Proc. Natl. Acad. Sci. USAVol. 90, pp. 2281-2285, March 1993Biochemistry

Identification of Ras farnesyltransferase inhibitors bymicrobial screening

(gpal mutant/manumycin/competltive inhibition/antitumor activity)

MITSUNOBu HARA*, KAZUHITO AKASAKA*, SHIRO AKINAGA*, MASAMI OKABE*, HIROFUMI NAKANO*t,RUBEN GOMEZt, DOUGLAS WOODt, MISOOK UHt, AND FUYUHIKO TAMANOIt**Tokyo Research Laboratories and Pharmaceutical Research Laboratories, Kyowa Hakko Kogyo Co., Ltd., Asahi-machi 3-66, Machida-shi, Tokyo 194,Japan; and tDepartment of Biochemistry and Molecular Biology, The University of Chicago, 920 East 58th Street, Chicago, IL 60637

Communicated by John A. Glomset, December 10, 1992 (receivedfor review October 8, 1992)

ABSTRACT A microbial screen using a yeast strain withconditional deficiency in the GPA1 gene was carried out tosearch for inhibitors of protein farnesyltransferase (PFT). Astrain of Streptomyces was found to produce active compoundsnamed UCF1-A, UCF1-B, and UCF1-C. Structural determi-nation of these compounds revealed that UCF1-C is identical tothe known antibiotic, manumycin, whereas UCF1-A andUCF1-B are structurally related to manumycin. AH threeUCF1 compounds suppress the lethality of gpal disruption,with UCF1-C exhibiting the strongest activity. UCF1 inhibitsyeast as well as rat brain PFT. Fifty percent inhibition of yeastPFT activity is observed with 5 pAM UCF1-C. Kinetic analysesof the inhibition suggest that UCF1-C acts as a competitiveinhibitor of PFT with respect to farnesyl pyrophosphate,exhibiting a K; of 1.2 ,uM, whereas the same compound appearsto act as a noncompetitive inhibitor of PFT with respect to thefarnesyl acceptor, the Ras protein. UCF1-C shows significantactivity to inhibit the growth of Ki-ras-transformed fibrosar-coma, raising the possibility of its use as an antitumor drug.

Since activation of Ras protein by a point mutation is foundin a large number of cancer cells, ras has been identified asa predominant oncogene in human cancer cell (1). Thus,inhibition of the Ras function is thought to be a crucial targetfor cancer chemotherapy (2). The Ras oncoprotein p21 mustbe localized in the plasma membrane in order to function andtransform cells (reviewed in ref. 3). This membrane localiza-tion is facilitated by posttranslational modifications occurringat the C terminus of Ras. The first step in a series ofmodifications is farnesylation ofa conserved cysteine residuepositioned four amino acids from the C terminus. Followingfamesylation, the three C-terminal amino acids are pro-teolytically removed, and the newly formed farnesylcysteineresidue is methyl esterified.One promising pharmacological approach for inhibiting

oncogenic Ras activity in human malignancies would be tointerfere with Ras membrane localization by inhibiting thefarnesylation reaction (2). Inhibition of Ras farnesylation inXenopus oocytes was demonstrated with compactin (4).However, this drug inhibits hydroxymethylglutaryl-CoA re-ductase and acts as a general inhibitor of the isoprenoidpathway and, thus, is unlikely to be useful for specificallyantagonizing Ras function. On the other hand, an inhibitor ofRas farnesyltransferase, the key enzyme that catalyzes thefarnesylation (5), would not perturb other elements of themevalonate pathway and, therefore, would be expected to bea more effective antagonist of Ras function.

Inhibitors of protein farnesyltransferase (PFT) should alsoserve as valuable tools for the study of this enzyme. PFT

purified from rat brain is a heterodimer of a and ( subunits(5). Crosslinking of Ras to the , subunit suggests that thissubunit recognizes the substrate protein (6). A similar en-zyme is detected in yeast and has been shown to consist oftwo subunits, DPR1/RAM1 and RAM2 (7-10). Sequencehomology has been detected between DPR1/RAM1 and 13aswell as between RAM2 and a (11, 12). PFT is a member of aprotein prenyltransferase family which also includes proteingeranylgeranyltransferase (PGGT) I (13-15) and II (16, 17).PFT and PGGT I share the a subunit (13, 14). PGGT I ofSaccharomyces cerevisiae consists of CDC43/CAL1 andRAM2, the latter being shared with PFT (18, 19).We have used a microbial screen in which inhibitors of

farnesylation can be detected (20) and have screened micro-organisms, isolated from soil and plants, for their ability toproduce inhibitory compounds. A culture of newly isolatedStreptomyces was found to produce active compounds des-ignated UCF1. Isolation and structural characterization ofthese active compounds assigned them to the manumycinfamily of antibiotics (21, 22) and resulted in the identificationof two new compounds. In this study, we report the effect ofthese compounds on Ras PFT and Ras-activated murinetumors.

MATERIALS AND METHODSMaterials. [1-3H(N)]Farnesyl pyrophosphate (FPP) (20 Ci/

mmol, 1 Ci = 37 GBq) and [1-3H(N)]geranylgeranyl pyro-phosphate (GGPP) (20 Ci/mmol) were obtained from Du-Pont/NEN. RAS2CT1 protein is a truncated form of yeastRAS2 protein ending with the C-terminal sequence Cys-Ile-Ile-Ser (7). GST-CIIL is a glutathione S-transferase fusionprotein ending with C-terminal Cys-Ile-Ile-Leu (19). S. cere-visiae KMG4-8C (MATa ura3 his3 trpl leu2 gpal::HIS3)carrying a plasmid, pG1501, containing the GPAI gene con-trolled by the GAL] promoter (23) was provided by K.Matsumoto (Nagoya University).

Isolation of UCF1. The filtered mycelial cake from 0.6 literof culture of Streptomyces sp. strain UOF-1 was suspendedin acetone and stirred to elute active compounds. After themycelium was removed by filtration, the filtrate was con-centrated by evaporation under reduced pressure. The con-centrate was then extracted with ethyl acetate and applied toa column of silica gel. The column was developed withchloroform/methanol, 50:1, and active compounds elutedwere concentrated to dryness. Further purification was car-ried out by HPLC [YMC SH363-5 ODS 30 mm (i.d.) x 250mm] using methanol/50 mM potassium phosphate (pH 7),7:3, as a mobile phase. Three active fractions were pooled

Abbreviations: FPP, farnesyl pyrophosphate; GGPP, geranylgeranylpyrophosphate; PFT, protein famesyltransferase; PGGT, proteingeranylgeranyltransferase.tTo whom correspondence should be addressed.

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H,NN

R

0

O HN YR

0

Oxidized UCFI

R

ow CH3 CH3

NH CH3 CH3Co ~~~~~~CH3U I CH3 CH3 CH3

UCF1 H

UCF1-A

UCF1-B

UCF1-C

FIG. 1. Structure of UCF1-A, UCF1-B and UCF1-C and theiroxidized derivatives. Stereochemistry at the C-4 position (of thecyclohexenone ring) of UCF1-A and UCF1-C is R, whereas that ofUCF1-B is S.

separately, desalted by Diaion HP-20 column chromatogra-phy, and concentrated to obtain pure compounds. UCF1-A(57 mg), UCF1-B (82 mg), and UCF1-C (63 mg) were obtainedas yellow powder. NMR and MS studies revealed thatUCF1-A and UCF1-B are related to manumycin (21, 22) (Fig.1). Details ofthe structure determination ofthese compoundswill be published elsewhere. Oxidation and reduction ofUCF1 were performed by the method of Zeeck and col-leagues (22, 24). The adduct with thiol was prepared bymixing UCF1-C with three equivalents of 2-mercaptoethanolin 20mM Tris HCl (pH 7.5)/methanol, 1:1, for 120 min at 4°Cand was purified by HPLC.

Suppression of Lethality of gpal Disruption. The yeaststrain KMG4-8C carrying pG1501 was grown at 30°C tostationary phase in galactose medium (25). Agar plates wereprepared by adding 50 ,ul of the above culture to 50 ml ofglucose agar (25). Paper disks soaked in drugs were placed onthe glucose agar plates, the plates were incubated at 30°C for

3 days, and the diameters of the zones of growth weremeasured.PFT Assay. PFT was purified from S. cerevisiae cells

overexpressing DPRI and RAM2 (8). Rat brain PFT waspartially purified by the method of Reiss et al. (5). PGGTpurified from bovine brain was a gift of M. Gelb and K.Yokoyama (University of Washington). PFT assays werecarried out using RAS2CT1 protein and [3H]FPP as described(7) except that Tris HCl (pH 8) was used instead ofphosphatebuffer. PGGT assays were carried out using GST-CIILprotein and [3H]GGPP as described (19) except that Tris HCl(pH 7.4) was used instead of phosphate buffer. Since UCF1was found to be inactivated by thiol, transferase assays wereperformed in the absence of dithiothreitol. Incubation wascarried out at 37°C.

Antitumor Activity. The K-BALB isograft line (26) wasestablished by subcutaneous inoculation of the culturedKi-ras-transformed murine fibrosarcoma cells into adult maleBALB/c mice (20-25 g; Japan Charles River, Atsugi). TheHT1080 xenograft line (26) was established by subcutaneousinoculation of cultured human fibrosarcoma HT1080 intoadult male BALB/c-nu/nu mice (23-27 g; Nippon Clea,Tokyo). For evaluation of antitumor activity, tumor volumewas calculated (27). Drug efficacy was expressed as thepercentage of the mean V/V0 value against that ofthe controlgroup, where V is the tumor volume at the day of evaluationand V0 is the tumor volume at the day of the initial treatmentwith the drug. Drugs were administered intraperitoneallydaily for 5 days from day 0 to day 4. Murine fibrosarcomaK-BALB and human fibrosarcoma HT1080 were trans-planted 5 days before the drug administration. Statisticalanalysis was done by Student's t test (two-tailed).

RESULTSMicrobial Screen for Inhibitor of PFT. In S. cerevisiae, the

posttranslational modifications of Ras protein, including far-nesylation, share a set of modifications with STE18 protein,the y subunit of the yeast guanine nucleotide-binding protein(G protein) that is involved in mating-pheromone signaltransduction (20). The same genetic and pharmacological

FIG. 2. Suppression of the lethality of gpal disruption by UCF1. Filters soaked in drugs were placed on plates seeded with the gpal mutant.After 3 days at 30°C, the plates were photographed. Drugs assayed: 50 ,g (A) and 10 ,ug (a) of UCF1-A; 50 ,ug (B) and 10 ,ug (b) of UCF1-B;50 ug (C) and 10 ,ug (c) of UCF1-C.

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treatments that block the processing ofRas protein also blockSTE18 processing and rescue gpal lethal mutants that aredisrupted in the a subunit of the yeast G protein (4, 20). Thisrescue occurs because the growth-inhibitory action of theG-protein fly subunit complex is blocked. As previouslyreported (20), this allows the development of a microbiallybased screen in which inhibitors of PFT can be detected bytheir ability to restore the growth of the gpal mutant. In thepresent study, we developed the following assay. A GPAIdisruption strain, KMG4-8C, carrying the GPAI gene con-trolled by the GAL] promoter was grown to stationary phasein galactose medium. Agar plates were then prepared byadding the above mycelia to glucose agar. In the presence ofglucose, GPAI was not expressed and the cells did not grow.Paper disks, soaked in samples to be tested, were placed onthe glucose agar plates and incubated at 30°C for 2 days.Active compounds could suppress the lethality of gpaldisruption and thereby allow a halo of growth around thepaper disks (Fig. 2). This method was used to screen micro-organisms isolated from soil and plants for their ability toproduce inhibitors. A strain of Streptomyces freshly isolatedfrom soil collected in Sakai, Japan, was found to produceactive compounds. Isolation and purification afforded threeactive components, UCF1-A, -B, and -C. NMR and MSstudies revealed that UCF1-C is identical to the knownantibiotic manumycin (21, 22), whereas UCF1-A andUCF1-B are structurally related to manumycin but differfrom manumycin in the structure of the acylamino side chainat C-2 of the cyclohexenone epoxide (Fig. 1). The stereo-chemistry at C-4 of UCF1-A is R, the same as that ofmanumycin, whereas that of UCF1-B is S. Details of thestructure determination of UCF1-A and UCF1-B will bepublished elsewhere.

Suppression of the Lethality of gpal Disruption by UCF1.Fig. 2 compares the suppression of the lethality of gpaldisruption by the UCF1 compounds. UCF1-C exhibits thestrongest activity. As seen in data for 50 ,ug of UCF1-C, thegrowth of cells close to the drug-containing paper disk wasinhibited by the high concentrations of drug. Regions furtheraway from the growth-inhibitory zone, however, show thehalo ofgrowth ofthe mutant strain. The zone ofgrowth of thegpal strain in the presence of UCF1-A and UCF1-B issmaller than that in UCF1-C, suggesting that UCF1-A andUCF1-B have weaker activity than UCF1-C. It is thereforelikely that acylamino side chain at C-2 of the cyclohexenoneepoxide contributes significantly to the activity of UCF1.To understand the structural constituents necessary for the

activity of UCF1, oxidized derivatives were synthesized andassayed (Table 1). The oxidized derivatives lack the trieneand the amide-bound C5N moiety (Fig. 1). These derivativesalso showed the halo ofgrowth. Thus, the triene chain and theamide-bound C5N moiety are not necessary for UCF1 action.They may, however, have some role in modulating the actionof these compounds, since the oxidized UCF1-A andUCF1-B showed a slightly larger halo of growth than theparent compounds. Reduction of the cyclohexenone epoxidemoiety of UCF1-C, on the other hand, afforded an inactive

Table 1. Effect of UCF1 on the lethality of gpal disruption inS. cerevisiae

Compound Suppression of gpal*UCF1-A 10UCF1-B 14UCF1-C 16Oxidized UCF1-A 15Oxidized UCF1-B 18

derivative (data not shown). Therefore, the cyclohexenoneepoxide moiety of UCF1 appears to be crucial.

Inhibition of Farnesyltransferase. UCF1-A, UCF1-B, andUCF1-C all inhibited the activity of yeast PFT in a concen-

tration-dependent manner (Fig. 3). Among these compounds,UCF1-C showed the strongest activity. The IC50 value forUCF1-C in this experiment was 5 ,uM, whereas the IC5ovalues for UCF1-A and UCF1-B were 13 and 7 ,iM, respec-

tively. A similar inhibition was observed with partially pu-

rified rat brain PFT (IC50 was 35 ,uM). The oxidized UCF1also inhibited rat brain PFT (data not shown). Modification ofthe cyclohexenone epoxide by treatment with 2-mercapto-ethanol destroyed the ability to inhibit PFH (data not shown).These results for the derivatives are consistent with theresults described above concerning their ability to suppress

the lethality of gpal cells.UCF1-C inhibits PGGT I much less efficiently than it does

PFH (Fig. 3). The IC50 value for PGGT I inhibition is 180 ,uM,and =30%o of the activity is still detectable even at 1 mMconcentration. Thus, UCF1-C appears to preferentially in-hibit PFH.To investigate the mechanism of inhibition by UCF1-C,

kinetic analyses were carried out. A Lineweaver-Burk plot ofPFT for various concentrations of FPP and UCF1-C and a

constant concentration of Ras (Fig. 4A) shows that theaddition of UCF1-C changes the Km value for FPP but doesnot change V.., suggesting that UCF1-C is a competitiveinhibitor with respect to FPP. A secondary plot derived byplotting slopes against UCF1-C concentration gave the Kivalue of 1.2 uM. In contrast, UCF1-C does not appear toaffect the Km value for the substrate protein RAS2 (Fig. 4B).

A

Concentration (PM)

*;:

B

1-

:]:E

1000

Concentration (PM)

FIG. 3. (A): Inhibition of yeast PFT by UCF1. PFT activity was

determined by measuring the amount of [3H]farnesyl transferred

from [3H]FPP to RAS2CT1 protein. Details of the assay are de-

scribed in Materials and Methods. Assays were carried out for 10

min at 37°C in the presence of various concentrations of UCF1-A (o),UCF1-B (x), and UCF1-C (a). The 100lo value corresponds to

54,800 cpm. (B) Inhibition of PGGT I (-) and PFT (o) by UCF1-C.

PGGT assays were carried out using [3H]GGPP and the glutathione

S-transferase fusion protein GST-CIIL as described in Materials and

Methods. Incubation was for 40 min at 37°C. PFT assay was for 10

min at 37°C. The 100o value corresponds to 12,142 cpm for the

PGGT assay and 49,419 cpm for the PFT assay.

Oxidized UCF1-C 16

*Halo of growth (mm) due to suppression of the lethality of gpaldisruption. Paper disks were soaked with 10 ,ug of drug.

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1/[FPP]

B

40

20

-0.1

a

0.0 0.11/[RAS2CT1]

0.2

FIG. 4. (A) Effect ofUCF1-C on the kinetics ofPFT with respectto the substrate FPP. Initial velocity (v) ofyeast PFT was determinedat various concentrations of FPP and UCF1-C and 26 ,uM RAS2protein. A plot of 1/v (pmol1min-1) versus 1/[FPP] (,uM-1) is shown.The amount of UCF1-C used was either 0 ,uM (o), 10 ,uM (A), 20 ,uM(m), or 40 ,uM (o). (B) Effect of UCF1-C on the kinetics of PFT withrespect to the substrate Ras. Initial velocity of yeast PFT wasdetermined under various concentrations ofRAS2CT1 and UCF1-C.A plot of 1/v (pmol min-1) versus 1/[RAS2CT1] (,.M-') is shown for1.1 ,uM [3H]FPP. The amount of UCF1-C used was either 0 ,uM (o),10 AM (A), or 20 ,uM (m). Variability between these assays was within10% of the values shown. Experiments were repeated three timeswith similar results.

The same Km value was observed when the Lineweaver-Burk plot was analyzed under different Ras and UCF1-Cconcentrations in the presence ofa constant concentration ofFPP. Thus, UCF1-C appears to act as a competitive inhibitorof PFT with respect to FPP and a noncompetitive inhibitorwith respect to Ras protein. The inhibition appears to bereversible, since PFT activity was detected even after thepreincubation ofPFT with 20 ,uM UCF1-C (data not shown).Growth Inhibition of Ras-Activated Murine Tumor in Vivo.

In Ras-activated tumor cells, administration of a PFT inhib-itor would be expected to decrease the amount of Ras in themembrane, and also generate a cytosolic form of activatedRas protein (28, 29), which can act as a dominant negativeinhibitor of Ras function. It is possible that the PFT inhibitorcould suppress outgrowth of Ras-activated tumors. We ex-amined the antitumor activity of UCF1 against Ki-ras-transformed fibrosarcoma inoculated in syngeneic mice (K-BALB). UCF1-C showed significant (P < 0.01) regression ofthe growth of solid tumor K-BALB, exhibiting a minimum

T/C value of 0.37 (Table 2). Under the same conditions,lovastatin, which is known to inhibit the farnesylation of Rasby decreasing the intracellular concentration of FPP, showedonly marginal activity. UCF1-A and UCF1-B did not exhibitany significant antitumor effect at the concentration used.Antitumor activity of UCF1-C was further supported bytesting the effects of the compound on HT1080 tumor inoc-

Table 2. Antitumor activity of UCF1 against Ki-ras-activatedmurine solid tumor K-BALB

Drug Dose,* mg/kg T/Ct No. dead*UCF1-A 3.3 0.61 0/5UCF1-B 6.3 0.72 1/5UCF1-C 6.3 0.37§ 0/5Lovastatin 150 0.46 0/5

*BALB/c mice (n = 5) transplanted s.c. with murine fibrosarcomaK-BALB on day 5 were treated i.p. with drug daily for 5 days ondays 0-4.tMinimum value of treated versus control: mean tumor volumeincrease of drug-treated group (T)/mean tumor volume increase ofuntreated group (C). Tumor volumes were calculated as (length xwidth2)/2.*No. ofdead mice per total mice on the day when minimal T/C valueswere obtained with the drug treatment.§P < 0.01 by Student's t test.

ulated into nude mice. HT1080 is a human fibrosarcoma thathas an activated N-ras oncogene. UCF1-C (6.3 mg/kg)showed a weak but significant growth inhibition with aminimum T/C value of 0.53. A minimum T/C value forlovastatin (150 mg/kg) was 0.65.

DISCUSSIONIn this paper, we report a type of PFT inhibitor we havenamed UCF1. This inhibitor is cell-permeant and appears toblock Ras function at the level ofRas membrane localization.Inhibitors of PFT so far reported include prenyl substrateanalogues (30, 31) or synthetic peptides that correspond tothe Ras CAAX motif (C, cysteine; A, aliphatic residue; X,methionine or serine) (32). However, intracellular delivery ofthese inhibitors may present a problem. For prenyl substrateanalogues, the diphosphate prevents cell penetration. As forpeptides, cellular uptake may also be inefficient and degra-dation in intestinal cells may be rapid.The most potent of the UCF1 compounds, UCF1-C, was

originally discovered as an antibiotic produced by Strepto-myces parvulus strain Tu64 (21, 22). This compound was oneof the manumycin family shown to exhibit antibacterialactivity (21, 22). The antibacterial activity appears to be aproperty distinct from the PFT inhibition, since anothermember of the manumycin family, asukamycin (33), exhibitsantibacterial activity comparable to that of manumycin butdoes not inhibit PFT (unpublished results). Our work alsorevealed that manumycin exhibits in vivo antitumor activity.Development ofan antitumor drug, however, requires furtherwork. As shown in Fig. 2, an optimum concentration isneeded to obtain suppression of gpal lethality, since con-centrations which are too high are lethal. Although we havenot observed significant mortality after the administration ofUCF1-C to mice (Table 2), various concentrations should betested to obtain the optimum condition for its antitumoractivity. Nevertheless, UCF1-C provides a promising leadcompound for developing therapeutically useful inhibitors ofRas farnesylation.UCF1 inhibits PFT from S. cerevisiae as well as from rat

brain. The inhibition appears to be specific to PFT, since an:40 times higher concentration of UCF1-C is needed toinhibit PGGT I. Preferential inhibition of PFT over PGGTagrees with its ability to rescue the gpal mutant, sinceinhibition of PGGT would have lethal effect. These obser-vations also point to the possibility that the prenyl diphos-phate binding site of PFT is different from that of PGGT I.Our kinetic analyses suggest that UCF1-C acts as a compet-itive inhibitor of PFT with respect to FPP and a noncompet-itive inhibitor with respect to the acceptor Ras protein. Thisagrees with a structural feature ofUCF1, a long carbon chain

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reminiscent of a farnesyl group. This side chain might con-tribute to its interaction with PFT at the FPP binding site. Onthe other hand, UCF1-C does not affect the Km ofPFT for thesubstrate Ras protein. This suggests that the binding of Rasmay not be affected by the binding of FPP and that the twosubstrates bind independently of each other. Such a mech-anism was observed by Pompliano et al. (31) with bovinebrain PFT.Our structure-activity study revealed that the cyclohex-

enone epoxide group is essential for the activity of UCF1.The acylamino side chain at C-2 also contributes significantlyto the activity. As discussed above, the reason why theacylamino side chain of UCF1-C exerts strongest PFT inhi-bition among related compounds may be explained by thestructural resemblance of the acylamino side chain to thefarnesyl group of the prenyl substrate. The triene chain andthe amide-bound C5N moiety are not essential, although theymay affect the activities of UCF1 to some extent. As forUCF1-A and UCF1-B, the removal of these structures led tostronger suppression of the lethality of the gpal mutant, andstronger PFT inhibition.Most of our success has been achieved through screening

natural sources by developing a high-throughput microbiallybased screen. The effectiveness of this approach is furtherconfirmed by the identification of another PFT inhibitor,UCF2000. This compound has an ICso value of 10 ,uM for PFT(unpublished data). Thus our microbial screen could yield abattery of inhibitors of Ras PFT that have potential applica-tion in cancer therapy.

We thank Dr. Howard Tager for critical reading of this paper andDr. Patrick Poullet for stimulating discussion. We are grateful to Dr.Kunihiro Matsumoto for providing the gpal strain and to Drs. KoheiYokoyama and Michael Gelb for providing bovine brain PGGT I.F.T. is supported by National Institutes of Health Grant CA41996and is an Established Investigator of the American Heart Associa-tion.

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