Structural homology among mammalian and Saccharomyces cerevisiae isoprenyl-protein transferases

Farnesyl-protein transferase (FTase) purified from rat or bovine brain is an alpha/beta heterodimer, comprised of subunits having relative molecular masses of approximately 47 (alpha) and 45 kDa (beta). In the yeast Saccharomyces cerevisiae, two unlinked genes, RAM1/DPR1 (RAM1) and RAM2, are required for FTase activity. To explore the relationship between the mammalian and yeast enzymes, we initiated cloning and immunological analyses. cDNA clones encoding the 329-amino acid COOH-terminal domain of bovine FTase alpha-subunit were isolated. Comparison of the amino acid sequences deduced from the alpha-subunit cDNA and the RAM2 gene revealed 30% identity and 58% similarity, suggesting that the RAM2 gene product encodes a subunit for the yeast FTase analogous to the bovine FTase alpha-subunit. Antisera raised against the RAM1 gene product reacted specifically with the beta-subunit of bovine FTase, suggesting that the RAM1 gene product is analogous to the bovine FTase beta-subunit. Whereas a ram1 mutation specifically inhibits FTase, mutations in the CDC43 and BET2 genes, both of which are homologous to RAM1, specifically inhibit geranylgeranyl-protein transferase (GGTase) type I and GGTase-II, respectively. In contrast, a ram2 mutation impairs both FTase and GGTase-I, but has little effect on GGTase-II. Antisera that specifically recognized the bovine FTase alpha-subunit precipitated both bovine FTase and GGTase-I activity, but not GGTase-II activity. Together, these results indicate that for both yeast and mammalian cells, FTase, GGTase-I, and GGTase-II are comprised of different but homologous beta-subunits and that the alpha-subunits of FTase and GGTase-I share common features not shared by GGTase-II.     

Genetic evidence for in vivo cross-specificity of the CaaX-box protein prenyltransferases farnesyltransferase and geranylgeranyltransferase-I in Saccharomyces cerevisiae.

Two protein prenyltransferase enzymes, farnesyltransferase (FTase) and geranylgeranyltransferase-I (GGTase-I), catalyze the covalent attachment of a farnesyl or geranylgeranyl lipid group to the cysteine of a CaaX sequence (cysteine [C], two aliphatic amino acids [aa], and any amino acid [X]. In vitro studies reported here confirm previous reports that CaaX proteins with a C-terminal serine are farnesylated by FTase and those with a C-terminal leucine are geranylgeranylated by GGTase-I. In addition, we found that FTase can farnesylate CaaX proteins with a C-terminal leucine and can transfer a geranylgeranyl group to some CaaX proteins. Genetic data indicate that FTase and GGTase-I have the same substrate preferences in vivo as in vitro and also show that each enzyme can prenylate some of the preferred substrates of the other enzyme in vivo. Specifically, the viability of yeast cells lacking FTase is due to prenylation of Ras proteins by GGTase-I. Although this GGTase-I dependent prenylation of Ras is sufficient for growth, it is not sufficient for mutationally activated Ras proteins to exert deleterious effects on growth. The dependence of the activated Ras phenotype on FTase can be bypassed by replacing the C-terminal serine with leucine. This altered form of Ras appears to be prenylated by both GGTase-I and FTase, since it produces an activated phenotype in a strain lacking either FTase or GGTase-I. Yeast cells can grow in the absence of GGTase-I as long as two essential substrates are overexpressed, but their growth is slow. Such strains are dependent on FTase for viability and are able to grow faster when FTase is overproduced, suggesting that FTase can prenylate the essential substrates of GGTase-I when they are overproduced.     

Conversion of protein farnesyltransferase to a geranylgeranyltransferase

Posttranslational modifications are essential for the proper function of a number of proteins in the cell. One such modification, the covalent attachment of a single isoprenoid lipid (prenylation), is carried out by the CaaX prenyltransferases, protein farnesyltransferase (FTase) and protein geranylgeranyltransferase type-I (GGTase-I). Substrate proteins of these two enzymes are involved in a variety of cellular functions but are largely associated with signal transduction. These modified proteins include members of the Ras superfamily, heterotrimeric G-proteins, centromeric proteins, and a number of proteins involved in nuclear integrity. Although FTase and GGTase-I are highly homologous, they are quite selective for their substrates, particularly for their isoprenoid diphosphate substrates, FPP and GGPP, respectively. Here, we present both crystallographic and kinetic analyses of mutants designed to explore this isoprenoid specificity and demonstrate that this specificity is dependent upon two enzyme residues in the beta subunits of the enzymes, W102beta and Y365beta in FTase (T49beta and F324beta, respectively, in GGTase-I).     

Thematic review series: lipid posttranslational modifications. Structural biology of protein farnesyltransferase and geranylgeranyltransferase type I

More than 100 proteins necessary for eukaryotic cell growth, differentiation, and morphology require posttranslational modification by the covalent attachment of an isoprenoid lipid (prenylation). Prenylated proteins include members of the Ras, Rab, and Rho families, lamins, CENPE and CENPF, and the gamma subunit of many small heterotrimeric G proteins. This modification is catalyzed by the protein prenyltransferases: protein farnesyltransferase (FTase), protein geranylgeranyltransferase type I (GGTase-I), and GGTase-II (or RabGGTase). In this review, we examine the structural biology of FTase and GGTase-I (the CaaX prenyltransferases) to establish a framework for understanding the molecular basis of substrate specificity and mechanism. These enzymes have been identified in a number of species, including mammals, fungi, plants, and protists. Prenyltransferase structures include complexes that represent the major steps along the reaction path, as well as a number of complexes with clinically relevant inhibitors. Such complexes may assist in the design of inhibitors that could lead to treatments for cancer, viral infection, and a number of deadly parasitic diseases.     

Crystallographic analysis of CaaX prenyltransferases complexed with substrates defines rules of protein substrate selectivity

Post-translational modifications are essential for the proper function of many proteins in the cell. The attachment of an isoprenoid lipid (a process termed prenylation) by protein farnesyltransferase (FTase) or geranylgeranyltransferase type I (GGTase-I) is essential for the function of many signal transduction proteins involved in growth, differentiation, and oncogenesis. FTase and GGTase-I (also called the CaaX prenyltransferases) recognize protein substrates with a C-terminal tetrapeptide recognition motif called the Ca1a2X box. These enzymes possess distinct but overlapping protein substrate specificity that is determined primarily by the sequence identity of the Ca1a2X motif. To determine how the identity of the Ca1a2X motif residues and sequence upstream of this motif affect substrate binding, we have solved crystal structures of FTase and GGTase-I complexed with a total of eight cognate and cross-reactive substrate peptides, including those derived from the C termini of the oncoproteins K-Ras4B, H-Ras and TC21. These structures suggest that all peptide substrates adopt a common binding mode in the FTase and GGTase-I active site. Unexpectedly, while the X residue of the Ca1a2X motif binds in the same location for all GGTase-I substrates, the X residue of FTase substrates can bind in one of two different sites. Together, these structures outline a series of rules that govern substrate peptide selectivity; these rules were utilized to classify known protein substrates of CaaX prenyltransferases and to generate a list of hypothetical substrates within the human genome.     

Protein farnesyl transferase target selectivity is dependent upon peptide stimulated product release

Protein farnesyl transferase (FTase) catalyzes transfer of a 15 carbon farnesyl lipid to cysteine in the C-terminal Ca1a2X sequence of numerous proteins including Ras. Previous studies have shown that product release is rate limiting and is dependent on binding of either a new peptide or isoprenoid diphosphate substrate. While considerable progress has been made in understanding how FTase distinguishes between related target proteins, the relative importance of the two pathways for product release on substrate selectivity is unclear. A detailed analysis of substrate stimulated product release has now been performed and provides new insights into the mechanism of FTase target selectivity. To clarify how FTase selects between different Ca1a2X sequences, we have examined the competition of various peptide substrates for modification with the isoprenoids farnesyl diphosphate (FPP) and anilinogeranyl diphosphate (AGPP). We find that reactivity of some competing peptides is correlated with apparent Kmpeptide, while the reactivity of others is predicted by the selectivity factor apparent kcat/Kmpeptide. The peptide target selectivity also depends on the structure of the isoprenoid donor. Additionally, we observe two peptide substrate concentration dependent maxima and substrate inhibition in the steady-state reaction which require a minimum of three peptide binding states for the steady-state FTase reaction mechanism. We propose a model for the FTase reaction mechanism that, in addition to FPP stimulated product release, incorporates peptide binding to the FTase-FPP complex and the formation of an FTase-product-peptide complex followed by product release leading to an inhibitory FTase-peptide complex as a natural consequence of catalysis to explain these results.     

Peptide specificity of protein prenyltransferases is determined mainly by reactivity rather than binding affinity

Protein farnesyltransferase (FTase) and protein geranylgeranyltransferase type I (GGTase I) catalyze the attachment of lipid groups from farnesyl diphosphate and geranylgeranyl diphosphate, respectively, to a cysteine near the C-terminus of protein substrates. FTase and GGTase I modify several important signaling and regulatory proteins with C-terminal CaaX sequences ("C" refers to the cysteine residue that becomes prenylated, "a" refers to any aliphatic amino acid, and "X" refers to any amino acid). In the CaaX paradigm, the C-terminal X-residue of the protein/peptide confers specificity for FTase or GGTase I. However, some proteins, such as K-Ras, RhoB, and TC21, are substrates for both FTase and GGTase I. Here we demonstrate that the C-terminal amino acid affects the binding affinity of K-Ras4B-derived hexapeptides (TKCVIX) to FTase and GGTase I modestly. In contrast, reactivity, as indicated by transient and steady-state kinetics, varies significantly and correlates with hydrophobicity, volume, and structure of the C-terminal amino acid. The reactivity of FTase decreases as the hydrophobicity of the C-terminal amino acid increases whereas the reactivity of GGTase I increases with the hydrophobicity of the X-group. Therefore, the hydrophobicity, as well as the structure of the X-group, determines whether peptides are specific for farnesylation, geranylgeranylation, or dual prenylation.     

Plant farnesyltransferase can restore yeast Ras signaling and mating.

Farnesyltransferase (FTase) is a heterodimeric enzyme that modifies a group of proteins, including Ras, in mammals and yeasts. Plant FTase alpha and beta subunits were cloned from tomato and expressed in the yeast Saccharomyces cerevisiae to assess their functional conservation in farnesylating Ras and a-factor proteins, which are important for cell growth and mating. The tomato FTase beta subunit (LeFTB) alone was unable to complement the growth defect of ram1 delta mutant yeast strains in which the chromosomal FTase beta subunit gene was deleted, but coexpression of LeFTB with the plant alpha subunit gene (LeFTA) restored normal growth, Ras membrane association, and mating. LeFTB contains a novel 66-amino-acid sequence domain whose deletion reduces the efficiency of tomato FTase to restore normal growth to yeast ram1 delta strains. Coexpression of LeFTA and LeFTB in either yeast or insect cells yielded a functional enzyme that correctly farnesylated CaaX-motif-containing peptides. Despite their low degree of sequence homology, yeast and plant FTases shared similar in vivo and in vitro substrate specificities, demonstrating that this enzymatic modification of proteins with intermediates from the isoprenoid biosynthesis pathway is conserved in evolutionarily divergent eukaryotes.     

A novel protein geranylgeranyltransferase-I inhibitor with high potency, selectivity, and cellular activity

Inhibiting protein prenylation is an attractive means to modulate cellular processes controlled by a variety of signaling proteins, including oncogenic proteins such as Ras and Rho GTPases. The largest class of prenylated proteins contain a so-called CaaX motif at their carboxyl termini and are subject to a maturation process initiated by the attachment of an isoprenoid lipid by either protein farnesyltransferase (FTase) or protein geranylgeranyltransferase type I (GGTase-I). Inhibitors of FTase, termed FTIs, have been the subject of intensive development in the past decade and have shown efficacy in clinical trials. Although GGTase-I inhibitors (GGTIs) have received less attention, accumulating evidence suggests GGTIs may augment therapies using FTIs and could be useful to treat a myriad of additional disease states. Here we describe the characterization of a selective, highly potent, and cell-active GGTase-I inhibitor, GGTI-DU40. Kinetic analysis revealed that inhibition by GGTI-DU40 is competitive with the protein substrate and uncompetitive with the isoprenoid substrate; the Ki for the inhibition is 0.8 nM. GGTI-DU40 is highly selective for GGTase-I both in vitro and in living cells. Studies indicate GGTI-DU40 blocks prenylation of a number of geranylgeranylated CaaX proteins. Treatment of MDA-MB-231 breast cancer cells with GGTI-DU40 inhibited thrombin-induced cell rounding via a process that involves inhibition of Rho proteins without significantly effecting parallel mobilization of calcium via Gbetagamma. These studies establish GGTI-DU40 as a prime tool for interrogating biologies associated with protein geranylgeranylation and define a novel structure for this emerging class of experimental therapeutics.     

Crystallographic analysis reveals that anticancer clinical candidate L-778,123 inhibits protein farnesyltransferase and geranylgeranyltransferase-I by different binding modes

Many signal transduction proteins that control growth, differentiation, and transformation, including Ras GTPase family members, require the covalent attachment of a lipid group by protein farnesyltransferase (FTase) or protein geranylgeranyltransferase type-I (GGTase-I) for proper function and for the transforming activity of oncogenic mutants. FTase inhibitors are a new class of potential cancer therapeutics under evaluation in human clinical trials. Here, we present crystal structures of the clinical candidate L-778,123 complexed with mammalian FTase and complexed with the related GGTase-I enzyme. Although FTase and GGTase-I have very similar active sites, L-778,123 adopts different binding modes in the two enzymes; in FTase, L-778,123 is competitive with the protein substrate, whereas in GGTase-I, L-778,123 is competitive with the lipid substrate and inhibitor binding is synergized by tetrahedral anions. A comparison of these complexes reveals that small differences in protein structure can dramatically affect inhibitor binding and selectivity. These structures should facilitate the design of more specific inhibitors toward FTase or GGTase-I. Finally, the binding of a drug and anion together could be applicable for developing new classes of inhibitors.     

Thiazole- and imidazole-containing peptidomimetic inhibitors of protein farnesyltransferase

Mimetics of the C-terminal CAAX tetrapeptide of Ras protein were designed replacing internal dipeptide AA with 4-amino-2-phenylbenzoic acid and cysteine (C) with 2-amino-4-thiazolyl-, 2-mercapto-4-thiazolyl-, 2-mercapto-4-imidazolyl- and 2-methylmercapto-4-thiazolyl-acetic or propionic acid. The compound in which C is replaced by 2-amino-4-thiazolylacetic acid inhibited FTase activity in the low nanomolar range and showed antiproliferative effect on rat aortic smooth muscle cells interfering with Ras farnesylation. On the basis of these results, 2-aminothiazole can be considered as an alternative to heterocycles, such as pyridine and imidazole, normally used in FTase inhibitors designed as non-thiol CAAX mimetics.     

Specific isoprenoid modification is required for function of normal, but not oncogenic, Ras protein.

While the Ras C-terminal CAAX sequence signals modification by a 15-carbon farnesyl isoprenoid, the majority of isoprenylated proteins in mammalian cells are modified instead by a 20-carbon geranylgeranyl moiety. To determine the structural and functional basis for modification of proteins by a specific isoprenoid group, we have generated chimeric Ras proteins containing C-terminal CAAX sequences (CVLL and CAIL) from geranylgeranyl-modified proteins and a chimeric Krev-1 protein containing the H-Ras C-terminal CAAX sequence (CVLS). Our results demonstrate that both oncogenic Ras transforming activity and Krev-1 antagonism of Ras transforming activity can be promoted by either farnesyl or geranylgeranyl modification. Similarly, geranylgeranyl-modified normal Ras [Ras(WT)CVLL], when overexpressed, exhibited the same level of transforming activity as the authentic farnesyl-modified normal Ras protein. Therefore, farnesyl and geranylgeranyl moieties are functionally interchangeable for these biological activities. In contrast, expression of moderate levels of geranylgeranyl-modified normal Ras inhibited the growth of untransformed NIH 3T3 cells. This growth inhibition was overcome by coexpression of the mutant protein with oncogenic Ras or Raf, but not with oncogenic Src or normal Ras. The similar growth-inhibiting activities of Ras(WT)CVLL and the previously described Ras(17N) dominant inhibitory mutant suggest that geranylgeranyl-modified normal Ras may exert its growth-inhibiting action by perturbing endogenous Ras function. These results suggest that normal Ras function may specifically require protein modification by a farnesyl, but not a geranylgeranyl, isoprenoid.     

The basis for K-Ras4B binding specificity to protein farnesyltransferase revealed by 2 A resolution ternary complex structures

Background: The protein farnesyltransferase (FTase) catalyzes addition of the hydrophobic farnesyl isoprenoid to a cysteine residue fourth from the C terminus of several protein acceptors that are essential for cellular signal transduction such as Ras and Rho. This addition is necessary for the biological function of the modified proteins. The majority of Ras-related human cancers are associated with oncogenic variants of K-RasB, which is the highest affinity natural substrate of FTase. Inhibition of FTase causes regression of Ras-mediated tumors in animal models. Results: We present four ternary complexes of rat FTase co-crystallized with farnesyl diphosphate analogs and K-Ras4B peptide substrates. The Ca(1)a(2)X portion of the peptide substrate binds in an extended conformation in the hydrophobic cavity of FTase and coordinates the active site zinc ion. These complexes offer the first view of the polybasic region of the K-Ras4B peptide substrate, which confers the major enhancement of affinity of this substrate. The polybasic region forms a type I beta turn and binds along the rim of the hydrophobic cavity. Removal of the catalytically essential zinc ion results in a dramatically different peptide conformation in which the Ca(1)a(2)X motif adopts a beta turn. A manganese ion binds to the diphosphate mimic of the farnesyl diphosphate analog. Conclusions: These ternary complexes provide new insight into the molecular basis of peptide substrate specificity, and further define the roles of zinc and magnesium in the prenyltransferase reaction. Zinc is essential for productive Ca(1)a(2)X peptide binding, suggesting that the beta-turn conformation identified in previous nuclear magnetic resonance (NMR) studies reflects a state in which the cysteine is not coordinated to the zinc ion. The structural information presented here should facilitate structure-based design and optimization of inhibitors of Ca(1)a(2)X protein prenyltransferases.     

A mutant form of human protein farnesyltransferase exhibits increased resistance to farnesyltransferase inhibitors.

Protein farnesyltransferase (FTase) is a key enzyme responsible for the lipid modification of a large and important number of proteins including Ras. Recent demonstrations that inhibitors of this enzyme block the growth of a variety of human tumors point to the importance of this enzyme in human tumor formation. In this paper, we report that a mutant form of human FTase, Y361L, exhibits increased resistance to farnesyltransferase inhibitors, particularly a tricyclic compound, SCH56582, which is a competitive inhibitor of FTase with respect to the CAAX (where C is cysteine, A is an aliphatic amino acid, and X is the C-terminal residue that is preferentially serine, cysteine, methionine, glutamine or alanine) substrates. The Y361L mutant maintains FTase activity toward substrates ending with CIIS. However, the mutant also exhibits an increased affinity for peptides terminating with CIIL, a motif that is recognized by geranylgeranyltransferase I (GGTase I). The Y361L mutant also demonstrates activity with Ha-Ras and Cdc42Hs proteins, substrates of FTase and GGTase I, respectively. In addition, the Y361L mutant shows a marked sensitivity to a zinc chelator HPH-5 suggesting that the mutant has altered zinc coordination. These results demonstrate that a single amino acid change at a residue at the active site can lead to the generation of a mutant resistant to FTase inhibitors. Such a mutant may be valuable for the study of the effects of FTase inhibitors on tumor cells.     

Total in vitro maturation of the Saccharomyces cerevisiae a-factor lipopeptide mating pheromone

The a-factor mating pheromone, produced by Saccharomyces cerevisiae a haploid cells, is post-translationally modified in a manner analogous to that of the ras proto-oncogene product. A consensus C-terminal amino acid sequence, -CAAX (C is cysteine, A is aliphatic amino acid, and X is any amino acid), is the target of these modifications, which include isoprenylation (essential for Ras function), proteolysis of the -AAX sequence, and carboxy methyl esterification. Recently, the RAM/DPR1 gene product was shown to be a component of the activity responsible for isoprenylation of both Ras and a-factor. In this report, we present an in vitro assay which not only detects a-factor isoprenylation, but also proteolysis and carboxy methyl esterification, and directly demonstrates, biochemically, the order of these processing events. This a-factor maturation assay may prove useful for screening agents which block any of the steps involved in the post-translational modification of the a-factor and Ras -CAAX sequences. Such agents would be potential anti-Ras-related cancer therapeutic drugs.     

Modification of nuclear lamin proteins by a mevalonic acid derivative occurs in reticulocyte lysates and requires the cysteine residue of the C-terminal CXXM motif.

The C-terminus of nuclear lamins (CXXM) resembles a C-terminal motif (the CAAX box) of fungal mating factors and ras-related proteins. The CAAX box is subject to different types of post-translational modifications, including proteolytic processing, isoprenylation and carboxyl methylation. By peptide mapping we show that both chicken lamins A and B2 are processed proteolytically in vivo. However, whereas the entire CXXM motif is cleaved from lamin A, at most three C-terminal amino acids are removed from lamin B2. Following translation of cDNA-derived RNAs in reticulocyte lysates, lamin proteins specifically incorporate a derivative of [14C]mevalonic acid (MV), i.e. the precursor of a putative isoprenoid modification. Remarkably, no MV is incorporated into lamin B2 translated from a mutant cDNA encoding alanine instead of cysteine in the C-terminal CXXM motif. These results implicate this particular cysteine residue as the target for modification of lamin proteins by an isoprenoid MV derivative, and they indicate that isoprenylation is amenable to studies in cell-free systems. Moreover, our observations suggest that C-terminal processing of newly synthesized nuclear lamins is a multi-step process highly reminiscent of the pathway elaborated recently for ras-related proteins.     

Protein farnesylation is critical for maintaining normal cell morphology and canavanine resistance in Schizosaccharomyces pombe

Protein farnesyltransferase (FTase) plays important roles in the growth and differentiation of eukaryotic cells. In this paper, we report the identification of the Schizosaccharomyces pombe gene cpp1(+) encoding the beta-subunit of FTase. The predicted amino acid sequence of the cpp1(+) gene product shares significant similarity with FTase beta-subunits from a variety of organisms. S. pombe FTase purified from E. coli exhibits high enzymatic activity toward the CAAX farnesylation motif substrates (where C represents cysteine, A represents aliphatic amino acid, and X is preferentially methionine, cysteine, serine, alanine, or glutamine) while showing little preference for CAAL geranylgeranylation motif substrates (where L represents leucine or phenylalanine). cpp1(+) is not essential for growth as shown by gene disruption; however, mutant cells exhibit rounded or irregular cell morphology. Expression of a geranylgeranylated mutant form, Ras1-CVIL, which can bypass farnesylation, rescues these morphological defects. We also identify a novel phenotype of cpp1(-) mutants, hypersensitivity to canavanine. This appears to be due to a 3-4-fold increase in the rate of arginine uptake as compared with wild-type cells. Expression of the geranylgeranylated mutant form of a novel farnesylated small GTPase, SpRheb, is able to suppress the elevated arginine uptake rate. These results demonstrate that protein farnesylation is critical for maintaining normal cell morphology through Ras1 and canavanine resistance through SpRheb.     

Carboxyl-terminal isoprenylation of ras-related GTP-binding proteins encoded by rac1, rac2, and ralA

Membrane localization of p21ras is dependent upon its posttranslational modification by a 15-carbon farnesyl group. The isoprenoid is linked to a cysteine located within a conserved carboxyl-terminal sequence termed the "CAAX" box (where C is cysteine, A is an aliphatic amino acid, and X is any amino acid). We now show that three GTP-binding proteins encoded by the recently identified rac1, rac2, and ralA genes also undergo isoprenoid modification. cDNAs coding for each protein were transcribed in vitro, and the RNAs were translated in reticulocyte lysates. Incorporation of isoprenoid precursors, [3H]mevalonate or [3H]farnesyl pyrophosphate, indicated that the translation products were modified by isoprenyl groups. A protein recognized by an antibody to rac1 also comigrated with a protein metabolically labeled by a product of [3H] mevalonate in cultured cells. Gel permeation chromatography of radiolabeled hydrocarbons released from the rac1, rac2, and ralA proteins by reaction with Raney nickel catalyst indicated that unlike p21Hras, which was modified by a 15-carbon moiety, the rac and ralA translation products were modified by 20-carbon isoprenyl groups. Site-directed mutagenesis established that the isoprenylated cysteines in the rac1, rac2, and ralA proteins were located in the fourth position from the carboxyl terminus. The three-amino acid extension distal to the cysteine was required for this modification. The isoprenylation of rac1 (CSLL), ralA (CCIL), and the site-directed mutants rac1 (CRLL) and ralA (CSIL), demonstrates that the amino acid adjacent to the cysteine need not be aliphatic. Therefore, proteins with carboxyl-terminal CXXX sequences that depart from the CAAX motif should be considered as potential targets for isoprenoid modification.     

Prenylated c17orf37 induces filopodia formation to promote cell migration and metastasis

Post-translational modification by covalent attachment of isoprenoid lipids (prenylation) regulates the functions and biological activities of several proteins implicated in the oncogenic transformation and metastatic progression of cancer. The largest group of prenylated proteins contains a CAAX motif at the C-terminal that serves as a substrate for a series of post-translational modifications that convert these otherwise hydrophilic proteins to lipidated proteins, thus facilitating membrane association. C17orf37 (chromosome 17 open reading frame 37), also known as C35/Rdx12/MGC14832, located in the 17q12 amplicon, is overexpressed in human cancer, and its expression correlates with the migratory and invasive phenotype of cancer cells. Here we show that C17orf37 contains a functional CAAX motif and is post-translationally modified by protein geranylgeranyltransferase-I (GGTase-I). Geranylgeranylation of C17orf37 at the CAAX motif facilitates association of the protein to the inner leaflet of plasma membrane, enhances migratory phenotype of cells by inducing increased filopodia formation, and potentiates directional migration. A prenylation-deficient mutant of C17orf37 is functionally inactive and fails to trigger dissemination of tail vein-injected cells in a mouse model of metastasis. These findings demonstrate that prenylation is required for the function of the C17orf37 protein in cancer cells and imply that the post-translational modification may functionally regulate metastatic progression of disease.     

Selective modification of CaaX peptides with ortho-substituted anilinogeranyl lipids by protein farnesyl transferase: competitive substrates and potent inhibitors from a library of farnesyl diphosphate analogues

Protein farnesyl transferase (FTase) catalyzes transfer of a 15-carbon farnesyl group from farnesyl diphosphate (FPP) to a conserved cysteine in the C-terminal Ca1a2X motif of a range of proteins ("C" refers to the cysteine, "a" to any aliphatic amino acid, and "X" to any amino acid), and the lipid chain interacts with, and forms part of, the Ca1a2X peptide binding site. Here, we employed a library of anilinogeranyl diphosphate (AGPP) derivatives to examine whether altering the interacting surface between the two substrates could be exploited to generate Ca1a2X peptide selective FPP analogues. Analysis of transfer kinetics to dansyl-GCVLS peptide revealed that AGPP analogues with substituents smaller than or equal in size to a thiomethyl group supported FTase function, while analogues with larger substituents did not. Analogues with small meta-substitutions on the aniline ring such as iodo and cyano increased reactivity with dansyl-GCVLS and provided analogues that were effective FPP competitors. Other analogues with ortho-substitutions on the aniline were potent dansyl-GCVLS modification FTase inhibitors (Ki in the 2.4-18 nM range). Both meta- and para-trifluoromethoxy-AGPP are transferred to dansyl-GCVLS while the ortho-substituted isomer was a potent farnesyl transferase inhibitor (FTI) with an inhibition constant Ki = 3.0 nM. In contrast, ortho-trifluoromethoxy-AGPP was efficiently transferred to dansyl-GCVIM. Competition for dansyl-GCVLS and dansyl-GCVIM peptides by FPP and ortho-trifluoromethoxy-AGPP gave both analogue and farnesyl modified dansyl-GCVIM but only farnesylated dansyl-GCVLS. We provide evidence that competitive modification of dansyl-GCVIM by ortho-trifluoromethoxy-AGPP stems from a prechemical step discrimination between the competing peptides by the FTase-analogue complex. These results show that subtle changes engineered into the isoprenoid structure can alter the reactivity and FPP competitiveness of the analogues, which may be important for the development of prenylated protein function inhibitors.