Prenylation of Ras GTPase Superfamily Proteins and Their Function in Immunobiology

ADVANCES I N I M M U N O L O G Y . V O L 68

Prenylation of Ras GTPase Superfamily Proteins and Their Function in lmmunobiology ROBERT B. LOBEU Merck Research labomtories, hporhnent of Cancer Research, Merck and Company, Inc., West Point, Pennsybanio 19486

I. Introduction

The Ras superfamily of GTPases comprises a diverse group of proteins that play critical regulatory roles in a variety of cellular processes involved in immune system function (see Fig. 1).Although members of this family play diverse roles in cells, they carry out their functions via similar biochemical mechanisms. First, these proteins cycle between GDP and GTP-bound states and rely on accessory proteins to regulate this GDP/GTP cycle. Second, many members of the Ras superfamily can regulate multiple signaling pathways through interactions with different downstream effector molecules. Third, these proteins all function at membrane surfaces and are localized to membranes via C-terminal lipid moieties that are added to the protein posttranslationally in a process commonly referred to as prenylation. Lipidation of the Ras superfamily of proteins involves a family of prenyltransferases, which attach isoprenoid-derived lipids consisting of 15 carbon units (farnesyl) or 20 carbons (geranylgeranyl) to C-terminal cysteine residues. In contrast to membrane insertion via transmembrane domains, membrane association via prenylation can be a transient and regulatable phenomenon. This transient association is essential to the function of some of the prenylated GTPases, in particular the Rab GTPases, which catalyze the intracellular flow of membrane compartments and that must cycle on and off membrane sites to function. This review will give an overview into the biochemistry and function of the Ras superfamily members, discuss the enzymology and functional consequences of protein prenylation, detail specific roles of the three major subfamilies of the Ras superfamily in immune system function, and discuss inhibitors of protein prenylation and their effects on the function of the Ras superfamily of proteins. II. The Ras Superfamily Members

The Ras superfamily can be subdivided into three major subfamilies, the Ras proteins, the Rho/Rac proteins, and the Rab proteins (Boguski and 145

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HORERT 8. LORELL

FIG.1. Some ofthe functions of Ras superfamily proteins in immune cells. Ras superfamily members are indicated by the solid circles.

McCormick, 1993).The Ras proteins play a key role in signal transduction processes that regulate cell proliferation, activation, and differentiation. The Ras subfamily includes the mammalian ras alleles Harvey (H), N-ras, and Kirsten (K). The K-ras gene codes for two alternatively spliced variants, K4A and K4B, which are distinguished by the presence of a highly charged C-terminal region in K-Ras4B known as the polybasic domain (Barbacid, 1987). The expression of the different rus alleles vanes in different tissues (Leon et al., 1987), and there is some evidence to suggest that these different forms of Ras have somewhat different biochemical activity because mutant forms of these proteins differ in their ability to transform cells (Maher et al., 1995). The Ras proteins are closely related to the Rap proteins (RaplA, -1B, -2A, and -2B) and the R-Ras, RalA, RalB, and TC21 proteins. The Rho/Rac subfamily of proteins play many regulatory roles, including regulation of the actin cytoskeleton (Ridley, 1995),and regulation of the c-Jun kinasehtress activated protein kinase ( JNWSAPK) pathway.

PRENYLATION OF KZis GTPdw PROTEINS

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The Rho/Rac family includes RhoA, RhoB, RhoC, RhoD, RhoE, RhoG, Racl, Rac2, CDC42Hs, and TC10, which are -50% identical with each other and share -30% identity with other Ras-like GTPases (Nobes and Hall, 1994). The Rab subfamily of proteins consists of -30 members, which regulate the trafficking of intracellular membrane compartments (Pfeffer, 1994);this includes a role in regulating endocytosis and exocytosis, two types of membrane transport that are particularly important in immune cell function. 111. The GTPase Cycle

The members of the Ras superfamily of GTPases function as on-off switches that cycle between GTP-bound and GDP-bound states. They are in the “resting state” or “off” position when bound to GDP and activate their respective cellular processes when in the GTP-bound state. Turning these molecular switches on and off requires accessory proteins that are specific for the different members of the family (reviewed in Boguski and McCormick, 1993). The activation step involves guanine nucleotide exchange factors (GEFs),which facilitate dissociation of the bound GDP. Dissociation of the bound GDP enables the GTP to bind due to the high concentration of GTP in the cell relative to GDP (Bourne et al., 1991). The signaling pathway leading from transmembrane receptors such as the EGF or platelet-derived growth factor (PDGF) growth factor receptors to the activation of Ras is fairly well understood and proceeds through the activation of the guanine nucleotide exchange factor SOS. The initiating event is the binding of ligand to the receptor, which induces tyrosine autophosphorylation of residues in the receptor’s intracellular domain. The phosphorylated tyrosines of the receptor serve as docking sites for adapter proteins, such as Grb2 or Shc, which bind to the phosphotyrosines via their SH2 domains (Burgering and Bos, 1995).These adapter proteins also contain SH3 domains that mediate a binding interaction with polyproline stretches found on SOS (Quilliam et ul., 1995).Thus, receptor activation recruits the Grb2-SOS complex to the membrane, leading to guanine nucleotide exchange and activation of membrane-bound Ras. GTP binding is thought to induce a change in conformation that exposes the so-called effector domain, allowing the Ras protein to interact with downstream signaling effectors. A fairly detailed molecular description of the activation of Ras and its interaction with downstream effectors has been provided from crystallographic studies of Ras and Ras-related proteins (for a review, see Wittinghofer and Nassar, 1996). The members of the Ras family of proteins remain activated until their bound GTP is hydrolyzed. The Ras family members have weak intrinsic

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GTPase activity and require an interaction with another auxiliary protein, called a GTPase activating protein (GAP), to hydrolyze the bound GTP. In the case of Ras, Ras-GAP can accelerate the Ras GTPase reaction by almost five orders of magnitude (Gideon et al., 1992). Rho family members are activated by Rho-GAP domains, which are found in a variety of large, multifunctional proteins (Lamarche and Hall, 1994). The crystal structure of the Ras-GAP domain of p12OGAP, and the Rho-GAP domain from p50rhoGAP, have been solved recently (Barrett et al., 1997; Scheffzek et al., 1996). The deactivated GDP-bound form of the protein remains dormant until the proper activation stimulus is received and the protein can repeat the GTPase cycle. In the case of the Rho and Rab proteins, a third type of auxiliary protein, called a guanine nucleo'ide dissociation inhibitor (GDI),is involved in the GTPase cycle. GDI binds the GDP-bound form of the protein and, as the name implies, inhibits the dissociation of GDP. More important, the interaction of GDI with the Rho or Rab proteins prevents their binding to cellular membranes and, additionally, GDI can extract the Rho or Rab proteins from membranes (Wu et al., 1996). The ability of GDI to extract Rab proteins from membranes is critical to Rab protein cycling. GDI functions in retrieving the Rab protein from an acceptor membrane after a membrane vesicle fusion event has occurred and in delivering the Rab protein back to the donor membrane for another round of transport (Pfeffer, 1994; Soldati et al., 1994; Ullrich et al., 1994). IV. Downstream Signaling Effectors: Ras and the Rho/Rac Connection

A common feature of the Ras superfamily of GTPases, exemplified by both the Ras and Rho/Rac proteins, is the ability to activate multiple downstream effector pathways. Proteins that interact with the Ras effector domain include the Raf serinekhreonine kinase, phosphoinositide 3'kinase, MEK kinase (a kinase in the JNUSAPK kinase cascade), Ras GAP, Ral-GEF, and two proteins of unknown function (Rin, for Ras interacting; and Rsb; for Ras binding, Marshall, 1995; Wittinghofer and Nassar, 1996). Although the physiological importance of many of these effector interactions to the function of Ras remain unclear, the activation of the mitogenactivated protein kinase (MAPK) pathway via the Ras-Raf interaction is clearly important in transducing growth proliferation signals. The Ras-Raf interaction serves to localize Raf to the plasma membrane (Moodie et al., 1993; Van Aelst et al., 1993; Vojtek et al., 1993), where Raf itself becomes activated (Stokoe et al., 1994).One mechanism of Raf activation is through phosphorylation by a ceramide-activated protein (CAP) kinase (Yao et al., 1995). Recently, CAP kinase was shown to be the KSR (kinase suppressor

PRENYLATION OF Rat; GTPaw PROTEINS

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of Ras) protein (Y. Zhang et al., 1997), which had been identified through genetic studies in Caenorhabditis elegans and Drosophila mlanogaster as being a modulator of the Ras-Raf pathway (Kornfeld et al., 1995; Sundaram and Han, 1995; Therrien et al., 1995).Once activated, Raf phosphorylates MEK (Dent et al., 1992; Howe et al., 1992; Kyriakis et al., 1992),a tyrosine/ threonine kinase that in turn phosphorylates MAP kinases such as Erk2. Phosphorylated Erk2 can then translocate to the nucleus, where it can phosphorylate transcription factors such as Elk-1. Elk-1 in turn can activate genes involved in cell growth, such as cfos. The ras genes, particularly K-ras and N-ras, are frequently mutated in a variety of human cancers; for example, N-ras in the case of acute myelogenous leukemia, and K-ras in carcinomas of the pancreas, lung, and colon (Bos, 1990).These mutations inactivate the GTPase activity of Ras, leaving the Ras switch stuck in the “on” position. The inability to turn off Ras leads to the transformed phenotype of the cancerous cells because they no longer require growth factor-induced transmembrane signals to initiate the signaling pathways leading to cell proliferation. In addition to stimulating uncontrolled proliferation via activation of the MAPK pathway, oncogenic versions of Ras also have a profound effect on cellular morphology. Ras-transformed cells growing in monolayer cell cultures are not contact inhibited as are normal cells, and they have a refractile appearance in the light microscope. The effect of Ras on cell morphology is most likely mediated through the Rho/Rac family. The involvement of Rho and Rac proteins in Ras-mediated cell transformation is illustrated by the ability of dominant-negative inhibitors of these proteins to block Ras transformation (Khosravi-Far et al., 1995; Prendergast et al., 1995; Qiu et al., 1995a,b). Although the exact molecular link between Ras and the Rho/Rac pathway is unclear, mutations in the Ras effector domain reveal that the Ras-Rho/ Rac connection is independent of the Ras-Raf interaction (White et al., 1995).One such Ras mutant is defective in its interaction with Raf and is unable to stimulate the MAP kinase pathway but still causes the change in cellular morphology typical of activated Ras (Joneson et al., 1996b; Khosravi-Far et al., 1996). Several mechanisms have been proposed to account for the effect of Ras on cell morphology. One possible mechanism is through stimulation of a Rho-GAP activity; the Ras effector domain can interact with Ras-GAP, which in turn has been shown to bind the p190 Rho-GAP protein (Foster and Hu, 1994). Alternatively, the Ras effector, phosphoinositide 3’ kinase (PIS-K), might be involved in linking Ras to the Rho/Rac pathway (Rodriguez-Viciana et al., 1997). This is indicated by the correlation between the ability of Ras effector domain mutants to affect the actin cytoskeleton and to bind to PI3-K, and by the finding that inhibition of PI3-K blocks Ras induction of membrane ruffling.

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V. Rho/Rac Effectors

Like Ras, the Rho family of proteins have multiple cellular effectors. These proteins were first noted for their effects on the cell cytoskeleton. Microinjection studies in fibroblasts have shown that Rho induces actin stress fiber formation, whereas Rac and Cdc42 induce membrane ruffling and the formation of filopodia and lamellipodia (Nobes and Hall, 1995). In addition to their regulation of the cytoskeleton, Rho family members regulate several different protein kinases. For example, CDC42, as well as Racl, regulates the JNWSAPK kinase cascade via an interaction with the P6EiPAK kinase (Coso et al., 1995; Minden et al., 1995). Racl and Cdc42 bind and activate the 70-kDa S6 kinase (Chou and Blenis, 1996),which has been shown to play an important role in cell cycle progression in many cell types including lymphocytes. Other downstream signaling targets of the Rho/Rac family include the pl2OAcKtyrosine kinase (Manser et al., 1993) and the p160HoCK serinekhreonine kinase (Ishizaki et al., 1996). Rho family members also function through signaling systems that involve lipid metabolism. For example, stress fiber formation induced by growth factors involves leukotriene generation via the metabolism of arachidonic acid. An activated (GTPase-defective) Rac mutant induces stress fiber formation and leukotriene generation in a growth factor-independent manner, and leukotriene synthesis inhibitors abrogate Rac-induced stress fiber formation (Peppelenbosch et al., 1995). Additionally, Cdc42Hs binds to the p85 subunit of PI3-K and regulates its activity (Zheng et al., 1994), whereas RhoA has been implicated in phospholipase D activation (see below). The list of Rho/Rac effectors is likely to grow because Burbelo et al. (1995) have identified a motif found in the GTPase binding sites of p120ACK and P65PAKthat is present in more than 25 proteins from a variety of eukaryotic species. Similar to what has been found for Ras, mutations in the effector domain of Rac and Cdc42 have been defined that prevent their ability to interact with P6SPAK and activate the JNK kinase pathway but do not affect their ability to regulate the cytoskeleton (Lamarche et al., 1996; Joneson et al., 1996a). VI. Prenylation of the Rar Superfamily Members

Since 1980 it had been known that Ras localized to the plasma membrane of cells and that this localization required posttranslational modification of the protein (Lowy and Willumsen, 1993). In 1984, it was shown that a cysteine residue in a CaaX motif found at the C terminus of Ras played a role in its membrane localization because a Cys to Ser substitution

151

PHEKYLATION OF R a GTP'sr PROTEINS

abolished membrane binding (Willumsen et al., 1984). Furthermore, this Cys to Ser mutation abolished the transforming ability of a GTPasedefective H-Ras mutant, suggesting that membrane localization was critical to the function of Ras. A number of observations made in the mid-1980s led to the definition of the nature of the Ras C-terminal posttranslational modification. It had been shown that an inhibitor of HMG-CoA reductase, the rate-limiting enzyme in the cholesterol biosynthetic pathway (Fig. Z), blocked entry of cells into the S phase of the cell cycle (Schmidt et al., 1982). Furthermore, metabolites of exogenously added [3H]mevalonate, an intermediate in the cholesterol synthetic pathway, were incorporated into proteins (Schmidt et al., 1984).In 1986, genetic studies in yeast showed that posttranslational modification of yeast Ras and the yeast a-mating Factor, which also contained a CaaX at its C terminus, was controlled by the same genes (Powers et al., 1986).In 1988, the precise chemical structure of yeast a-mating factor was elucidated and shown to contain a C-terminal cysteine that was farnesylated via a thioether linkage and methylesterified

1

HMG-COA

1 I--

Lovastatin

Mevalonate

Cholesterol

1 1

Other

FTI

GGTl

- FTase

e GGPP

I

11

RPI

Methyl

___*

GGTase-l -CxC

GGTase-lI -cc

Methyl Transferase

~

,

-c-

s-9s

S - geranylgeranyl -C-OMe

s-99 s-99 , -C-X-C-OMe

s-99 C -OH

Frc;. 2. Biosynthetic pathway of prenylated proteins. The pathway is shown initiating from an early intermediate in the cholesterol biosynthetic pathway (HMG-CoA), leading to the prenylation enzyme snbstrdtes farnesyl diphosphate (FPP) and geranylgeranyldiphosphate (GGPP).The pathway branches through the three different prenyltransferase enzyrues and the subsequent processing enzymes. Sites of inhibition by existing pharinacologic agents, including HMG-CoA reductase inhibitors, FTIs, GCTIs, the CaaX protease inhibitor BPI, and the methyltrailsferase inhibitor AFC, are indicated. Abbreviations used: S, serine: M, rnethionine; Q, glutamine; L, leucine: X, any amino acid; gg, geranylgeranyl: -OMe, methylated carboy terminus.

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ROBERT B. LOBELL

on its carboxylate group (Anderegg et al., 1988). From this precedent, similar modifications on the Ras C-terminal CaaX were demonstrated (Casey et al., 1989; Hancock et al., 1989; Schafer et al., 1989). VII. Prenylation and Processing of CaaX Substrates

Protein prenylation involves the covalent addition of two types of isoprenoids, farnesyl pyrophosphate or geranylgeranyl pyrophosphate, to cysteine residues at or near the C terminus. The farnesyl isoprenoid, a 15-carbon lipid, is an intermediate of the cholesterol biosynthetic pathway and is derived from the basic 5-carbon isoprenoid unit, isopentyl pyrophosphate (Fig. 2). Geranylgeranyl pyrophosphate contains an additional isoprenoid unit and is derived directly from farnesyl pyrophosphate. Three different enzymes, or prenyltransferases, have been identified that carry out these modifications (Zhang and Casey, 1996). Farnesyltransferase ( FTase) and geranylgeranyltransferase type-I (GGTase-I) are sometimes referred to as the CaaX prenyltransferases, because they catalyze the addition of farnesyl diphosphate (FPP) and geranylgeranyl diphosphate (GGPP), respectively, to the cysteine residue in the sequence CaaX found at the C terminus of prenyltransferase substrates. A variety of proteins are substrates for the CaaX prenyltransferase enzymes (Table I), many of which are members of the Ras superfamily of small GTP-binding proteins. The third prenyltransferase enzyme, known as Rab geranylgeranyltransferase or geranylgeranyltransferase type I1 (GGTase-I1), adds geranylgeranyl groups to each cysteine in the XXCC, CCXX, and XCXC motifs at the C terminus of Rab proteins. VItI. CaaX Prenyltransferases

FTase and GGTase-I are both heterodimeric proteins that share a common 48-kDa a! subunit (Reiss et al., 1990; Seabra et al., 1991). The p subunits of FTase and GGTase-I are 46 and 43 kDa, respectively, and are approximately 30% identical at the amino acid level (Zhang et al., 1994). The genes for the yeast and mammalian prenyltransferases have been cloned and expressed in heterologous systems (Chen et al., 1991a,b; Fujiyama et al., 1987; Kohl et al., 1991; Omer et al., 1993; Powers et al., 1986). The a and /3 subunits of mammalian FTase are 30 and 37% identical to the corresponding yeast enzyme, encoded by the RAM2 and RAMl genes, respectively (Chen et a!., 1991a,b; Kohl et al., 1991; Omer et al., 1993). Mutations in either RAMl or RAM2 abolish the activity of FTase in yeast. FTase and GGTase-I both recognize the cysteine in CaaX motifs as the site for prenylation. In general, whether a protein is prenylated by FTase

PRENYLATION OF Ras CTPase PROTEINS

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or GGTase-I is defined by the X residue in the C a d motif; proteins with = serine, methionine, or glutamine are FTase substrates, whereas X = leucine for GGTase-I substrates (Casey et d.,1991; Moores et al., 1991; Yokoyama et al., 1991). The specificity of prenylation of CaaX substrates by FTase or GGTaseI is not always absolute because some proteins, such as K-Ras4B, can be both farnesylated by FTase and geranylgeranylated by GGTase-I in vitro (James et al., 1995; Moores et al., 1991). However, the catalytic efficiency (ke&,,,) for farnesylation of K-Ras4B by FTase is -140-fold greater than that for the geranylgeranylation of the protein by GGTase-I (F. L. Zhang et al., 1997). The preference for farnesylation of K-Ras4B is also reflected in vim, in which the protein is normally found in the farnesylated state (Casey et al., 1989). Other Ras isoforms, including K-Ras4A and N-Ras but not H-Ras, are also prenylated by both enzymes in uitro, with farnesylation being the preferred reaction (F. L. Zhang et al., 1997). The RhoB protein is another exception to the prenyltransferase specificity “rules” because this protein is both farnesylated and geranylgeranylated in vivo (Adamsonet al., 1992). RhoB is not an FTase substrate in vitro but rather is both farnesylated and geranylgeranylated by GGTase-I, with farnesylation being the preferred reaction (Armstrong et aZ., 1995). Some proteins, such as the heterotrimeric Gia subunit, contain an apparent CaaX motif but are not prenylated (Mumby et al., 1990). In the case of Gia, sequences upstream of its CGLF CaaX box apparently inhibit prenylation because the CGLF sequence confers prenylation when transferred to rus sequences (Cox et al., 1993). These data illustrate potential inaccuracies in predicting the nature of the prenyl group attached to a putative CaaX substrates based solely on prediction from the sequence of the CaaX. Characterization of the prenyl group on a CaaX substrate can be suggested by analysis of FTase and GGTase-I prenylation reactions on the substrate in vitro but should ultimately rely on characterization of the protein from cells or tissues. One commonly used method for characterization of the prenyl group involves labeling cells with [3H]mevalonate,which will incorporate into both farnesylated and geranylgeranylated proteins. After isolation of the labeled protein of interest, its labeled isoprenoid can be released via chemical means and identified by chromatographic separation and coelution with known standards (Casey et al., 1989). The potential for cross-prenylation of CaaX substrates in vivo, i.e., the farnesylation of GGTase-I substrates by FTase and vice versa, is suggested by the ability of proteins such as K-Ras4B to be prenylated by both FTase and GGTase-I. Studies in yeast illustrate the potential for cross-prenylation. It was shown that overexpression of the GGTase-I p subunit partially suppressed the growth defect of cells lacking FTase-P (Trueblood et al.,

X

TABLE I A CATALOG OF PRENYLATED PROTEINS Protein Ras proteins H-Ras N-Ras K-Ras4A K-Ras4B Ras-related proteins RaplA RaplB Rap2A Rap2B R-ras RalA RalB TC21 Rheb Rho proteins RhoA RhoB RhoC RhoD RhoE RhoG Cdc42Hs Racl Rac2 TClO

C terminus

Prenylation

CVLS CWM CIIM CVIM

F F F F

CLLL CQLL CNIQ CVIL CVLL CCIL CCLL CVIF CSVM

GG GG F GG GG GG GG GG F

CLVL CCKVL CPIL CCLAT CTVM CILL CVLL CLLL CSLL CLIT

GG FIGG GG F F GG GG GG GG GG

Protein Heterotrimeric G proteins yl (bovine, transducin) Y2 (ui2 a i3 Nuclear lamins Lamin A Lamin B cGMP phosphodiesterase (asub.) Phosphorylase kinase (rabbit)

C terminus

Prenylation

CVIS CAIL CGLF CGLF

F GG Not prenylated Not prenylated

CSIM CAIM

F F

CCIQ

F

CAMQ PXF (CHO cell) CLIM Interferon-inducible GTP binding proteins GBPl CTIS GBP2 CNIL Yeast YDJl CASQ Human HDJ2 CQTS Hepatitis delta virus large antigen CRPQ Protein tyrosine phosphatase, PRL-1 CCIQ Inositol triphosphate 5' phosphatase CWQ 2',5'-oligo(A) synthetase CTIL

F F F GG F F F F F GG

Rab proteins (selected members) Rabla cc cc Rab2 Rab3a CAC Rab3b csc Rab4a CGC Rab5 CCSN Rab5b CCSN Rab6 csc Rab’i dog csc Rab8 CVLL Rab9 dog CC RablO dog cc Rabll CCQNI

cli-GG di-GG di-GG di-GG di-GG di-GG di-GG di-GG di-GG GG di-GG di-GG di-GG

Note. The C-terminal amino acids (in standard amino acid code) and the prenylation state of each protein [farnesyl ( F ) or geranylgermyl (GG)]are indicated

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ROBERT B. LOBELL

1993). Similarly, overexpressionof two essential GGTase-I substrates, Rho1 and Cdc42, allows for growth of GGTase-I P-deficient cells: presumably, overexpression allows the cells to survive due to at least some level of farnesylation of these proteins (Ohya et al., 1993; Trueblood et al., 1993). Cross-prenylation has important implications in the context of pharmacological inhibition of prenyltransferases; alternative protein prenylation might rescue protein function in cells treated with a specific prenylation inhibitor (see Section XX). Structural information on prenyltransferases will further our understanding of the determinants of substrate specificity of FTase and GGTase-I and, in this regard, the structure of Rat FTase was recently solved by X-ray crystallography (Park et al., 1997). Although the crystal structure was determined in the absence of either substrate, the location of the active site of the enzyme was surmised based on the location of a bound zinc atom. It has been shown that the cysteine thiol of a CaaX peptide substrate coordinates to the zinc metal in a ternary complex consisting of enzyme, peptide, and FPP, indicating a direct role of the zinc in catalysis (Huang et al., 1997). The structure showed that the zinc atom of FTase is in close proximity to a hydrophobic pocket found in the P subunit of FTase. This hydrophobic pocket is likely the binding site for FPP because it is of sufficient length to accommodate FPP but not the larger GGPP molecule, consistent with the observation that FPP binds 15-fold tighter than GGPP to FTase (Yokoyama et al., 1997). In addition, Park et al. (1997) proposed a model for the interaction of the CaaX motif with active site residues of FTase. The model was based on the location of the nine C-terminal amino acids of the P subunit, which for some reason inserted into the active-site region of the adjacent aJP dimer in the crystal structure. Although this model provides a useful starting point for further studies, it is not supported by recent site-directed mutational data, which showed that mutation of three residues, Ser159, Tyr362, and Tyr366, changed the substrate specificity of yeast FTase to that of GGTase-I (DelVillar et al., 1997). The crystal structure model did not implicate these residues as being directly involved in CaaX substrate binding, although it cannot be ruled out that mutation of these residues changes substrate specificity through indirect effects on neighboring residues. Evidence from circular dichroism analysis indicates that FTase undergoes conformational changes upon binding CVIM peptide, FPP analogs, or tetrapeptide inhibitors of the enzyme (Wallace et al., 1996). Thus, the structure of the apoenzyme might not accurately reflect the structure of the active site with ligands bound. Further structural information, particularly data derived from enzyme-substrate or enzyme-inhibitor complexes, will enable a more precise

PRENYLATION OF Ras GTPase PROTEINS

157

definition of the molecular interactions involved in substrate recognition by FTase and GGTase-I. IX. CaaX Protease and Carboxymethyltransferase

Proteins modified by FTase and GGTase-I undergo additional Cterminal processing steps (Fig. 2). The C-terminal aaX is cleaved from the protein by a microsomal protease and the resulting C-terminal prenylated cysteine is carbolo/methylated. A protein activity that binds both farnesylated and geranylgeranylated proteins that contain an intact aaX C terminus has been postulated to play a role in these additional processing steps by localizing prenylated proteins to the membrane surfaces where the CaaX protease and methyltransferase activities reside (Thissen and Casey, 1993). Two Saccharomyces cerevisiae genes, Rcel and Afcl, are required for the C-terminal proteolysis of prenylated proteins (Boyartchuk et al., 1997). The AFCl protein is a zinc-dependent metalloprotease that is required for proteolysis of the yeast mating pheromone, a-factor, but is not essential for processing of yeast Ras. RCEl is essential for processing of both afactor and Ras. The mammalian protease activity responsible for processing of prenylated proteins has only been partially characterized. Proteolytic activity capable of releasing the Val-Ile-Met tripeptide from the tetrapepis localized to memtide substrate, N-acetyl-S-farnesyl-L-Cys-Val-Ile-Met, branes, is not affected by standard protease inhibitors, and displays properties consistent with a serine or cysteine protease (Ma et al., 1993). This proteolytic activity requires detergent for its solubilizationfrom membranes and chromatographs as a single peak of activity over gel filtration and anion exchange chromatography (Chen et al., 1996). Tetrapeptide inhibitors of the CaaX protease, such as RPI (Fig. 3), have been developed and have been reported to block Ras processing and function in cells (Chen et al., 1996). The mammalian enzyme(s) responsible for C-terminal methylation of prenylated proteins is also incompletely characterized. In yeast, the methyltransferase for CaaX proteins has been identified as Stel4 (Hrycyna and Clarke, 1990; Marr et al., 1990).Prenylcysteine-directed mammalian methyltransferase enzyme(s) is associated with microsomal membranes and utilizes S-adenosylmethionine as the methyl donor (Stephenson and Clarke, 1992). Methylation reactions are potentially reversible and, in this regard, a methylesterase has been described that is selective for prenylated cysteines containing methylesters (Tan and Rando, 1992). The ability to add or remove methyl groups from the C terminus of prenylated proteins suggests that these events could regulate the function of prenylated proteins, and there is some evidence to suggest that this does occur (see Section XIV).

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FPP

P

7--

0 P-0-P-0 I

0

1

0

PPP-Competitive m ' s

L-704,272

Manurnycin 0

L bi substrate FTi

BMS-186511 Fic;. 3. Representative inhibitors of prenyltransferases and other enzymes in the biosynthesis of prenylated proteins. Shown at the top are the substrates for the farnesylation of k-Ras (FPP) and the k-Ras CaaX (CVIM). See text for details and references.

X. Rab GGTase-ll

Rab proteins are digeranylgeranylated by GGTase-11, a heterodimeric enzyme containing a 50-kDa a subunit and a 38-kDa 6 subunit that share approximately 30% identity in amino acid sequence with the cdfi subunits

CVlM

CaaX Competitive FTl‘s

I

L-731,734

I L-739,749 (kCH3) L-744,832 (R=CHz(CH&

FTI-276 (R=H) sFTI-277 (R=CH3)

I

SOzCH,

sHwc

HzN

% “O H

FTI-265

NHz

n

8956 (R=H) 81086 (R=CHa)

L-745,631

0

SCH44342

/

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ROBERT B. LOBELL

GGTase-I lnhlbitor

GGTI-286: R=OCHB

I

GGTI-287: R=O-

HMG-CoA Reduction Inhibitor

Lovastatin

L

CaaX Proteare Inhibitor

Carboxymbthyitransferaselnhibltor

t?+n L-AFC(1)

"y 0

AFC

FIG.3-Continued

of FTase and GGTase-I (Armstrong et al., 1993).GGTase-I1 adds geranylgeranyl groups to each cysteine residue at the C terminus of Rab proteins, which end in CCXX, XXCC, or CXC sequences (Farnsworth et al., 1994).

PRENYLATION OF

Ray

GTPase PROTEINS

161

Rab proteins are not proteolyzed at their C termini, and only Rab proteins

with the CXC motif are carboxymethylated (Smeland et al., 1994). Prenylation of Rab proteins by GGTase-I1 requires a third protein called

Rab escort protein or Rep. Rep functions by presenting the unprenylated Rab protein to the catalytic GGTase-I1 d p heterodimer (Andres et al., 1993; Seabra et al., 1992). Mutational analysis of Rab proteins has shown that in addition to the C-terminal cysteines, internal Rab protein sequences are involved in the prenylation reaction (Wilson and Maltese, 1993). The effect of these mutations may be due to effects on the Rab-Rep interaction. Rep binds both unprenylated and prenylated Rab proteins; due to this property, the GGTase-I1 reaction in uitro is limited by the concentration of Rep. MonogeranylgeranylatedRab protein remains tightly bound to Rep, even in the presence of detergents, ensuring that the second geranylgeranyl group can be added by GGTase-11. Although digeranylgeranylated Rab exhibits a somewhat greater propensity to dissociate from Rep in the presence of detergents or phospholipids (Shen and Seabra, 1996) in vim the prenylated Rab likely remains bound to Rep until it is delivered to the correct intracellular membrane compartment (Alexandrovet al., 1994). In uiuo, delivery of the Rab protein to the correct membrane compartment presumably facilitates the dissociation of the prenylated Rab protein from Rep, although it is not known what directs the Rab protein to its correct membrane compartment. Another aspect of the Rab-Rep interaction that is not well understood concerns the low affinity of Rep for Rab-GTP. Because GTP is found at much higher concentrations than GDP in cells, newly synthesized, unprenylated Rab might bind GTP and then would be less able to bind Rep. One possibility is that a chaperone protein might bind newly synthesized Rab in a conformation that prevents GTP binding and allows the Rab to bind Rep (Desnoyers et al., 1996). Two Rep proteins, Repl and Rep2, have been identified. A defect in Repl function is responsible for choroideremia, a human retinal degenerative disease (Andres et al., 1993). Repl and Rep2 are 75% identical and are generally redundant in activity except for two known examples. Rab27, a protein found in high levels in the retina, has a somewhat higher affinity for Repl compared to Rep2, which might explain why the effects of choroideremia are limited to the retina (Seabra et al., 1995). Additionally, the prenylation rate of Rab3a is lower when Rep2 is the escort in the reaction (Cremers et al., 1994). Rep proteins are 30%identical in amino acid sequence to the Rab-GDI protein (Wu et al., 1996). X-ray crystallography of Rab-GDI has revealed that two of the most highly conserved regions between Rep and GDI are found on one face of GDI (Schalk et al., 1996). Mutational analysis of GDI suggests that this protein surface is involved in the interaction of

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GDI, and presumably Rep, with Rab proteins (Wu et al., 1996). Although there are similarities to the interaction of Rep and GDI with Rab proteins, there must be significant differences because only Rep can bind unprenylated Rab and thus only Rep can facilitate the geranylgeranylation reaction (Pfeffer et nl., 1995). In addition, GDI but not Rep apparently interacts with the effector domain on the Rab protein. A single mutation in the RablB effector domain abolishes the GDI-Rab binding interaction but does not affect the geranylgeranylation reaction, indicating that the mutant protein retains the ability to interact with Rep (Wilson et al., 1996). In cells, this RablB effector domain mutant was targeted to the correct intracellular compartment but was unable to cycle between membrane and cytosolic compartments (Wilson et al., 1996). This result is consistent with a model in which Rep functions in the delivery of prenylated Rab proteins to donor membranes, whereas GDI functions specifically in the recycling of Rab back to donor membranes after the vesicle fusion process has occurred. XI. Role of Prenylation in Membrane Binding and in Prokin-Prokin Interactions

Ras proteins containing Cys to Ser mutations in the CaaX are not prenylated and are not membrane bound (Willumsen et nl., 1984). Although farnesylation of Ras is clearly a critical component to its membrane localization, the proteolytic cleavage of the a& and the methylation of the farnesyl cysteine at the mature C terminus are also important. An in vitro system utilizing rabbit reticulolysates, reconstituted with or without microsomal membranes containing CaaX protease and methyltransferase activity, can produce Ras in various states of posttranslational processing. Utilizing this system, it was found that farnesylation of K-Ras4B in the absence of proteolysis and methylation results in only 20% of the K-Ras4B protein being associated with membrane fractions (Hancock et al., 1991a). Forty percent of farnesylated and proteolyzed K-Ras4B associated with membranes, whereas addition of the carboxymethylation activity led to 80% of the fully processed protein associated with the membrane. Similarly, a KRas4B CaaX mutant that can be farnesylated but not further processed is approximately 50% membrane associated in cells compared to a >90% association of the wild-type protein (Kato et ul., 1992). The importance of the carboxymethylation event to membrane binding is further illustrated by in vitro analysis of the binding of prenylated peptides to liposomes. Farnesylated peptides bind poorly to liposomes unless the farnesyl cysteine is methylated, whereas geranylgeranylated peptides bind reasonably well in the absence of methylation (Silviusand L’Heureux, 1994).The difference between farnesylated and geranylgeranylatedpeptides in their requirement

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for carboxymethylation for binding to membranes reflects the greater lipophilicity of the geranylgeranyl group. In addition to C-terminal farnesylation, proteolysis, and methylation, other mechanisms, including the addition of other lipid moieties in the case of H-Ras or the presence of multiple-charged amino acid residues in the case of K-Ras4B, contribute to the binding of Ras and other prenylated proteins to cellular membranes. A specific palmitoyltransferase covalently modifies H-Ras and N-Ras on cysteine residues in their C-terminal region with the 16-carbon lipid, palmitate (Liu et al., 1996). The palmitoylation reaction apparently requires that the proteins are first farnesylated and further processed because nonfarnesylated, bacterial-expressed H-Ras is not a substrate for the palmitoyltransferase (Liu et al., 1996). C h a n p g the two cysteines in H-Ras that are normally palmitoylated to serine residues prevents palmitoylation, results in a 10-foldreduction in membrane binding compared to the palmitoylated protein (Hancock et al., 1990), and significantly impairs its signaling ability (Dudler and Gelb, 1996).The C-terminal polybasic domain of K-Ras4B, which contains a stretch of six lysine residues adjacent to the CaaX, was shown to contribute significantlyto its membrane binding in cells (Hancock et al., 1990).Changing these lysines to glutamine impairs the membrane binding of K-Ras4.B; as the number of lysines in the polybasic region is progressively decreased, the affinity of the protein for membranes is also progressively decreased. Other prenylated proteins in the Ras superfamily also contain polylysine stretches adjacent to their Cadi. These studies indicate that all the C-terminal processing events, including prenylation, proteolysis, carboxymethylation, palmitoylation, as well as the polybasic domain of K-Ras4B, all contribute to the membrane binding of Ras proteins. However, mutations that abolish palmitoylation, proteolysis, or the polybasic domain only slightly impair the transforming ability of Ras proteins, whereas Cys to Ser mutations in the CaaX completely abolish the transforming ability (Hancock et al., 1990; Kato et al., 1992). These mutational studies show that the only processing event that is absolutely critical to the transforming ability of Ras is the farnesylation step. However, these transformation studies should be interpreted with caution because they involve overexpression of the Ras protein. A mutant version of Ras that is partially impaired in its membrane binding might reach the threshold level of signaling at the plasma membrane that is required for transformation of the cell only when the protein is overexpressed. Although the C-terminal lipidation of Ras is critical for its function, this requirement can be abrogated by artificially targeting the protein to membranes via introduction of a lipid functionality at the N terminus of the protein. This has been accomplished by introducing the v-Src N-

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myristoylation sequence at the N terminus of Ras (Buss et at., 1989). Furthermore, it has been suggested that the sole function of Ras is to tether the signaling molecules downstream of Ras to the plasma membrane. This is supported by the finding that a Raf kinase construct containing a CaaX motif is transforming to cells even in the presence of a dominantnegative Ras protein that can normally inhibit transmembrane signaling to the MAPK pathway (Leevers et al., 1994; Stokoe et al., 1994). Although the processed, prenylated C terminus of Ras mediates its membrane association, it is unclear what directs Ras to the plasma membrane rather than to other intracellular membrane compartments. It would seem that other signals are required for targeting Ras to the proper membrane compartment because farnesylated proteins other than Ras can be localized to other membrane structures such as the nucleus in the case of the lamins (Brown et al., 1992; Chen et al., 1991b; Reiss et aE., 1991) and the cytoplasmic surface of peroxisomes in the case of a protein of unknown function known as PXF (James et al., 199413). Although prenylation of Ras is important in localization of the protein to membrane surfaces, prenylation also plays a role in protein-protein interactions. For example, yeast Ras2 regulates the enzyme adenylyl cyclase, and farnesylation is required for this interaction. The interaction of unprocessed Rase with solubilized adenylyl cyclase is approximately 100fold less than when Ras2 is farnesylated (Kuroda et al., 1993). The interaction of H-Ras and K-Ras with the guanine nucleotide exchange protein, SOS, is influenced by prenylation (Porfiri et al., 1994; McGeady et al., 1997). SOS fails to catalyze nucleotide exchange of unprocessed H-Ras and K-Ras, and addition of the 10-carbon geranyl group fails to reconstitute the interaction. The exchange reaction occurs with Ras modified with farnesyl, analogs of farnesyl such as tetrahydrofarnesyl, and geranylgeranyl groups, and proceeds to a greater extent when it is fully processed (McGeady et al., 1997). Although these results indicate that SOS interacts, at least in part, with the prenyl group of Ras, they do not exclude the possibility that the prenyl group induces a structural change in Ras that enables it to interact with SOS. The contribution of prenylation and carboxymethylation to membrane binding is further illustrated from studies of other prenylated proteins, notably the heterotrimeric G-proteins. These GTPases are localized to the plasma membrane via myristoylation and palmitoylation of the G, subunit and prenylation, either farnesylation or geranylgeranylation, of the Gy subunit (see Table I and Higgins and Casey, 1996). Transducin, a Gprotein found in the retina, contains a farnesylated G, that is found in both methylated and unmethylated forms. Both farnesylation and methylation

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of G, contribute to the membrane binding of this protein (Parish and Rando, 1994). Prenylation also plays an important role in many aspects of proteinprotein interactions involving heterotrimeric G-proteins, including intrasubunit interactions, interactions between the G-protein and the transmembrane receptor, and perhaps interaction of the G-protein subunits and downstream signaling effectors. Heterotrimeric G-proteins dissociate into G, and Gpysubunits when ligand binds to the seven-transmembrane receptor to which the G-protein was originally bound. The G, dimer is extremely stable, and its initial assembly appears to be influenced by prenylation. The assembly of the Gpycomplex is thought to occur prior to prenylation and proteolytic processing of G,; this is suggested by the finding that proteolysis of the a& of prenylated G, impedes complex formation with G, (Higgins and Casey, 1994). The high-affinity interaction of Gp, with G, requires the prenylation of G, (Higgins and Casey, 1994) and the myristoylation of G, (Linder et al., 1991). Farnesylated peptides correspondmg to the C terminus of G, can inhibit the interaction of Gpywith G, and the degree of inhibition increases as the hydrophobicity of the prenyl group is increased by either methylation of the farnesylated peptide or geranylgeranylation of the peptide (Matsuda et al., 1994). The prenylation and methylation state of the G, subunit can also influence the interaction of the heterotrimeric G-protein with the seven-transmembrane receptor. In the case of transducin, both farnesylation and methylation are required for high-affinity binding to the receptor rhodopsin (Fukada et al., 1994), and as with the G,, and G, interaction, a farnesylated peptide corresponding to the C terminus of G, can disrupt the rhodopsin-transducin complex (Kisselevet al., 1994). Prenylation and methylation of G, is also critical for the interaction of G, with downstream effectors, as has been demonstrated in the regulation of a phospholipase CP, (Parish et al., 1995), although it is not clear if this effect is due to enhanced membrane binding of the fully processed Go, or to a specific G,,-phospholipase CP, interaction. In the case of Rab proteins, prenylation plays a role in the interaction of Rab with Rep and GDI. In addition to promoting the association of Rab with membranes (Overmeyer and Maltese, 1992), prenylation of Rab is required for binding to GDI (Musha et al., 1992).Although both monoand digeranylgeranylated Rab proteins can associate with GDI, the length of the prenyl group affects the ability of Rab to bind GDI because a Rab5 mutant with a farnesylation site in place of the geranylgeranylation sites binds weakly to GDI (Ullrich et al., 1993).Another system in which prenylation has been shown to affect protein-protein interactions is in the biosynthesis of hepatitis delta virus. Prenylation of the large antigen of hepatitis

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delta virus is required for its assembly with the hepatitis B surface antigen in the formation of hepatitis D virus particles (Hwang and Lai, 1993). XII. Role of Ras GTPase Family Members in Immunobiology: The Ras Pathway

Many of the components of the Ras signaling pathway, which were originally defined from work involving growth factor signaling in fibroblasts, have now been demonstrated in cells of the immune system. In T lymphocytes, the Ras pathway has been shown to be important in the immediate activation events triggered via the T cell antigen receptor (TCR), which leads ultimately to IL-2 secretion and upregulation of IL-2 receptors (IL2R). The Ras pathway is also involved in the proliferative events in T cells triggered by binding of IL-2 to its receptor (IL-2R) (Pastor et al., 1995). The Ras pathway is activated in other antigen receptor signaling systems related to the TCR, including the B cell antigen receptor (Cambier et al., 1994), and in mast cell activation via the high affinity receptor for IgE, FceRI (Fukamachi et al., 1993; Turner et al., 1995). The Ras pathway is involved in other aspects of lymphocyte biology, including the regulation of B cell function by the CD40 receptor (Gulbins et al., 1996a) and T cell activation via engagement of the GD3 disialoganglioside (Ortaldo et at., 1996). As is the case in growth factor receptor signaling in fibroblasts, signaling from lymphocyte antigen receptors and the IL-2R involves activation of Ras via the SOS guanine nucleotide exchange factor through a series of protein-protein interactions that initiates with tyrosine phosphorylation events (Quilliam et al., 1995). In the case of growth factor receptors, intrinsic tyrosine kinase domains in the receptor autophosphorylate receptor tyrosine residues which serve as adapter sites for the GrbYSOS complex, which in turn activates Ras. The TCR and the IL-2R lack intrinsic kinase activity but initiate the Ras pathway through receptor-associated kinases of the Src family, including ZAP-70, p56Ick,and ~ 5 9 s in " the case of the TCR, and through activation of ~ 5 6 'in ' ~the case of the IL-2R (Weiss and Littman, 1994).These kinases phosphorylate multiple substrates, including proteins that couple to the GrbYSOS complex. The phosphorylated proteins that link SOS/Grb2 to the TCR and IL-2R appear to be different. In the case of the TCR, a 36-kDa protein serves as the phosphoprotein adapter to Grb2 (Buday et al., 1994; Reif et al., 1994; Sieh et al., 1994), whereas for IL-2R, the Shc protein is phosphorylated by p56Ick,coupling the activated kinase to the GrbYSOS complex(Ravichandran and Burakoff, 1994). FcsRI activation of Ras in mast cells has also been shown to involve the GrbYSOS pathway (Turner et al., 1995); it is not clear which phospho-

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protein adapter couples the Src kinases associated with this receptor to GrbWSOS. The events downstream of Ras are fairly well understood in the case of 1995). Ras activation in response to signaling via the TCR (Pastor et d., TCR activation leads ultimately to upregulation of a transcription factor that activates the IL-2 promoter, known as nuclear factor of activated T cells (NFAT). NFAT is a complex of AP-1, itself a complex of the Fos/ Jun factors, and NF-ATp, a member of the c-re1 family of transcription factors, (€320, 1994). The pathway leading to activation of NFAT by Ras most likely involves activation of the MAPK pathway. Activation of Erk2 via Raf and MEK has been demonstrated in T lymphocytes (Izquierdo et al., 1993, 1994; Franklin et d., 1994). It is likely that NFAT activation by Ras ultimately involves activation of AP-1 through the induction of the cfos gene; this could occur via activation of the Elk-1 transcription factor by the Erk2 map kinase (Marais et al., 1993).As in the T cell, IgE receptor activation in the mast cell leads to activation of NFAT via the Ras-RafMek-map kinase-Elk-1 cascade (Turner and Cantrell, 1997). Additionally, another member of the Ras superfamily, Rac-1, has been implicated in NFAT activation in mast cells (Turner and Cantrell, 1997). In granulocytes such as the neutrophil, Ras is activated in response to proinflainmatory mediators. For example, Ras and its downstream effectors, Raf and MAPK, are activated in human neutrophils in response to the chemoattractants FMLP and C5a (Buhl et al., 1994; Worthen et al., 1994). The FMLP and CSa receptors are seven-transmembrane spanning G-protein coupled receptors, and the activation of the Ras pathway in neutrophils via these receptors is sensitive to pertussis toxin. The linkage between the G-protein coupled receptors and the Ras pathway in the neutrophil has not been firmly established but appears to involve the Src-family kinase Lyn. FMLP stimulates Lyn, which in turn binds and phosphorylates Shc; this could lead to Ras activation via phosphorylated Shc binding to GrbWSOS (Ptasznik et al., 1995). There is some data to suggest that the expression of proteins in the Ras pathway can be modulated in the neutrophil in viva in response to inflammatory stimuli. Neutrophils from bum patients contain elevated levels of Ras and Ras-GAP but reduced levels of Rapl, a Ras superfamily member that regulates the NADPH oxidase (Brom et al., 1993). Neutrophils from burn patients exhibit impaired chemotactic and phagocytic function, although it is not clear what role, if any, the elevation of Ras protein levels has in this impaired function. Although Ras activation plays a growth stimulatory role in T cells, there is evidence that in some settings, it can actually transduce growth inhibitory and apoptotic signals. For example, in a recent study it was shown that Ras negatively regulates calcium-dependent immediate early gene induction in

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lymphocytes (Chen et al., 1996). Furthermore, FAS-induced apoptosis in lymphocytes involves Ras activation through a pathway involving ceramide generation via a sphingomyelin signaling pathway (Gulbins et al., 1995). This was indicated by an increase in Ras-GTP levels upon FAS stimulation of Jurkat cells and by the inhibition of FAS-induced apoptosis by a dominant-negative Ras mutant. FAS-induced events downstream of Ras involve the generation of superoxide anions (Gulbins et al., 1996b), which have also recently been implicated in signaling events downstream of Ras induced by mitogenic stimulation of NIH-3T3 fibroblasts (Irani et al., 1997). Ras activation is also involved in apoptosis induced by the cytokine, tumor necrosis factor (TNF) (Trent et al., 1996). Like FAS, TNF activates a sphingomyelin pathway leading to ceramide production, which has been shown to cause phosphorylation and activation of Raf via a CAP kinase (Yao et al., 1995). It remains to be seen whether FAS-induced apoptosis, which involves ceramide generation, also results in CAP kinase and Raf activation. It is apparent that further studies are needed to sort out the tangle of signaling pathways in lymphocytes and other cells that involve Ras, which can result in a variety of responses, including activation, growth, or cell death. XIII. The Rho/Rac Pathway and leukocyte Function

Most cells of the immune system are motile and migrate in response to specific chemotactic stimuli. Leukocyte migration involves integrindependent adhesioddeadhesion events and changes in the cell cytokeleton, including actin polymerization and membrane ruffling (Stossel, 1993). There is direct evidence from studies in immune cells that integrindependent adhesion events and changes in the cytoskeleton involve Rho proteins. The involvement of Rho proteins in chemoattractant-induced effects on integrin-dependent adhesion was illustrated in a recent paper in which a lymphoid cell line, transfected with the FMLP or IL-8 chemoattractant receptors, showed agonist-stimulated activation of nucleotide exchange on RhoA within seconds (Laudanna et al., 1996). Furthermore, in this paper it was demonstrated that Clostridium botulinurn toxin C3 ADP ribosyltransferase, an enzyme that inhibits Rho function through ADP ribosylation, blocked agonist-induced lymphocyte a 4 p l integrin-mediated adhesion to vascular cell adhesion molecule-1 and also blocked neutrophil p2 integrin-mediated adhesion to fibrinogen. The involvement of Rho proteins in cytoskeletal organization in leukocytes is further supported by the finding that the C3 ADP ribosyltransferase inhibits actin microfilament formation and chemoattractant-induced motility in neutrophils (Stasia et al., 1991). The C3 ADP ribosyltransferase also inhibits events that involve

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leukocyte cell-cell interactions, including the CD1ldCD18-dependent homotypic aggregation of B cells (Tominaga et al., 1993) and the cytolytic function of cytotoxic T cells (Lang et al., 1992). Additionally, CDC42Hs is required for the polarization of T cells toward antigen presenting cells (Stowers et al., 1995). Further evidence for the involvement of Rho family members in leukocyte cytoskeletal organization and cell motility comes from studies of the Wiskott-Aldrich syndrome (WAS).WAS is a hematopoietic disorder characterized by thrombocytopenia, recurrent infections, and eczema (Ammann and Hong, 1989).The cellular abnormalities in WAS patients include cytoskeletal defects in T cells and platelets (Molina et al., 1992) and defective neutrophil chemotaxis (Ochs et al., 1990). The genetic defect in WAS has been mapped by positional cloning (Derry et al., 1994). The involvement of the WAS protein (WASP) in regulation of actin polymerization was demonstrated by the recent finding that WASP binds to CDC42Hs (Symons ef al., 1996). Overexpression of WASP produced intracellular clusters of the protein that were highly enriched in polymerized actin; formation of these clusters was inhibited by coexpression of dominantnegative CDC42Hs-Nl7. Thus, mutation of WASP, a downstream effector of CDC42Hs, can have profound effects on immune cell functions that involve regulation of the cytoskeleton. XN. Regulation of the Neutrophil NADPH Oxidose by Roc and Rap

Activation of neutrophils by proinflammatory mediators, including the chemoattractant peptides FMLP and C5a, leads to a number of cellular responses including the generation of toxic and microbiocidal oxygen metabolites such as superoxide anion and hydrogen peroxide. This event, termed the respiratory burst, is due to activation of the multisubunit NADPH oxidase complex (Chanock et al., 1994). The oxidase consists of a heterodimeric flavocytochrome b that consists of 22- and 91-kDa transmembrane protein components (Parkos et al., 1987). The oxidase is also composed of two cytoplasmic proteins, p47P""' and p67P"", that bind tightly to the transmembrane oxidase components upon cell activation (Clark et al., 1990) . The inability to generate the respiratory burst seriously compromises the host defense system, as evidenced by individuals with chronic granulomatous disease, a hereditary condition caused by a mutation in one of the NADPH oxidase components (Dinauer, 1993). The NADPH oxidase is regulated by two geranylgeranylated GTPbinding proteins, RaplA and Rac. The involvement of these GTP-binding proteins was first suggested by the requirement for guanine nucleotides in the activation of the oxidase in a cell-free system (Gabig et al., 1987),

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and the involvement of RaplA in the NADPH oxidase system was first suggested by its association with purified neutrophil flavocytochrome b (Quinn et al., 1989).This interaction is functionally important in NADPH oxidase function because cytosol immunodepleted of RaplA is unable to reconstitute oxidase activity unless recombinant RaplA is added back (Eklund et al., 1991). Furthermore, dominant inhibitory mutants of RaplA inhibit the NADPH oxidase when expressed in differentiated HL-60 cells and EBV-transformed B cells (Gabig et al., 1995; Maly et al., 1994). Rac was first shown to play a role in regulation of the NADPH oxidase by experiments in the cell-free system (Abo d. al., 1991; Knaus et al., 1991).In unstimulated cells, Rac is present in the cytosol and is complexed with RhoGDI (Aboet al., 1994). Upon immunologic activation, Rac dissociates from RhoGDI and translocates to the plasma membrane (Abo et al., 1994; Quinn et al., 1993). Rac translocates independently of the p47PhoX and p67ptiox proteins and interacts with both the p2Wp91 flavocytochrome subunits and with p67P’’0xvia its effector domain (Diekmann et al., 1994; Heyworth et al., 1994). The role of the Rac geranylgeranyl group in activation of the NADPH oxidase has been examined. Unprenylated Racl was found to activate the oxidase in the cell-free system, but only when it was preloaded with GTPyS (Heyworth et al., 1993).This suggested that prenylation of Rac is required only in the activation of Rac itself, presumably through an interaction of Rac with a guanine nucleotide exchange protein, but that prenylation is not absolutely required for the activation of the oxidase by Rac. However, recent evidence suggests that prenylation of Rac is an important determinant in the activation of the oxidase. It was shown that prenylated Racl and Race are significantly more effective in activating the oxidase in vitro than the nonprenylated forms (Kreck et nl., 1996). Racl is a more effective activator than Race; this is likely due to the presence of a polybasic domain near the CaaX of Rac, as is found in K-Ras4B. The polybasic domain of Racl is an important determinant of membrane binding because elimination of only one of the charged residues markedly reduces its activation of the oxidase. The polybasic domain contributes to the membrane binding of Rac via electrostatic interactions. This is indicated by the finding that the activation of the oxidase by Racl but not by Rac2 is sensitive to salt concentration, and that addition of acidic phospholipids to reconstituted oxidase subunits enhances the activation by Racl but not by Rac2. The exact role that Rac plays in the regulation of the NADPH oxidase remains unclear, although it is reasonable to propose that its function may be to anchor the soluble p47Phohat the plasma membrane in proper orientation to the transmembrane oxidase components (Kreck et al., 1996).

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XV. Regulation of Phospholipase D by RhoA

There may be another level of regulation of the NADPH oxidase by a Rho protein. Phosphatidic acid, a product of the hydrolysis of phospholipids by phospholipase D (PLD),enhances oxidase activity (Agwu et al., 1991). PLD activity in human neutrophils is activated by GTPyS and this effect was suggested to be mediated by a Rho protein because Rho-GDI can inhibit the activation of PLD (Bowman et nl., 1993). Depletion of Rho from membranes with Rho-GDI, followed by add back of recombinant Rho to the membrane, showed that RhoA but not Cdc42Hs could reconstitute PLD activity in rat liver membranes and in human neutrophil membranes(Kwak et al., 1995; Malcolm et nl., 1994). Evidence for Rho protein involvement in receptor-mediated PLD activation in intact cells has been obtained through the inactivation of Rho proteins with either the Clostridiurn botulinurn C3 ADP ribosyltransferase or the Clostridiurn dificile toxin B, a Rho glucosylation enzyme (Malcolm et al., 1996; Schmidt et al., 1996). RhoA activation of PLD has also been implicated in IgE receptor-mediated mast cell activation because the C. dificile toxin B abolishes antigeninduced PLD activation and granule enzyme release (Ojio et al., 1996). However, the involvement of RhoA in the activation of PLD has been questioned by a recent study involving HL-60 cells; this study reported that depletion of RhoA from membranes with Rho-GDI had no effect on PLD activity and attributed the activation of PLD by GTPyS to the GTPbinding protein, Arf (ADP ribosylatioii factor) (Martin et al., 1996). XVI. Role of C-Terminal Methylation of Prenylated Proteins in NADPH Oxidase Regulation and Other Leukocyte Functions

It has been suggested that the C-terminal inethylation of Rac and Rap proteins is regulated and plays a role in the translocation of these proteins to the plasma membrane upon FMLP-induced activation of the NADPH oxidase in neutrophils. The amount of carboxymethylation of Ras-related proteins in neutrophils, including Rac and Rap, increases in response to FMLP or nonhydrolyzable GTP analogs, both in intact cells and in cell lysates (Philips et al., 1993). Furthermore, N-acetyl-S-trans,trans-farnesylr,-cysteine (AFC) (see Fig. 3), an inhibitor of the carboxyinethyltransferase, effectively inhibits FMLP-induced superoxide generation, whereas N acetyl-geranylcysteine, a poor inhibitor of the methylase, does not inhibit superoxide generation. In neutrophils, prenylcysteine-directed carboxymethyltrarisferase activity is localized to the plasma membrane (Pillinger et nl., 1994). This methylase activity is dependent on phosphatidic acid, a lipid that increases in concentration upon neutrophil activation. These data suggest that upon activation of neutrophils with FMLP, Rac is released

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from its interaction with the GDI protein and then translocates to the plasma membrane where it becomes carboxymethylated and participates in the activation of the NADPH oxidase. Carboxymethylation of prenylated proteins has also been suggested to play a role in other aspects of leukocyte activation. The heterotrimeric Gprotein subunit Gy2 is carboxymethylated in response to FMLP, and AFC inhibits the reaction (Philips et al., 1995). In addition to its inhibition of FMLP-mediated superoxide generation in neutrophils, AFC inhibits FMLP-mediated homotypic aggregation but enhances both the FMLPinduced upregulation of CDllbICD18 and the granule enzyme release in these cells (Philips et al., 1995). Furthermore, agonist-mediated activation of human platelets (Huzoor-Akbar et al., 1993) and the chemotaxis of mouse peritoneal macrophages toward lipopolysaccharide (LPS)-activated serum are also inhibited by AFC (Volker et al., 1991). Although AFC inhibits a variety of leukocyte activation-dependent responses, the role of the carboxymethyltransferase in these processes has been questioned (Ma et al., 1994). These authors show that several AFC analogs that are not inhibitors of the methyltransferase can nonetheless inhibit agonist-induced platelet aggregation. Furthermore, they found that the KM of farnesylcysteine for the platelet methyltransferase in vitro is -28 PM, whereas AFC inhibits platelet aggregation in the range of 1-10 PM (Huzoor-Akbar et al., 1993), suggesting that the methyltransferase is not the target of AFC in platelets. Additional studies to delineate the mechanism of inhibition of leukocyte activation by AFC are clearly necessary. XVII. Role of Rab Proteins in Membrane Transport in Leukocytes

Membrane transport functions that play particularly important roles in the biology of the immune system include endocytosis mediated via immunoglobulin Fc receptors and complement receptors. These types of receptor-mediated endocytosis are important in the clearance of foreign antigens by phagocytic cells and in the presentation of antigens via the MHC class I1 pathway by antigen presenting cells. At least four distinct Rab proteins, Rab4, Rab5, Rab7, and Rab9, play a role during various stages of endocytosis (Bottger et al., 1996; Rybin et al., 1996; Soldati et al., 1995). However, studies on the role of these proteins in the endocytic process in cells of the immune system are lacking. Another important immune system function that involves membrane transport is exocytosis, also known as degranulation or regulated secretion. For example, many inflammatory events including allergic reactions are triggered by antigen binding to high-affinity IgE receptors on mast cells, resulting in the rapid release of proinflammatory mediators from preformed

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secretory granules. Other granulocytes, including basophils, neutrophils, and eosinophils, degranulate in response to inflammatory stimuli. There is significant data from studies in leukocytes demonstrating that Rab proteins play an important role in exocytosis. The involvement of Rab proteins in exocytosis was first suggested by the ability of nonhydrolyzable GTP analogs to trigger mast cell degranulation when delivered through a patch pipette (Fernandez et al., 1984). GTP analogs have also been shown to trigger exocytosis in other granulocytes, including neutrophils (Nusse and Lindau, 1988) and eosinophils (Nusse et al., 1990). Several studies have implicated the Rab3A protein in the GTP-dependent exocytotic process. Mast cell degranulation is triggered by the injection of peptides corresponding to the effector domain of Rab3A (Oberhauser et al., 1992). Rab3A appears to play a role in regulated exocytosis in other cells because application of the Rab3A effector domain peptide to permeabilized pancreatic acini, chromafin cells, and insulinsecreting cells also triggers secretion (Nuoffer and Balch, 1994). Further pharmacological evidence for the involvement of Rab3A in exocytosis comes from the finding that prenylcysteine analogs stimulate exocytosis in permeabilized HIT-T15 cells (Regazzi et al., 1995). These authors suggest that Rab3A might normally inhibit exocytosis, and that the Rab3A effector peptide or the prenylcysteine analogs stimulate exocytosis by disrupting an inhibitory interaction between the prenylated Rab3A protein and a Rab effector protein. That Rab3A might act as a negative regulator of exocytosis is supported by the finding that microinjection of Rab3A antisense oligonucleotides enhanced exocytosis in adrenal chromaffin cells (Johannes et al., 1994). However, another study found the opposite result for a different isoform of Rab; in anterior pituitary cells microinjection of Rab3B antisense oligonucleotides inhibited regulated exocytosis but did not affect constitutive secretion or endocytosis (Liedo et al., 1993). The involvement of the Rab3 protein in exocytosis is equivocal. Although rat peritoneal mast cells express the Rab3B and Rab3D isoforms (Oberhauser et al., 1994), guinea pig eosinophils do not express any known isoforms of Rab3, even though they degranulate in response to nonhydrolyzable GTP analogs (Lacy et al., 1995). It has been suggested that other Rab proteins could play a role in exocytosis. In resting human neutrophils, Rab5A is localized to both membranes and cytosol, and upon challenge with PMA there is increased membrane association of the protein and a concomitant decrease in the cytosolic pool (Vita et al., 1996). The time course for the increased membrane association parallels the time course of exocytosis, suggesting that Rab5A might play a role in the secretory process. Alternatively, regulation of exocytosis by GTPases might involve proteins other than, or in addition to, Rab proteins because some members

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of the heterotrimeric G-protein family have been implicated in this process (Aridor et al., 1993). Although there is significant evidence to suggest a role of Rab proteins in exocytosis, further studies are required to understand their exact involvement in this process. XVIII. Regulation of Vesicular Transport by Rho Proteins

Although the Rab family of GTP-binding proteins is well known for its function in vesicular transport, there is an increasing appreciation for the involvement of Rho family members in these transport processes. For example, activated mutants of RhoA and Racl impair the formation of clathrin-coated vesicles in cells and in a reconstituted cell-free system (Lamaze et al., 1996). Cdc42Hs may also be involved in membrane transport because it is localized to the Golgi apparatus and its intracellular distribution is affected by brefeldin A, an agent that has profound effects on vesicular transport (Erickson et al., 1996).Additionally, a newly discovered Rho family member, RhoD, was shown to regulate cell morphology and endosome dynamics in a variety of mammalian cell types, including the macrophage cell line J774 (Murphy et al., 1996). Overexpression of wild-type RhoD or a GTPase-defective RhoD caused striking changes in cell morphology, including the formation of extended membrane processes that protruded from the body of the cell that were enriched in F-actin. This was accompanied by the disappearance of actin stress fibers and the disassembly of focal adhesion complexes in the cell body. Furthermore, wild-type RhoD and the RhoD mutant were localized to the plasma membrane and endosomes, and the RhoD mutant dramatically reduced the motility of endosomes in the cell. These studies indicate that RhoD regulates the movement of endosomes via a process that may depend on actin stress fibers. XIX. Other Prenylated Proteins

Several other prenylated proteins that might play roles in leukocyte function have been identified. Two interferon-? ( IFN-.)I)inducible GTPbinding proteins of unknown function have been identified in human fibroblasts (Cheng et al., 1991). One of these proteins, huGBP1, contains a C-terminal CTIS sequence predictive of modification by farnesyltransferase, whereas the other, huGBP2, contains a CNIL sequence at its C terminus that predicts modification by GGTase-I. HuGBPl and its murine homolog are induced by IFN-y and LPS in human monocytes, the human promyelocytic HL-60 cell line, and in murine macrophages, and their prenylation is sensitive to farnesyltransferase inhibitors (Nantais et al.,

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1996).Another potentially important prenylated protein is the yeast YDJl protein and its human homolog, hDJ2. The yeast YDJl protein is a farnesylated protein that functions as a molecular chaperone and is involved in cell cycle regulation (Yaglom et al., 1996).Other prenylated proteins that could be involved in immune cell function are two protein tyrosine phosphatases referred to as PTPcm (Cates et al., 1996).These phosphatases can transform human epithelial cells when overexpressed, and thus may normally play a role in regulating cell growth. XX. Prenyltransferase Inhibitors

The importance of the Ras pathway in cellular transformation and cancer, and the discovery that Ras requires farnesylation for its function, sparked the development of FTase inhibitors as potential chemotherapeutic agents. HMG-CoA reductase inhibitors such as lovastatin that inhibit prenylation of both farnesylated and geranylgeranylated proteins through their inhibition of isoprenoid synthesis, existed even before the discovery of protein prenylation and have been valuable tools for understanding the biological roles of prenylation. However, HMG-CoA reductase inhibitors have been considered unsuitable as clinically useful inhibitors of Ras function because they inhibit the biosynthesis of downstream metabolites in the mevalonate pathway, including cholesterol, dolicliol, and ubiquinone, as well as the prenylation of both farnesylated and geraiiylgeranylatedproteins. The findings that tlie CaaX motif itself is the minimal essential element for substrate recognition and catalysis by FTase and GGTase-I and that substitution of tlie second aliphatic amino acid within the CaaX with an aromatic amino acid converts the CaaX peptide substrate into a competitive inhibitor (Brown et al., 1992; Goldstein et al., 1991; Reiss et al., 1990; Schaber et al., 1990) serve as a starting point for the development of potent, cell active inhibitors of FTase. A number of CaaX peptidomimetic compounds that display excellent selectivity for FTase inhibition compared to GGTase-I inhibition have been reported, including L-731,734,BZA-SB, and BS81 (see Fig. 3) (Garcia et al., 1993;James et al., 1993; Kohl et al., 1993).These farnesyltransferase inhibitors ( FTIs) are modified CaaX peptides that lack peptide bonds and are therefore resistant to hydrolysis by proteases. Additionally, many of the first peptidomimetics were made as prodrugs, containing an esterified C-terminal carboxylate group that eliminates the charged nature of the molecule, making it permeable to cell membranes. They are prodrugs because significant activity against FTase requires generation of the free carboxylate through the action of cellular esterases. Nonpeptide mimetics that lack the C-terminal carboxylate and/or tlie sulfhydryl moeity of the

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cysteine residue in the CaaX have also been developed (Bishop et al., 1995; Hunt et al., 1996; Vogt et al., 1995;Williams et al., 1996).In addition to CaaX competitive inhibitors, compounds competitive with FPP such as manumycin, as well as bisubstrate analogs competitive with both CaaX and FPP, have been developed (Fig. 3) (Haraet al., 1993;Pate1 et al., 1995). The CaaX competitive FTIs can block the famesylation of Ras and other FTase substrates in cells (Garcia et al., 1993; James et al., 1993; Kohl et al., 1993) and, in general, are more potent in cells than FPP competitive compounds (Hara et al., 1993).The efficacy of these compounds in inhibiting farnesylation in cells is illustrated by their effects on the Ras signaling pathway. FTIs inhibit many aspects of the transformed phenotype that are induced through the introduction of oncogenic H-ras into rodent fibroblasts, including anchorage-independent cell growth, rapid growth in monolayer culture, and alterations in cell morphology (James et al., 1993; Kohl et al., 1993; Prendergast et al., 1994). FTIs inhibit the formation and growth of rodent and human xenograft tumors in nude mice (Hara et al., 1993; Kohl et al., 1994; Sun et al., 1995). Additionally, the FTI L-744,832 is efficacious in a transgenic mouse model of mammary cancer (Kohl et al., 1995). In this model, oncogenic H-ras is expressed under control of the MMTV promoter, which induces mammary and salivary carcinomas (Sinn et al., 1987). Daily treatment of tumorbearing mice with L-744,832 induced a rapid regression of the tumors, and continual treatment prevented the reappearance of new tumors (Kohl et al., 1995). Cultured cells growing under anchorage-independent conditions undergo apoptosis in response to FTI treatment, suggesting that the rapid tumor regression induced by FTI treatment in the H-ras oncomouse model might also be due to apoptosis (Lebowitz et al., 1997). No detectable toxicity has been reported in animal studies involving FTI treatment. The lack of toxicity was unanticipated, given the ubiquitous importance of Ras in cell proliferation. One explanation for the lack of global toxicity in the face of dramatic effects on tumor growth is that many of the published studies used tumors that are driven by activated H-Ras, which is a relatively poor substrate for FTase and is thus easily inhibited. For example, the &, of FTase for H-Ras, which has a CaaX where X = ser, is much higher than the K,,, of FTase for K-Ras, where X = met (James et al., 1995). Additionally, 10-fold higher concentrations of the BZA-5B FTI are required to block farnesylation of the nuclear lamins compared to those for H-Ras (Dalton et al., 1995). Another explanation for the lack of toxicity in normal tissues is that transduction of growth proliferative signals in normal cells may rely on other forms of Ras, such as K-Ras and N-Ras, or Ras-related proteins such as R-RasmC21. This is suggested by studies that showed that FTIs did not inhibit the EGF-

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stimulated activation of MAPK in nontransformed cells but did inhibit the MAPK activation induced by oncogenic H-ras (James et al., 1994a). Additionally, cross-prenylation of some farnesylated proteins by GGTaseI in the presence of an FTI blockade might explain the lack of toxicity. R-RasZflC21, which is capable of triggering malignant transformation (Graham et al., 1994), as well as K-Ras4B are prenylated by both FTase and GGTase-I in vitro (James et al., 1995; Carboni et al., 1995) and might remain functional and transduce growth signals in normal tissues treated with an FTI. The finding that K-Ras4B, which is the predominant form of mutated Ras associated with cancer (Barbacid, 1987), is prenylated by GGTase-I as well as FTase in vitro (James et al., 1995) has raised important questions concerning the development of FTIs as chemotherapeutic agents. Although K-Ras4B is found as a farnesylated protein in vivo (Casey et al., 1989),it remains prenylated in FTI-treated cells (James et al., 1996), and preliminary data suggest that this is due to cross-prenylation by GGTase-I (Lerner et al., 1997; Pai et al., 1996; Rowel1 et al., 1997). Furthermore, K-Ras4B containing an altered CaaX sequence (CVIL) that is presumed to be exclusively a GGTase-I substrate is transforming to cells (Hancock et al., 1991b; Kato et al., 1992), suggesting that geranylgeranylated K-Ras4B in FTItreated cells would be functional. Sebti, Hamilton, and coworkers have further explored the issue of K-Ras4B cross-prenylation through their development of prenylation inhibitors that are more specific for GGTase-I compared to FTase. These compounds were derived from FTI peptidomimetics by replacing the methionine residue of an FTI peptidomimetic with leucine in the X position of the CaaX (Fig.3) (Lerner et al., 1995). Although one of these compounds was reported to block K-Ras processing in NIH-3T3 cells and to inhibit MAP kinase activation, recent data suggest that a combination treatment with both an FTI and a GGTase-I inhibitor is required to effectively inhibit K-Ras4B prenylation in human tumor cell lines (Lerner et al., 1997). Although FTIs alone may not inhibit K-Ras4B prenylation, FTIs can inhibit the anchorage-independent growth of a variety of cell lines derived from human tumors including those containing K-Ras4B mutations (SeppLorenzino et al., 1995; Nagasu et al., 1995). The sensitivity of these tumor lines to growth inhibition by the FTI varied greatly and was independent of the ras mutational status of the cell. This suggests that human tumor cell proliferation can be regulated by farnesylated proteins in addition to Ras. One such protein may be RhoB, a member of the Ras superfamily of GTPases that can be both farnesylated and geranylgeranylated in vivo (Adamson et al., 1992). FTI treatment of cells disrupts the intracellular localization of this protein, and cells transformed with an FTI-resistant

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form of RhoB containing an N-myristylation site require 10-fold higher concentrations of FTI to be growth inhibited (Lebowitz et al., 1995). Growth inhibition by FTIs might involve multiple mechanisms because the processing of at least 18 cellular proteins is affected by FTI treatment (James et al., 199413). In addition to the potential of FTIs for cancer treatment, GGTase-I inhibitors might also have potential as chemotherapeutics. HMG-CoA reductase inhibitors and the GGTase-I inhibitor, GGTI-287 (Fig. 3),inhibit the proliferation of cultured cells through a mechanism that involves growth arrest in the GI phase of the cell cycle (Vogt et al., 1996). Progression of a cell from GI into S phase involves the ubiquitin-dependent degradation of the p27 cyclin-dependent kinase inhibitor (Pagan0 et al., 1995), and the HMG-CoA reductase inhibitor pravastatin prevents the elimination of p27 through a mechanism that appears to involve geranylgeranylated Rho proteins (Hirai et d., 1997). The involvement of geranylgeranylated Rho proteins in p27 elimination is suggested by the finding that in pravastatintreated cells, addition of liposomes containing the GGTase-I substrate GGPP but not the FTase substrate FPP results in a decrease in p27 protein levels and progression of the cells through GI into S. Furthermore, the Rho inactivator, C3 ADP ribosyltransferase,prevents the ability of GGPP to stimulate progression into S phase in pravastatin-treated cells. Additionally, both lovastatin and a GGTase-I inhibitor block the PDGF-induced tyrosine phosphoylation of the PDGF receptor (McGuire et al., 1996). A Rho protein could be involved in this aspect of signaling because the PDGF type B receptor has been found to associate with Rho (Zubiaur et al., 1995). The ability of GGTase-I inhibitors to induce G, arrest, to inhibit multiple aspects of signal transduction, and to block the prenylation of KRas4B in conjunction with FTI treatment suggests that these agents might also be suitable as chemotherapeutics. In this regard, preliminary studies indicate that GGTase-I inhibition can block the growth of several human tumor lines in nude mice (Sun et al., 1997). MI. Effects of Prenylation Inhibitors on Leukocyte Function

Although FTase inhibitors show little toxicity in animal models, the potential effect of these inhibitors on the function of the immune system has not been adequately addressed. The HMG-CoA reductase inhibitor lovastatin inhibits both proximal and distal signaling events in the human Jurkat T cell line and in normal human peripheral blood mononuclear cells activated through the TCR (Goldman et al., 1996). In Jurkat cells, lovastatin inhibited both the processing of Ras and the activation of MAPK. Additionally, TCR signaling events that are presumably independent of

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Ras were inhibited, including mobilization of intracellular calcium, inositol phosphate production, and tyrosine phosphorylation, suggesting the involvement of a prenylated protein other than Ras in these aspects of TCR-mediated signaling. The effect of lovastatin on these Ras-independent signaling events was specific to the T cell receptor because calcium signaling and inositol metabolism triggered by transfected type-1 muscarinic receptors were unaffected in these cells. The potential for prenylation inhibitors to affect diseases involving lymphocyte proliferation and/or differentiation is suggested by several other studies involving HMG-CoA reductase inhibitors. For example, lovastatin showed some efficacy in inhibiting chronic allograft rejection in an animal model (O’Donnell et al., 199.5). IIMG-CoA reductase inhibitors and zaragozic acid, an FTI isolated from natural products, can inhibit signaling, specifically inositol lipid metabolism, in human keratinocytes induced by inflammatory mediators such as PAF and bradykinin (Alaei et al., 1996). This suggests that prenylation inhibitors could ameliorate the symptoms of inflammatory skin diseases. Another cell of the immune system that is responsive to HMG-CoA reductase inhibition is the human macrophage; it has been shown that lovastatin inhibits the expression of the type I lipoprotein scavenger receptor gene in these cells (Umetani et al., 1996). The inhibition of lipoprotein scavenger receptor expression in niacropliages is likely not related to the efficacy of this cholesterol-lowering agent in cardiovascular disease management because the inhibition of gene expression occurred at concentrations (5-15 ~ L ) Mfar higher than the peak plasma concentration commonly achieved in patients treated with this agent. XXII. Conclusion

Members of the Ras superfainily of GTP-binding proteins regulate a wide variety of cellular processes, and many of members of this family have been shown to play an iinportant role in tlie function of irninune system cells. I expect that appreciation of the importance of these proteins in immunobiology will only continue to grow as studies of these proteins and tlie discovery of new family members progress. The C-terminal processing of the Ras superfainily of proteins, which depends on the action of prenyltransferases and other processing enzymes including the CaaX protease, the prenylcysteine-directed methyltransferase, and in some cases palmitoyltransferase, is critical to the function of these proteins. The development of specific inhibitors of these C-terminal processing enzymes, in particular, inhibitors of farnesyltransferase, has proceeded rapidly in recent years and has helped to illustrate the importance of protein prenylation in various cell functions. Studies of these prenylation inhibitors in irninune

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system function should be expanded. These studies would be valuable not only from a clinical standpoint but also to aid in our understanding of the importance of Ras superfamily members in the proper functioning of the immune system. ACKNOWLEDGMENTS I thank Dr. Jay Gibbs and Dr. Charles Omer of the Merck Research Labs for their advice and suggestions on the manuscript.

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