©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
A Novel GTPase-activating Protein for R-Ras (*)

(Received for publication, August 14, 1995; and in revised form, October 3, 1995)

Takaharu Yamamoto (1) Takeshi Matsui (1) Masato Nakafuku (1) Akihiro Iwamatsu (2) Kozo Kaibuchi (1)(§)

From the  (1)Division of Signal Transduction, Nara Institute of Science and Technology, Ikoma 630-01 and (2)Central Laboratories for Key Technology, Kirin Brewery Co. Ltd., Yokohama 236, Japan

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

R-Ras, belonging to the Ras small GTP-binding protein superfamily, has been implicated in regulation of various cell functions such as gene expression, cell proliferation, and apoptotic cell death. In the present study, we purified an R-Ras-interacting protein with molecular mass of about 98 kDa (p98) from bovine brain cytosol by glutathione S-transferase (GST)-R-Ras affinity column chromatography. This protein bound to GTPS (guanosine 5`-(3-O-thio)triphosphate, a nonhydrolyzable GTP analog)bulletR-Ras but not to GDPbulletR-Ras, GTPSbulletR-Ras with a mutation in the effector domain (R-Ras), GTPSbulletHa-Ras, or GTPSbulletRalA. We obtained a cDNA encoding p98 on the basis of its partial amino acid sequences. The predicted protein consists of 834 amino acids whose calculated mass, 95,384 Da, is close to the apparent molecular mass of p98. The amino acid sequence shows a high degree of sequence similarity to the entire sequence of Gap1^m, one of the GTPase-activating proteins (GAP) for Ha-Ras. A recombinant protein consisting of the GAP-related domain of p98 fused to maltose-binding protein stimulated GTPase activity of R-Ras, and showed a weak effect on that of Ha-Ras but not that of Rap1 or Rho. These results clearly indicate that p98 is a novel GAP for R-Ras. Thus, we designated this protein as R-Ras GAP.


INTRODUCTION

Accumulating evidence indicates that Ras (Ha-Ras, Ki-Ras, N-Ras) serve as downstream molecules for tyrosine kinase-type receptors such as epidermal growth factor receptor, as well as Src family members (for reviews, see (1) and (2) ). Ras appears to transmit its signal to influence expression of genes that control cell cycle, proliferation, and differentiation(1, 2) . Ras has a GDP-bound inactive form and GTP-bound active form, the latter of which recognizes target proteins including c-Raf-1. The GDP-bound form is converted to the GTP-bound form by GDP/GTP exchange reaction, which is regulated by GDP/GTP exchange proteins such as Smg GDS, mSos, and mCdc25(3, 4, 5, 6) . The GTP-bound form is converted to the GDP-bound form by intrinsic GTPase activity, which is regulated by GTPase-activating proteins such as p120 GAP, (^1)NF1, and Gap1^m(7, 8, 9) .

R-Ras was originally identified as the gene product of a Ras homologue (10) . R-Ras has been reported to physically associate with Bcl-2, which is known to be a blocker of apoptotic cell death(11, 12) . Recently, the activated R-Ras that presumably remains mostly in the GTP-bound form due to impaired GTPase activity has been reported to enhance the apoptotic cell death in cytokine-deprived 32D.3 cells and serum-deprived NIH/3T3 cells(13) . Bcl-2 abrogates most of the effects of R-Ras, indicating that R-Ras promotes apoptosis caused by growth factor deprivation via a Bcl-2- suppressible mechanism. In NIH/3T3 cells, R-Ras confers the ability to proliferate under low serum conditions, to form colonies in soft agar, and to form tumors in nude mice, although its ability is weaker than that of the activated Ha-Ras(14) . R-Ras as well as Ha-Ras have been shown to interact with c-Raf-1, p120 GAP, and NF1, to induce MAP kinase activation, and to stimulate Ras response elements in certain cells(15, 16) . On the other hand, R-Ras does not induce maturation of Xenopus oocytes or differentiation of PC12 cells. Taken together, although Ras and R-Ras share some biochemical and cellular functions, these proteins seem to play different biological roles.

To understand the specific functions of R-Ras, we attempted to identify proteins that specifically interact with R-Ras in the present study, and have purified an R-Ras-interacting protein with molecular mass of about 98 kDa by GST-R-Ras affinity column chromatography, cloned its cDNA, determined its primary structure, and identified it as a novel R-Ras GAP.


EXPERIMENTAL PROCEDURES

Materials and Chemicals

Polyvinylidene difluoride membranes (Problott, 0.45 µm pore size) were purchased from Applied Biosystems. Achromobacter protease I was from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). All materials used in the nucleic acid study were purchased from Takara Shuzo Co., Ltd. (Kyoto, Japan). Prokaryotic expression plasmid pGEX-2T and pMal-c2 were obtained from Pharmacia Biotech Inc. and New England Biolabs, respectively. In vitro transcription plasmid pGEM-3zf(+) was obtained from Promega Corp. Other materials and chemicals were obtained from commercial sources. [-P]GTP and [S]methionine were purchased from Amersham Corp.

Plasmids and Protein Preparation

pGEX-R-Ras was constructed as follows. Human R-Ras cDNA was amplified by PCR from the pTRGA (a kind gift from Drs. David G. Lowe and Alan Hall) with the primers 5`-ATAAGGTACCATGAGCTCTGGTGCTGC-3` and 5`-TATAGGTACCAAGGCACACAGTGGCAG-3`, and cloned into the KpnI site of pGEX2T-KpnI, which was made by introducing the KpnI site to the BamHI site of pGEX-2T. The R-Ras mutant was produced by PCR of pGEX-R-Ras with the primers 5`-TCCTACACGAAGATCTGC-3` and 5`-TCGTGTAGGAGGCCTCAATAGTG-3`, and cloned into pGEX-2T-KpnI. Human RalA cDNA was amplified by PCR from human brain cerebral cortex Quick Clone cDNA (Clontech) with the primers 5`-TATAGGTACCATGGCTGCAAATAAGCCCAAG-3` and 5`-AATTGGTACCTTATAAAATGCAGCATCTTTCTC-3`, and cloned into pGEX-2T-KpnI. pGEX-BH2 harboring NF1-GRD was a kind gift from Dr. K. Tanaka (Osaka University). pMal-c2 harboring R-Ras GAP-GRD was constructed as follows to produce R-Ras GAP-GRD fused to MBP (MBP-R-Ras GAP-GRD). R-Ras GAP-GRD cDNA was amplified by PCR from R-Ras GAP cDNA in gt10 with the primers 5`-TATAGGATCCGGCTCCCTGCGCTTGAATGTG-3` and 5`-TTATGGATCCCTAGAACCCTTCCTTAAGCAGGAT-3`, and cloned into the BamHI site of pMal-c2. GST, GST fusion proteins, and MBP-R-Ras GAP-GRD were expressed in Escherichia coli DH5alpha and purified according to the manufacturer's instructions. For in vitro translation, the R-Ras GAP cDNA was cut at the BamHI sites, and cloned into the BamHI site of pGEM-3Zf(+).

Cytosol Preparation

Bovine brain gray matter (100 g) was cut into small pieces with scissors and suspended in 300 ml of homogenization buffer (25 mM Tris/HCl at pH 7.5, 1 mM DTT, 5 mM EGTA, 10 mM MgCl(2), 10 µM (p-amidinophenyl)methanesulfonyl fluoride, 1 µg/ml leupeptin, and 10% sucrose). The suspension was homogenized with a Potter-Elvehjem Teflon-glass homogenizer and filtered through four layers of gauze. The homogenate was centrifuged at 20,000 times g for 30 min at 4 °C and then at 100,000 times g for 60 min at 4 °C. The supernatant was stored at -80 °C as the crude cytosolic fraction.

GST-R-Ras Affinity Column Chromatography

The guanine nucleotide-bound forms of GST-small G proteins were made by incubating small G proteins (1.5 nmol) for 1 h at 30 °C with 15 µM GDP or GTPS in 1 ml of a reaction mixture (20 mM Tris/HCl at pH 7.5, 10 mM EDTA, 1 mM DTT, 5 mM MgCl(2), 1 mML-alpha-dimyristoylphosphatidylcholine, and 0.3% CHAPS(17) . GST-small G proteins (each 30 nmol) were immobilized on 1.25 ml of glutathione-Sepharose 4B, which packed into columns. Then, 300 ml of brain cytosolic fraction was preabsorbed to remove the native GST with 1 ml of glutathione-Sepharose 4B and was loaded onto the GST-small G protein affinity columns. The columns were washed with 12.5 ml (10 volumes) of buffer A (20 mM Tris/HCl at pH 7.5, 1 mM EDTA, 1 mM DTT, and 5 mM MgCl(2)), followed by washing with 12.5 ml (10 volumes) of buffer A containing 50 mM NaCl. The proteins bound to the affinity columns were eluted four times by addition of 1 ml (0.8 volumes) of buffer A containing 200 mM NaCl.

Purification of p98 and Peptide Sequence

To purify p98, 3 liters of brain cytosolic fraction was used for GTPSbulletGST-R-Ras affinity column chromatography as described above. The second and third fractions of the 200 mM NaCl-eluates were dialyzed three times with distilled water and concentrated by freeze-drying. The concentrated samples were subjected to SDS-PAGE and transferred onto polyvinylidene difluoride membranes(18) . The immobilized p98 was reduced and S-carboxymethylated, followed by in situ digestion with Achromobacter protease I and Asp-N as described previously(18) . The digested peptides were fractionated by C18 column chromatography and subjected to amino acid sequencing(18) .

Molecular Cloning and Determination of Nucleic Acid Sequence of Bovine R-Ras GAP cDNA

To amplify a partial fragment of R-Ras GAP cDNA, we performed PCR from bovine quick clone cDNA (Clontech) using degenerate oligonucleotide primers corresponding to the peptide sequences indicated by double underlines in Fig. 2. The amplified fragment was labeled with [alpha-P]dCTP using a Random Primer DNA labeling kit (Takara Shuzo Co.) and used to screen a bovine brain cDNA library (1.2 times 10^6 independent plaques in total)(19) . The cDNA inserted into gt10 phage DNA was cloned into pUC18 for the nucleotide sequencing with an Applied Biosystems model 373S DNA sequencer.


Figure 2: Deduced amino acid sequence of p98. Amino acid sequences determined from native p98 are indicated by single underlines. Amino acid sequences used for amplification of p98 cDNA are indicated by double underlines.



Interaction of in Vitro Translated Recombinant R-Ras GAP with GST Small G Proteins

In vitro translation of pGEM-R-Ras GAP was performed using the TNT T7-coupled reticulocyte lysate system (Promega) under the conditions described in the instruction manual. GST-small G proteins (0.75 nmol each) were immobilized onto 31 µl of glutathione-Sepharose 4B beads and washed with 310 µl (10 volumes) of buffer A. The immobilized beads were added to 40 µl of in vitro translated mixture and incubated for 1 h at 4 °C with gentle mixing. The beads were washed three times with 102 µl (3.3 volumes) of buffer A, and the bound proteins were eluted with GST-small G proteins by addition of 102 µl (3.3 volumes) of buffer A containing 10 mM glutathione.

GTPase Assay

GTPase activity of Ha- or R-Ras was assayed by measuring the decreased radioactivity of [-P]GTPbulletGST-Ha- or R-Ras(20) . [-P]GTPbulletGST-Ha- or R-Ras (10 pmol) was incubated at 30 °C for appropriate periods in the presence or absence of MBP-R-Ras GAP-GRD or GST-NF1-GRD in 100 µl of reaction mixture (20 mM Tris/HCl at pH 7.5, 5.35 mM EDTA, 1 mM DTT, 10 mM MgCl(2), 1 mM GTP, and 0.15% CHAPS). The reaction was stopped by adding 3 ml of ice-cold stopping buffer (20 mM Tris/HCl at pH 8.0, 100 mM NaCl, and 25 mM MgCl(2)), followed by rapid filtration using nitrocellulose filters. The filters were washed three times with the same ice-cold buffer, and bound radioactivity was determined using a liquid scintillation counter.

RT-PCR Analysis

To design rat R-Ras GAP primers for RT-PCR, we determined partial sequence of the rat R-Ras GAP cDNA as follows. A partial fragment of rat R-Ras GAP cDNA was amplified by PCR from rat brain Quick Clone cDNA (Clontech) with the same degenerate oligonucleotide primers as used for amplification of bovine R-Ras GAP partial cDNA. The amplified fragment of about 400 base pairs was cloned into pCRII vector (Invitrogen) and subjected to nucleotide sequencing as described above. Total RNA (1 µg) from various adult rat tissues was subjected to RT-PCR with the following primers, 5`-ACACAGAAGACCACGTCTTCTC-3` and 5`-ATGGCTGCCTCTTGCTTATCTC-3` which were designed from rat R-Ras GAP partial cDNA sequences. The PCR products were subjected to 6% polyacrylamide gel electrophoresis(21) .

Other Procedures

SDS-PAGE was performed as described previously(22) . Protein concentrations were determined with bovine serum albumin as a reference protein as described(23) . The BLAST program was used for protein homology search(24) .


RESULTS

Purification of GTPSbulletR-Ras-interacting Molecule

To detect R-Ras-interacting molecules, bovine brain cytosolic fraction was loaded onto a GST-R-Ras affinity column. The proteins bound to the affinity column were eluted by addition of 200 mM NaCl. A protein with mass of about 98 kDa (p98) was detected in the 200 mM NaCl-eluate from GTPSbulletGST-R-Ras affinity column but not from GST or GDPbulletGST-R-Ras affinity column (Fig. 1A). p98 was not detected in the eluate of GTPSbulletGST-R-Ras affinity column. R-Ras is structurally equivalent to Ha-Ras, which has a mutation in the effector-interacting domain(1, 2) . To further confirm the specificity of the interaction, affinity column chromatography using GST-Ha-Ras and GST-RalA was performed. p98 was eluted from neither GST-Ha-Ras nor GST-RalA affinity columns (Fig. 1B).


Figure 1: Purification of R-Ras-interacting molecule. A, detection of R-Ras-interacting molecule by GST-R-Ras affinity column chromatography. Bovine brain cytosolic fraction was loaded onto GST-small G protein affinity columns. The proteins bound to the affinity columns were eluted by addition of 200 mM NaCl. Aliquots (40 µl) of the second fraction of the 200 mM NaCl eluates were subjected to SDS-PAGE and silver-stained. Lane 1, GST; lane 2, GDPbulletGST-R-Ras; lane 3, GTPSbulletGST-R-Ras; lane 4, GTPSbulletGST-R-Ras. B, specific interaction of p98 with GTPSbulletGST-R-Ras. Affinity column chromatography using various small G proteins was carried out as described in the legend for panel A. Lane 1, GST; lane 2, GDPbulletGST-R-Ras; lane 3, GTPSbulletGST-R-Ras; lane 4, GDPbulletGST-Ha-Ras; lane 5, GTPSbulletGST-Ha-Ras; lane 6, GDPbulletGST-RalA; lane 7, GTPSbulletGST-RalA. An arrow denotes the position of p98. The results shown are representative of three independent experiments.



Amino Acid Sequence Analysis of p98

To identify the GTPSbulletR-Ras-interacting molecule, p98 was subjected to amino acid sequencing as described under ``Experimental Procedures.'' Thirteen peptide sequences derived from p98 were determined. Two sequences of the peptides were used to design the degenerate oligonucleotide primers for amplification of the specific DNA fragments derived from the p98 cDNA. A fragment of about 400 base pairs was obtained and used as a probe for library screening. Of 1.2 times 10^6 recombinant phage plaques from a bovine brain cDNA library, two clones hybridized with the probe. The nucleotide sequence of one of the cloned cDNAs of about 3.9 kilobase pairs was determined. The cDNA contained an open reading frame encoding a protein consisting of 834 amino acids. The calculated molecular mass was 95,384 Da, which is close to the apparent molecular mass of p98 estimated by SDS-PAGE. The deduced amino acid sequence is shown in Fig. 2. All of the 13 peptide sequences obtained were found within the deduced amino acid sequence. The neighboring sequence around the initiation codon was consistent with the translation initiation start site proposed by Kozak (25) but we found no termination codon in the preceding region. To confirm whether the first ATG is the real initiation codon, in vitro translation was performed using p98 cDNA cloned downstream of the T7 promoter of pGEM-3zf(+). In vitro translated protein migrated with an apparent size of about 98 kDa, which was the same size as native p98, and co-migrated with native p98 purified from GTPSbulletGST-R-Ras affinity column (data not shown).

Structural Characteristics of p98

As a result of homology search in GenBank protein data base, p98 showed a high degree of sequence similarity with rat Gap1^m, which is thought to be the mammalian counterpart of Drosophila Gap1 (9, 26) . The identity of nucleic acid and amino acid sequences was 62.5% and 59.7%, respectively. To examine relationship between p98 and rat Gap1^m, we determined partial sequence of rat p98 using of a cDNA fragment amplified by PCR (data not shown). The partial amino acid sequences of rat p98 showed identities of 95.2% with bovine p98 but only 55.4% with Gap1^m. Thus, we concluded that p98 is a homologue of rat Gap1^m rather than its counterpart. Similarly to rat Gap1^m, p98 contained two C2 domains(27) , GRD (28) , and PH domain (29) (Fig. 3). The alignment of amino acid sequences in each domain are shown in Fig. 3. Since p98 shows a high degree of amino acid sequence similarly with Gap1^m and exhibits GAP activity toward R-Ras (see below), we designated it as R-Ras GAP.


Figure 3: Schematic representation of p98 and rat Gap1^m. The numbers indicate the amino acid sequence identities in each domain. C2, C2 domain; GRD, GAP-related domain; PH, pleckstrin homology domain.



Interaction of Recombinant R-Ras GAP with GTPSbulletGST-R-Ras

To address whether recombinant R-Ras GAP interacts with GTPSbulletR-Ras, immobilized GST-small G proteins were mixed with in vitro translated R-Ras GAP and interacting proteins were eluted with GST small G proteins by addition of glutathione. In vitro translated R-Ras GAP was co-eluted strongly with GTPSbulletGST-R-Ras but weakly with GST, GDPbulletGST-R-Ras, GTPSbulletGST-R-Ras, GST-Ha-Ras, and GST-RalA (Fig. 4). The weak bands detected in the eluates other than that from GTPSbulletGST-R-Ras may result from nonspecific interaction. The slightly faster migrated band may be a degraded product of R-Ras GAP.


Figure 4: Interaction of in vitro translated R-Ras GAP with GTPSbulletGST-R-Ras. In vitro translated R-Ras GAP was mixed with GST-small G proteins immobilized to glutathione-Sepharose 4B beads. The interacting proteins were eluted with GST-small G proteins by addition of glutathione. Aliquots (40 µl) were subjected to SDS-PAGE and vacuum-dried followed by autoradiography. Lane 1, in vitro translated R-Ras GAP; lane 2, GST; lane 3, GDPbulletGST-R-Ras; lane 4, GTPSbulletGST-R-Ras; lane 5, GTPSbulletGST-R-Ras; lane 6, GDPbulletGST-Ha-Ras; lane 7, GTPSbulletGST-Ha-Ras; lane 8, GDPbulletGST-RalA; lane 9, GTPSbulletGST-RalA. An arrow denotes the position of R-Ras GAP. The results shown are representative of three independent experiments.



GAP Activity of R-Ras GAP

We examined whether R-Ras GAP-GRD stimulates intrinsic GTPase activity of R-Ras. As described previously (15) , GST-NF1-GRD stimulated GTPase activity of R-Ras in both a time- and dose-dependent manner ( Fig. 5and 6). The rate constant of GST-NF1-GRD for Ha-Ras was about 5-fold higher than that for R-Ras. MBP-R-Ras GAP-GRD also stimulated GTPase activity of R-Ras in a time- and dose-dependent manner ( Fig. 5and Fig. 6). In contrast with the GAP activity of GST-NF1-GRD, the rate constant of MBP-R-Ras GAP-GRD for R-Ras was about 5-fold higher than that for Ha-Ras. MBP-R-Ras GAP-GRD did not stimulate the GTPase activities of Rap1 or Rho (data not shown). It has been reported that p120 Ras GAP and NF1 do not stimulate GTPase activity of Ha-Ras, which has a mutation in the effector-interacting domain. The GTPase activity of R-Ras was not stimulated by GST-NF1-GRD as described for Ha-Ras (Fig. 5A). We examined the GAP activity of MBP-R-Ras GAP-GRD toward the effector mutant of R-Ras. MBP-R-Ras GAP-GRD weakly stimulated the GTPase activity of R-Ras but the activity was much lower than that toward wild-type R-Ras (Fig. 5B). Under the similar conditions, MBP-R-Ras GAP-GRD did not affect guanine nucleotide release from R-Ras (data not shown).


Figure 5: Time course for the GAP activity of R-Ras GAP. [-P]GTPbulletGST-small G proteins (100 nM each) were incubated at 30 °C for the indicated periods of time with respective GRDs (30 nM). circle, bullet, with [-P]GTPbulletGST-Ha-Ras; box, , with [-P]GTPbulletGST-R-Ras; up triangle, , with [-P]GTPbulletGST-R-Ras. A, circle, box, up triangle, in the absence of GST-NF1-GRD; bullet, , , in the presence of GST-NF1-GRD. B, circle, box, up triangle, in the absence of MBP-R-Ras GAP-GRD; bullet, , , in the presence of MBP-R-Ras GAP-GRD. The values shown are means ± S.E. of three independent experiments.




Figure 6: Dose-dependent effect of R-Ras GAP on GTPase activity. [-P]GTPbulletGST-small G proteins (100 nM each) were incubated for 3 min at 30 °C with indicated amounts of respective GRDs. bullet, with [-P]GTPbulletGST-Ha-Ras; , with [-P]GTPbulletGST-R-Ras. A, GST-NF1-GRD. B, MBP-R-Ras GAP-GRD. The values shown are means ± S.E. of three independent experiments.



Tissue Distribution of R-Ras GAP

To examine the tissue distribution of R-Ras GAP, RT-PCR was performed using mRNA prepared from various rat tissues (Fig. 7). Primers were designed from partial nucleotide sequences of rat R-Ras GAP. R-Ras GAP mRNA was expressed highly in cerebrum and cerebellum; moderately in heart, spleen, thymus, lung, liver, kidney, and pancreas; and hardly in skeletal muscle, small intestine, adrenal grand, and testis.


Figure 7: RT-PCR analysis of R-Ras GAP expression in various rat tissues. Total RNAs (1 µg of each) were subjected to RT-PCR using rat R-Ras GAP or beta-actin primers as indicated. The PCR products were electrophoresed on a 6% polyacrylamide gel. Lane 1, cerebrum; lane 2, cerebellum; lane 3, heart; lane 4, skeletal muscle; lane 5, spleen; lane 6, thymus; lane 7, lung; lane 8, liver; lane 9, kidney; lane 10, pancreas; lane 11, small intestine; lane 12, adrenal gland; lane 13, testis. The results shown are representative of three independent experiments.




DISCUSSION

In the present study, we purified an R-Ras-interacting protein, p98, by GST-R-Ras affinity column chromatography. p98 interacts with GTPSbulletR-Ras but not with GDPbulletR-Ras, GTPSbulletR-Ras, GTPSbulletHa-Ras, or GTPSbulletRalA. We determined partial amino acid sequences of peptides derived from p98, cloned its cDNA, and determined its primary structure. p98 shows a high degree of amino acid sequence similarity to Gap1^m, and recombinant GRD of p98 showed GAP activity toward R-Ras higher than that toward Ha-Ras. Taken together, these results clearly indicate that p98 serves as GAP for R-Ras. Since GAP specific for R-Ras was identified here for the first time, we designated p98 as R-Ras GAP.

Among the small G protein-interacting proteins, both target proteins and GAP appear to interact with small G proteins in a GTP-dependent fashion, and not to interact with their effector mutants. Since R-Ras GAP is the first molecule that specifically interacts with R-Ras in a GTP-dependent fashion, we speculate that R-Ras GAP may serve as a downstream target for R-Ras rather than GAP. However, this possibility seems unlikely, because genetic evidence indicates that Gap1, which shows a high degree of amino acid sequence similarity with R-Ras GAP, functions as a GAP rather than a downstream target for Ras in Drosophila(26) .

The GAP activity for R-Ras was first detected in human spleen(30) . This protein has been partially purified and shown to be the same as p120 Ras GAP. NF1 was also reported to exhibit GAP activity toward R-Ras(15) . We have shown here that R-Ras GAP exhibits higher GAP activity toward R-Ras than toward Ha-Ras. Although we cannot rule out the possibility that p120 Ras GAP and NF1 serve as GAP for R-Ras as well as for Ha-Ras, it is more likely that p120 Ras GAP and NF1 primarily serve as GAP for Ha-Ras, and that R-Ras GAP primarily serves as GAP for R-Ras in vivo. Further studies are necessary to estimate how much R-Ras GAP contributes to the regulation of R-Ras in vivo.

RT-PCR experiments indicate that R-Ras GAP is highly expressed in cerebrum and cerebellum, moderately in heart, spleen, thymus, lung, liver, kidney, and pancreas and hardly in skeletal muscle, small intestine, adrenal grand, and testis, suggesting that R-Ras GAP plays important roles in brain. On the other hand, R-Ras is expressed in most tissues including skeletal muscle, small intestine, adrenal grand, and testis (data not shown). From these observations, it is conceivable that isoforms or different types of R-Ras GAP are expressed in the tissues where R-Ras GAP is hardly expressed.

R-Ras GAP has unique structural features such as C2 domains and PH domain which are also observed in Gap1 and Gap1^m. This suggests that Gap1^m and R-Ras GAP may share some functions or be regulated in a similar way in vivo. The C2 domain, which is observed in protein kinase C, synaptotagmin, and Rabphilin-3A, is believed to be involved in the binding to Ca and phospholipid(27, 31, 32) . It is possible that R-Ras GAP is recruited to membranes via the C2 domains upon influx of Ca into cells. The PH domain is assumed to be involved in the binding to phosphatidylinositol-4,5-bisphosphate or beta subunits of trimeric G proteins(33, 34) . It is speculated that R-Ras GAP associates with these molecules through the PH domain in vivo. Further studies may provide insight into roles of the C2 and PH domains of R-Ras GAP, leading to better understanding of modes of action and activation of R-Ras GAP.

R-Ras has been reported to interact with Bcl-2(12) . Furthermore, it has been shown that R-Ras increases the rate of apoptotic cell death in the setting of growth factor withdrawal, and that Bcl-2 completely abrogates this effect of R-Ras(13) . R-Ras has been shown to interact with c-Raf-1, to activate MAP kinase cascade, and to induce transformation of NIH/3T3 cells(14, 15, 16) . However, it is not yet clear how R-Ras accelerates apoptotic cell death in growth factor-deprived cells and promotes transformation of some types of cells such as NIH/3T3 cells, and how R-Ras is activated presumably downstream of receptors for some extracellular signals. Several groups have demonstrated that Ras is involved in regulation of a variety of cell functions including cell transformation, proliferation, and differentiation by utilizing p120 Ras GAP as a probe(35, 36) . Overexpression of p120 Ras GAP suppresses growth factor- and Ras-mediated responses leading to cell transformation, proliferation, and differentiation. Similarly to p120 Ras GAP, R-Ras GAP will enable us to dissect how R-Ras regulates various cell functions and how R-Ras is regulated during actions of certain extracellular signals.


FOOTNOTES

*
This investigation was supported by grants-in-aid for scientific research and for cancer research from the Ministry of Education, Science, and Culture, Japan(1994), and by a grant for research on metabolic disease from the Yamanouchi Foundation(1994). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U30857[GenBank].

§
To whom correspondence should be addressed. Tel.: 81-7437-2-5440; Fax: 81-7437-2-5449.

(^1)
The abbreviations used are: GAP, GTPase-activating protein; NF1, neurofibromatosis type 1; PCR, polymerase chain reaction; GRD, GAP-related domain; GST, glutathione S-transferase; MBP, maltose-binding protein; DTT, dithiothreitol; G protein, GTP-binding protein; GTPS, guanosine 5`-(3-O-thio)triphosphate; PAGE, polyacrylamide gel electrophoresis; RT-PCR, reverse-transcription PCR; PH domain, pleckstrin homology domain; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid.


ACKNOWLEDGEMENTS

We thank Drs. David G. Lowe (Genentech, Inc.) and Alan Hall (University of London) for their kind gift of human R-Ras cDNA (pTRGA). We are indebted to Dr. Kazuma Tanaka (Osaka University) for the kind gift of pGEX-BH2, and we are grateful to Masako Nishimura for secretarial assistance.


REFERENCES

  1. Satoh, T., Nakafuku, M., and Kaziro, Y. (1992) J. Biol. Chem. 267, 24149-24152 [Medline]
  2. McCormick, F. (1994) Curr. Opin. Genet. Dev. 4, 71-76 [Medline]
  3. Mizuno, T., Kaibuchi, K., Yamamoto, T., Kawamura, M., Sakoda, T., Fujioka, H., Matsuura, Y., and Takai, Y. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 6442-6446 [Medline]
  4. Bowtell, D., Fu, P., Simon, M., and Senior, P. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 6511-6515 [Medline]
  5. Martegani, E., Vanoni, M., Zippel, R., Coccetti, P., Brambilla, R., Ferrari, C., Sturani, E., and Alberghina, L. (1992) EMBO J. 11, 2151-2157 [Medline]
  6. Shou, C., Farnsworth, C. L., Neel, B. G., and Feig, L. A. (1992) Nature 358, 351-354 [Medline]
  7. Trahey, M., Wong, G., Halenbeck, R., Rubinfeld, B., Martin, G. A., Ladner, M., Long, C. M., Crosier, W. J., Watt, K., Koths, K., and McCormick, F. (1988) Science 242, 1697-1700 [Medline]
  8. Xu, G., Lin, B., Tanaka, K., Dunn, D., Wood, D., Gesteland, R., White, R., Weiss, R., and Tamanoi, F. (1990) Cell 63, 835-841 [Medline]
  9. Maekawa, M., Li, S., Iwamatsu, A., Morishita, T., Yokota, K., Imai, Y., Kohsaka, S., Nakamura, S., and Hattori, S. (1994) Mol. Cell. Biol. 14, 6879-6885 [Medline]
  10. Lowe, D. G., Capon, D. J., Delwart, E., Sakaguchi, A. Y., Naylor, S. L., and Goeddel, D. V. (1987) Cell 48, 137-146 [Medline]
  11. Tsujimoto, Y., Cossman, J., Jaffe, E., and Croce, C. (1985) Science 228, 1440-1443 [Medline]
  12. Fernandez-Sarabia, M. J., and Bischoff, J. R. (1993) Nature 366, 274-275 [Medline]
  13. Wang, H.-G., Millan, J. A., Cox, A. D., Der, C. J., Rapp, U. R., Beck, T., Zha, H., and Reed, J. C. (1995) J. Cell Biol. 129, 1103-1114 [Medline]
  14. Cox, A. D., Brtva, T. R., Lowe, D. G., and Der, C. J. (1994) Oncogene 9, 3281-3288 [Medline]
  15. Rey, I., Taylor-Harris, P., van Erp, H., and Hall, A. (1994) Oncogene 9, 685-692 [Medline]
  16. Spaargaren, M., Martin, G. A., McCormick, F., Fernandez-Sarabia, M. J., and Bischoff, J. R. (1994) Biochem. J. 300, 303-307 [Medline]
  17. Shimizu, K., Kuroda, S., Yamamori, B., Matsuda, S., Kaibuchi, K., Yamauchi, T., Isobe, T., Irie, K., Matsumoto, K., and Takai, Y. (1994) J. Biol. Chem. 269, 22917-22920 [Medline]
  18. Iwamatsu, A. (1992) Electrophoresis 13, 142-147 [Medline]
  19. Kaibuchi, K., Mizuno, T., Fujioka, H., Yamamoto, T., Kishi, K., Fukumoto, Y., Hori, Y., and Takai, Y. (1991) Mol. Cell. Biol. 11, 2873-2880 [Medline]
  20. Maekawa, M., Nakamura, S., and Hattori, S. (1993) J. Biol. Chem. 268, 22948-22952 [Medline]
  21. Shuldiner, A. R., Perfetti, R., and Roth, J. (1993) Methods in Molecular Biology: PCR Protocols , Vol. 15, pp. 169-176, Humana Press Inc., Totowa, NJ
  22. Laemmli, U. K. (1970) Nature 227, 680-685 [Medline]
  23. Bradford, M. M. (1976) Anal. Biochem. 72, 248-254
  24. Altshul, S. F., Gish, W., Miller, W., Myers, E. W., and Lipman, D. J. (1990) J. Mol. Biol. 215, 403-410 [Medline]
  25. Kozak, M. (1987) Nucleic Acids Res. 15, 8125-8148 [Medline]
  26. Gaul, U., Mardon, G., and Rubin, G. M. (1992) Cell 68, 1007-1019 [Medline]
  27. Nishizuka, Y. (1988) Nature 334, 661-665 [Medline]
  28. Martin, G. A., Viskochil, D., Bollag, G., McCabe, P. C., Crosier, W. J., Haubruck, H., Conroy, L., Clark, R., O'Connell, P., Cawthon, R. M., Innis, M. A., and McCormick, F. (1990) Cell 63, 843-849 [Medline]
  29. Musacchio, A., Gibson, T., Rice, P., Thompson, J., and Saraste, M. (1993) Trends Biochem. Sci. 18, 343-348 [Medline]
  30. Garrett, M. D., Self, A. J., van Oers, C., and Hall, A. (1989) J. Biol. Chem. 264, 10-13 [Medline]
  31. Perin, M. S., Brose, N., Jahn, R., and Südhof, T. C. (1991) J. Biol. Chem. 266, 623-629 [Medline]
  32. Yamaguchi, T., Shirataki, H., Kishida, S., Miyazaki, M., Nishikawa, J., Wada, K., Numata, S., Kaibuchi, K., and Takai, Y. (1993) J. Biol. Chem. 268, 27164-27170 [Medline]
  33. Harlan, J. E., Hajduk, P. J., Yoon, H. S., and Fesik, S. W. (1994) Nature 371, 168-170 [Medline]
  34. Touhara, K., Inglese, J., Pitcher, J. A., Shaw, G., and Lefkowitz, R. J. (1994) J. Biol. Chem. 269, 10217-10220 [Medline]
  35. Yatani, A., Okabe, K., Polakis, P., Halenbeck, R., McCormick, F., and Brown, A. M. (1990) Cell 61, 769-776 [Medline]
  36. Zhang, K., DeClue, J. E., Vass, W. C., Papageorge, A. G., McCormick, F., and Lowy, D. R. (1990) Nature 346, 754-756 [Medline]

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.