Posttranslational

modification

isoprenoids WILLIAM

in mammalian Geisinger

Clinic,

Danville,

Abstract Isoprenylation is a posttranslational modification that involves the formation of thioether bonds between cysteine and isoprenyl groups derived from pyrophosphate intermediates of the cholesterol biosynthetic pathway. Numerous isoprenylated proteins have been detected in mammalian cells. Those identified include K-, N-, and H-p21, ras-related GTP-binding proteins such as G25K (G), nuclear lamin B and prelamin A, and the ‘y subunits of heterotrimeric G proteins. The modified cysteine is located in the fourth position from the carboxyl terminus in every protein where this has been studied. For p2l, the last three amino acids are subsequently removed and the exposed cysteine is carboxylmethylated. Similar processing events may occur in lamin B and G protein ‘y subunits,

but

the

upstream

proteolytic

cleavage

in prelamin

from the modified cysteine. modified by C15 farnesyl

Lamin

A B

and p2lras are groups, whereas other proteins such as the G protein ‘y subunits are modified by C20 geranylgeranyl chains.

Separate enzymes may catalyze these modifications. The structural features that govern the ability of particular proteins to serve as substrates for isoprenylation by C or C20 groups are not completely defined, but studies of the p21ras modification using purified farnesyl:protein transferase suggest that the sequence of the carboxyl-terminal tetrapeptide is important. Isoprenylation plays a critical role in promoting the association of p2l and the lamins with the cell membrane and nuclear envelope, respectively. Future studies of the role of isoprenylation in the localization and function of ras-related GTP-binding proteins and signaltransducing G proteins should provide valuable new insight into the link between isoprenoid biosynthesis and cell growth. MALTESE, W. A. Posttranslational modification of proteins by isoprenoids cells.FASEBJ. 4: 3319-3328; 1990. Key Words: isoprenylation mevalonate nucleotide binding pmteins#{149}lamins

in mammalian

ras

guanine

THE LATE 1970s, INVESTIGATORS studying fungal peptide pheromones discovered a novel lipid modification involving thioether derivatization of carboxyl-terminal..

IN

flRq-FlRIqnInnnLl-uqIcn1

by

cells

A. MALTESE

The Weis Center for Research,

occurs

of proteins

n

n

Pennsylvania

17822,

USA

cysteine residues by farnesyl or hydroxyfarnesyl isoprenoid groups (1, 2). This type of posttranslational modification, which has been termed isoprenylation, is now known to occur in a broad spectrum of mammalian cell proteins. Recent developments, such as identification of oncogenic ras proteins (3-6), G proteins (7-9), and nuclear lamins (10-14) as targets for isoprenylation, have stimulated considerable interest. This review is a summary of current knowledge regarding isoprenylated proteins in mammalian cells, highlighting issues that seem likely to emerge as focal points for future research. THE MEVALONATE CELL CYCLING

REQUIREMENT

FOR

The discovery of protein isoprenylation in mammalian cells grew from studies aimed at defining the relationship between mevalonate metabolism and cell proliferation. As shown in Fig. 1, mevalonate occupies a central position in a complex branched pathway for isoprenoid biosynthesis (for review, see ref 15). Cholesterol, a major lipid component required for membrane replication, is only one of several end products derived from mevalonate. Others include the dolichyl phosphates, which carry oligosaccharides in the process of N-linked protein glycosylation, isopentenyladenine, which is found in some types of tRNA, and the polyisoprenoid side chains of ubiquinone and heme-a, which transport electrons in the mitochondrial respiratory chain. Evaluation of the potential roles of these diverse isoprenoid products in cell cycling was facilitated by the development of drugs, such as compactin (16) and mevinolin (lovastatin) (17), which inhibit 3-hydroxy-3methylglutaryl-CoA (HMG-CoA)’ reductase (see Fig. 1). Early studies with these compounds clearly established that blocking mevaionate synthesis could arrest cell cycling in vitro (18-20) and suppress tumor growth in vivo (21). In some cases, arrest of cell cycling was preceded or accompanied by striking changes in cell morphology, such as cell rounding (22) or neurite outgrowth (23). However, the precise mechanisms underlying these effects remained elusive. Although changes

1Abbreviations: SDS, sodium dodecyl sulfate; IEF, isoelectric focusing; HMG-CoA, 3-hydroxy-3-methylglutaryl-CoA; MEL, munne erythroleukemia.

lo

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.

2 Ac.tyl-CoA AcStoIo.tyi-COA

ISOPRENOID BIOSYNTHESIS IN MAMMALIAN CELLS

iNcises

Ac.toac.tyl-CoA Aostyl-COA ---‘

MEVALONATE

M.valonat.-P

-* Mivalonat#{149}-PP-* Phosphen,svsiensts Phosphem.vsionsts Kiness D.csrboxyl.se

-* M.vsien.t. Kin,,.

Isop.nt.nyl-tRNA

sop#{149}nt#{149}nyl-PP I

DIm.thylallyl-PP

4Olmsthyisilyl Trsnstsrss.

isopentlny-Pe isom.rso.

DIm.thylallyl-PP

Ad.nOsln.

+

#{149} lsopsnt.nyl-PP Synthas.

O.ranyl-PP

Farn.syI-PP

G.ranyl-PP

lsepsnt.nyi-PP Syntheos

E

:

;:-

I

Farn.syI-PP

#{149} Psrn.syl-PP Sqoslsns Synthes.

l.opsnt.nyi-PP G.rsnylgeranyi-PP

Synthass

Squel.n. Farn..ylatsd

H#{149}m.-a DoIIchyI-PP

Prot.ins

D.capr.nyl-PP

of the pathway,

growth

and

see ref

morphology

Ublquinon.s

ChoI.st.rol

could

ISOPRENYLATED CELLS

December

1990

cells.

Mevalonate,

which

is frequently

used

as a precursor

or in part from mevalonate are shown in red. Major in green. For additional information concerning the

15.

be prevented

by

PROTEINS

ditions a significant amount of the labeled mevalonate was incorporated covalently into cellular proteins. Through the use of mevalonate radiolabeled at different carbon positions, the modifying group was shown to be an isoprenoid derivative rather than mevalonate itself. Later reports established that isoprenylation of proteins is a general phenomenon in mammalian cells (26-29), and that the electrophoretic pattern of proteins visualized by incorporation of radiolabeled mevalonate is generally similar from one cell line to another (for example, see Fig. 2A). Subcellular fractionation studies suggested that the multiple mevalonate-labeled polypeptides were unique gene products, as isoprenylated proteins with different molecular masses were distributed in several compartments, including the nuclear matrix, plasma membrane, microsomes, and cytosol (28,

Vol. 4

Ito.

in mammalian

New insight into the mechanism underlying the cellular requirement for mevalonate was provided by a groundbreaking study in which Schmidt Ct al. (25) traced the fate of radiolabeled mevalonate taken up by cultured 3T3 fibroblasts whose endogenous mevalonate synthesis had been blocked with mevinolin. Under these con-

3320

Dsmsthyistlon

derived completely steps are indicated

adding mevalonate to the culture medium, a similar protective effect was not achieved by supplying the cells with cholesterol, dolichol, ubiquinone, or isopentenyladenine (22, 23). Nor did it appear that cellular cholesterol/phospholipid ratios (23), mitochondrial respiratory function (24), or glycoprotein synthesis (23) were significantly impaired before the onset of growth arrest. Thus, although it was clear that one or more nonsterol isoprenoid derivatives of mevalonate was critically important for cell cycling and maintenance of cell shape, the identities of these derivatives were not immediately obvious. DISCOVERY OF IN MAMMALIAN

1?)

Prot.ins

Oxldosquslsn.

Lanost.rol

1. Overview of the pathway for isoprenoid biosynthesis in metabolic labeling studies, is shown in blue. End products intermediates are in black. Enzymes catalyzing the individual

Protein

Tr.nsfsrss.

Cycles,

Polypr.nyl-4hydroxyb.nzoat.

Figure

Oersnylgeranyi

2,3-Epoxysqual#{149}n#{149} G.ranylg.ranylat.d

Tr$nsl.rss.

in cell

$

Or

$

regulation

G.ranylg.ranyl-PP

Sqosl.n.

Nonapr.nyl-PP

#{149} lssp.nt.nyl-PP Synthese

- - Farn.syI-PP

C ;.

$

Farn.syi-PP

5. S

29).

The posttranslational nature of the isoprenoid modification was established by experiments using inhibitors of protein synthesis (25, 26, 30). However, the precise temporal relationship between translation and isoprenylation varied, depending on mevalonate availability (30). Under normal culture conditions, short-term

The FASEB lournal

MALTESE

www.fasebj.org by Kaohsiung Medical University Library (163.15.154.53) on November 12, 2018. The FASEB Journal Vol. ${article.issue.getVolume()}, No. ${article.issue.getIssueNumber

.

B kDa

C

SOLUBLE IEF

PARTICULATE IEF

kDa Figure malian

2. Electrophoretic

profiles of isoprenylated visualized by incorporation A) Several established mammalian

cells,

mevalonate. cubated in vitro with [‘4C]mevalonate, tein

from

each

cell line

were

subjected

of pro-

to one-dimensional

SDS-

46-

polyacrylamide gel electrophoresis. The fluorograph shows the positions of the radiolabeled isoprenylated proteins relative to the molecular mass markers (from ref 28, with permission). B, C) Friend MEL cells labeled with [3H]mevalonate were subjected to

!

subcellular fractionation to obtain total soluble and particulate (nuclei, membranes, mitochondria) components. The fluorographs show the complex patterns of isoprenylated proteins typically obtained when these fractions are resolved by two-dimensional dcctrophoresis (IEF, isoelectric focusing; SDS, sodium dodecyl sulfate gel electrophoresis. Taken from an illustration in ref 55, with permission).

suppression of protein synthesis with cycloheximide abolished the incorporation of exogenous [3H]mevalonate into most cellular proteins, implying that isoprenylation is an immediate posttranslational event. In contrast, when cells were preincubated briefly with an inhibitor of mevalonate synthesis,the subsequent incorporation of exogenous [3H]mevalonate into cellular proteins was rendered insensitive to cycloheximide (30). This indicated that transient interruption of the synthesis of mevalonate and its isoprenyl pyrophosphate derivatives could cause a rapid accumulation of unmodified substrate proteins. It is not yet certain whether the isoprenoid groups attached to specific proteins undergo turnover that is independent of protein degradation. However, preliminary evidence derived from pulse-chase experiments with murine erythroleukemia (MEL) cells suggests that isoprenylation is a stable modification. For example, when proteins were prelabeled with [3H]mevalonate and growth-related dilution of the labeled proteins was taken into account, no appreciable turnover of radiolabeled protein-bound isoprenyl groups was detected during an 8-h chase period (30).

ISOPRENOID

MODIFICATION

OF PROTEINS

+

Ga-v

proteins in mamof radiolabeled cell lines were in-

and equal amounts

+

14.6

-

STRUCTURES ISOPRENYL

OF PROTEIN-BOUND GROUPS

Recent characterization of the isoprenoid moieties involved in the modification of mammalian cell proteins has gone hand in hand with identifying the proteins themselves. Progress in both areas can be attributed largely to the recognition of structural parallels between the mammalian isoprenylated proteins and the aforementioned fungal peptide pheromones, where farnesyl or hydroxyfarnesyl groups are linked to carboxylterminal

cysteine

residues

via

thioether

bonds.

Disruption of the isoprenyl thioether bonds in the fungal peptides was accomplished by the formation of S-methylsulfonium derivatives with methyl iodide and subsequent cleavage of the sulfonium salts to release free isoprenyl alcohols (1, 2, 31). Application of the same approach to [3H]mevalonate-labeled proteins from MEL cells resulted in release of most of the isoprenoid (32). Chromatographic analysis of the radiolabeled material revealed the presence of C15 isoprenoids, such as farnesol and nerolidol (a rearrangement product of farnesol), as well as unidentified compounds

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(32). More recent work using gas chromatography-mass spectroscopy to identify isoprenoids released by Raneynickel cleavage of proteins from Chinese hamster ovary cells (33) and HeLa cells (34) has established the existence of a second type of modifying group (i.e., C20 geranylgeranyl), which appears to be the predominant protein-bound isoprenoid in these cells. The C15 and C20 isoprenoid groups attached to mammalian proteins are derived from pyrophosphate intermediates of the cholesterol pathway (35-38) (see Fig. 1). The sequential condensation reactions involved in forming farnesyl pyrophosphate are catalyzed by farnesyl pyrophosphate in some detail (39,

synthetase, 40). Much

which has been studied less is known about the

biogenesis of geranylgeranyl pyrophosphate. Farnesyl pyrophosphate functions poorly as a substrate for elongation by farnesyl pyrophosphate synthase (39), and at least in the liver, a separate enzyme appears to catalyze the synthesis of geranylgeranyl pyrophosphate (41) (see Fig. 1). The discovery of proteins that are modified by geranylgeranyl

groups

should

stimulate

interest

in

greater characterization of the latter enzyme. The heterogeneity in the chain-lengths of the isoprenoid groups attached to mammalian cell proteins raises a number of important questions: Are there different isoprenyl:protein transferases with specificities for farnesyl and geranylgeranyl pyrophosphates? Can the same proteins

be

modified

by

either

farnesyl

or

geranyl-

geranyl groups, depending on the particular pyrophosphate substrates or isoprenyl:protein transferases that predominate in a particular cell type, or is each protein able to accept only a single type of isoprenyl group? If there is specificity to the modification, is it dictated by discrete structural features of the acceptor proteins? Are the functional consequences of farnesyl vs. geranylgeranyl modification similar or unique? As reviewed in the following sections, answers to some of these questions have begun to emerge with identification of specific isoprenylated proteins and their modifying groups. IDENTIFICATION

OF

ISOPRENYLATED

PROTEINS

The ated

prospect proteins

INDIVIDUAL

of trying to identify all of the isoprenylin mammalian cells initially seemed

daunting, based on the complexity of the electrophoretic patterns typically seen when [3H]mevalonatelabeled proteins were resolved on two-dimensional gels (Fig. 2B and Fig. 2C). However, examination of the structures of the farnesylated fungal mating factors suggested that the task might be simplified by looking for particular cysteine-containing carboxyl-terminal sequences in mammalian proteins. Specifically, the yeast a-mating factor terminates with a farnesylated, carboxylmethylated cysteine (31), whereas its predicted amino acid sequence contains an extension of three amino acids beyond the cysteine (-CVIA) (42). This raised the possibility that the terminal Cxxx motif (often referred to as a Caax box, because the two amino acids distal to the cysteine quence for

are

aliphatic)

cysteine

might

isoprenylation

serve

as a signal

and

proteolytic

se-

removal of the amino acid extension. As shown i Table 1, a substantial number of proteins in vertebrat cells contain a Cxxx motif in their predicted sequences. ras

Proteins

Clarke et al.(43) first reported that the terminal cysteine of p2111#{176} was methyl esterified, and noted the parallel between the predicted carboxyl-terminal sequences o the farnesylated yeast a-mating factor and the rasencoded proteins. Then, in an elegant series of studies, Hancock et al. (3) demonstrated that cysl86 of the ras proteins is modified by a derivative of mevalonic acid rather than palmitate, as was previously thought. Isoprenylation is the first step in a series of posttranslational processing events required for the stable association of p2lras with the cell membrane (3) (Fig. .3). Blocking mevalonate synthesisresultsin accumulation of nonisoprenylated p2iras precursor (3-6) and loss of transforming activityof oncogenic ras proteins (4, 5). Preliminary chromatographic analysis of the isoprenoid released from K-ras 4B by sulfonium salt cleavage suggested that p2lraS belongs to the class of proteins modified by C15 farnesylresidues (4). More recent evidence for a farnesyl modification comes from studies in which recombinant ras proteins have been used as substrates for farnesyl:protein transferase in vitro (36-38). The enzymes involved in the proteolytic processing and carboxylmethylation steps that follow isoprenylation have not been isolated or characterized. However, current interest in developing novel therapeutic strategies aimed at interfering with the posttranslational processing of oncogenic ras proteins undoubtedly will stimulate research in this area. Nuclear

lamins

The association of isoprenylated proteins with the nuclear matrix (28) raised the possibility that these proteins were components of the nuclear lamina. On the basis of immunoblotting and immunoprecipitation criteria, two of the 66- to 72-kDa [3H]mevalonatelabeled proteins in cultured HeLa and Chinese hamster ovary cells were identified as lamin B (10, 11) and lamin A (11). More recently, Vorburger et al. (13) showed that cysteines in the carboxyl-terminal Cxxx sequences of chicken lamins A and B2 can be modified by products of [3H]mevalonate in reticulocyte lysates. Although the proteolytic processing of lam in B2 resembles that of the ras proteins (i.e., removal of three amino acids distal to the isoprenylated cysteine), the cleavage of lamin A actually occurs upstream from the cysteine, resulting in the loss of a 2-kDa fragment (including the isoprenylated Cxxx) in the mature lamin A. Using specific inhibitors of mevalonate synthesis, Beck et a!. (14) showed that this proteolytic processing of the lamin A precursor

is dependent

on the initial

isoprenylation

step.

Isoprenylation of the lamins appears to play an important role in determining their association with the nuclear envelope. Forms of lamin A or Xenopus lamin L1 (a B-type lamin) that have been altered by removal

3322 Vol. 4 December 1990 The FASEBjournal MALTESE www.fasebj.org by Kaohsiung Medical University Library (163.15.154.53) on November 12, 2018. The FASEB Journal Vol. ${article.issue.getVolume()}, No. ${article.issue.getIssueNumber

TABLE

1. Examples

Low molecular

of vertebrate proteins containing Cxxx carboxyl-ter7ninal amino acid sequence motifs

mass GTP-binding

proteins

Nuclear

ras Family

lamins

Lamin

A (human)

-TQSPQNCSIM

-GCMSCKCVLS

Lamin

A (chicken)

-APAPQCCSIM

K-rasA (human) K-rasA (mouse) K-racE (human)

-CVKIKKCIIM

Lamin

A (Xenopus)

-QVAPQNCSIM

K-rasB (mouse) N-ras (human) N-ras (mouse)

H-ras (human,

mouse)

-C VKIKKC

VIM

Lamin

B (mouse)

-KKSKTKC

VIM

Lamin

B1 (chicken)

-KKSRTRCTVM

Lamin

B2 (chicken)

-RAWNKSCAIM -RKPERSCVVM -RTTSRGCLVM

-GCMGLPCVVM

Lamin

L1 (Xenopus)

-KSGNKNCAIM

-GCMGSPCVLM

Heterotrimeric

rho Family rhoA (rhol2) rhoB (rho 6)

-GKKKSGCLVL -NGCINCCKVL -NKRRRGCPIL

rhoC (rho 9)

G11,

p21/Krev

Go,, G,,1,

-KPKKKSCLLL -ARKKSSCLQL -DPCCSACNIQ.

-KKRKRKCLLL

rac2

-RQQKRACSLL

-KNNLKECGLY

-ANNLRGCGLY -KENLKDCGLF

Gta2

‘y Subunits

rac Family racl

-KNNLKDCGLF

G1n2

G1a3

rap Family raplAlsmg rapl B rap2

G-proteins

a Subunits

76

(G1, G0, G,)

-REKKFFCAIL

76

(G,)

-KELKGGCVIS

cGMP

phosphodiesterases

a’ Subunit a Subunit

(retinal (retinal

cones) rods)

-DKKSKTCLML -GPASKSCCVQ

ral Family ralA

-KRIRERCCIL

ralB

-KSFKERCCLL

rab Family rab5

-QPTRNQ#{231}CSN

of the Cxxx sequence or by substitution of another amino acid in place of the cysteine fail to localize to the nuclear envelope (44, 45). Mammalian lamin C, which normally does not have a Cxxx sequence, becomes tightly associated with the nuclear envelope when lamin A or B sequences containing this domain are added to the protein (45). Variations in the proteolytic

processing of lamin B and lamin A suggest that the specific mechanisms underlying their assembly into the nuclear envelope may be quite different. For instance, the isoprenyl group is retained on lamin B after processing, and may therefore contribute to the relatively stable association of this protein with the nuclear membrane. In the case of lamin A, isoprenylation of the translation product (prelamin A) initially directs the protein to the nuclear membrane, but the subsequent removal of the entire carboxyl-terminal region containing the isoprenoid implies that continued association of mature lamin A with the nuclear envelope depends on protein-protein interactions, perhaps involving the a helical domain (44). The isoprenoid group linked to lamin B in HeLa cells has been characterized by gas chromatographymass spectroscopy as a C15 farnesyl moiety (12). This finding is consistent with the similarity between the carboxyl-terminal tetrapeptides of the lamin and rcs ISOPRENOID

MODIFICATION

OF PROTEINS

protein methyl whether

families esterification this occurs

(see

Table

1). Lamin

B undergoes

(46), although it is not yet known specifically at the farnesylated cys-

teine residue. Finally, the determination that HeLa cell lamin B is modified by a C15 farnesyl group (12), although most isoprenylated HeLa cell proteins appear to contain C20 geranylgeranyl groups (34), raises the possibility that a given cell type may contain more than one kind of isoprenyl:protein transferase, and that the chain length of the group attached to a particular protein may be determined by specific protein sequence elements. With the recent development of assays to study isoprenylation in vitro (13, 36-38), it is now feasible to test this possibility. ras-Related GTP-binding

low molecular proteins

mass

Mammalian cells contain many low molecular mass GTP-binding proteins that are structurally distinct from H-, K-, and Np21ras, but nevertheless exhibit ras homologies in the regions contributing to the guanine nucleotide-binding site. The predicted sequences of many of these proteins, including rho (47, 48), rap (49, 50), rab (51), ral (52), and rac (53), have carboxylterminal Cxxx motifs, which is consistent with the possi-

3323

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TSTEPSIN

THE CARBOXYL-TERMINAL tee

ISO

PROCESSING

too

G-C-M-S-C-k-C-v-L-S

--

OF

tee

G-C-M-S-C-K-C-v-L-s thlosthsr farn.sylation of cysl8S

tee

ISO

tee

G-C-M-8-C-IC-C-v-L-s

G-C-M-S-C-K-C-

COON

protsolytic r.moval of 3 amino acid, from C-t.rminal teO

tee

G-C-M-S-C-K-C-

,es

COON

- -----

G-C-M-S-C-K-C-

-

S.. ,es

G-C-M-S-C-K-C-

OCH

5

tee

carboxyl m.thylation

t86

OCH

G-C-M-S-C-K-C-

\

,//

palmitat.

OCH3

,

palmltylation of upstr#{149}am cy.t.in.

Figure 3. Proposed sequence of events in the posttranslational processing of 21 H-rae as described by Hancock et al. (3). Before

the final step the p21

H-rae

translation

ble fraction. The mature palmitylated ized in the cell membrane.

product is found in the soluform of the protein is local-

undergoes phosphorylation by epidermal growth facto receptor tyrosine kinase in reconstituted vesicles (60). Therefore, future studies aimed at determining whethe isoprenylation may affect the interaction of G25K with the EGF-receptor will be of great interest. Preliminary evidence exists for the isoprenylation o two additional low molecular mass GTP-binding proteins, racl (61) and rho (38), both of which have carboxyl-terminal sequences fitting the general Cxxx pattern. Information concerning the structures of the modifying groups in these proteins probably will be forthcoming. A major unresolved issue pertaining to the low molecular mass GTP-binding proteins is whether isoprenylation of terminal cysteines can occur in proteins such as rabl and rab2 (xxCC) or rab3 (xCxC) (51), which contain variations of the standard Cxxx motif. Although studies with altered p2l proteins have suggested that changing the position of cysteine within the terminal tetrapeptide greatly diminishes its abilityto undergo farnesylation(36-38), little isknown about the structural requirements for modification by geranylgeranyl groups or possibly other unidentified isoprenoid moieties. Because the proteins ending with xxCC motifs are structural and functional homologs of yeast GTP-binding proteins involved in vesicular transport (i.e., YPT1), their isoprenylation could have important implications for the regulation of intracellular protein traffic and secretion in mammalian cells. Heterotrimeric

bility that they undergo isoprenylation (see Table 1). Accumulating evidence suggests that this is indeed the case. Most 20- to 30-kDa proteins labeled with [3H]mevalonate in cultured cells are not recognized by ras-specific antibodies, although they undergo isoprenoid-dependent carboxylmethylation and bind GTP when transferred to nitrocellulose membranes (54). On the basis of twodimensional immunoblots, one of these proteins has recently ben identified as G25K (formerly called G) (55). Originally isolated from human placental membranes (56) and bovine brain (57), this protein is now known to be present in a variety of normal and transformed cell lines (55, 58). Unlike p21’, most of the isoprenylated G25K is released in the soluble fraction when MEL cells are lysed in detergent-free buffer (55). This implies that factors other than isoprenylation may ultimately determine the proportion of this protein that becomes stably associated with the cell membrane or other organelles. A preliminary report indicates that a brain protein similar or identical to G25K is modified by a geranylgeranyl moiety (59). The full sequence of G25K has not yet been reported, but unpublished information indicates that at least two forms of the protein may exist; one ending with a carboxyl-terminal sequence of-C-V-L-L,2 and another with C-C-I-F3. At present it is not known whether one or both forms of the protein undergo isoprenoid modification, or whether further carboxyl-terminal processing occurs. A possible role for G25K in transduction of growth-modulating signals has been suggested by the recent finding that it

C proteins

The trimeric guanine nucleotide-binding proteins (G proteins) consisting of a, /3, and ‘y subunits play essential regulatory roles in receptor-mediated signal transduction pathways in mammalian cells (for review, see ref 62). The presence of Cxxx elements in the predicted sequences of several of the a subunits, and two subunits (see Table 1), suggested that these proteins might be subject to isoprenylation. A C20 geranylgeranyl modification has recently been confirmed in two G protein ‘y subunits purified from the brain (7, 8). Studies using reticulocyte lysates to modify the brain 76 translation product in vitro have further shown that isoprenylation occurs at the predicted cysteine in the carboxyl-terminal Cxxx sequence (9). Because the isoprenylated fragment of the subunit also appears to be methyl esterified (8), the geranylgeranyl modification probably triggers a ras-like proteolytic processing event that exposes the carboxyl-terminal cysteine residue. Until now, the mechanism underlying the strong association of the brain G protein f3y subunit complexes with the cell membrane has been obscure, as neither subunit contains extensive hydrophobic domains in its primary sequence. It is tempting to speculate that isoprenylation confers membrane affinity on the /3-y

2Personal communication University, Ithaca, N.Y. tPersonal communication Emeryville, Calif.

from from

Richard Paul

Cerione,

Polakis,

Cetus

Cornell Corp.,

MALTESE 3324 Vol. 4 December 1990 The FASEB journal www.fasebj.org by Kaohsiung Medical University Library (163.15.154.53) on November 12, 2018. The FASEB Journal Vol. ${article.issue.getVolume()}, No. ${article.issue.getIssueNumber

complex. Support for this idea has been provided by studies of yeast, which have shown that genetic or pharmacologic disruption of isoprenoid biosynthesis alters the biological activity of the f3’y subunits of Gm (63). In contrast to the subunits, the a subunits of the mammalian G proteins do not appear to be isoprenylated, although their carboxyl-terminal sequences fit the general pattern of Cxxx (9). The absence of isoprenylation may be related to the fact that the carboxylterminal cysteine undergoes a different type of modification in some of the a subunits, i.e., pertussis toxinmediated ADP-ribosylation (64). Single amino acid substitutions in the carboxyl-terminal tetrapeptide of p2lrss can alter its ability to serve as a substrate for farnesylation (36), and so it is worth noting that the carboxyl-terminal tetrapeptides of the a subunits of G1, G0, and G1 differ from those of other known isoprenylated proteins in having glycine residues immediately distal to the cysteine, and aromatic amino acids in the terminal position (see Table 1). It remains to be seen whether these differences are responsible for inhibiting isoprenylation, and whether they may confer specificity for ADP-ribosylation in these G protein subunits. ISOPRENYL:PROTEIN

TRANSFERASES

In vitro assays for farnesyl:protein transferase activity have been developed using recombinant p2lHras (36, 37) or yeast RAS protein (38) as acceptors (isoprenylation and processing do not occur in bacterial expression systems) and [3H}farnesyl pyrophosphate as the isoprenoid donor. Enzyme activity is present exclusively in the soluble fractions of a variety of tissues and cultured cell lines (36-38). Peptide inhibition studies suggest that recognition of acceptor proteins by the farnesyl:protein transferase may be determined largely by the amino acids of the terminal Cxxx motif (36). For instance, tetrapeptides based on the carboxyl-terminal sequences of p2lKras, p2lNras, and human lamins A and B act as potent inhibitors of the enzyme, apparently by competing as alternative substrates. The tetrapeptide of p21 H-ras, which ends with serine, also competes in this assay, but is somewhat less effective than the tetrapeptides ending with methionine. Consistent with findings described in the preceding section, the carboxyl-terminal tetrapeptide of Gia was completely ineffective in the competition assay (36). The availability of these in vitro assay systems should result in accelerated progress toward defining the specific amino acids that constitute functional farnesyl or geranylgeranyl acceptor sites in different proteins. Reiss et a!. (36) recently purified a brain farnesyl:protein transferase to apparent homogeneity by affinity chromatography. The purified protein elutes at 70- to 100-kDa upon gel filtration, and gives rise to two closely spaced bands at 50 kDa when subjected to electrophoresis in sodium dodecyl sulfate (SDS) gels. It is not yet known whether the native enzyme exists as a monomer or dimer. Based on the existence of discrete farnesylated and geranylgeranylated proteins within a given tis-

sue or cell type, it seems likely that additional nyl:protein transferases with different substrate ficities may be identified. However, the ability farnesyl:protein transferase to use geranylgeranyl phosphate as an alternative substrate remains thoroughly tested.

FUNCTIONAL ISOPRENYLATION

SIGNIFICANCE

isoprespedof the pyroto be

OF

Subcellular fractionation studies have shown that many isoprenylated proteins are found in the cytosol (28, 29, 54, 55, see Fig. 2B). This argues against the idea that the isoprenoid group necessarily serves to anchor proteins into the membrane lipid bilayer. A more subtle role is suggested by the diverse and sometimes paradoxical observations derived from recent studies of individual isoprenylated proteins. For example, isoprenylation appears to be essential for association of oncogenic ras proteins with the cell membrane, as evidenced by the loss of transforming activity when isoprenylation is blocked (3-5). However, for p2lH-raS, an intermediate form of the protein (cp21F5), which is isoprenylated but not yet processed to the mature palmitylated form (m-p21”), is found in the soluble fraction (3). Another isoprenylated GTP-binding protein, G25K, is capable of stable association with membranes, but behaves as a predominantly soluble protein in some cell lines (55). At the opposite end of the spectrum, the isoprenylated brain G protein y subunits exist exclusivelyin complexes with /3 subunits tightly bound to membranes (62). The association of lamin B with the nuclear matrix is so strong that it is resistant to detergents used to solubilize membrane proteins (28, 45). Most puzzling is the finding that different isoprenylated proteins are found in a variety of subcellular compartments (28, 29). How can attachment of an isoprenoid moiety, albeit one that is heterogeneous with respect to chain length, specify the localization of proteins to different membrane systems? One possible explanation for the differential solubility of various isoprenylated proteins is that the proportion of a particular protein associated with the plasma membrane, nucleus, or other organelle may depend on additional hydrophobic modifications such as carboxylmethylation

or palmitylation.

However,

this

concept

is difficult to generalize. Although palmitylation of p2lF increases its affinity for the cell membrane, currently there is no evidence for palmitylation of other isoprenylated proteins such as the G protein 7 subunit or lamin B. Nor is it certain that all isoprenylated proteins undergo carboxylmethylation. Evidence for phosphorylation

of

isoprenylated

proteins

has

been

pre-

sented (29), and it is conceivable that the equilibrium between soluble and membrane-bound forms of these proteins is governed by the activity of specific kinases and phosphatases. It is possible that phosphorylation may account for multiple isoforms of particular isoprenylated proteins such as G25K (55). Nevertheless, these issues remain to be addressed experimentally.

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A model that might provide a plausible explanation for the localization of particular isoprenylated proteins to different subcellular sites would envision the initial isoprenoid modification as a relatively nonspecific membrane targeting mechanism. Thus, attachment of a farnesyl or geranylgeranyl moiety may simply confer sufficient hydrophobicity to bring the modified proteins into proximity with the cellular membrane network. Whether this results in stable association of the proteins with a particular membrane system may depend on interactions between the isoprenylated proteins and specific receptor proteins distributed nonuniformly throughout the cell. Variations in the avidity and dynamics of these protein-protein interactions could account for the differences observed in the relative proportions of soluble vs. membrane-bound forms of isoprenylated proteins such as G25K and p2lras. Experimental evidence to support such a model is sparse. Binding of lamin B to a specific receptor in the nuclear envelope has been described (65), and G25K has been reported to undergo EGF-receptor-mediated phosphorylation in reconstituted vesicles (60). However, it is not yet known whether isoprenylation may influence these interactions. Perhaps the best support for the notion that posttranslational lipid modifications can promote specific interactions between proteins and their membrane receptors comes not from work on isoprenylation, but from studies of myristylation of p6ov-src. As with p2lras, association of this protein with the plasma membrane is required for transforming activity. Recent work has shown that stable association of p6#{216}src with the membrane is mediated by its interaction with a specific Src receptor protein, and that this interaction depends on myristylation of p6otrc at its N terminus (66). The possibility that isoprenylation may play a similar

role

specific

receptors

FUTURE

in facilitating

merits

the

docking

of proteins

with

consideration.

DIRECTIONS

Recognition of the Cxxx carboxyl-terminal motif as a potential signpost for isoprenoid modification should lead to identification of an increasing number of farnesylated or geranylgeranylated proteins in the near future. It should also become clear whether cysteine residues that are not localized within a Cxxx domain may be targets for isoprenoid modification in some proteins. With the development of in vitro assays and the purification of isoprenyl:protein transferases, it will be possible to determine whether changes in the activities of these enzymes are important in regulating the state of protein modification in relation to cell growth and differentiation. Progress can also be expected in mapping the structural features that determine whether proteins can serve as substrates for modification by C15 or C20 isoprenoids. With this knowledge, it may be feasible to design inhibitors capable of blocking the isoprenylation and transforming activity of oncogenic ras proteins without affecting the modification of proteins involved in normal cellularphysiology (e.g., lamins, G proteins). 3326

Vol. 4

December

1990

The most challenging issues for future consideratio center on defining the influence of isoprenoid modific tion on the localization and biological activity specific proteins in the cell. Emerging evidence alread suggests that studies of isoprenylation may provide ne insights into the assembly of heterotrimeric G protei complexes, the association of p2lras and related pro teinswith effectorsystems involved in signal transduc tion, and the interaction of lamins with the nuclea envelope. However, in view of the structural diversity o the proteins undergoing isoprenoid modification, an the wide range of cellular activities in which they par ticipate, considerable time probably will be require for a comprehensive picture of the functional sig nificance of isoprenylation in mammalian cells t emerge. F During National

preparation Institutes

of this of Health

review, grant

the author ROl

was supported

b

CA34569.

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