the human guanylate binding protein, huGBP1 - CiteSeerX

Prenylation of an interferon-y-induced GTP-binding the human guanylate binding protein, huGBP1 David E. Nantais, Janice E. Buss

Martin

Department ofBiochemtstry Medical Microbiology and institute,

La Jolla,

the expression guanylate binding

ducible GTP-binding sess a CaaX motif they

might

be

human

GBP1

cos-1

cells,

of

precursor expressed

IFN-y-treated were also

found

a prenyl proteins

protein (GBPs)

Deborah

J. Vestal,t

Ames; Department Republic ofGermany;

and

of Virology, and

for

kDa that indicating

prenyltransferases. expressed radioactivity

In,s:i:we

for

Burnham

human fibroblasts to be isoprenoid

In

induction and down-regulation In human cells, huGBP1

The

dant

the

addition, genes

in

or monocytic cells modified. IFN-y-in-

the

farnesyl transferase that huGBPs in HL-60 by a C15 farnesyl rather

activities by binding

posthat

in transfected from

[3H]mevalonate. from the endogenous

and antimicrobial initiates its effects

ceptor found on the surface of including macrophages, initiating suiting in tyrosine phosphorylation

of 65 kDa. are IFN-in-

duced huGBPs in HL-60 cells were not the specific C20 isoprenoid, [3Hgerany1geranio1, did show decreased isoprenoid incorporation treated with 5B, indicating ably modified

tumor, IFN-’y

and lipopolysaccharide in isoprenoid-modified the most dramatic

protein, when incorporates

isoprenoid huGBPs

T. Stickney,

and Biophysics, Iowa State University, Hygiene, University ofFreiburg, Federal

proteins of -65 at their C-terminus,

substrates

John

California

Abstract: Interferons (IFN) (LPS) cause multiple changes proteins in murine macrophages, being The

Schwemmle,

protein:

mRNAs

potent GBP1

induced

by but in cells

inhibitor BZAcells are probthan the more

transcription tivator

and

factor

great

deal

is

the

activating genes,

known

is the most

induce JAK-i

induce growth

the protein and JAK-

from the IFN-y receptor of human GBP1 [5-7]. (signal

transducer

is phosphorylated into

to gamma of selected

IFN-y

do not kinases,

STAT-i/p9i

translocates

binds regions

[4].

but IFN-a can also such as epidermal

serum protein

of transcription)

then

many mammalian cells, numerous changes, reof proteins, and both

by interferons

inducer of GBP1 protein, expression. Other agents

factor (EGF), insulin, [4]. The tyrosine-specific

[1]. re-

of many genes [2, 3]. mRNA is one of the most abun-

2, are involved in signaling subsequently in the induction

labeled

in macrophages to a glycoprotein

nucleus

and

by

of the

sites including

(GAS) that

and The

the

cell,

ac-

JAKs,

where

it

in the promoter for GBP1 [7]. A

about

the

promoter

regions

the

GAS

sequence

was

of

the

common C2olipid. Differentiated HL-60 cells treated with IFN-y/LPS showed no change in the profile of constitutive isoprenylated proteins and the IFN-

GBP1 gene, in which identified [8, 9]. The protein sequences

y/LPS-induced spite being endogenous

chicken [10], rat [1 1], murine, and human cells [12, 13], have been elucidated from their cDNAs. However, the biological function of the GBP proteins is not yet known. One

(-85%) select tightly dant

cytosolic. group

Key proteiru

of

regulated protein

farnesyl prevent

Biol.

huGBPs prenylated, huGBPs in Human prenyl by whose

GBPs proteins

a cytokine. prenylation

transferase farnesylation

inhibitors of Ras

60: 423-431; Words:

remained huGBP1 HL-60

prenylated. Dein COS cells and cells were primarily are

thus

whose HuGBP1 may

among

the

synthesis

that are proteins.

designed J. Leukoc.

[13-15].

GBPs

to

proteins,

but

to

size being

.

farp.isylitin . cytokine

.

lipid

modflcation

of

INTRODUCTION (IFN-y) natural

(-65 able

far

shared

is the are form

kDa)

relaxed GTP,

to

family

all bind

members

a distinct

and

to bind

by

ability thus

GBP

GBP

and

members family

on their

binding GMP

from

nucleotides

broad

based

nucieotide GDP,

family

guanine

of the

subgroup

members

[13].

of G larger

specificity, The

human

1996.

isoprenoid prenyl traruferase

Interferon-y T cells and

so

property

tested

is an abun-

be vulnerable

biochemical

is

of

originally

is an important killer cells that

cytokine induces

produced antiviral,

by anti-

Abbreviations: GBPs, guanylate binding proteins; IFN, interferon; LI’S, lipopolysaccharide; EGF, epidermal growth factor, GAS, gamma activating sites; PMA, phorbol myristate acetate; LPC, lysophosphatidylcholine; [3HJMVA, R,S[5-3Hlmevalonolactone; DM EM, Dulbecco’s modified Eagle’s medium; [3H]GGOH, [3H]geranylgeraniol; TBS, Tris-buffered saline; HFLC, human lung fibroblast cells. Correspondence: Dr. Janice E. Buss, 3212 Molecular Biology Building, Iowa State University, Ames, IA 50011. Current address of Martin Schwemmle: Department of Neurology, University of California, Irvine, CA 92717. Received December 8, 1995; revised July 1, 1996; accepted July 2, 1996.

Journal

of Leukocyte

Biology

Volume

60,

September

1996

423

than [3H]geranylgeranyl Recently, however,

GBP1 protein seems to be unusual, in that its major product of hydrolysis is GMP rather than GDP [14]. In addition to GTP-binding consensus motifs, the predicted sequences of six of the seven cloned GBPs (human GBP1, human GBP2, mouse chicken

murine

GBP1/mag-1,

GBP-2 GBP,

the

rat

p67

protein

[D. J. Vestal et al., unpublished but apparently not murine

Both the C-terminal domain immediately

and re-

portant

veal cysteine residues that are four amino acids from the carboxyl terminus of the protein, designating these GBPs as possible substrates for protein:prenyl transferases [17,

which geranyl

is

a 15-carbon isoprenoid

nus of a protein are isoprenylated studied of these

a posttranslational

modification

in the

thermore, was found

18].

Isoprenylation

in

farnesyl group or a 20-carbon geranylis attached to a cysteine at the C-termi-

and

tein

tion

acid]

defined

the

[21, 22]. are likely ate and

cysteine

as the

site

Although numerous to exist, isoprenoid allow Ras proteins

plasma ability

membranes, to regulate

[19-22].

For

Mutational

studies

C-terminal modification,

of isoprenoid

on

attachment

or Rab

proteins

instead may participate the GTPase and additional factors or inhibitors) and hence, activation,

(other

members

directly in interactions proteins (nucleotide

that control their state [23-25].

its

proteins

outside

of this

fled. Among these are the subunits of the hetemtnmeric nases

involved

family

been

of adrenergic

have

The

provides

MATERIALS of

Human

identi-

and

and

and

cells

LPS

charged prenylation

the

huGBP1 might This possibility which showed

424

Journal

[17].

Such

studies

have of the [18],

shown

that

The

Leukocyte

Biology

Volume

September

huGBP1

to many other with cellular is prenylated

modification intervention,

study

of GBP

is preand

function.

exposed

International

phorbol from

for

Sigma

18-24

10

Radiolabeled from

(St.

h with

1g/mL

and

from

in the

was

obtained MO),

.

(Arlington

and

Heights,

from GIBCO-BRL

Calbiochem Dr.

In some of IFN-y

Louis,

.

Sciences

was purchased from

(PMA),

MO).

or as indicated (St.

.

Life

Pansorbin gift

Louis,

a combination

LPS

Chemicals Amersham

a generous

acetate

([3H]MVA)

for transfections

MD)

(Camarillo,

myristoyl

R,S-[5-3H]mevalonolactone

promyelocytic

RPMI

(San

Donald

Graves

Diego,

CA).

(Iowa

State

medium

cytic for

cells 24

in

lines

HFLC

(293) medium

1996

h [31,

begun

the

lines (Iowa

HL-60 State

(GIBCO-BRL) of the

the

antibody

cell

McCloskey

differentiation

CaaX box is a indicating that

60,

of the

huGBP1

BioSource

(LPC)

were

was

Michael

be modified with a C15 farnesyl isoprenoid. is supported by previous in vitro studies, specific incorporation of [3H]farnesyl rather

of

from (LPS),

Cells and huGBP1

residues (italsite and with

signal

senne at the last position for the farnesyl transferase

are trans-

University).

protein

nu-

sequence

for the

purchased

14

Lovastatin

the residues of its CaaX motif (CTIS) predicted from merous in vitro studies to be suitable as a prenyltransferase amino acid good signal

status

that

U/mL IFN-’y and

(300

(Gaithersburg,

family, with a number of positively ics) immediately upstream of the

was

L C]mevalonolactone IL). Lipofectamine

location of a prenyl protein. of huGBP1 (-Lys-Met-Arg-Argconforms to the pattern Ras-related

cells

famesyl

METHODS

lysophosphatidylcholine

experiments

receptors

avenue

lipopolysaccharide

mation on the subcellular The C-terminal domain Arg-Lys-Ala-Cys-Thr-Ile-Ser)

of the

IFN-exposed developed

demonstration

AND

IFN-y

CA),

American

members

by a C15 prenyla-

Materials

from

many

intact,

newly

the prenylation

a unique

legends).

the

when

of the

From

may not to a pro-

is particularly

by a farnesyl group, although in contrast prenylated proteins it is not highly associated

figure

in

affected

examined

and rhodopsin [26]. Prenyl modification of these proteins is also required for their biological activities. Thus, detection of an isoprenoid does not provide unambiguous infor-

found

Fur-

30].

if modified target whose

membranes.

nuclear lamins, the gamma G proteins, and protein ki-

in desensitization

be

[29,

information

protein, potential

will now permit studies in which lipid vented by mutation or pharmacological

nucleotide

have

such

of the but

Although numerous members of the Ras superfamily GTPases are now known to be prenylated, few isoprenoidmodified

huGBP1

as the huGBP1 be an abundant

with any inhibitors.

We

between exchange

guanine

For

cells

that in vitro assays isoprenoid attached

of the

prenylated C-terminus in membrane binding,

protein.

protein in intact COS-1 cells as well as of huGBPs induced in various IFN-’y-treated cells. In this study we report that human GBP1 can indeed be prenylated in vivo, apparently

functions for isoprenylation modification appears to initito associate with cellular

superfamily) the not be involved

K-RasB

with a C15 signal (CYIS) both C15- and C2o-modified

in intact

it is clear the actual

,

may

treated ferase

amino and

a localization that is crucial to cellular growth or differentiation

Rac

Ras GTPase protein may

[20].

these four isoprenoid

of the

‘y-subunit to be

geranylgeranyl

in vivo.

acids

amino

residue and the polybasic of the CaaX motif were im-

C20 modification

these precedents accurately reveal important lipid, will

another

[14]. the

when expressed and labeled in insect cells [2W . Conversely, the RhoB protein, with a CKVL motif that was predicted to be C20-modified, was found to incorporate both

tains what is referred to as a CaaX box at its C-terminus [a cysteine residue followed by two (usually) aliphatic amino and

methionine upstream

a G protein unexpectedly

farnesyl

via a thioether linkage [18]. Many proteins in eukaryotic cells, the most thoroughly being the p2lRas protein [19]. Ras con-

Ras proteins have identified acids as being essential for

huGBP1 Ras family,

K-RasB protein, which has a CaaX motif (CYIM) that was predicted to be farnesylated, was shown to be capable of modification in vitro by either C15 or C20 isoprenoids [27].

[16],

results], mag-2 [12])

into recombinant member of the

one

HL-60

cultures 32]

were then

continued [13]

and

and

with promyelocytes treated

presence

were and

10% with

radiolabeling 1523,

THP-1

University)

or

monkey

kidney cells were maintained (DMEM; GIBCO-BRL) with

fetal

calf

into

more

0.5

of both

obtained were

nM

(COS-1)

developed plus with

agents.

The and

in Dulbecco’s 10% fetal calf

To

serum.

PMA

treatment

from

Dr.

maintained

in cause mono-

50

j.LM LPC

IFN/LPS

was

fibroblastic human

modified serum.

cell

embryonic Eagle’s

The

anti-GBP1

Histj-tagged

mouse

His-huGBP1

was

chromatography

column

human

purified

0.1%

NP4O,

12%

nized

[14]. (20

1 mM

-mercaptoothanol)

the

peritoneum

injections forms

at of both

Human used

10-day

intervals.

This

huGBP1

and

pressed

that

with

Hind

and

2.0

j.tg were were

antibody

cells

after

three

PVDF

cDNA,

expression

in

EcoRV

site

of the

[13].

This

10.9-kb

CoA types.

promoter,

The

gel

anion-exchange

the

GBP1

mammalian was

cDNA

produced

was

present,

three

bands

can and

and

protocol

the

the

cells

transfected

using

Qiagen.

DNA

expected

was

sizes

cells

were

10 tg

of pHMG-huGBP1

DNA

on a 60-mm

plate)

5 h, after which time Twenty-four allowed

in serum-free

were

incubated

1 mL of medium hours

to grow

later,

the

digested

Cells

3.3,

for another

24

with

reagent

medium.

The

in 1 mL

of this

20%

fetal

medium

was

h before

further

were

rubber

and

cells

(200

on a sterile

serum and

was

the

compactin The

was

fetal (gift

of Dr.

plates

were

directly

in

containing

DNA

6 or 15%

The

Akira then

and exposed GBP1 protein alkaline

iL

with

separated membrane,

was

detected kit

fetal reduced

h) at 37#{176}C,after

and

the

gel

were

the

the

fractions the

times.

Following by

use

Laboratories,

0.5%

anti-human secondary

Tween

20

GBP1 mouse antibody and

Laboratories,

Burlin-

fraction

and

of

was

and

P100

also

g)

(-120,000 and

membrane

tubes.

The

pellet sample

4 volumes

of acetone

precipitated

5100

dissolved

were

and to 150

electrophoresis

with

samples

rpm

individual

The

10

strength

(5100)

protein

a

cell

containing

ionic

at 50,000

treated

proteins.

with The

homogenizer,

the

cytosol into

iL

centrifugation

S100

mm

plates

Prefabloc, 1 tg/mL Cells were allowed

to a Dounce

The

200

the

centrifugation. solution

to adjust

separated

in

from

by

of a hypotonic

for 30

concentrate by

Both

scraped

1% Trasylol, 1 mM 1 mM dithiothreitol.

rotor.

then

S100

and

collected

lysates

spun

were

TBS,

and

transferred

100.3

dissolved

while

Four

in 200

.tL

on

a 15%

separated

to

proteins sample SDS-

gel.

90-mm

of

[1

of human

with

CJMVA

1000

either

time

of huGBP1

GBP

antibody. on

a

10%

Amplify

fluorographic

lysate

expoed

to film

loaded

the

legends.

.tCi

using

human

IFN-y,

lung

and

two

(two

protein

plates).

GBPs

The

Sepharose buffer

was

(30 washed

and

released

SDS-polyacrylamide enhancer for the

[ 5S]methionine-labeled

plates) were

(HFLC),

labeled or

captured coated

with

three

times

then

which

was

Sciences)

[14C]MVA-laheled

sample,

iCi

by

j.tL)

Life

over20

radiolabeled

gel,

(Amersham

cells

were

(two

A Sepharose

sample

for 8 weeks

fibroblast

control,

[35S]methionine

lovastatin

electrophoresis

separated

U/mL

200

without

munoprecipitation in

of subconfluent

with

with and

plates

treated

night

buffer

figure

im-

10 tL boiled proteins

soaked

in

dried,

and

or 1 h for

sample.

by elec-

sprayed

DuPont-NEN,

ice,

lysates

immediately

transferred

membrane

membrane

(VECTOR

as described

plate

sample

in the

with

milk

antibody

proteins.

with .tL

to the

TLA

collected

two

j.tM

(TBS)

were

j.tL of

25-SO

in the

50

which

saline

as indicated

(En3Hance, on

or

to each

lysates

nonfat

with

a

Boston,

MA),

fluorography,

the

of the

RESULTS

VectaStain

Burlingame,

CA)

and

below.

Prenylation cells

of human

GBP1

expressed

in COS-1

of cells with BZA-5B

HL-60

of the

and

proteins

lovastatin added

on

Immunoprecipitation

.tCi,

8 h (for experiments

cell

im-

evaporated

[34], and the or RPMI plus

electrophoresis

The

enhancer

tM

Tris-buffered

gels

25 was

flow hood of DMEM

and

of protein

to film for the indicated

Treatment

dialyzed

50

(-18

times

a PVDF

antibody

Control

with Tokyo)

by vortexing,

phosphatase

anti-GBP1

1 mL

or overnight three

sheared

fluorography

in

to incubate

SDS-polyacrylamide onto

surface

all

Endo, allowed

100-200

radiolabeled

troblotting

9 mM

serum

-mercaptoethanol.

boiled,

on

in a laminar up

and

nonspecific

(VECTOR the

MgC12, and

added

cell

directly

buffer

times in 850

mm

10 was

the

buffer.

in ethanol

plate

tL

SDS-

fractions

if needed,

polyacrylamide

[33]

taken

bovine

BZA-5B or [3H}GGOH) the cells were rinsed lysed

for

(P100)

or

three

a Beckman

was

cells

experimentation

([3H]GGOH,

Inc.)

to

(w/v)

any in TBS

phosphatase

resuspended

and

in

and

[3H]geranylgeraniol

culture

precursor

dialyzed

cells.

or

Chemicals,

tissue

radioactive 10%

j.tCi)

a 2.5%

block

washed

of subcellular

NaC1

were

Radiolabeled

then

to visualize

pH 7.4, 1 mM 1 .tM pepstatin,

precipitate

[‘H]MVA

in

4#{176}C to

was

used

rinsed

was

mM

for

harvesting.

American

in 100

subjected

fluorography,

a 1:1000 dilution of the A biotinylated anti-mouse

policeman,

lysed.

(80%

mixture

bovine

replaced

FadiolabeIing with [3H]mevalonolactone [ Hjgeranylgeraniol

of which

To

of 5.6,

j.LL of Lipofectamine

incubated at

membrane

were

mM Tris, leupeptin, 25

were

overnight

Preparation

a 100

to swell COS-1

concentration directly

tL

(PAGE),

mM

of BZA-

for 8 h, after

lysed

20-30

electrophoresis

alkaline

CA)

pellet

of COS-1

for a final

a 100

ex-

using

from

game,

by-

constitutively

TBS

avidin-conjugated

be

can

to

purified

isolated

of the

poly

vector due

be

isolated

column

pHMG-SV

cells

which

DNA

and

solution

incubated

by centrifugation, buffer,

membranes The

the

media were

into

.tM

as described.

binding.

Transfection

were

collected gel

above

all plates

1:10 500

lmmunoblotting The

into

the

diluted

resulting

recombinant

huGBP2.

ligated

into

sample

munoblotting

kb.

added.

were

and incubated with polyclonal antiserum.

III and

confluent

1:10

then

the

injected

bled

recognizes

the

was and

resuspension,

of human GBP1 cDNA

in all cell

verify

animals

diluted After

polyacrylamide

homoge-

solution solution

electrophoresis

in 1.5

and

of -4.5

The

time

at --300

polymerized

further

50 tM.

5 mM

stock

in

protein

silica

was

7.6,

eluted

This glutathione

solution

droxymethylglutaryl .Lm

Portions

was used for all transfections

for

mL)

SB

to a MonoQ

pH and

DMSO.

reduced

The

nickel-agarose

bound

Na-phosphate, (1.5

saline. mice.

GBP1

with

was

in

using

antigen.

(SDS)-polyacrylamide

of BALB/c

Purification

A vector,

mM

sulfate

in phosphate-buffered

into

coli

buffer

dodecyl

prepared as

His-huGBP1

of the His-huGBP1

sodium

was

(His-huGBP1)

Escherichia

from

and

mM KCI. A portion

antiserum

GBP1

as described

in a phosphate

MgCl2, mL

polyclonal

full-length

cells bovine

were serum

glutathione,

above

BZA-5B-treated including IFN/LPS, sters, Genentech).

resuspended containing and

added

plus

cells

were

in

100

200

50 iM

tCi

of

human

with

all

RPMI

[3H]MVA,

compactin.

U/mL

treated

1 mL

IFN-treated IFN-y

and

of

above

the

with 1%

10% DMSO,

cells 1 jig/mL

had LPS.

compounds

plus 50 jiM BZA-5B [35] (a gift from James MarThe BZA-5B was prepared as a 5 mM stock solution

To study whether a selected vivo, cDNA coding for the

huGBP could entire protein

man

into

GBP1

was

were incubated labeled proteins and The

introduced

with [3H]MVA by SDS-PAGE,

a combination of fluorography polyclonal antiserum made

Naruais

et a!.

Isoprenoid

COS-1 followed transfer

be prenylated in sequence of hucells

and

the

cells

by separation to a membrane,

of

and immunodetection. to human GBP1 detected

modification

of huGBP1

425

a protein of the predicted size for human GBP1 (60-65 kDa) in the transfected cells (Fig. 1A). This protein was

A fluorograph protein at -65

not present in the lysate from untransfected COS cells. This verified that the antiserum recognized the authentic huGBP1 protein and that there were no cross-reactive pro-

cells. brane grated

teins and

immunoblotting. This indicated expressed in COS cells from the

that the transfected

modified

isoprenoid.

of similar transfected

[3H]MVA

molecular COS

and

weight cells

cellular

in COS cells. Both were then labeled

proteins

separated +GBP-1

A

control with

by SDS-PAGE.

C

of these lysates kDa only in the

by a [3H]MVA-derived

The endogenous studied in four mia cell types and

GBPs

embryonic kidney a combination

the expression

cells (293). of IFN-’y

of GBPs

35,100--

cell lines (Fig. 2). The basal amount of huGBP although

B

tion

Blot

incubation

+GBP-l

1.

(A)

Detection

cells,

in electrophoresis polyacrylamide

gel

on the

human

GBP1

tion

left.

labeled,

The

arrow

with

anti-huGBP

ment

of the

film

indicates

the

65-kDa

in COS-1

cells.

or control gel

then

After

antibody

to detect

([3H]MVA)

with

Journal

of

with the the

huGBP1

transferred

exposure,

expressed immunoblot

cells

Biology

were

was

huGBP1

Volume

separated membrane.

enhancer

membrane

and

incubated

protein. revealed

Alignthat

band transfected

60,

September

caused

Because

currently

immunoblots

the

on the COS

1996

a large

induc-

available

an-

the highly homologous induced protein(s) de-

Both huGBPs have nearly identical and contain a CaaX motif [13]. However,

is

simply

identified

as

molecular previous

that huGBP2 mRNA in IFN-treated cells as abundant as huGBP1 mRNA [13] so that the predominant species detected

is huGBP1.

of huGBPs

y/LPS.

with

As

in IFN-’y-treated

of IFN-y and LPS for 24 [3H]MVA in the continued

seen

band appeared present in the tein comigrated

(B) Prenyla-

to a PVDF

(Blot)

the

indicated

[3Hjmevalonate-

a fluorographic the

huGBP1

are

IFN/LPS

on

combination overnight

SDS-

against

protein. from

with the new [3H]MVA-laheled the huGBP1 expressed in the

Leukocyte

The made

markers

(C) COS-1

and

sprayed

for 4 weeks.

membrane.

Lysates

directly

on a 15%

an antibody

ofmolecularweight

huGBP1 protein comigrated fluorogram, indicating that cells is prenylated.

426

with

werelysed

separated

the

mobility as huGBP1 exby IFN-’y/LPS in all four

HL-60 cells were selected to examine were prenylated when endogenous GBP duced by IFN-y/LPS. HL-60 cells were

by immunoblotting.

(C),

to a PVDF

(+GBP1), was

to film

proteins

Positions

expressed

cells

orcontrol

detected

SDS-polyacrylamide

membrane

exposed

in COS

the

transferred was

protein.

transfected

The

and

buffer,

membrane

of huGBP1

on a 15%

GBP1 (+GBP1)

sample

protein on the

of human

eithertransfected

treatment by SDS-PAGE, Antibody-reac-

huGBP. weights

Prenylation cells

Fig.

also leuke(1523),

HL-60 and to induce

tected

here

COS-1

IFN/LPS

do not distinguish between and huGBP2 proteins, the these

were

1523 cells appeared to have some present before IFN/LPS treatment,

huGBP.

work has shown is about one-tenth that it is probable

huGBP-1

I-

with

of additional

tibodies huGBP1

protein could be

in IFN-y-treated

proteins separated by immunoblotting.

tive GBP protein with the same pressed in COS cells was induced

+GBP-l

huGBP1 DNA

memcomiby

Cells were treated for and LPS. These condi-

[4, 8]. After

cells were lysed, their and huGBPs detected

C

same protein detected

tions were chosen to maximally activate the THP-1 cells, although IFN-’y alone is sufficient

50,000--

[3H]MVA

that

GBPs induced by IFN-y/LPS human cell lines: two promyelocytic (HL-60, and THP-1), fibroblasts

48 h with

huGBP-1

1B) revealed a new from the transfected

Alignment of the film image with showed that this [3H]MVA-labeled precisely with the huGBP1

Expression of endogenous human cell lines

97,200--

(Fig. lysate

in Figure

3A,

substantial, prenylated

whether gene(s) incubated

huGBPs were inwith a

h and then presence

labeled of IFN-

a -65-kDa

[3H]-labeled

in the IFN-y-treated lysate that was not untreated cells. This [3H]MVA-labeled prowith a huGBP detected by immunoblotting

of the same sample, indicating that huGBP was prenylated. Incorporation -65-kDa observed labeled

human

at least one endogenous of [3H]MVA into

an

IFN-’y-induced protein in THP-l cells was also (data not shown). The amount of the [3H]MVAhuGBP expressed in an IFN-y-treated cell was making proteins

huGBPs among in the IFN-y-treated

the

most plentiful cell, with a con-

cos +GBP-l

clearly did not prevent induction portantly, the major, IFN-’y-induced

HL-60

1523

+IFN

+IFN

C

a major, therefore

GBP

293

induced, appeared cells

those more

in the appeared

GBP 2.

lines. and

Expression

The

of endogenous

indicated

10 pgfmL

cell

LPS

for 48

a 15%

SDS-polyacrylamide

probed

with

with 75-kDa

centration proteins proteins with

h. Cells

of the

for

in

treated were

IFN-y-treated with

lysed,

immunoblots

from

proteins COS-1

comparison

human

units/mL

to a PVDF

A lysate size

300

the

transferred

antiserum.

is included

region

were gel,

anti-GBP1

huGBP1

GBPs

lines

cell

of IFN-y separated

on

membrane, cells

(arrow).

after

and

transfected

Only

the

55-

to

treatment,

including

sizes between 20 of the Ras super-

family (see prenylation

of general treatment

with [35] tion

similar

data

and indicates is not visibly

from

persistence after IFN-y

IFN-y-treated

mouse

that the machinery altered by IFN-y/LPS.

Neither general protein prenylation specific proteins has been examined man myeloid cells treated with whether induced

isoprenoid proteins

modification such as GBPs

of constitutive was influenced

cell line that macrophages)

can differentiate into either were again used. To initiate

into

a monocytic

phorbol ester time the cells shape to cells

cell

numerous (data

cells

incubated

out or with IFN-y/LPS, rated by SDS-PAGE. in the treated, amount

type

cells

HL-60

ruffles and not shown). overnight

cells

then labeled with Protein A Sepharose

some mature

constitutive cells, but

that certain

expression differentiation

cells. found that, in mouse IFN-’y can induce ex-

65-kDa

protein

that

is

not

the

was used to immunoprecipitate cellular lysates. A [‘4C]MVA-labeled from prenylated

A

proteins the

or [‘4C]MVA. GBP1 antibody

from the labeled protein was detected

IFN-’y-treated cells protein comigrated

and pre-

[3H]MVA

Blot +IFN

C

+IFN

C

GBP

B

[3H]MVA

Blot PMA/IFN

PMA

PMAIIFN

PMA

(a promyelocytic granulocytes differentiation were

[3H]MVA,

undifferentiated cells was induced differentiated HL-6O cells (Fig. of an immunoreactive protein was cells

HL-60 cells, although in the differentiated

either [35S]-methionine coated with human

or IFN-yby the stage or

treated

either

withsepaas that

in the IFN-y3B). A small present in the

had not been exposed if this protein might of a huGBP of HL-60

GBP-

with

processes characteristic of The differentiated HL-60 with

to

prenyla-

then were lysed and proteins A huGBP of the same mobility

sample from differentiated to IFN-y. It is not presently reflect more

HL-60

similar

protein agrees

(PMA) and LPC for 24 h [32]. During this changed visibly from a nonadherent, round that adhered to the culture dish and that

possessed macrophages were

cells,

are

murine GBP1, it was important to verify that the IFN-y-induced prenylated protein in human cells was a human GBP and not simply a prenylated protein of similar size. For this experiment HFLC cells from which the original huGBP1 cDNA clones were obtained were treated with IFN-y and

nor prenylation of in more mature huIFN-y/LPS. To learn

of the

HuGBPs in the dif-

macrophages

of protein

of maturation

results

Imwith

the

many prenylated proteins with molecular and 25 kDa, most of which are members Figure SB). This in human cells

These

undifferentiated to be expressed

in the immunoprecipitate this newly induced,

is shown.

IFN-’y/LPS

3B).

of a prenylated

equal to or greater than that of the cellular Ras (data not shown). The majority of the prenylated in HL-60 cells continued to incorporate [3H]MVA

no change

(Fig.

protein. prenylated

cells than in the bipotential precursor Because Vestal et al. [35] have bone marrow-derived macrophages,

THP-1

pression

Fig

[3H]MVA-labeled capable of being

ferentiated seen GBP

of huGBP by IFN-y. GBP comigrated

in

the cells

3. (A) Prenylation

Fig.

ofa

lysates

from

treated

for 24

h with

IFN-’y

SDS-polyacrylamide membrane

gel

was

for 6 weeks.

sprayed

After Alignment

revealed

a

GBP

only and

the

in the

(PMA) 18

h. Cell

lamide

gel,

transferred

weeks.

After

exposure

with the ([3H]MVA).

Nantais

huGBP1

et al

or presence lysates

were

to a PVDF the

GBP

antibody

Isoprenoid

was in

exposed while

the

with

ofhuGBP labeled

of IFN-y

and and

on the

modification

and

LPS

membrane

for 24

for an

SDS-polyacry-

exposed with

h

[3H]MVA

(PMA/IFN) to film

membrane

of

present

LPC with

a 15%

aligned

which

also

in differentiated

were on

huGBP

cells, band

separated

the

([3H]MVA)

IFN-treated

cells

and

The to film

fluorogram

ester

the

or cells

on a 15%

exposed

incubated and

Proteins

(C)

membrane. and

to phorbol

proteins (Blot)

separated

a PVDF

(B) Prenylation

were

were

[ HIMVA-labeled

new

sample. cells continued

absence

additional

the

cells.

[control

enhancer (Blot)

exclusivel’

with

HL-60

to

membrane

immunoblot

protein

treatment

transferred

the

HL-60 cells

(+IFN)]

a fluorographic

ofthe

IFN-treated

cells.

HL-60 LPS

and with

precisely

in the

HL6O

in IFN-’y-treated

and

exposure

antibody. comigrated

huGBP

[ H]MVA-labeled

in cell

were the

huGBP1

film

for 4

detected image

427

[35S]Met C

teins in the 20- to 25-kDa indicated that the majority HL-60 cells were modified

[14C]MVA C

+IFN

+IFN

Subcellular Although membranes, the induced subcellular membrane

GBP

cells

4.

Fig. cells.

Immunoprecipitation

HFLC

cells,

labeled

were

captured

by

to

huGBPl

overnight

.

with

.

The

induced 4).

prenylated

Sjmethionine

gel

was

by the

to film

protein

GBP

recognition

antibody

of this

indicated

was

CIMVA.

that

indeed

GBPs and

on for

a

10%

1 h for

[‘4C]MVA-labeled

[35S]-labeled The

(C),

A-Sepharose

separated

exposed

for the

[

or

the

sample.

protein

from

[‘4C]MVA-lathe

of [3H]MVA

many prenylated proteins are associated with early work on human GBPs had suggested that proteins were cytosolic [4, 13]. To examine the location of huGBP1 specifically, cytosolic and fractions were prepared from transfected COS-1 the

into

an inhibitor

SB,

which

of the

farnesyl

selectively

by IFN-’y in HL-6O farnesyl, or the cells were treated

transferase

decreases

enzyme,

farnesyl

radioactivity

transferase-specific into

several

ished by the inhibitor, 20 and 25 kDa, most

inhibitor. proteins

other

whereas of which

proteins are Ras

of

Incorporation of was also diminwith family

sizes between members that

are C20-modified, continued to be radiolabeled (data shown). This selective in vivo inhibition of isoprenoid corporation by BZA-SB suggested that in HL-60 cells predominant IFN-y-induced huGBP was modified by a nesyl. GBPs cells

Second, to determine if any portion of the was modified by C20 isoprenoid, IFN-treated were labeled with [3H]geranylgeraniol. This

not inthe far-

induced HL-60 method

Journal

of

Leukocyte

Biology

Volume

60,

was

tions contained the prenylated cells

are

that that

tested

from HL-6O cells Figure 6B shows

cells. precursor forms it was possible a nonprenylated

assessed

of many prenythat the soluble precursor and

was prenylated was was membrane-associated.

by

that had that both

preparing

primarily

the

subcellular

small This

fractions

been labeled with [3H]MVA membrane and cytosolic frac-

radiolabeled huGBP, indicating forms of induced human GBPs

that even in HL-60

cytosolic.

September

DISCUSSION The guanylate merous cell

binding types after

abundantly

induced

(GBPs) are with IFNs

the

most

[4]. The human GBP1 protein has been as a GTPase in vitro, with the somewhat

proteins

induced and are

in nuamong

in an IFN-y-treated shown to funcunusual prop-

erty of formation of GMP rather than GDP as its major product [14]. A second class ofIFN-inducible proteins, the MxA proteins, also bind GTP [36], but differ from GBPs in that they lack the C-terminal CaaX motif and are therefore not likely to be prenylated. Of the GBP family members whose sequences have been determined, six have C-termi-

as a prenyl

1996

proteins treatment

cell tion

nal motifs that by isoprenoids.

would also detect the huGBP2 protein, whose CaaX motif (CTIL) is predicted to be modified by a C20 isoprenoid. No incorporation of radiolabeled C20 isoprenoid could be detected in the induced huGBPs (Fig. 5B), although good labeling was observed for the numerous C20-modified pro-

428

fraction

BZA-

incorporation

[3H]MVA-derived isoprenoid into farnesylated proteins. As shown in Figure 5A, huGBPs could still be IFN-induced in BZA-SB-treated cells, but the amount of radioactive isoprenoid incorporated was diminished by the presence of the

in each

differentiated HL-60 cells (data not shown). Thus huGBPs do not appear to interact tightly with membranes (or proteins in membranes) of untreated COS-1 or IFN-’y-treated HL-60 cells, nor to gain that ability if HL-60 cells differ-

that the only form amount of huGBP

There was not sufficient incorporation of radiolabel to allow us to determine chemically the isoprenoid attached to huGBPs in vivo. Two alternative approaches were therefore

with

of huGBP1

although there was a small amount (-15%) in the membrane-containing P100 fraction. In HL-6O cells the same large degree of solubility of induced huGBPs was observed (Fig. 6A), and this cytoplasmic location was retained in

possibility

used to evaluate if the huGBPs induced cells was modified by the C15 isoprenoid, C20 lipid, geranylgeranyl. First, HL-6O

amount

entiate into macrophage-like Because non-prenylated lated proteins are cytosolic, majority of huGBP1 was

IFN-y-in-

a GBP.

Effect of BZA-5B on incorporation huGBPs in HL-60 cells

and

of huGBPs

by immunoblotting. The majority of the huGBP1 protein was detected in the cytosolic (S100) fraction (Fig. 6A),

HFLC

or control 14

Protein

proteins

from

(IFN) .

with

or 8 weeks

the

duced

[

huGBP IFN-y .

radiolabeled

(Fig.

protein

U/mL 35

either

and

sample

beled

1000 .

with

gel.

L35SlMet-labeled

I-IFLC cells

with

immunoprecipitation

SDS-polyacrylamide

cisely

of [14CJMVA-labeled

treated

were antibody

location

size range. Both approaches thus of the IFN-induced huGBPs in by the C15 isoprenoid, farnesyl.

suggest that these proteins may be modified Although recombinant huGBP1 could act

transferase

substrate

in vitro,

this

did

not

ad-

dress whether in an intact cell, particularly an IFN-ytreated one, the GBP CaaX motif was prenylated. The possibility that a protein with a CaaX motif might not be prenylated in vivo has an important precedent. Another GTP-binding protein, the alpha subunit of the pertussis

Blot

A

[3H]MVA

each

CIFNBZA

CIFNBZA

expressed huGBP three cell lines,

,Y. In protein

comigrated

protein(s) after an IFN-induced

with

the

13j11MV4

H1GGOH

CIFN 82

was tein

tightly being

regulated, expressed

with

IFN-y.

of huGBP

plus

49

-

33

-

occur after IFN-’y of these is a prenyl

29

-

of similar

proteins whose et al. [35] have

with

BZA-SB.

LPS

were

membrane,

on the

(IFN) exposed

a 6% were

the

image

[3HIGGOH

into

huGBPs

treated

lysates

were

exposed GBPs

100

U/mL on

for 21 days.

is given

on the

on the

and

either plus

The left.

gel,

The

the huGBP1

size

of changes

treatment. protein

that the altered

in prenylated

in

proteins

The most abundantly of 65 kDa, p65, which,

to GBPs,

does

not

appear

induced expression

left

in

induced although

to be the

strains [38].

of The

murine

mice that IFN-y-in-

transferred

G

protein,

G1a,

any

[37].

Cys

results

fected COS cells, where the selectively and unambiguously, an isoprenoid attached. Thus

has

human

the

[3H]MVA

position

lines

of

P

S

lysate

P

S

shown).

a C-terminal

that,

were

Blot

in

CaaX

GBP

is the site for pertussis and is not modified by demonstrate

HL-60

and

markers,

in trans-

protein could be expressed huGBP1 does indeed have huGBP1 resembles Ras-like

cell

B

for 8 h. Cell

weight

in two crucial aspects: the ability GTP, and in having an isoprenoid several

of with

to a membrane,

(not

S

(Blot)

labeled

(IFN)

indicates

P

S

GBP

GBP

(C) or simultane-

of molecular arrow

P

to a the

were

LPS

cos

HL-60

for 8 h. Cell

antibody

untreated

A

with

of incorporation

cells

1 .tg/mL

dashed

in which the ADP-ribosylation,

In addition,

cells, indicating is not appreciably

IFN-y

transferred

exposure,

by immunoblotting

motif (CGLF), toxin-mediated

proteins hydrolyze terminus.

After

HL-60

U/mL

(BZA)

(B) Lack

position

membrane

100

gel,

days. with

IFN-y a 15%

Our

21

cells.

toxin-sensitive

isoprenoid

in HL-60

expression is specifically regulated. Vestal shown that in mouse bone marrow-derived a number

in HL-60 labeled

with

BZA-SB

([3H}MVA).

in HL-60

separated

detected

plus

for

huGBPs

simultaneously

or treated

detected

or [3H]GGOH with

to film

thousands,

film

into

polyacrylamide

to film

membrane

[3H]mevalonate

were

(C)

or IFN-y/LPS on

with

ously

cells

untreated

aligned

and

of [3H]MVA

HL-60

left

separated and

proteins

incorporation

either

1 tg/mL

occurred

procells

-

Decreased and

lysates

in HFLC

during differentiation of HL-60 into more macrophage-like cells. There have been few studies of protein prenylation cells treated with cytokines, and even fewer that examined

-

19

treated

also

be prenylated in the differentiated activity of prenyl transferases

CIFN

GBP1 protein, as it is genetically lack mGBP1

[HJMVA,

and

had been compelled to differentiate into macrocells, but again, only after IFN-y/LPS exposure. constitutively expressed proteins continued to

macrophages

cells

GBP,

with no detectable prenylated without prior treatment of the

Prenylation

cells that phage-like In addition,

B

5. (A)

induced

fibroblasts, this prenyl protein was shown to he a GBP by successful capture with an anti-GBP antibody. Thus the major huGBPs produced in response to IFN-y also appear to contain isoprenoid. With the possible exception of 1523 cells, the synthesis of huGBPs in these various cell types

GBP

Fig.

treatment with IFN[3H]MVA-labeled

to bind and at its carboxy used

to study

the prenylation of endogenous huGBPs expressed in cells treated with their natural inducer, IFN-y. HL-60 promyelocytic cells, HFLC lung fibroblasts, the THP-1 monocytic cell line, 1523 fibroblasts, and 293 embryonic kidney cells

6.

Fig.

(A)

Subcellular

location

Membrane

(P) and

transfected

with

IFN-y/LPS

for 24 h. Equivalent

separated

huGBP1

on a 15%

by immunoblotting. proteins

(P),

beled

HL-60

and

Equivalent was weeks. GBP1

sprayed After antibody

Nantais

or from

cells. cytosolic

cells

(S)

that

that

been

treated

with

fraction

were

ofeach

et al.

treated

Isoprenoid

membrane with the

subcellular and

cellular

been

a fluorographic

exposure, the and aligned

had GBP

proteins

ofprenylated

of total

transferred

cells.

cells gel

fraction

HL-60 COS-1

were

had

and from

fractions

of each

gel and with

Samples

COS

prepared

HL-60 amounts

in

were

SDS-polyacrylamide

amounts

polyacrylamide

of huGBPs

(5) fractions

(B) Subcellularlocation

in HL-60

brane

cytosolic

were

with

separated

on

enhancer

and

modification

of

IFN-yILPS. a

The

exposed

was incubated film image.

mem-

[ H]MVA-la-

overnight membrane.

of huGBP

(l;sate).

from

to a PVDF

detected

forms

proteins

prepared

cells

15%

to film with

huGBP1

SDS-

membrane the

for human

429

6

ducible appear p65

prenylated protein(s) in the to be homologous to the murine binds

rather

well

to membranes

cells do not as prenylated

(-80%),

ducible [J. E. member,

human prenyl protein(s) do not comigrate with p65 Buss, unpublished data]. One other GBP family the rat p67 protein, has recently been shown to in transfected macrophages

GBPs under

whose

are thus stringent

synthesis

Our

results

huGBP on the

COS treated

regulated

suggest

is modified observations

that

much

more the in-

cells and rat bone with IFN-y/LPS

among a select transcriptional

is tightly also

kDa are Furthermore,

the

prenylated (-.- 15%

Human proteins

of 60-65 bound).

whereas

human soluble

be prenylated row-derived

protein(s) membrane

human p65,

mar[16].

group of prenyl regulation, and

major

by the C15 isoprenoid that its prenylation

IFN-y-induced farnesyl, based is diminished in

cells exposed to a farnesyl transferase inhibitor and that it fails to incorporate detectable C20 isoprenoid. Because isoprenoid addition to a protein occurs only once, shortly after a protein is synthesized [18], and is then retained for the lifespan pected

of the protein, to be particularly

Other previously ing pool of protein ably

newly

synthesized that will

functional.

While

farnesyl change

transferase in abundance

[39-41],

synthesis

the

synthesized vulnerable

proteins are exsuch inhibitors.

to

Ras

proteins,

of GBPs

toward

are targeted, a variety of increases

rapidly

which

functions

purpose For

this small K4B-Ras ing

and

be a valuable

tool

of GBP induction. Ras proteins, prenylation

six

GTPase protein protein a domain

the

contiguous

lysine

to its prenylation near its C-terminus

motif, (see

of the

human

proteins

huGBP 1 protein with an isoprenoid

in

fibroblasts

Rab proteins are held nucleotide dissociation

nine nucleotide nine nucleotide GTPase [24,

430

Journal

exchange factor) binding and 25]. Importantly,

of

Leukocyte

Biology

Dr. James

Marsters,

Genentech,

for

Staeheli, University of Dr. Richard Maki, The discussions. This work

was

supported

by Hatch

of For-

Act and

the of

REFERENCES

In the includto

1.

the

2.

3.

also contains Introduction). 4.

Thus

5.

the 6.

7.

8.

cytosol by GDI or GEF (gua-

60,

to thank

the BZA-SB, Dr. Peter for generous support, and Institute, for many helpful

and

proteins that control guahence, activation of the these regulatory proteins

Volume

like

Station, Project 3179, State of Iowa funds.

cytosolic, even available to assist

in the inhibitor)

would

paper J-16583 Experiment

possible membrane association. Similar to huGBP1, members of the Rae and Rab families of GTPases have also been found to be largely cytosolic despite the presence of an isoprenoid. In these cases it appears that the prenylated Rae and (guanine

ACKNOWLEDGMENTS

This study is journal and Home Economics

just

[13].

remai ns predominantly and basic residues

designed

schungsgemeinschaft. of the Iowa Agriculture

Despite the presence of these motifs, human GBP1 in COS cells and the huGBPs induced in HL-60 cells associate very poorly with membranes. Early work on the GBPs showed this same point through immunofluorescence studies

the newly [39-41].

in response

amino-terminal side of the CaaX motif and isoprenoid, provides supplementary ionic interactions that further support membrane interactions [22, 42]. The human GBP1 protein, in addition five basic residues

during treatments with transferase inhibitors

Institutes Deutsche

association

located

tor to toxicity farnesyl:protein

of huGBP1 or contnbu-

was funded by a grant from the National Health to J. E. B. (CAS189O) and from the

with cell membranes. rich in basic residues, residues,

group suggests that interruption may be a significant limiting factor

show little conditions

for deciphering

promotes

a farnesyl prenylation

supplying Freiburg, Bumham

to a4 IFNs as well as to IFN-’y [4, 12]. Therefore exposure of cells to farnesyl transferase inhibitors during the initial phases of IFN responses may selectively compromise huGBP

prenylated protein may be a useful approach for identifying partner proteins that interact with huGBP1. HuGBP1 is now the third identified IFN-y-inducible protein shown to be isoprenoid modified, the others being the murine p65 protein [35] and another GBP family member, the rat p67 protein [16]. The dramatic induction and abundance of the huGBP1 mRNA and protein in IFN-y-

We

proteins will have a preexistremain prenylated and presum-

inhibitors under

at the C-terminus of construction of a noncomparison with the

treated cells [4, 8, 9, 12, 13] suggests that prenylation of huGBP1 will be important to IFN-y responses in human cells. In addition, the indication that huGBP is modified by

by a cytokine. the

appear to interact with the isoprenoid the GTPase. Based on this model, prenylated form of huGBP1 and

September

9.

Farrar, M. A., Schreiber, R. D. (1993) The molecular cell biology of interferon-gamma and its receptor. Annu. Rev. Immunol. 1 1, 571-611. Darnell, J. E., Kerr, I. M., Stark, G. R. (1994) Jak-STAT pathways and transcriptional activation in response to IFNs and other extracellular signaling proteins. Science 264, 1415-1421. Kotenko, S. V., Izatova, L. S., Pollack, B. P, Mariano, T. M., Donnelly, R. J., Muthukumaran, G., Cook, J. R., Garotta, G., Sivennoinen, 0., Ihle, J. N., Pestka, S. (1995) Interaction between the components of the interferon gamma receptor complex. J. Biol. C/tern. 270,20915-20921. Cheng, Y. E., Colonno, R. J., Yin Fay, H. (1983) Interferon induction of fibroblast proteins with guanylate binding activity. J. BiO1. C/tern. 258, 7746-7750. Watling, D., Guschin, D., Muller, M., Silvennoinen, 0., Witthuhn, B. A.,Quelle, F. W., Rogers, N. C., Schindler, C., Stark, G. R, IhIe, J. N., Kerr, I. M. (1993) Complementation by the protein tyrosine kinase JAK2 of a mutant cell line defective in the interferon-y signal transduction pathway. Nature 366, 166-170. Muller, M., Briscoe, J., Laxton, C., Guschin, D., Ziemiecki, A., Silvennoinen, 0., Harpur, A. G., Barbieri, G., Witthuhn, B. A., Schindler, C., Pellegrini, S., Wilks, A. F., Ihle, J. N., Stark, C. R., Kerr, I. A. (1993) The protein tyrosine kinase JAK1 complements defects in interferon-W and -y signal transduction. Nature 366, 129-135. lhle, J. N., Witthuhn, B. A., Quelle, F. W., Yamamoto, K., Thierfelder, W. E., Kreider, B., Silvennoinen, 0. (1994) Signalling by the cytokine receptor superfamily: JAKS and STATs. Trends Bid. Sci. 19, 222-227. Lew, D. J., Decker, T., Strehlow, I., Darnell, J. E. (1991) Overlapping elements in the guanylate-binding protein gene promoter mediate transcriptional induction by alpha and gamma interferons. Mol. Cell. Biol. 11, 182-191. Briken, V., Ruffner, H., Schultz, U., Schwai-z, A., Rica, L F. L, Streblow, I., Decker, T., Staeheli. P. (1995) Interferon regulatory factor 1 is required for mouse CBP gene activation by gamma interferon. Mol. Cell. Blot. 15, 975-982.

1996

10.

11.

12.

13.

Schwemmle, M., Kaspers, B., Irion, A., Staeheli, P., Schultz, U. (1996) Chicken guanylate-binding protein: conservation of GTPase activity and induction by cytokines.J. Biol. Chem. 271, 10304-10308. Asundi, V. I., Stahl, R. C., Showalter, L., Conner, K. J., Carey, D. J. (1994) Molecular cloning and characterization of an isoprenylated 67 kDa protein. Biochem. Biophys. Acta 121, 257-265. Wynn, T. A., Nicolet, C. M., Paulnock, D. M. (1991) Identification and characterization ofa new gene family induced during macrophage activation. J. Immunol. 147, 4384-4392. Cheng, Y.-S. E., Petterson, C. E., Staeheli, P. (1991) Interferon-induced guanylate-binding

14.

15.

16.

17.

18. 19. 20. 21.

22.

23. 24.

25. 26.

proteinslack

an N(T)KXD

consensus

motifand

bind

27.

C..

Goldstein,

sequences

28.

29. 30.

GMP

in addition to GDP and GTP. Mol. Cell. Biol. 1 1, 4717-4725. Schwemmle, M., Staeheli, P. (1994) The interferon-induced 67-kDa guanylate-binding protein (hGBP1) is a GTPase that converts GTPto GMP.J. Biol. Chem. 269, 11299-11305. Cheng, Y-S. E., Becker-Manley, M. F., Chow, T. P., Horan, D. C. (1985) Affinity purification of an interferon-induced human guanylate-binding protein and its characterization. J. Biol. Chem. 260, 15834-15839. Vestal, D.J., Buss,J. E., Kelner, G. S., Maciejewski, D., Asundi, V. K., Maki, R. A. (1996) Rat p67 GBP is induced by interfemn-yand is isoprenoid-modifled in macrophages. Biochem. Biophys. Res. Commun., in press. Clarke, S. (1992) Protein isoprenylation and methylation at carboxyl-tenninal cysteine residues. Annu. Rev. Biochem. 61, 355-386. Casey, P. (1993) Biochemistry of protein prenylation. J. Lipid Res. 33, 1731-1740. Kato, K., Der, C. J., Buss, J. E. (1992) Prenoids and palmitate: Lipids that control biological activity of Ras proteins. Semin. Cancer Biol. 3, 179-188. Der, C. J., Cox, A. D. (1991) Isoprenoid modification and plasma membrane association: critical factors for ras oncogenicity. Cancer CelLs 3, 331-340. Kato, K., Cox, A. D., Hisaka, M. M., Graham, S. M., Buss, J. E., Der, C. J. (1992) Isoprenoid addition to Ras protein is the critical modification for its membrane association and transforming activity. Proc. Nail. Acad. Sd. USA 89, 6403-6407. Jackson, J. H., Cochrane, C. C., Boume,J. R., Solski, P. A., Buss, J. E., Der, C. J. (1994) Farnesol modification of Kirsten-ras exon 4B protein is essential for transformation. Proc. Nail. Acad. Sci. USA 87, 3042-3047. Marshall, C. J. (1993) Protein prenylation: a mediator of protein-protein interactions. Science 259, 1865-1866. Pfeffer, S. R., Dirac-Svejstrup, A. C., Soldati, T. (1995) Rab GDP dissociation inhibitoc putting Rab GTPases in the right place. J. Biol. Chem. 270, 17057-17059. Chanock, S. J, Benna, J. E., Smith, R. M., Babior, B. M. (1994) The respiratory burst oxidase. J. Biol. C/tern. 269, 24519-24522. Newman, C. M. H., Magee, A. I. (1993) Posttranslational processing of the ras superfamily of small GTP-binding proteins. Biochem. Biophys. Acta 1 155, 79-96.

James,

31.

32.

protein Crick, aniol

34.

36.

37.

38.

39.

for

41.

42.

A2 in HL-60

protein

cell

differentiation

isoprenylation

S. M., Buss,

Vestal,

D. J, Maki,

protein

in macrophages

and

R. A., Buss,

C6

fatty

and

and

confer

to macrophages

glial

CVIM

resistance

induced

by

USA 90, 4917-4921. Utilization of geranylgerBiochem. Biophys. Res.

cells.

Metabolic

L. Z., Spiegel, occurs only

proteins.

J. E. (1995)

Induction

orlipopolysaccharide.J.

M., Herrmann, MxA protein:

270,

radiolabeling techniques for Methods [San Diego]

acylated

by interferons

58, 607-622. Richter, M. F., Schwemmle, (1995) Interferon-induced properties. J. Biol. Chem. Jones,T. a subunit

in

J. E. (1990)

of prenylated

ofa prenylated65 Leukoc.

C., Wittinghofer, GTP binding and

kDa Biol.

A., Staeheli, CII’

P.

hydrolysis

13512-13517.

A. M. (1990)Isoprenylation upon mutagenesis of the

ofan carboxyl

inhibitory terminus.

C protein

J. Biol. Chem. 265, 19389-19392. Staeheli, P., Prochazka, M., Steigmeier, P. A., Hailer, 0. (1984) Genetic control of interferon action: Mouse strain distribution and inheritance of an induced protein with guanylate-binding property. Virology 137, 135-142. Marsters, J. C., Jr., Buss, J. E. (1995) Farnesyl transferase inhibitors: the and

surprises

ofa

new

class

ofpotential

cancerchemotherapeutics.

C/tern. Biol. 2, 787-791. Gibbs, J. B., Oliff, A., Kohl, N. E. (1994) Farnesyitransferase inhibitors: Ras research yields a potential cancer therapeutic. Cell 77, 175-178. Kohl, N. E., Omer, C. A., Conner, M. W. et al. (1995) Inhibition of farnesyitransferase induces regression of mammary and salivary carcinomas in ms transgenic mice. Nature Med. 1, 792-795. Hancock, J. F., Paterson, H., Marshall, C. J. (1990) A polybasic domain or paimitoylation to the plasma

Journal

Polylysine

of prenylation

205,955-961.

Commun. Mumby,

successes 40.

M. S. (1995)

specificity

kinase C activation. Proc. Nail. Acad. Sci. D. C., Waechter, C. J., Andres, D. A. (1994)

identification 1, 216-220.

35.

Brown,

dictate

to benzodiazepine peptidomimetic in vitro.). Biol. Chem. 270, 6221-6226. Kalman, V. K., Erdman, R. A., Maltese, W. A., Robishaw, J. D. (1995) Regions outside ofthe C4AX motifinfluence the specificity ofprenylation of C protein ysubunits.J. Biol. Chem. 270, 14835-14841. Adamson, P., Marshall, C. J., Hall, A., Tilbrook, P. A. (1992) Post-translational modifications of p2lrho proteins. J. Biol. Chem. 267, 20033-20038. Armstrong, S. A., Hannah, V. C., Goldstein,j. L., Brown, M. S. (1995) CAAX Geranylgeranyl transferase transfers farnesyl as efficiently as geranylgeranyl to RhoB. J. Biol. Chem. 270, 7864-7868. Bushkin, I., Roth, J., Heffetz, D., Zick, Y. (1991) pp7S: a novel tyrosinephosphorylated protein that heralds differentiation of HL-60 cells. J. Biol. Chem. 266, 11890-11895. Asaoka, Y., Yoshida, K., Sasaki, Y., Nixhizuka, Y. (1993) Potential role of phospholipase

33.

J. L.,

of K-RasB

of

Leukocyte

is required membrane.

Biology

in addition

Cell 57,

to the

CAAX

motif

to localize

p2lras

1167-1177.

Volume

60,

September

1996

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