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

THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1991 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 266, No. 15, Issue of May 25, pp. 9786-9794,1991 Printed in U. S. A .

Carboxyl-terminal Isoprenylation of ras-related GTP-binding Proteins Encoded by rad , rac2, and ralA*

(Received for publication, October 10,1990)

B. Therese Kinsella, Robert A. Erdman, and William A. Maltese$ From the Weis Center for Research, Geisinger Clinic, Danuille, Pennsylvania 17822

Membrane localization of p21'" is dependent upon its posttranslational modification by a 15-carbon farnesyl group. The isoprenoid is linked to a cysteine located within a conserved carboxyl-terminal sequence termed the "CAAX" box (where C is cysteine, A is an aliphatic amino acid, and X is any amino acid). We now show that three GTP-binding proteins encoded by the recently identified racl, rac2, and ralA genes also undergo isoprenoid modification. cDNAs coding for each protein were transcribed in vitro, and the RNAs were translated in reticulocyte lysates. Incorporation of isoprenoid precursors, [3H]mevalonate or [3H]farnesyl pyrophosphate, indicated that the translation products were modified by isoprenyl groups. A protein recognized by an antibody to rac l also comigrated with a protein metabolically labeled by a product of [3H] mevalonate in cultured cells. Gel permeation chromatography of radiolabeled hydrocarbons released from the racl, rac2, and ralA proteins by reaction with Raney nickel catalyst indicated that unlike p21Hr"", which was modified by a 15-carbon moiety, the rac and ralA translation products were modified by 20- carbon isoprenyl groups. Site-directed mutagenesis established that the isoprenylated cysteines in the r ac l , rac2, and ralA proteins were located in the fourth position from the carboxyl terminus. The three-amino acid extension distal to the cysteine was required for this modification. The isoprenylation of r a c l (CSLL), ralA (CCIL), and the site-directed mutants r a c l (CELL) and ralA (CSIL), demonstrates that the amino acid adjacent to the cysteine need not be aliphatic. Therefore, proteins with carboxyl-terminal CXXX sequences that depart from the C A M motif should be considered as potential targets for isoprenoid modification.

The formation of thioether bonds between cysteine residues and isoprenyl groups derived from pyrophosphate intermedi- ates of the cholesterol biosynthetic pathway is an important type of posttranslational protein modification in mammalian cells (1, 2). Protein-bound isoprenyl groups of two different chain-lengths have been described; 15-carbon farnesyl (3-8) and 20-carbon geranylgeranyl (9-13). Proteins known to undergo isoprenylation include members of the K-, N-, and H-p2lrnS family (5 , 6, 14-19), G25K (20, 21), smg p21B

* This work was supported by United States Public Health Service Grant CA-34569. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ To whom correspondence should be addressed Weis Center for Research, 26-16 Geisinger Clinic, Danville, PA 17822. Tel.: 717-271- 8258; Fax: 717-271-6701.

(raplB) (13), nuclear lamin B (4, 22-25), the precursor of lamin A (23, 24, 26), and the y subunits of transducin (7, 8) and the heterotrimeric brain G-proteins (11, 12, 27). In all cases where the site of isoprenylation has been established, the modifying group is attached to a cysteine located in the fourth position from the carboxyl terminus, with the two amino acids distal to the cysteine usually having aliphatic side chains. Studies with ~21'"" suggest that the signal for modification of proteins by farnesyl groups may reside within this carboxyl-terminal "CAAX" box (where C is cysteine, A is an aliphatic amino acid, and X is any amino acid) (5,6, 14, 15, 18). For example, tetrapeptides based on the carboxyl- terminal sequences of K-, N-, and H-p21'"" can inhibit the farnesylation of recombinant ras protein by acting as alternative substrates for purified farnesy1:protein transferase in vitro (5).

When cultured cells are labeled with an isoprenoid precursor such as [3H]mevalonolactone (MVA),' most of the radioactivity is incorporated into cellular proteins with molecular masses between 21 and 28 kDa (28-33). Several of these proteins bind GTP when transferred to nitrocellulose but are immunologically distinct from p21'"" (33). Therefore, it is likely that they are members of a large family of diverse low molecular mass GTP-binding proteins that are homologous to p21ra5 in regions contributing to the GTP binding site and contain C A A X or C X X X sequences at their carboxyl termini

We recently identified one of the major low molecular mass isoprenylated proteins in murine erythroleukemia cells as G25K (G,) by means of two-dimensional immunoblotting (20). However, this approach is limited insofar as it depends on the availability of an antibody that is highly specific for the protein in question and the ability to resolve a number of closely related proteins on two-dimensional gels. In situations where cDNAs are available, an alternative strategy involving in vitro translation of specific gene products in the presence of [3H]MVA has been used to demonstrate the isoprenylation of nuclear lamins (24, 25) and the 7 6 subunit of brain G- proteins (27). In the present study we have utilized the latter approach to establish that three low molecular mass GTP- binding proteins encoded by racl, rac2, and ralA, undergo isoprenylation at cysteines located within their carboxyl-terminal sequences. Under conditions that result in the modification of ~ 2 1 ~ " " by a 15-carbon isoprenyl group, these proteins appear to be modified by a 20-carbon group. Most importantly, results obtained with the natural rac and ralA translation products and site-directed mutants containing variations in the C A A X motif indicate that the amino acids

(1, 34-36).

~~~~ ~ ~ ~

The abbreviations used are: MVA, mevalonic acid lactone; PCR, polymerase chain reaction; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; FPP, farnesyl pyrophosphate; DTT, ditbiothreitol; HPLC, high performance liquid chromatography; CHO, Chinese hamster ovary.

9786

Isoprenylation of racl, rac2, and ralA Proteins 9787

distal to the cysteine need not be aliphatic in order for isoprenylation to occur.

EXPERIMENTAL PROCEDURES

MateriaL~-(l-~H]Farnesyl pyrophosphate, triammonium salt (20 Ci/mmol), and ~-[~~S]methionine (1186 Ci/mmol) were obtained from Du Pont-New England Nuclear. Deoxyadenosine 5’-[c~-~~S]thiotri- phosphate triethylammonium salt (1338 Ci/mmol), AmplifyTM fluorography reagent, and pre-stained “Rainbow” protein molecular weight markers were purchased from Amersham Corp. [5-3H]Meva- lonolactone was prepared by reduction of mevaldic acid precursor (Sigma) with sodium b~ro[~H]hydride (Amersham) as described by Keller (37). The identity and purity of the labeled product were confirmed by thin layer chromatography (31). Lovastatin was provided by Alfred Alberts of the Merck, Sharp and Dohme Research Laboratories (Rahway, NJ). 2,6,10-Trimethyldodecane (farnesane) was purchased from Wiley Organics (Coshocton, OH) and 2,6,10,14- tetramethylhexadecane (phytane) was custom-synthesized by Far- chan Laboratories (Gainesville, FL). Rabbit reticulocyte translation system (minus methionine), T7 RNA polymerase, T4 polynucleotide kinase, T4 DNA ligase, DNA polymerase I (Klenow fragment), calf intestinal alkaline phosphatase, RNasin, and all restriction endonu- cleases were purchased from Promega. The Sequenase (Version 2) DNA sequencing kit was from United States Biochemical and the GeneAmp kit was from Perkin Elmer-Cetus. All other reagents were purchased from Sigma.

Construction of Plasmids-cDNAs coding for human racl, rac2, and ralA, all of which had been cloned previously (38, 39), were obtained from Dr. Richard F. Weber, Genentech Inc. (South San Francisco, CA). The human Hras (wild type) cDNA was provided by Dr. Channing Der, La Jolla Cancer Research Foundation (La Jolla, CA). To facilitate in vitro transcription, the cDNAs coding for Hras, racl, rac2, and ralA were subcloned into either pGEM3 or pGEM4 vectors. pGEM4-Hras was constructed by subcloning a 1.2-kb BamHI-EcoRI Hras cDNA fragment into pGEM4. pGEM4-racl was generated by subcloning a 660-bp HindIII-BamHI racl cDNA fragment into pGEM4, pGEM3-rac2 was constructed by subcloning a 720-bp EcoRI-BarnHI rac2 cDNA fragment into pGEM3. pGEM3- ralA was constructed by subcloning a 760-bp cDNA fragment encod- ing ralA into the SmaI site of pGEM3. Routine DNA manipulations, including restriction endonuclease digestions, DNA ligations, dephos- phorylations, bacterial transformations, plasmid isolations, and agarose gel electrophoresis were carried out using standard procedures, essentially as described by Sambrook et al. (40).

Site-directed Mutagenesis-The general strategy used for site- directed mutagenesis by means of the polymerase chain reaction (PCR) was essentially as described by Hemsley et al. (41). Fig. 1 outlines the various mutations that were created in the carboxyl- terminal regions of racl, rac2, and ralA using the plasmid templates pGEM4-rac1, pGEM3-rac2, and pGEM3-ralA. The sequences of the specific oligonucleotides used as primers in the individual PCR reactions are also shown. Oligonucleotides were synthesized on an Applied Biosystems 381A DNA synthesizer. Specific oligonucleotide primers were constructed so as to be “back to back” on the duplex DNA to facilitate “inverse PCR” around the plasmid templates (41). Standard PCR amplification reactions were performed with 1 ng of template DNA and 100 pmol of each primer, using 35 amplification cycles consisting of denaturation at 94 “C (1 min), annealing at 45 ‘C (2 min), and primer extension at 72 “C (2 min). Following amplification, PCR products were treated with DNA polymerase I (Klenow fragment), 5’-end-phosphorylated using T4 polynucleotide kinase, and ligated using T4 DNA ligase (41). The ligated products were transformed into CaC12-competent E. coli DH5a. All mutations were confirmed by nucleotide sequence analysis, using the double-stranded plasmids as templates (42).

In Vitro Transcription and Translation-Prior to in vitro transcrip- tions, pGEM4 plasmids containing Hras or racl inserts were linearized by digestion with EcoRI. The pGEM3 plasmids containing rac2 or ralA inserts were linearized by digestion with BamHI. The linearized plasmids (5 pg) were transcribed with T7 RNA polymerase as described previously (27). Aliquots from each transcription reaction were analyzed by agarose gel electrophoresis to confirm the size and integrity of the RNA products.

In vitro translations of the RNA transcripts were carried out in a methionine-deficient rabbit reticulocyte lysate system (Promega) ac- cording to the manufacturer’s instructions. Standard 25-pl reactions containing 2.5 pg of RNA and 20 pCi of ~-[~~S]methionine (1186 Ci/

5’ AAG AGA AM TGC CTG CTG l l G TAA ATGTCTCAGCCCCTCGllCll 3 K R K C L L L * (UP)

3’ TC TCT Tll ACT GAC GAC AAC A l l 5‘ K R K a

(MP)

3’ T f TE! l% A 8 &C GkC P!k A: 5‘

(MP)

3’ TC TCT Til ACG ACG ACA ACG A l l 5 K R K C C C C *

( W

3’ TC TCT T l l ACG GCC GAC AAC A l l 5’ K R K C R L L *

W”

Ired) 5‘ M G CGC GCC TGC AGC CTC CTC TAG GGGllGCACCCAGCGCTCC 3’

K R A C S L L *

K R A O (UP)

3’ TC GCG CGG ACT TCG GAG GAG ATC 5’

(MP)

K R A I S L L * 3’ TC GCG CGG ACC TCG GAG GAG ATC 5’

( W

3’ TC GCG COG ACG ATC GGA QGA GATC 5‘ K R A C O

(MP)

S~TGG AM AGT TTA GCC AAG AGA ATC AGA GAA AGA TGC TGC ~ll TTA TAA 3’ R K S L A K R I R E R C C I L *

3’ TCC Tll TCA AAT COG l l C TCT TAG T 5’

(UP) 5‘ GA GAA AGA TGC TCC A l l l l A TAA 3’

R E R C S I L * WP)

5‘ GA GAA AGA TCC TGC A l l l l A TAA 3’ R E R S C I L *

( W

FIG. 1. Site-directed mutagenesis of racl, rac2, and ralA. Carboxyl-terminal amino acid sequences of racl, rac2, and ralA are shown immediately below the corresponding nucleotide sequences of the coding strand at the top of each panel. The specific alterations introduced into the amino acid sequences of the racl, rac2, and ralA proteins are shown in outlined letters. For each alteration, the corresponding sequence of the mutant oligonucleotide primer ( M P ) used to create the alteration by PCR is shown immediately below the protein sequence. The nucleotide sequences of the oligonucleotides used as second or “universal” primers (UP) in each set of PCR amplifications are underlined. “Inverse” PCR reactions were carried out with pGEM4-rac1, pGEM3-rac2 or pGEM3-ralA plasmids serving as template DNAs. Throughout the figure, nucleotide sequences shown above the amino acid sequences represent coding strands. Nucleotide sequences shown below the amino acid sequences represent complementary strands. *, a termination codon.

mmol) were incubated for 1 h at 30 “C. Procedures used for analysis of translation products by SDS-PAGE and fluorography have been described previously (27, 33). In some instances, regions of the gel containing translation products labeled with [35S]methionine or [3H] MVA were excised, solubilized, and counted in a liquid scintillation spectrometer as described previously (31).

In Vitro Isoprenylation of Translation Products-Initial assessment of the ability of translation products to undergo isoprenylation was accomplished by adding 25 pCi of [3H]MVA (3.15 Ci/mmol for rac studies and 4.91 Ci/mmol for ralA studies) to a standard 25 pl of translation reaction and substituting an equimolar amount of unla- beled L-methionine for the radiolabeled methionine. Following incubation at 30 “C for 1 h, the reactions were supplemented with 5 pl of brain extract and incubated for an additional 1 h at 37 “C. The brain extract, which represents a good source of protein:farnesyl transferase activity (5), was prepared by homogenizing cerebral tissue from two adult Balb/c mice in 2.5 volumes of 100 mM Tris-HC1, pH 7.5, 5 mM MgC12, 2 mM MnC12, 30 mM nicotinamide, and removing all particu- late material by centrifugation at 100,000 X g for 1 h at 4 “C. In later studies (i.e. see Fig. 5) incorporation of [3H]MVA into translation

9788 Isoprenylation of racl, rac2, and ralA Proteins

46-

14.3- . *

1 2 3 4 5 6 7 8 9 1 0 1 1 " ..& . 1

k D a 200-.

9 7 - . 6 9 -

46-

. -

30- . ' .

2 1 . 5 -

14.3- 1 2 3 4 > b I 8 Y l l I 1 1

FIG. 2. Incorporation of radiolabeled precursors into Hrae, racl, and rac2 translation products. RNAs coding for Hrm, racl, rac2, and various site-directed mutants (see Fig. 1) were translated in oitro in standard reticulocyte lysate reactions (25 pl) containing either ["S]methionine ( A ) or ["HjMVA ( R ) as described under "Experimental Procedures." Aliquots were removed from each translation reaction ( 3 p1 from reactions containing [""Sjmethionine and 1 5 . ~ 1 from reactions containing ["HIMVA) and the proteins were subjected to SDS-PAGE and fluorography. The fluorograph in A was exposed for 16 h, whereas that shown in R was exposed for 14 days. Translation products were loaded on the gels as follows: lane I , roc2 (CSLL); lane 2, rac2 (WSLL); lane 3, rac2 (*); lane 4, rac2 (C*); lane 5, racl (CLLL); lane 6 , racl (WLLL); lane 7, racl (*); lune 8, roc1 ( C E ) ; lane 9, racl (CELL); lone 10, no RNA; lone 1 1 , Hrav. Positions of prestained molecular mass marker-proteins are indicated on the left of each fluorograph. The arrow in R points to the ['HI MVA-labeled hands that were aligned with the major [""Slmethio- nine-labeled translation products indicated by the arrow in A. The radiolabeled band a t approximately 28 kDa in all lanes in R is an endogenous component of the reticulocyte lysate (see "Experimental Procedures"). In a separate experiment, the radioactivity in gel slices containing the labeled translation products was quantitated by liquid scintillation counting. Rased on the incorporation of ['HIMVA and ["S]methionine, an estimate of the stoichiometry of isoprenylation' was obtained for each wild-type protein; p21'"*, 0.93 mol of 15-carbon isoprenoid/mol of protein; racl, 0.90 mol of 20-carbon isoprenoid/ mol of protein; rac2, 1.0 mol of 20-carbon isoprenoid/mol of protein.

* Molar ratios are only approximations. In calculating these ratios, it was assumed that p21'"" was modified by a 15-carhon isoprenyl group, whereas the racl, rac2, and ralA proteins were modified by 20- carbon isoprenyl groups (see Fig. 4). Calculations took into account the decay of "S, the dilution of radiolabeled methionine with unla- beled methionine in the reticulocyte lysate (estimated as 5 p~ by the supplier), and the number of methionines in the predicted sequences for each protein; i.e. 5 for p21'"", 3 for racl, 2 for rac2, and 6 for ralA. Since the translation reaction is essentially complete after the first hour of incubation, dilution of ["S]methionine by any methionine present in the brain c.ytosol added during the second hour of incuhation was assumed to have little if any influence on the incorporation of ["Slmethionine into the translation product. No attempt was made to adjust the specific radioactivity of the ['HIMVA to compensate for the concentrations of endogenous MVA in the reticulocyte lysates or brain extracts, since the latter were assumed to be relatively small compared to the concentrations of added ["HIMVA (200-260 p ~ ) .

products was observed in reticulocyte lysates without brain extract and it was therefore omitted from subsequent reaction mixtures. For studies aimed at estimating the chain-lengths of the radiolaheled protein-hound isoprenoids, translation reactions containing ['HI MVA were scaled up to a final volume of 137 pl.

T o further compare the abilities of the Hrm and roc1 proteins to serve as acceptors for farnesyl groups, translation reactions (137-pI final volume) were allowed to proceed without radiolaheled isoprenoid precursor for 1 h a t 30 "C. Reactions were then optimized for farnesylation by adding 6.6 pl of 0.5 M MgCI,, 5.5 pl of 0.1 M DTT. and 5 pI (6.9 pCi) of [l-'H]FPP (20 Ci/mmol) (18), and incuhation was continued for 1 h a t 37 "C. Identical reactions were carried out in parallel without addition of MgC12 and DTT. Translation reactions containing no exogenous RNA served as controls for hackground incorporation.

Esfirnafion of Chain-length of Radioloheled Isoprenoids-In order to determine the approximate size of the isoprenyl group attached to a particular translation product, radiolabeled protein from a Iarge- scale translation reaction containing ['HIMVA or [ 'HJFPP was suh- jected to SDS-PAGE on a 12.5% polyacrylamide slah gel. One lane of the gel was dried and fluorographed to localize the isoprenylated translation product, while the remainder of the gel was frozen at -80 'C. Using the fluorogram as a guide, the zone of the frozen gel containing the isoprenylated protein was excised and the laheled protein was electroeluted. For determination of hackground radioactivity, a parallel zone of a gel containing protein from a reaction without added RNA was treated identically. A yellow-colored component of the reticulocyte lysate migrated as a discrete hand at approximately 28 kDa in all lanes of the gel. With some lots of reticulocyte lysate ( e .g those used for the studies of roc1 and roc2 in Fig. 2) this hand was laheled with ['HIMVA (for example, see Fig. 2H), whereas with other lots of lysate (e.#. those used for translation of ralA Fig. 3) no laheling of this endogenous component was evident. Therefore, in studies aimed a t determining the size of the radiolaheled MVA derivative associated with the rac proteins. the labeled material comigrating with the yellow component of the lysate was eluted separately as an additional control. 1 mg of hovine serum albumin was added to each protein solution (1.5-2 ml) and SDS was removed by precipitation with potassium phosphate (43). The protein was precipitated with ice-cold acetone and dissolved in 0.4 ml of 8 M guanidine HCI, 200 mM sodium phosphate, pH 7.0. The tritium- labeled isoprenoid was released from the protein by reaction with Raney nickel-activated catalvst at 100 "C for 16 h and extracted into

kDa A B 46 - . 4 6

30- = .30

21.5 - .21.5

14.3- . 14.3 1 2 3 4 1 2 3 4

FIG. 3. Incorporation of radiolabeled precursors into ralA translation products. RNAs coding for ralA, and various site- directed mutants (see Fig. 1) were translated in oifro in standard reticulocyte lysate reactions (25 pl) containing either ["'S]methionine ( A ) or ['HIMVA ( R ) as descrihed under "Experimental Procedures." Aliquots were removed from each translation reaction (15 pl from reactions containing [""Sjmethionine and 10 pl from reactions containing ['HJMVA) and the proteins were subjected to SDS-PAGE on a 10-18% polyacrylamide gradient followed hy fluorography. The fluorograph in A was exposed for 3 h, whereas that shown in R was exposed for 21 days. Translation products were loaded on the gels as follows: lone I , ralA (CCIL); lane 2, ralA ( C ~ I L ) ; l a n ~ 3 , ralA (SCIL); lane 4, no RNA. Positions of prestained molecular mass marker proteins are indicated on the edge of each fluorograph. The ['HI MVA-labeled endogenous component of the reticulocyte lysate that was evident in Fig. 2 R was not detected with the hatch of lysate used in the translations shown above. After the fluorographic exposure was completed, the radioactivity in gel slices containing the laheled translation products was quantitated hy liquid scintillation counting. Rased on the incorporation of ["HIMVA and ["'Slmethionine into the combined 25-kDa and 29-kDa translation products, the stoichiometry of isoprenylation? of the wild-type ralA protein was estimated to he 0.95 mol of 20-carhon isoprenoid/mol of protein.

Isoprenylation of racl, rac8 and ralA Proteins 9789

pentane (4). To avoid potential loss of volatile hydrocarbons that might have occurred upon concentration of the organic phase by evaporation, extraction of the radiolabeled isoprenoid with pentane was carried out in two steps. The aqueous phase was first extracted with 200 pl of pentane. This small amount of pentane routinely contained 80-90% of the total radioactivity ultimately recovered. The remaining pentane-extractible material was removed from the aqueous phase by a second extraction with 1 ml of pentane. Total recoveries of radiolabeled isoprenoid ranged from 60 to 85%. The first and second pentane extracts were hydrogenated separately over plat- inum oxide (Adams' catalyst) for 2 h essentially as described by Farnsworth et al. (4). The size of the radiolabeled saturated hydrocarbon was then estimated by gel permeation chromatography of a 50-pl aliquot of the first pentane extract. Pilot studies confirmed that the chromatographic behavior of the more dilute radiolabeled material in the second pentane extract was qualitatively the same as that in the first extract. Prior to injection on the column, the radiolabeled hydrocarbon derived from the protein was mixed with standard hydrocarbons that would be expected to be generated by hydrogena- tion of farnesyl and geranylgeranyl isoprenoids; i.e. 2,6,10-trimethyldodecane (farnesane; 2.5 pl) and 2,6,10,14-tetramethylhexadecane (phytane; 2.5 pl) . HPLC was performed on a Beckman "System Gold" chromatograph, using tandem columns (300 X 7.8 mm) of 5-pm Phenogel matrix (Phenomenex Inc.). The first column had a pore size of 50 A, and the second had a pore size of 100 A. Radiolabeled hydrocarbons were eluted with tetrahydrofuran at a flow rate of 0.25 ml/min. Fractions were collected at 0.5-min intervals, mixed with ReadySafeTM (Beckman) and counted for tritium in a liquid scintillation spectrometer. The elution positions of the standards in each run were determined with a Beckman model 156 refractive index detector. Raney nickel treatment of the material eluted from the control gel segments (i.e. reactions without added RNA) yielded negligible amounts of pentane-extractible radioactivity. The radioactivity extracted from the 28-kDa yellow-colored band after reaction with Raney nickel was less than 10% of that recovered from the ruc and ralA translation products. When chromatographed, this material failed to elute as a discrete peak of radioactivity.

Metabolic Labeling, Two-dimensional Electrophoresis, and Immu- noblotting of Zsoprenylated Proteins-Simian COS-M6 cells, obtained from Dr. David Russell, University of Texas Southwestern Medical Center (Dallas, TX) were grown in monolayer culture as described previously (20). Isoprenylated proteins were labeled by incubating cells in medium containing t3H]MVA (200 pCi/ml medium) and lovastatin (25 p ~ ) for 20 h. Cellular protein was solubilized in Garrels' electrophoresis sample buffer (44), and subjected to two-dimensional gel electrophoresis as described (20). Proteins were then electroblot- ted onto nitrocellulose and the [3H]MVA-labeled proteins were local- ized by fluorography of the blot. After washing out the fluorography reagent, the region of the same nitrocellulose membrane containing proteins between 17 and 32 kDa was immunoblotted with an affinity- purified antibody against racl protein. The antibody, which was developed by Dr. Tony Evans, Genentech Inc. (South San Francisco, CA) and kindly provided by Dr. Paul Polakis, Cetus Corp. (Emery- ville, CA), was generated against a peptide based on the carboxyl- terminal sequence upstream from the CAAX box of the racl protein. The bound antibody was detected with 9-labeled goat anti-rabbit IgG secondary antibody. Detailed descriptions of the methods used for fluorography and immunoblotting can be found in Ref. 20.

RESULTS

Translation and Isoprenylation of racl and rac2 Proteins in Vitro-The carboxyl-terminal sequences of two newly identified low molecular mass GTP-binding proteins encoded by racl and rac2, suggested that they might be capable of undergoing isoprenylation (see Fig. 1). To examine this possibility, RNAs coding for each protein were translated in reticulocyte lysates containing either [35S]methionine to label the polypeptide chains or [3H]MVA to label the isoprenyl groups. Parallel translations containing RNA coding for p2lHr"", a known farnesylated protein, served as positive controls. The molecular masses of the racl and rac2 translation products labeled with [35S]methionine (Fig. 2A) were consistent with values reported previously for racl protein (24-25 kDa) in the Laemmli SDS-PAGE system (39). As shown in Fig. 2B, incorporation of [3H]MVA into the translation prod-

ucts of Hras (lune 11), racl (lane 5) , and rac2 (lane 1) was readily detected by fluorography. In contrast, a parallel translation reaction without exogenous RNA (lane 10) showed no labeling at the same position in the gel. Stoichiometries estimated on the basis of a separate study in which the radioactivity in gel slices was quantitated by liquid scintillation counting (see Fig. 2, legend) suggest that in uitro isoprenylation was reasonably efficient in the case of each protein. A 28-kDa [3H]MVA-labeled band was seen in all lanes of the gel, and was observed to overlap with a yellow-colored zone that was visible in unstained gels. Since this [3H]MVA-labeled band did not appear to be labeled with [35S]methionine and was present in reactions that did not contain exogenous RNA (lane IO), it was assumed to be generated by isoprenoid derivatization of an endogenous component of the reticulocyte lysate. The amount of [3H]MVA incorporated into this material varied greatly with different lots of reticulocyte lysate.

Several classes of site-directed mutants were constructed in order to explore the structural requirements for isoprenylation of the rac proteins (see Fig. 1). The first set of mutants was designed to evaluate the consequences of removing the carboxyl-terminal cysteine from the proteins. Deletion of the sequences coding for the last four amino acids of racl (Fig. 2, lane 7) or rac2 (Fig. 2, lane 3 ) abolished the ability of each protein to incorporate [3H]MVA, although [35S]methionine labeling indicated that the amount of each protein synthesized in the lysate was comparable to that obtained with the corresponding unaltered RNA. Similarly, when the carboxyl- terminal sequences of the racl and rac2 proteins were left intact, except for the substitution of tryptophan in place of cysteine in the CAAX box (see Fig. l), the mutated racl (WLLL) (Fig. 2, lane 6) and rac2 (WSLL) (Fig. 2, lane 2) proteins failed to incorporate [3H]MVA.

A second set of mutants was constructed for the purpose of assessing the importance of the three amino acids distal to the cysteine for isoprenylation of the racl and rac2 proteins. The proteins were altered so as to terminate with a cysteine, in one case (rac2) by eliminating the last three amino acids, and in another case (racl) by replacing the terminal three amino acids of the CAAX box with three additional cysteines (see Fig. 1). As shown in Fig. 2, neither the truncated rac2 protein (C*) (lune 4 ) nor the racl protein ( C E ) containing multiple carboxyl-terminal cysteines (lane 8) incorporated detectable [3H]MVA-derived isoprenyl groups. However, when the carboxyl-terminal CAAX box of the racl protein was altered so that the leucine immediately distal to the target cysteine was replaced with a nonaliphatic residue (CBLL), the translation product was still able to undergo isoprenylation (Fig. 2, lane 9).

Translation and Isoprenylation of ralA protein in Vitro- Since the carboxyl-terminal cysteine of the racl protein re- tained the ability to serve as an isoprenyl acceptor after substitution of an arginine for the normal aliphatic residue adjacent to the cysteine, we considered the possibility that low molecular mass GTP-binding proteins with other variations in this position within the CAAX motif might undergo isoprenylation. The product of ralA (CCIL) was selected as a representative of this class of proteins.

As shown in Fig. 3A, translation of the ralA RNA in vitro resulted in the generation of two major [35S]methionine- labeled products with molecular masses of 29 and 25 kDa. The molecular mass of the larger product (29 kDa) was most similar to that previously reported for ralA (38). The results depicted in Fig. 3B, clearly show that [3H]MVA was incorporated into both the 29-kDa and the 25-kDa ralA translation products (lane 1 ), but not in the reaction without exogenous


RNA (lune 4 ) , indicating that the ralA protein was isoprenylated. Since isoprenylation occurs at the carboxyl terminus (see below), and since there are no potential initiation codons within the 5"untranslated region of the ralA insert, the efficient [3H]MVA-labeling of both translation products suggests that the 25-kDa translation product probably represents an amino-terminal truncated ralA protein formed by initiation of translation at an internal ATG codon (e.g codon 22; see Ref. 39 for complete sequence) or by amino-terminal proteolytic cleavage of the primary translation product.

Site-directed mutants of ralA were constructed in order to determine whether the protein could be isoprenylated with only one cysteine occupying either the third or fourth position from the carboxyl terminus (see Fig. 1). Substitution of a serine for the cysteine in the third position from the carboxyl terminus (CSIL) did not abolish isoprenylation of the 29-kDa and 25-kDa translation products (Fig. 3, lane 2 ) . However, substitution of a serine in place of the cysteine in the fourth position from the carboxyl terminus (SCIL) prevented the incorporation of [3H]MVA into both the 29-kDa and 25-kDa protein bands (Fig. 3, lune 3 ) . Thus, as in the cases of ~21'"' and the rac proteins, it appears that a cysteine residue must occupy the fourth position from the carboxyl terminus in order for the ralA protein to undergo isoprenylation.

Sizes of the [3H]MVA-derived Isoprenyl Groups Transferred to ras, rac, and ralA Proteins in Vitro-In an attempt to determine whether the radiolabeled [3H]MVA derivatives transferred to the racl, rac2, and ralA translation products (see Figs. 2B and 3B) were of the 15-carbon farnesyl class reported to be associated with the ras proteins, the isoprenyl groups were released from the proteins by Raney nickel- catalyzed desulfurization, converted to saturated hydrocarbons, and subjected to gel permeation chromatography. As expected, the [3H]MVA-derived isoprenoid associated with the Hras translation product was eluted along with the 15- carbon standard, farnesane (Fig. 4). In contrast, the racl, rac2, and ralA (25-kDa band) translation products, as well as their site-directed mutants racl (CRLL) and ralA (CSIL), were modified by [3H]MVA derivatives that were coeluted with the 20-carbon standard (Fig. 4). Similar differences in the sizes of the [3H]MVA-derived modifying groups associated with Hras (15 carbon) versus rac2 (20 carbon) were also observed when brain extract was excluded from the reticulocyte lysate translation/isoprenylation reaction (Fig. 5).

Determination of the Sizes of [3H]FPP-deriued Isoprenyl Groups Attached to ras and racl Proteins in Vitro-The initial results described above, coupled with observations published previously (24, 25, 27), indicate that reticulocyte lysate con- tains the enzymatic apparatus necessary for converting [3H] MVA to radiolabeled isoprenyl pyrophosphates, as well as one or more pr0tein:isoprenyl transferases. However, the concentrations of different isoprenyl pyrophosphates generated in this system were unknown. We therefore reexamined the chain-lengths of the isoprenyl groups transferred to the Hras and racl translation products, using [3H]FPP (2.2 PM) instead of [3H]MVA as the isoprenyl donor in the translation reaction. This concentration of FPP was previously shown to be optimal for ~21'"" pr0tein:farnesyl transferase activity in cell and tissue lysates (18). Reactions were further optimized for ~ 2 1 ' " ~ pr0tein:farnesyl transferase activity by adjustment of the Mg2+ and DTT concentrations to 21 and 3.5 mM, respectively (18).

As shown in Fig. 6, the Hras translation product was found to contain a 15-carbon radiolabeled moiety after incubation with [3H]FPP (Fig. 6A). When additional Mg2+ and DTT were added to the reaction mixture (Fig. 6B), there was no

change in the apparent chain-length of the radiolabeled isoprenyl group extracted from the modified ras protein, but the total amount of radioactivity incorporated into the protein from [3H]FPP increased approximately 9-fold. Thus, unlike the coupled translation/isoprenylation reaction employing [3H]MVA as a precursor (Figs. 2 and 3), the posttranslational modification of preformed p21'"" with [3H]FPP appeared to occur at suboptimal levels unless Mg2+ and DTT concentrations were raised to approximate those previously described for assays of pr0tein:farnesyl transferase activity in cell and tissue lysates (18). In contrast to the results obtained with the ras protein, most of the [3H]FPP-derived material recovered from the racl translation product migrated with the 20-carbon standard (Fig. 6C). When racl protein was incubated with [3H]FPP under optimal conditions for protein:farnesyl transferase (Fig. 6D), there was no overall stim- ulation of isoprenylation such as that seen with the Hras translation product under the same conditions (Fig. 6B), although there was a small shift in the proportion of radiolabeled material migrating with the 15-carbon standard uersus 20-carbon standard.

[3H]MVA Labeling of a rucl-immunoreactive Protein in Viuo-In two separate cases (i.e. lamin B and a G-protein ys subunit) proteins that were shown to undergo isoprenylation in reticulocyte lysate translation systems (24, 25, 27) were also found to undergo isoprenylation in vivo (4, 11, 12). To assess this possibility in the case of the rac proteins, cultured COS cells were metabolically labeled with [3H]MVA and proteins were separated by two-dimensional gel electrophoresis. The immunoblot depicted in Fig. 7 shows that a 24-25- kDa [3H]MVA-labeled protein comigrated with a protein that was recognized by an antibody directed against a racl peptide. Under these conditions only one isoprenylated protein out of many 21-28-kDa proteins resolved by two-dimensional electrophoresis appeared to react with the racl antibody. More- over, the particular protein recognized by the racl antibody was not recognized by antibodies to G25K (20) or the ras family of proteins (33) (not shown). Nevertheless, it should be noted that the racl antibody cross-reacted with several nonisoprenylated proteins in the high molecular mass region of the blot (not shown in Fig. 7).

DISCUSSION

Mammalian cells contain several GTP-binding proteins in the range of 21-28 kDa that undergo posttranslational modification by isoprenyl groups derived from mevalonate (33). With the exceptions of H-, K-, and N-p21'"" (14-19), G25K (20, 21), and raplB (13) the identities of these isoprenylated proteins have not been established. Proteins of the rac and ral families are among the most recent additions to the list of low molecular mass GTP-binding proteins in mammalian cells. The predicted sequences for the racl and rac2 proteins show only 26-30% homology with ras (38). The ralA and ralB genes code for a separate family of proteins with approximately 50% homology with ras (39, 45, 46). The possibility that these proteins might undergo posttranslational modification by isoprenoids was suggested by two observations. First, the predicted sequences for the racl, rac2, and ralA proteins all contain cysteines in the fourth position from their carboxyl termini. Cysteines in this position are known to be isoprenylated in p21'"" (14, 15), rap1B (13), nuclear lamins (24), and the G-protein y subunits (27). Second, human rac and ralA cDNAs were isolated from an HL-60 cell library with the aid of oligonucleotide probes based on the sequence of G25K (38, 39), a related low molecular mass GTP-binding protein whose isoprenylation was recently established by two-

Isoprenylation of racl, rac2, and ralA Proteins 9791

FIG. 4. Gel permeation chromatography of [‘HIMVA-derived hydrocarbons associated with Hras, racl, rac2, and ralA proteins in reticulocyte lysates supplemented with brain extract. Following translation, the proteins indicated in each panel were incubated with [3H]MVA in reticulocyte lysates supplemented with brain extract (see “Experimental Proce- dures”). Specific proteins were eluted from SDS-polyacrylamide gels and the radiolabeled isoprenyl groups, released by Raney nickel cleavage, were extracted into pentane and hydrogenated. Gel permeation chromatography was performed on a 5O-pl aliquot of the first 2004 pentane extract, as described under “Ex- perimental Procedures.” Elution of the radiolabeled hydrocarbons was monitored by liquid scintillation counting. The retention times of the farnesane (C15) and phytane (C20) standards included in each run (monitored by a refractive index detector) are shown at the top of each chromatogram.

C20 C15

RETENTION TIME (mln)

C20 C15 2000

racl

RETENTION TIME (min)

C20 C15 I

ralA

RETENTION TIME (mln)

C20 C15 1 0 0 0

rac2

RmNTION TIME (mln)

C20 C15 I I 2500

racl IR)

RmNTlON TIME (mln)

C20 C15 2500,


dimensional immunoblotting (20) and analysis of the protein purified from brain (21).

The studies described in this report show that rucl, rac2, and rulA proteins can be modified by derivatives of MVA in vitro. Results obtained with termination mutants of rucl and ruc2 lacking the last four amino acids, as well as substitution mutants such as rucl (WLLL), ruc2 (WSLL), and rulA (SCIL), demonstrate that isoprenoid modification of the ruc and rulA proteins occurs at a cysteine residue in the fourth position from the carboxyl terminus, as it does in the ras proteins.

Studies of farnesylated fungal mating factors (47,48), ~ 2 1 ” ~ (5, 6, 14-19), nuclear lamins (4, 23-26), and G-protein y subunits (7, 8, 11, 12), have led to a general perception that a carboxyl-terminal motif common to these proteins (i.e. CAAX) constitutes a signal that marks proteins as substrates for isoprenoid modification. However, the recent finding that CY subunits of Gi do not incorporate radiolabeled isoprenoid in uitro or in transfected cells, despite having carboxyl-terminal sequences fitting the CAAX pattern, implies that the recognition mechanism for isoprenylation is more complex than initially thought (27, 49). The present studies with rucl demonstrate that replacement of the natural carboxyl-terminal sequence (CLLL) with ( C a ) abolishes isoprenylation of this protein. The ruc2 termination mutant (C*) was also

unable to serve as an acceptor for isoprenyl groups. These findings reinforce the concept that the three amino acids comprising the carboxyl-terminal extension adjacent to the target cysteine play an important role in the recognition of proteins as substrates for isoprenylation. However, it is noteworthy that rucl (CSLL), rulA (CCIL), and the site-directed mutants, rucl(R) (CRLL) and ralA(S) (CSIL), were able to undergo isoprenylation despite having amino acids with hy- droxyl, basic or thiol side chains in the third position from the carboxyl terminus. Therefore, the definition of carboxyl- terminal structures that potentially may undergo isoprenylation ( i e . CAAX) should be expanded to include protein sequences with nonaliphatic amino acids distal to the cysteine.

Chromatographic characterization of the MVA derivative attached to p21ra8 in 3T3 fibroblasts suggests that the modifying group is a 15-carbon farnesyl chain, linked to the protein via a thioether bond (15). This is supported by recent studies showing that recombinant ras proteins can serve as specific substrates for pr0tein:farnesyl transferase in uitro (5, 6, 18). Nevertheless, in light of reports that the predominant isoprenoid associated with HeLa and CHO cell proteins is a 20- carbon geranylgeranyl moiety, rather than a 15-carbon farnesyl group (9, lo), we considered the possibility that the ruc and rul proteins might be modified by isoprenyl groups differ- ing from that found in p21“”. Based on gel permeation chro-


matography, the estimated size of the [3H]MVA derivative transferred to p2lHraB in the reticulocyte lysate was consistent with a 15-carbon farnesyl modification. However, under identical conditions the racl, rac2, and ralA proteins appeared to be modified by a 20-carbon MVA derivative. The differences

C20 C15 I I 1 M O

H-ras

.n (cvLs’ I I \ A \ l

4 8 4 9 5 0 5 1 5 2 U 5 4 5 6 y I 5 7 5 8 5 9 8 0 8 l I ~ 8 1 8 5


C20 C15

’” I

l _ I 800 n (CSLL) rac2

4 8 4 S 5 0 5 1 5 2 ~ ~ 5 6 ~ ~ 7 ~ ~ 8 0 ~ 1 ~ g ) ~ 8 5


FIG. 5. Gel permeation chromatography of [‘HIMVA-derived hydrocarbons linked to Hras and racl proteins in reticulocyte lysates without brain extract. Following translation, the proteins indicated in each panel were incubated with [3H]MVA in reticulocyte lysates (see “Experimental Procedures”). Specific proteins were eluted from SDS-polyacrylamide gels and the radiolabeled isoprenyl groups, released by Raney nickel cleavage, were extracted into pentane and hydrogenated. Gel permeation chromatography was performed on a 50-gl aliquot of the first 200-~1 pentane extract, as described under “Experimental Procedures.” Elution of the radiolabeled hydrocarbons was monitored by liquid scintillation counting. The retention times of the farnesane (C15) and phytane (C20) standards included in each run (monitored by a refractive index detector) are shown at the top of each chromatogram.

FIG. 6. Gel permeation chromatography of [aH]FPP-derived hydrocarbons transferred to Hrm and racl proteins in reticulocyte lysates. Hras and racl translation products were incubated with [3H]FPP and radiolabeled isoprenyl groups were removed from the gel-eluted proteins by Raney nickel cleavage. Following hydro- genation, the radiolabeled material was subjected to gel permeation chromatography. The retention times of the farnesane ( C 1 5 ) and phytane (CZO) standards included in each run (monitored by a refractive index detector) are shown at the top of each chromatogram. Radiola- beled hydrocarbons were derived from the following [3H]FPP/protein incuba- iion mixtures: A , Hras, no Mg2f or DTT added; B, Hras, optimized for protein:farnesyl transferase by addition of Mg2+ and DTT; C , racl, no Mg2+ or DTT added; D, racl, optimized for protein:farnesyl transferase by addition of Mg2+ and DTT.

in the isoprenoid modifications of the Hrus and racl translation products were further highlighted when [3H]FPP was used as an isoprenoid donor. Whereas the modification of ~ 2 1 ~ ‘ “ ” by a 15-carbon [3H]FPP derivative was greatly enhanced when the reaction was optimized for protein:farnesyl transferase activity, this was not true for racl, which continued to show only a basal level of incorporation of radioactivity derived from [3H]FPP (mostly 20-carbon) under optimal conditions for farnesylation. Thus, racl does not behave as a specific substrate for pr0tein:farnesyl transferase in this system.

Although the precise structure of the 20-carbon moiety transferred to the rac and ral proteins cannot be established solely on the basis of the results of gel permeation chromatography, a likely possibility is geranylgeranyl, the only 20- carbon isoprenoid thus far established to be linked to mammalian proteins (9-13). The transfer of radiolabeled 20-carbon isoprenyl groups to proteins incubated with [3H]MVA or [3H]FPP implies that reticulocyte lysates contain enzymes capable of converting these precursors to the putative 20- carbon isoprenyl donor, geranygeranyl pyrophosphate. How- ever, it is presently unclear whether this occurs via conden- sation of farnesyl pyrophosphate with isopentenyl pyrophosphate, catalyzed by farnesyl pyrophosphate synthase (50), or a separate reaction catalyzed by a specific geranylgeranyl pyrophosphate synthase, such as that described in porcine liver (51).

Didsbury et al. (52) have recently demonstrated that over- expression of racl or rac2 in transfected COS cells is corre- lated with enhanced [3H]MVA-labeling of a 24-kDa protein band on one-dimensional SDS gels. In the present study we show comigration of a racl-immunoreactive protein and a [3H]MVA-labeled protein on a two-dimensional blot of COS cell proteins (Fig. 7). Taken together, these observations strongly suggest that the isoprenylation of racl, rac2, and ralA proteins that occurs in the reticulocyte lysate translation system is representative of a posttranslational modification that occurs i n vivo.

Whether the modification of the rac and ral proteins i n vivo actually involves a 20-carbon geranygeranyl group remains to be determined, but the size of the [3H]MVA derivative asso-

C20 C15 I I BDO, I

600-

n . 200-

REIENTION TIME (min)

C20 C15

C20 C15 I I

I

REIENTION TIME (min)

RmNTION TIME (min) RETENTION TIME (min)

Isoprenylation of racl , rac2. a n d ralA Proteins 9793

...-

'H - MVA

- IEF - +

antl- rac l

FIG. 7. A [3H]MVA-labeled protein in cultured cells comi- grates with a protein recognized by an antibody against racl. ['HIMVA-labeled proteins from COS cells were separated by isoelec- tric focusing ( I E F ) in the first dimension and SDS-PAGE in the second dimension. Proteins were transferred to nitrocellulose and the region of the blot containing proteins between 17 and 32 kDa was fluorographed to visualize the isoprenylated proteins (upper panel). The same nitrocellulose membrane was then immunoblotted with antibody to racl protein, and the immunoreactive protein was detected by '2sI-labeled secondary antibody (lower panel). The exposure times were 12 days and 17 h for the fluorograph and immunoblot, respectively. The radiolabeled spots indicated by the arrows in the upper and lower panels overlapped.

ciated with the racl, rac2, and ralA translation products in reticulocyte lysates is consistent with the results of a recent study of isoprenylated proteins in three different mammalian cell lines. Specifically, gel permeation chromatography of ["HI MVA-labeled isoprenyl groups extracted from proteins in discrete molecular-mass categories revealed that unlike p21'"", which contained a 15-carbon moiety, most of the other proteins in the 21-28-kDa category were modified by isoprenyl groups that migrated with a 20-carbon standard (53). This supports the notion that the nature of the isoprenoid modification (geranylgeranyl versus farnesyl) of specific low molecular mass GTP-binding proteins is determined primarily by intrinsic structural features which mark these proteins as substrates for different pr0tein:isoprenyl transferases.

Although the recent studies of p21'"" farnesy1:protein transferase (5) imply that the specific structural signals for farnesylation reside in the last four amino acids of the carboxyl terminus, the precise combinations of amino acids that are permissive for 15-carbon versus 20-carbon isoprenoid modifications have not yet been defined. In this regard, it is noteworthy that the carboxyl-terminal sequences of the brain G-protein 7 6 subunit (CAIL; 54, 55), smg p21B (raplB) (CQLL; 56), and one form of G25K (CVIL; 57), resemble the terminal sequences of the racl (CLLL), rac2 (CSLL), and ralA (CCIL) proteins, insofar as they all end with leucine. All of these proteins have been reported to undergo geranygeranyl modification (11-13, 21). In comparing these sequences with those present at the carboxyl termini of farnesylated proteins such as human p2lKraSB (CVIM), human ~ 2 1 ~ ' " " (CVLS), human lamin B (CAIM), and the y subunit of transducin (CVIS), the presence of a leucine rather than methionine or serine in the terminal position stands out as a consistent feature of the proteins modified by 20-carbon isoprenoids. Thus, it is tempting to speculate that the amino acid occupying the terminal position plays an important role in determining the isoprenoid substrate specificity of the CXXX sequence. Future studies with site-directed mutants of the above-mentioned proteins should help to clarify this issue.

Perhaps the most challenging issue that remains to be addressed concerns the functional significance of protein isoprenylation. In an immediate sense, isoprenylation appears to be a critical permissive step for further posttranslational processing (proteolytic cleavage and carboxyl methylation) of

proteins such as p21'"" (14-17, 58-61) and the G-protein -1 subunits (12). Therefore, it seems likely that this will be true for a t least some of the other isoprenylated low molecular mass GTP-binding proteins. Studies of p21'"' (14-16) and nuclear lamins (23, 26, 62, 63) also suggest that the increased hydrophobicity conferred on these proteins by addition of an isoprenyl chain plays an important role in targeting these proteins to the plasma membrane and nuclear envelope, respectively. However, the fact that several isoprenylated GTP- binding proteins, including G25K, can be found predomi- nantly in the cytosol of some cells (20, 33), argues that isoprenylation is not the only factor involved in promoting the association of these proteins with membranes. In the cases of rac and ral, assessment of t,he functional significance of isoprenylation is further complicated by the fact that little is known about the normal subcellular localization and phys- iological roles of these proteins. Although there is 92% identity between the protein sequences of racl and ruc2, Northern blot analyses of various tissues and cell lines indicate that racl is expressed ubiquitously, whereas rac2 is expressed primarily in cells of hemopoietic lineage (38,64). Increases in the amount of rac2 mRNA in U937 and HL-60 cells treated with cyclic AMP, suggest a possible role for rac2 in myeloid differentiation (38). The ralA gene was originally cloned from a cDNA library of immortalized simian B-lymphocytes (451, and the subsequent isolation of additional ral cDNAs from human pheochromocytoma (46) and HL-60 leukemia (39) libraries has revealed the existence of two forms of the gene, ralA and ralB, which encode proteins that are approximately 85% homologous. Recent studies have established that members of the ral family are represented prominently among the low molecular mass GTP-binding proteins in human platelets (65), but little is known about the distribution of these proteins in other tissues. Based on current knowledge concerning the functions of low molecular mass GTP-binding proteins of the ras, rho, rap, and rub families (for review see Refs. 35 and 36) it is conceivable that the racl, rac2, and ralA proteins could be involved in any one of a number of important cellular processes, including signal transduction, growth regulation, cytoskeletal organization, or vesicular transport of proteins. As more becomes known about these proteins, it should become easier to design meaningful studies aimed at assessing the functional implications of their posttranslational modification.

Acknowledgments-We thank Dr. Richard Weber for generously providing the cDNAs for racl, race, and ralA, Dr. Channing Der for the Hras cDNA, and Drs. Paul Polakis and Tony Evans for the racl antibody.

1. 2.

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