated Rap1B, was also blocked. The absence of endoproteolytic processing and carboxymethylation caused Ras proteins to be mislocalized within cells. These studies indicate that Rce1 is responsible for the endoproteolytic processing of the Ras proteins in mammals and suggest a broad role for this gene in processing other prenylated CAAX proteins.     

Differential membrane localization of ERas and Rheb, two Ras-related proteins involved in the phosphatidylinositol 3-kinase/mTOR pathway

Two Ras-related proteins, ERas and Rheb, which are involved in the phosphatidylinositol 3-kinase pathway, display high GTP affinity and have atypical CAAX motifs. The factors governing the intracellular localization of ERas and Rheb are incompletely understood. In the current study, we show by confocal microscopy that ERas is localized to the plasma membrane, whereas Rheb is confined to the endomembranes. Membrane localization of the two proteins was abolished by mutation of the cysteine of the CAAX motif. Membrane targeting was also abolished by a farnesyltransferase inhibitor but not by a geranylgeranyltransferase inhibitor. In mouse fibroblasts deficient in either Rce1 (Ras converting enzyme 1) or Icmt (isoprenylcysteine carboxyl methyltransferase), ERas was mislocalized mainly to the Golgi apparatus, whereas Rheb showed diffuse localization. Mutation of cysteines in the hypervariable region of ERas prevented the plasma membrane localization of ERas, very strongly suggesting that palmitoylation of the cysteines is essential for membrane targeting. The hypervariable region of Rheb does not contain cysteines or polybasic residues, and when it was replaced with the hypervariable region of H-Ras, Rheb displayed plasma membrane localization. These data indicate that ERas shares the same posttranslational modifications with H-Ras and N-Ras and is localized at the plasma membrane. Rheb also shares the same membrane-targeting pathway but because of the absence of palmitoylation is located on endomembranes.     

Targeted inactivation of the isoprenylcysteine carboxyl methyltransferase gene causes mislocalization of K-Ras in mammalian cells

After isoprenylation and endoproteolytic processing, the Ras proteins are methylated at the carboxyl-terminal isoprenylcysteine. The importance of isoprenylation for targeting of Ras proteins to the plasma membrane is well established, but the importance of carboxyl methylation, which is carried out by isoprenylcysteine carboxyl methyltransferase (Icmt), is less certain. We used gene targeting to produce homozygous Icmt knockout embryonic stem cells (Icmt-/-). Lysates from Icmt-/- cells lacked the ability to methylate farnesyl-K-Ras4B or small-molecule Icmt substrates such as N-acetyl-S-geranylgeranyl-L-cysteine. To assess the impact of absent Icmt activity on the localization of K-Ras within cells, wild-type and Icmt-/- cells were transfected with a green fluorescent protein (GFP)-K-Ras fusion construct. As expected, virtually all of the GFP-K-Ras fusion in wild-type cells was localized along the plasma membrane. In contrast, a large fraction of the fusion in Icmt-/- cells was trapped within the cytoplasm, and fluorescence at the plasma membrane was reduced. Also, cell fractionation/Western blot studies revealed that a smaller fraction of the K-Ras in Icmt-/- cells was associated with the membranes. We conclude that carboxyl methylation of the isoprenylcysteine is important for proper K-Ras localization in mammalian cells.     

AtFACE-2, a functional prenylated protein protease from Arabidopsis thaliana related to mammalian Ras-converting enzymes

Eukaryotic proteins containing a CAAX (A is aliphatic amino acid) C-terminal tetrapeptide sequence generally undergo a lipid modification, the addition of a prenyl group. Proteins that are modified by prenylation, such as Ras GTPases, can be subsequently modified by a proteolytic event that removes a C-terminal tripeptide (AAX). Two distinct proteases have been identified that are involved in the CAAX proteolytic step, FACE-1/Ste24 and FACE-2/Rce1. These proteases have different enzymatic properties, substrate specificities, and biological functions. However, a proposal has been made that plants lack a FACE-2/Rce1-type protease. Here, we describe the isolation of a cDNA from Arabidopsis thaliana that encodes a 311-aa protein with characteristics that are similar to the FACE-2/Rce1 group of enzymes. Northern blot analysis demonstrates widespread expression of this gene in plant tissues. Heterologous expression of the A. thaliana cDNA in yeast restores CAAX proteolytic activity to yeast lacking native CAAX proteases. The recombinant protein produced in this system displays an in vivo substrate specificity profile distinct from AtSte24 and cleaves a farnesylated CAAX tetrapeptide in vitro. These results provide evidence for the existence of a previously unsuspected plant FACE-2/Rce1 ortholog and support the evolutionary conservation of dual CAAX proteolytic systems in eukaryotes.     

Membrane trafficking of heterotrimeric G proteins via the endoplasmic reticulum and Golgi

Membrane targeting of G-protein alphabetagamma heterotrimers was investigated in live cells by use of Galpha and Ggamma subunits tagged with spectral mutants of green fluorescent protein. Unlike Ras proteins, Gbetagamma contains a single targeting signal, the CAAX motif, which directed the dimer to the endoplasmic reticulum. Endomembrane localization of farnesylated Ggamma(1), but not geranylgeranylated Ggamma(2), required carboxyl methylation. Targeting of the heterotrimer to the plasma membrane (PM) required coexpression of all three subunits, combining the CAAX motif of Ggamma with the fatty acyl modifications of Galpha. Galpha associated with Gbetagamma on the Golgi and palmitoylation of Galpha was required for translocation of the heterotrimer to the PM. Thus, two separate signals, analogous to the dual-signal targeting mechanism of Ras proteins, cooperate to target heterotrimeric G proteins to the PM via the endomembrane.     

The C-terminal polylysine region and methylation of K-Ras are critical for the interaction between K-Ras and microtubules

After synthesis in the cytosol, Ras proteins must be targeted to the inner leaflet of the plasma membrane for biological activity. This targeting requires a series of C-terminal posttranslational modifications initiated by the addition of an isoprenoid lipid in a process termed prenylation. A search for factors involved in the intracellular trafficking of Ras has identified a specific and prenylation-dependent interaction between tubulin/microtubules and K-Ras. In this study, we examined the structural requirements for this interaction between K-Ras and microtubules. By using a series of chimeras in which regions of the C terminus of K-Ras were replaced with those of Ha-Ras and vice versa, we found that the polylysine region of K-Ras located immediately upstream of the prenylation site is required for binding of K-Ras to microtubules. Studies in intact cells confirmed the importance of the K-Ras polylysine region for microtubule binding, as deletion or replacement of this region resulted in loss of paclitaxel-induced mislocalization of a fluorescent K-Ras fusion protein. The additional modifications in the prenyl protein processing pathway also affected the interaction of K-Ras with microtubules. Removal of the three C-terminal amino acids of farnesylated K-Ras with the specific endoprotease Rce1p abolished its binding to microtubules. Interestingly, however, methylation of the C-terminal prenylcysteine restored binding. Consistent with these results, localization of the fluorescent K-Ras fusion protein remained paclitaxel-sensitive in cells lacking Rce1, whereas no paclitaxel effect was observed in cells lacking the methyltransferase. These studies show that the polylysine region of K-Ras is critical for its interaction with microtubules and provide the first evidence for a functional consequence of Ras C-terminal proteolysis and methylation.     

8-Hydroxyquinoline-based inhibitors of the Rce1 protease disrupt Ras membrane localization in human cells

Ras converting enzyme 1 (Rce1) is an endoprotease that catalyzes processing of the C-terminus of Ras protein by removing -aaX from the CaaX motif. The activity of Rce1 is crucial for proper localization of Ras to the plasma membrane where it functions. Ras is responsible for transmitting signals related to cell proliferation, cell cycle progression, and apoptosis. The disregulation of these pathways due to constitutively active oncogenic Ras can ultimately lead to cancer. Ras, its effectors and regulators, and the enzymes that are involved in its maturation process are all targets for anti-cancer therapeutics. Key enzymes required for Ras maturation and localization are the farnesyltransferase (FTase), Rce1, and isoprenylcysteine carboxyl methyltransferase (ICMT). Among these proteins, the physiological role of Rce1 in regulating Ras and other CaaX proteins has not been fully explored. Small-molecule inhibitors of Rce1 could be useful as chemical biology tools to understand further the downstream impact of Rce1 on Ras function and serve as potential leads for cancer therapeutics. Structure–activity relationship (SAR) analysis of a previously reported Rce1 inhibitor, NSC1011, has been performed to generate a new library of Rce1 inhibitors. The new inhibitors caused a reduction in Rce1 in vitro activity, exhibited low cell toxicity, and induced mislocalization of EGFP-Ras from the plasma membrane in human colon carcinoma cells giving rise to a phenotype similar to that observed with siRNA knockdowns of Rce1 expression. Several of the new inhibitors were more effective at mislocalizing K-Ras compared to a potent farnesyltransferase inhibitor (FTI), which is significant because of the preponderance of K-Ras mutations in cancer.     

The polybasic region of Rho GTPases defines the cleavage by Yersinia enterocolitica outer protein T (YopT)

Pathogenic Yersinia strains evade the innate immune responses of the host by producing effector proteins ( Yersinia outer proteins [Yops]), which are directly injected into mammalian cells by a type III secretion system (TTSS). One of these effector proteins (YopT) disrupts the actin cytoskeleton of the host cell resulting in cell rounding. YopT is a cysteine protease that cleaves Rho proteins directly upstream of the post-translationally modified cysteine. Thereby, it releases the GTPases from the membrane leading to inactivation. Small GTPases are modified by isoprenylation of the cysteine of the CAAX box, cleavage of the -AAX tripeptide, and methylation of the cysteine. We have shown that isoprenylation and the endoproteolytic cleavage of the tripeptide of Rho GTPases are essential for YopT-induced cleavage, whereas carboxyl methylation is not required. In the present study, we post-translationally modified RhoA, Rac, Cdc42, and several mutants in vitro and characterized the YopT-induced cleavage with recombinant YopT. We show that farnesylated RhoA is a preferred substrate of YopT compared with the geranylgeranylated GTPase. Geranylgeranylated RhoA, however, is the preferred substrate for YopT-catalyzed cleavage with a threefold faster turnover rate over Rac and Cdc42. Moreover, our data indicate that the composition of the polybasic region of the GTPases defines the specificity and efficiency of the YopT-induced cleavage, and that a space between the polybasic stretch of amino acids at the C terminus and the CAAX box enhances the turnover rate of YopT-catalyzed cleavage.     

Thematic review series: lipid posttranslational modifications. CAAX modification and membrane targeting of Ras

Proteins that terminate with a consensus sequence known as CAAX undergo a series of posttranslational modifications that include polyisoprenylation, endoproteolysis, and carboxyl methylation. These modifications render otherwise hydrophilic proteins hydrophobic at their C termini such that they associate with membranes. Whereas prenylation occurs in the cytosol, postprenylation processing is accomplished on the cytoplasmic surface of the endoplasmic reticulum and Golgi apparatus. Among the numerous CAAX proteins encoded in mammalian genomes are many signaling molecules such as monomeric GTPases, including the Ras proteins that play an important role in cancer. In the course of their processing, nascent Ras proteins traffic from their site of synthesis in the cytosol to the endomembrane and then out to the plasma membrane (PM) by at least two pathways. Recently, retrograde pathways have been discovered that deliver mature Ras from the PM back to the Golgi. The Golgi has been identified as a platform upon which Ras can signal. Thus, the subcellular trafficking of Ras proteins has the potential to increase the complexity of Ras signaling by adding a spatial dimension. The complexity of Ras trafficking also affords a wider array of potential targets for the discovery of drugs that might inhibit tumors by interfering with Ras trafficking.     

On the physiological importance of endoproteolysis of CAAX proteins: heart-specific RCE1 knockout mice develop a lethal cardiomyopathy

Proteins terminating with a CAAX motif, such as the Ras proteins and the nuclear lamins, undergo post-translational modification of a C-terminal cysteine with an isoprenyl lipid via a process called protein prenylation. After prenylation, the last three residues of CAAX proteins are clipped off by Rce1, an integral membrane endoprotease of the endoplasmic reticulum. Prenylation is crucial to the function of many CAAX proteins, but the physiologic significance of endoproteolytic processing has remained obscure. To address this issue, we used Cre/loxP recombination techniques to create mice lacking Rce1 in the heart, an organ where Rce1 is expressed at particularly high levels. The hearts from heart-specific Rce1 knockout mice manifested reduced levels of both the Rce1 mRNA and CAAX endoprotease activity, and the hearts manifested an accumulation of CAAX protein substrates. The heart-specific Rce1 knockout mice initially appeared healthy but died starting at 3-5 months of age. By 10 months of age, approximately 70% of the mice had died. Pathological studies revealed that the heart-specific Rce1 knockout mice had a dilated cardiomyopathy. By contrast, liver-specific Rce1 knockout mice appeared healthy, had normal transaminase levels, and had normal liver histology. These studies indicate that the endoproteolytic processing of CAAX proteins is essential for cardiac function but is less important for the liver.     

The isoprenoid substrate specificity of isoprenylcysteine carboxylmethyltransferase: development of novel inhibitors

Isoprenylcysteine carboxylmethyltransferase (Icmt) is an integral membrane protein localized to the endoplasmic reticulum of eukaryotic cells that catalyzes the post-translational alpha-carboxylmethylesterification of CAAX motif proteins, including the oncoprotein Ras. Prior to methylation, these protein substrates all contain an isoprenylcysteine residue at the C terminus. In this study, we developed a variety of substrates and inhibitors of Icmt that vary in the isoprene moiety in order to gain information about the nature of the lipophilic substrate binding site. These isoprenoid-modified analogs of the minimal Icmt substrate N-acetyl-S-farnesyl-L-cysteine (AFC) were synthesized from newly and previously prepared farnesol analogs. Using both yeast and human Icmt enzymes, these compounds were found to vary widely in their ability to act as substrates, supporting the isoprenoid moiety as a key substrate recognition element for Icmt. Compound 3 is a competitive inhibitor of overexpressed yeast Icmt (K(I) = 17.1 +/- 1.7 microm). Compound 4 shows a mix of competitive and uncompetitive inhibition for both the yeast and the human Icmt proteins (yeast K(IC) = 35.4 +/- 3.4 microm, K(IU) = 614.4 +/- 148 microm; human K(IC) = 119.3 +/- 18.1 microm, K(IU) = 377.2 +/- 42.5 microm). These data further suggest that differences in substrate specificity exist between the human and yeast enzymes. Biological studies suggest that inhibition of Icmt results in Ras mislocalization and loss of cellular transformation ability, making Icmt an attractive and novel anticancer target. Further elaboration of the lead compounds synthesized and assayed here may lead to clinically useful higher potency inhibitors.     

Mechanism of isoprenylcysteine carboxyl methylation from the crystal structure of the integral membrane methyltransferase ICMT

The posttranslational modification of C-terminal CAAX motifs in proteins such as Ras, most Rho GTPases, and G protein γ subunits, plays an essential role in determining their subcellular localization and correct biological function. An integral membrane methyltransferase, isoprenylcysteine carboxyl methyltransferase (ICMT), catalyzes the final step of CAAX processing after prenylation of the cysteine residue and endoproteolysis of the -AAX motif. We have determined the crystal structure of a prokaryotic ICMT ortholog, revealing a markedly different architecture from conventional methyltransferases that utilize S-adenosyl-L-methionine (SAM) as a cofactor. ICMT comprises a core of five transmembrane α helices and a cofactor-binding pocket enclosed within a highly conserved C-terminal catalytic subdomain. A tunnel linking the reactive methyl group of SAM to the inner membrane provides access for the prenyl lipid substrate. This study explains how an integral membrane methyltransferase achieves recognition of both a hydrophilic cofactor and a lipophilic prenyl group attached to a polar protein substrate.     

Role of Isoprenylcysteine Carboxylmethyltransferase-catalyzed Methylation in Rho Function and Migration

A number of proteins that play key roles in biological regulatory events undergo a process of post-translational modifications termed prenylation. The prenylation pathway consists of three enzymatic steps; the final processed protein is isoprenoid-modified and methylated on the C-terminal cysteine. This protein modification pathway plays a significant role in cancer biology because many oncogenic proteins undergo prenylation. Methylation of the C terminus by isoprenylcysteine carboxylmethyltransferase (Icmt) is the final step in the prenylation pathway. Cysmethynil, a specific Icmt inhibitor discovered in our laboratory, is able to inhibit Ras-mediated signaling, cell growth, and oncogenesis. We sought to examine the role of Icmt-mediated methylation on the behaviors of cancer cells associated with metastatic potential. Our results indicate that inhibition of methylation reduces migration of the highly metastatic MDA-MB-231 breast cancer cell line. In addition, cell adhesion and cell spreading are also significantly impacted by cysmethynil. To examine the mechanism of Icmt-dependent migration we focused on RhoA and Rac1, prenylated proteins that are important mediators of cell migration through their control of the actin cytoskeleton. Inhibition of Icmt significantly decreases the activation of both RhoA and Rac1; an increase in Rho GDP-dissociation inhibitor (RhoGDI) binding in the absence of methylation appears to contribute to this effect. Furthermore, in the absence of Icmt activity the addition of exogenous RhoA or Rac1 is able to partially rescue directed and random migration, respectively. These findings establish a role for Icmt-mediated methylation in cell migration and advance our understanding of the biological consequences of Rho methylation.Post-translational modifications of proteins play vital roles in many aspects of cell biology. Hence, identifying and understanding the biological impact of these processes is crucial to furthering our basic understanding of how cells function. Numerous proteins that control important biological regulatory events undergo a complex series of post-translational modifications that are directed by the presence of a so-called CaaX motif at their C terminus. This post-translational pathway, termed protein prenylation, is initiated by the attachment of an isoprenoid lipid to an invariant cysteine residue, the C of the CaaX motif (1, 2). Either a 15-carbon farnesyl or 20-carbon geranylgeranyl isoprenoid is covalently attached to this cysteine by protein farnesyltransferase (FTase)² or protein geranylgeranyltransferase-I (GGTase-I), respectively (3). The prenylation step is followed by cleavage of the three C-terminal amino acids (the -AAX) by an endoplasmic reticulum (ER)-bound protease termed Rce1. Finally, the prenylated cysteine, which is now located at the C terminus, is methylated by isoprenylcysteine carboxylmethyltransferase (Icmt), another integral ER membrane protein (4, 5). The final result of these modifications is a protein that contains a prenylated and methylated cysteine at its C terminus. Numerous studies have demonstrated that this post-translational processing not only facilitates protein association with cellular membranes, but also can play important roles in protein-protein interactions and protein stability (1, 6, 7). Thus, it is clear that CaaX processing is necessary for the biological activities of these proteins.The prenylation pathway has been targeted for potential anticancer therapy because most members of the Ras superfamily, which contains many known oncogenes, undergo CAAX processing. The Ras superfamily consists of five large subfamilies; the two most well-characterized are the Ras and Rho subfamilies (8). Both Ras and Rho proteins are processed by the CaaX pathway; Ras family members are farnesylated, while most Rho family members are geranylgeranylated. These monomeric GTPases cycle between a GDP-bound inactive state and a GTP-bound active state. In their active states, Ras and Rho subfamily members control numerous cell signaling pathways that are involved in cell proliferation, differentiation, migration, polarity, and morphology (9).Abnormally high activity of Ras and Rho signaling pathways contribute to initiation and progression of many types of cancer (10, 11). For example, many breast cancers that are highly metastatic express abnormally high levels of Rho proteins (12). Rho proteins control migration and invasion of cells by tightly coordinating changes in the actin and microtubule cytoskeletons. Acting through their effectors, Rho proteins rearrange the actin cytoskeleton to respond to chemo-attractant gradients, polarize cells, and control migration and invasion. While cell migration is necessary for development, leukocyte function, and other normal cell biologies, dysregulation of migration and invasion results in cancer metastasis (13). Metastasis is an important and deadly progression of cancer and understanding the biology of migrating cancer cells is crucial for therapeutic targeting of this aspect of cancer.Pharmacologic targeting of the enzymes involved in the CaaX-processing pathway has emerged as a promising anticancer strategy. In particular, there has been much effort in designing inhibitors against the protein prenyltransferases, most notably FTase (14, 15). There is also recent evidence that inhibition of geranygeranylation of Rho proteins also impacts oncogenesis and metastasis (16–18). However, the overall success of the FTase inhibitors (FTIs) in the clinical setting has been somewhat disappointing. One possible reason is a phenomenon termed “alternate prenylation” in which some FTase substrates, most notably K- and N- Ras, are modified by GGTase and escape inhibition by FTIs (19–21). Because the Rce1 protease and Icmt methyltransferase act on all CaaX proteins, problems such as alternate prenylation would not arise if these enzymes were targeted. Hence, while protein prenyltransferase inhibitors still show some promise as anticancer agents, the emerging view that global attenuation of CaaX protein function may be advantageous in blocking cancer cell growth has increased interest in studying the two downstream enzymes involved in CaaX processing.While the biological consequences of prenylation are fairly well understood, the precise roles of C-terminal methylation in CaaX protein function are still elusive. Depending on the CaaX protein, methylation has been ascribed to roles in localization, protein-protein interactions and protein stability (11). The development of an Icmt knock-out mouse model has furthered our understanding of Icmt function (22, 23). Localization studies conducted in cells with genetically deleted Icmt have shown that methylation is important for proper membrane association of Ras proteins. However, the localization of Rho proteins in the absence of Icmt activity appears to be more complicated and may vary depending on family member and activation status (24–26). Importantly, inhibition of CaaX protein methylation via either genetic or pharmacologic targeting has shown a clear impact on oncogenic transformation and tumor growth (23, 27, 28).Defining the role of Icmt-mediated methylation in complex cellular behaviors such as migration and invasion is crucial for furthering our understanding of the impact of CaaX protein methylation on the biology of normal and cancer cells. In the current study, we have assessed the impact of Icmt inhibition on cell biological processes associated with the function of Rho proteins, specifically cell adhesion, morphology, and migration. We found that inhibition of Icmt results in a disruption of the actin cytoskeleton and impairs ligand-mediated activation of RhoA and Rac1, a potential consequence of increased RhoGDI binding to both RhoA and Rac1 when their methylation is impaired. Further, we show that the impact of Icmt inhibition on cell migration is due at least in part to impairment of RhoA and Rac1function. These findings establish a role for Icmt-mediated methylation in cell migration and further elucidate the role that methylation plays in the function of Rho GTPases.     

Identification of a novel human Rho protein with unusual properties: GTPase deficiency and in vivo farnesylation.

We have identified a human Rho protein, RhoE, which has unusual structural and biochemical properties that suggest a novel mechanism of regulation. Within a region that is highly conserved among small GTPases, RhoE contains amino acid differences specifically at three positions that confer oncogenicity to Ras (12, 59, and 61). As predicted by these substitutions, which impair GTP hydrolysis in Ras, RhoE binds GTP but lacks intrinsic GTPase activity and is resistant to Rho-specific GTPase-activating proteins. Replacing all three positions in RhoE with conventional amino acids completely restores GTPase activity. In vivo, RhoE is found exclusively in the GTP-bound form, suggesting that unlike previously characterized small GTPases, RhoE may be normally maintained in an activated state. Thus, amino acid changes in Ras that are selected during tumorigenesis have evolved naturally in this Rho protein and have similar consequences for catalytic function. All previously described Rho family proteins are modified by geranylgeranylation, a lipid attachment required for proper membrane localization. In contrast, the carboxy-terminal sequence of RhoE predicts that, like Ras proteins, RhoE is normally farnesylated. Indeed, we have found that RhoE in farnesylated in vivo and that this modification is required for association with the plasma membrane and with an unidentified cellular structure that may play a role in adhesion. Thus, two unusual structural features of this novel Rho protein suggest a striking evolutionary divergence from the Rho family of GTPases.     

Studies with recombinant Saccharomyces cerevisiae CaaX prenyl protease Rce1p

Eukaryotic proteins with carboxyl-terminal CaaX motifs undergo three post-translational processing reactions-protein prenylation, endoproteolysis, and carboxymethylation. Two genes in yeast encoding CaaX endoproteases, AFC1 and RCE1, have been identified. Rce1p is solely responsible for proteolysis of yeast Ras proteins. When proteolysis is blocked, plasma membrane localization of Ras2p is impaired. The mislocalization of undermodified Ras in the cell suggests that Rce1p is an attractive target for cancer therapeutics. Homologous expression of plasmid-encoded Saccharomyces cerevisiae RCE1 under the control of the GAL1 promoter gave a 370-fold increase in endoprotease activity over an uninduced control. Yeast Rce1p was detected by Western blotting with a yRce1p antibody or with an anti-myc antibody to Rce1p bearing a C-terminal myc-epitope. Membrane preparations were examined for their sensitivity to a variety of protease inhibitors, metal ion chelators, and heavy metals. The enzyme was sensitive to cysteine protease inhibitors, Zn(2+), and Ni(2+). The substrate selectivity of yRce1p was determined for a variety of prenylated CaaX peptides including farnesylated and geranylgeranylated forms of human Ha-Ras, Ki-Ras, N-Ras, and yeast Ras2p, a-mating factor, and Rho2p. Six site-directed mutants of conserved polar and ionic amino acids in yRce1p were prepared. Four of the mutants, H194A, E156A, C251A, and H248A, were inactive. Results from the protease inhibition studies and the site-directed mutagenesis suggest that Rce1p is a cysteine protease.