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[Cancer Research 60, 1720-1728, March 15, 2000]
© 2000 American Association for Cancer Research

Molecular Biology and Genetics

Both Normal and Transforming PCPH Proteins Have Guanosine Diphosphatase Activity But Only the Oncoprotein Cooperates with Ras in Activating Extracellular Signal-regulated Kinase ERK11

Juan A. Recio, J. Guillermo Páez, Baishali Maskeri, Mark Loveland, Juan A. Velasco and Vicente Notario2

Laboratory of Experimental Carcinogenesis, Department of Radiation Medicine, Georgetown University Medical Center, Washington, DC 20007


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Previous reports from our laboratory described the activation of the PCPH gene into the PCPH oncogene (mt-PCPH, reported previously as Cph) by a single point mutational deletion. As a consequence, the mt-PCPH oncoprotein is a truncated form of the normal PCPH protein. Although both proteins have ribonucleotide diphosphate-binding activity, only mt-PCPH acted synergistically with a human H-Ras oncoprotein to transform murine NIH3T3 fibroblasts. We report here the expression of the PCPH and mt-PCPH proteins in Escherichia coli and the finding that the purified bacterial recombinant proteins have intrinsic guanosine diphosphatase (GDPase) activity. However, expression of the Syrian hamster PCPH and mt-PCPH proteins in haploid yeast strains engineered to be GDPase deficient by targeted disruption of the single GDA1 allele did not complement their glycosylation-disabled phenotype, suggesting the existence of significant functional differences between the mammalian and yeast enzymes. Results from transient cotransfections into NIH3T3, COS-7, or 293T cells indicated that, in mammalian cells, both PCPH and mt-PCPH cause an overall down-regulation of the stimulatory effect of epidermal growth factor or the activated ras or raf oncogenes on the Ras/mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) signaling pathway. However, despite this overall negative regulatory role on Ras signaling, mt-PCPH, but not PCPH, cooperated with the Ras oncoprotein to produce a prolonged stimulation of the phosphorylation of ERK1 but had no effect on the phosphorylation levels of ERK2. These results represent a clear difference between the mechanisms of action of PCPH and mt-PCPH and suggest that the ability to cause a sustained activation of ERK1 may be an important determinant of the transforming activity of mt-PCPH.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The PCPH oncogene (initially reported as Cph and termed mt-PCPH here) was originally isolated in our laboratory from chemically initiated, neoplastic Syrian hamster embryo fibroblasts on the basis of its ability to transform NIH3T3 cells upon serial cycles of transfection (1 , 2) . We further demonstrated that mt-PCPH synergizes with the human H-ras oncogene in transforming NIH3T3 cells (2) , and that the PCPH proto-oncogene (termed PCPH here) is highly conserved in eukaryotic cells, from yeast to humans, and it is expressed in most normal adult tissues in Syrian hamsters, mice, and humans (3, 4, 5) , suggesting that the normal PCPH protein may have an important cellular function. Recently, we isolated full-length cDNA clones of the Syrian hamster mt-PCPH and PCPH, determined their nucleotide sequence, and demonstrated that mt-PCPH was activated by a point mutational deletion (6) within the coding region of the proto-oncogene. This single bp change causes both a shift in the normal ORF3 of PCPH and the translation of the mutated protein to terminate 33 residues downstream in the new ORF. Consequently, the mt-PCPH oncoprotein is a truncated form (246 versus 469 amino acids) of its normal counterpart and contains an additional, rather hydrophobic COOH-terminal tail (6) .

Biochemical analyses of the in vitro translated products of mt-PCPH and PCPH and functional analyses of cells transformed by mt-PCPH oncogene or transfected with expression constructs of PCPH allowed us to demonstrate (6) that: (a) the products encoded by both PCPH and mt-PCPH genes are ribonucleotide-binding proteins; (b) although they share partial homology with GTP/GDP exchange factors, they do not catalyze nucleotide exchange on the H-Ras protein or any other small G proteins tested; (c) steady-state levels of PCPH and particularly mt-PCPH mRNA are up-regulated in serum-deprived cells; and (d) the mt-PCPH oncoprotein, and to a lesser extent the PCPH protein, provide the cells with enhanced stress-survival functions against a variety of stress factors. Although these results suggest that PCPH participates in cellular mechanisms of response to stress, its biochemical activity remains unknown.

Most recently, we completed the cDNA cloning, sequencing, and chromosomal mapping of the mouse (4) and human (5) PCPH proto-oncogenes and determined that PCPH expression is frequently altered in human neoplasms (5) . Indeed, PCPH was not expressed in 16 of 16 primary human renal carcinomas, although it was highly expressed in matched normal kidney, and PCPH mRNA was also undetectable in the majority (67.4%) of the human tumor cell lines tested. In addition, recent data (7) showed that some human tumor cell lines expressed both the normal Mr 47,000 PCPH protein and a smaller immunoreactive polypeptide with the size (Mr ~30,000) of the mt-PCPH protein. These data suggested that PCPH loss and perhaps truncation may be involved in the development of some human tumors. Therefore, it becomes essential to identify the biochemical activity of the normal and transforming PCPH proteins.

We report here the expression of the PCPH and mt-PCPH proteins in Escherichia coli and the finding that the recombinant proteins have GDPase activity. However, expression of the Syrian hamster PCPH and mt-PCPH proteins in yeast strains engineered to be GDPase deficient by targeted disruption of the single GDA1 allele did not complement their glycosylation-disabled phenotype, suggesting the existence of significant functional differences between the mammalian and yeast enzymes. Results indicate that, in mammalian cells, both PCPH and mt-PCPH cause an overall down-regulation of the Ras/MEK signaling pathway. Despite this, mt-PCPH, but not PCPH, activated ERK1 but not ERK2 when cotransfected with an activated human H-ras oncogene.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Plasmids and General Methods.
The cytomegalovirus-based vector pcDNA3 (Invitrogen, Carlsbad, CA) containing the cDNA inserts (6) of PCPH (pcDNA3-PCPH) and mt-PCPH (pcDNA3-mt-PCPH) was used for the expression of the PCPH and mt-PCPH proteins in mammalian cells, using insertless pcDNA-3 as control. Expression of oncogenic Ras and Raf was accomplished using constitutive vectors with Ha-rasVal12 (pRasV12) and v-raf (pvRaf) under the transcriptional control of the Rous sarcoma virus promoter (8) . Luciferase reporter vectors pAP1-Luc and pSRE-Luc were obtained from Stratagene Cloning Systems (La Jolla, CA). Protein determinations were done using the BCA Protein Assay System (Pierce, Rockford, IL). Luciferase determinations were carried out 48 h after cotransfections with pAP1-Luc and pSRE-Luc using the Promega (Madison, WI) assay system, with the help of a Lumat LB9501 luminometer (Berthold Analytical Instruments, EG&G Wallac, Gaithersburg, MD).

Mammalian Cells, Culture, and Transfection Conditions.
Normal (84-3) and neoplastic (81C39) Syrian hamster cells, murine NIH3T3 fibroblasts, PCPH-derived stable NIH3T3 transfectants, and mt-PCPH-derived NIH3T3 transformants were cultured as described (6) . Human kidney 293T cells were maintained in DMEM supplemented with 10% fetal bovine serum. The calcium-phosphate precipitation technique (9) was used for the generation of stable NIH3T3 transfectants. Transient transfections of NIH3T3 and COS-7 cells were carried out with up to 5 µg of DNA/plate using SuperFect (Qiagen, Inc., Valencia, CA) as recommended by the manufacturers. Transient transfections of 293T cells were carried out using Lipofectamine (Life Technologies, Inc., Gaithersburg, MD) with up to 10 µg of DNA/plate. All transfections were performed in six-well plates containing 3.5 x 105 cells/well. To control for transfection efficiency, we used cotransfection with pSV-ß-galactosidase (10) , and data normalizations after all transient transfections were carried out using triplicate cultures, repeating every cotransfection protocol at least three times and keeping the total amount of DNA constant by the addition of appropriate amounts of empty pcDNA-3 vector DNA.

GDPase Activity Assays.
Precautions were taken to avoid contamination with inorganic phosphate, a nucleotide phosphatase inhibitor, in all manipulations. Cells were washed three times in TBS [25 mM Tris-HCl (pH 7.5), 140 mM NaCl], collected by trypsinization, and resuspended in TBS. Cell extracts were prepared by sonication for 1 min (three 20-s treatments, with 1-min cooling intervals), followed by addition of Triton X-100 to a final concentration of 1.5% and incubation at room temperature for 30 min. Three different methods were used to assay GDPase activity. The standard GDPase assays were carried using a modification of the method described by Abeijon et al. (11) . Briefly, incubation mixtures contained, in a final volume of 50 µl, 0.75 M Tris-HCl (pH 7.5), 0.1 M CaCl2, 0.1 M MgCl2, 1% Triton X-100, 2 mM GDP (Sigma Chemical Co., St. Louis, MO), and 20 µg of cell extracts or purified recombinant proteins. Incubations were done for 20 min at 37°C and stopped by the addition of SDS to a final concentration of 2%, and the inorganic phosphate released was determined from the absorbance at 820 nm, as described (12) . In-gel GDPase activity assays were performed by native gel electrophoresis of the extracts, followed by an incubation of the gels for 20 min at 37°C in a solution of 0.2 M imidazole buffer (pH 7.6), containing 20 mM GDP, 0.1 M CaCl2, and 3.5 mM Pb(NO3)2, and staining for inorganic phosphate as described (13) . Alternatively, GDPase was determined as described above but using 20 µM GDP and 1 µCi of [8,5'-3H]GDP (25–50 Ci/mmol; DuPont NEN, Wilmington, DE). After incubation, one-tenth of the reaction mixtures was loaded onto Cellulose MN300 polyethyleneimine TLC plates impregnated with fluorescent indicator (Macherey-Nagel Gmbh & Co., Düren, Germany), and the nucleotides were separated by ascending chromatography in 0.75 M KH2PO4 (pH 3.5). Then the TLC plates were sprayed with EN3HANCE (DuPont NEN) and exposed to X-OMAT AR films (Eastman Kodak, Rochester, NY). These three methods were also used to determine the phosphatase activity on UDP and IDP.

In Vitro Transcription/Translation.
A one-reaction system was used for the simultaneous transcription and translation of the PCPH and mt-PCPH proteins from the original cDNA clones (6) . The system uses T7 RNA polymerase for transcription and a rabbit reticulocyte extract for in vitro translation (TNT; Promega). A wheat-germ extract was used instead of the rabbit reticulocyte extract whenever GDPase activity assays were based on the colorimetric determination of inorganic phosphate to avoid interference from the color of the lysate on the absorbance measurements. Reactions were performed in the presence of L-[35S]methionine (Amersham, Arlington Heights, IL), as described previously (14) . Radiolabeled translation products were resolved by SDS-PAGE (12.5%), and dried gels were exposed to X-ray films.

Construction of Bacterial Expression Vectors.
The inducible pET-30a(+) bacterial expression vector (Novagen, Madison, WI) was used to subclone the inserts from the Syrian hamster PCPH and mt-PCPH cDNA clones (6) . The PCPH and mt-PCPH inserts were excised from the original cDNA cloning vector by cleavage with BspHI and XhoI, and pET-30a(+) was linearized by cleavage with NcoI and XhoI. The unique BspHI in the PCPH and mt-PCPH sequences is located at the ATG codon in position 13. Because BspHI and NcoI generate compatible 5' overhangs, ligation of the PCPH inserts with the linearized pET-30a(+) vector allowed directional cloning and regenerated the original ATG codon 13, keeping the PCPH or mt-PCPH sequences in-frame with the 5' sequences of the pET-30a(+) vector (Fig. 3A)Citation . Thus, both PCPH and mt-PCPH expression constructs lacked the first 12 codons of the PCPH or mt-PCPH sequence and encoded PCPH and mt-PCPH recombinant proteins containing a poly-His tag at their NH2 termini. Additional expression constructs encoding full-length, poly-His tagged PCPH and mt-PCPH proteins were generated by cloning the PCR-isolated, entire ORFs of PCPH and mt-PCPH into the appropriate restriction sites of the pET-30a(+) vector. Restriction endonucleases were from New England Biolabs (Beverly, MA).



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  		Fig. 3. Expression of PCPH and mt-PCPH in E. coli. A, scheme of the construction of the bacterial expression vectors. B, induction and purification of bacterial recombinant PCPH and mt-PCPH: SDS-PAGE analysis of extracts from bacteria transformed with empty vector DNA (control), the PCPH construct or the mt-PCPH construct, uninduced (U) or induced (I) by addition of IPTG for 1 h to the medium. Arrows, the mobility of recombinant PCPH and mt-PCPH before (left panel) and after (right panel) purification by Ni2+ chelation chromatography and refolding. C, TLC determination of the GDPase activity of purified recombinant (RP) PCPH and mt-PCPH. Controls included reactions without enzyme (C), with total extracts (TCE) of IPTG-induced bacteria expressing PCPH (shown here) or mt-PCPH (data not shown), and with heat-denatured (HD) recombinant proteins. The migration of the substrate (GDP) and the reaction product (GMP) is indicated.

 
Induction and Purification of Bacterial Recombinant Proteins.
E. coli BL21 (DE3) cells were transformed with the pET-30(+)-PCPH or pET-30(+)-mt-PCPH constructs, and expression of the recombinant PCPH and mt-PCPH proteins was induced by addition of 1 mM IPTG for various periods of time. For the extraction of the recombinant proteins, induced cells were processed following the instructions recommended in the His-Bind Buffer kit (Novagen), but 6 M urea was added during the extraction because both proteins fractionated with inclusion bodies. Recombinant proteins were purified by one-step Ni2+ chelation chromatography on His-Bind Resin (Novagen), and the purified proteins were then step-wise dialyzed against a solution containing TBS (pH 7.4), 0.1% Triton X-100, 5% glycerol, and progressively decreasing concentrations of urea (4, 2, 1, and 0.5 M) and without urea in the final step. After dialysis the protein concentrations were determined, and the preparations were aliquoted and frozen at -70°C.

Yeast Transformation, Northern, Western, and Glycosylation Analysis.
The Syrian hamster PCPH and mt-PCPH cDNA inserts (6) were directionally subcloned into the EcoRI and XhoI sites of the Saccharomyces cerevisiae shuttle expression vectors p416GPD and p426GPD (15) . Transcription of sequences inserted in these vectors is driven by the GAPDH promoter. The presence of a CEN6/ARS4 replicon makes p416GPD behave as a low copy number plasmid in S. cerevisiae, whereas p426GPD carries a yeast 2-µm replicon and behaves as a high copy number plasmid. The PCPH and mt-PCPH recombinant constructs were transformed as described (16) into haploid S. cerevisiae strains containing wild-type or disrupted alleles of GDA1, the GDPase encoding gene. Yeast strains and plasmids carrying the wild-type (p13H) or the disrupted (pGD{Delta}1) GDPase genes, generously provided by Dr. C. B. Hirschberg (Boston University, Boston, MA) (17) , were used for positive and negative transformation controls, respectively. Expression of PCPH and mt-PCPH was ascertained by the level of mRNA and protein extracted from exponentially growing cells. Conditions for RNA extraction, Northern analysis, and protein extraction were essentially as described (18) . The full-length Syrian hamster PCPH cDNA insert was used as the probe for the Northern analyses. Conditions for Western analyses are given below. Antibodies used were: a rabbit anti-Gda1p antiserum (72-515) provided by Dr. Hirschberg (19) , and a polyclonal antiserum (#367–10W) raised in rabbits against bacterial-recombinant Syrian hamster PCPH protein purified to near homogeneity (Fig. 3B)Citation . The glycosylation status was monitored by studying the electrophoretic mobility of purified extracellular chitinase as described (17) .

Western Analysis.
Mammalian cells were scraped into harvest buffer [20 mM Tris-HCl (pH 7.5), 20 mM p-nitrophenyl phosphate, 1 mM EGTA, 50 mM sodium fluoride, 50 µM sodium orthovanadate, and 5 mM benzamidine] and sonicated for 5 s. Yeast cells were resuspended in harvest buffer and broken with glass beads by vortexing as described above. Cell extracts (30–50 µg of protein) were subjected to SDS-PAGE on 4–15% gradient gels (20) and blotted onto polyvinylidene difluoride membranes by electrotransfer, and the membranes were probed with primary antibodies: polyclonal anti-MEK1/2, anti-phospho-MEK1/2, anti-phospho-ERK1/2 antibodies (New England Biolabs), or anti-GAPDH (Trevigen, Inc., Gaithersburg, MD). Visualization of immunoreactive polypeptides was accomplished by using a peroxidase-conjugated secondary antibody and development with chemiluminescence (ECL; Amersham Pharmacia Biotech, Inc., Piscataway, NJ). Linearity in the densitometric data analysis was controlled by scanning films corresponding to various developing times for each set of experimental data and matching GAPDH loading controls. Densitometry was performed using the NIH Image 1.61 software.

Determination of GDP:GTP Bound to RAS.
The ratio of GDP:GTP bound to p21Ras was determined essentially as described (21) . Cells were serum starved for 18 h and subsequently labeled for 4 h with 300 µCi of [32P]Pi per 100-mm dish in phosphate-free medium (Life Technologies, Inc.). Cells were then stimulated with EGF (50 ng/ml) for 5 min, after which they were put on ice, washed rapidly with ice-cold TBS, and lysed in 50 mM HEPES (pH 7.4), 1% Triton X-114, 100 mM NaCl, 5 mM MgCl2, 1 mM BSA, 10 mM benzamidine, 10 µg/ml leupeptin, 10 µg/ml aprotinin, 10 µM GTP, 10 µM GDP, 1 mM ATP, and 1 mM sodium phosphate, included to prevent postlysis labeling of p21ras. Nuclei were removed by centrifugation, and the Triton X-114 and aqueous phases were separated at 37°C for 2 min, followed by a brief spin (22) . The detergent phase was diluted 10-fold with lysis buffer without Triton X-114. The lysate was precleared for 5 min with protein G-Sepharose beads and further incubation with agarose-conjugated anti-p21Ras monoclonal Y13–259 (Calbiochem-Novachem, La Jolla, CA). Immunoprecipitates were collected and washed eight times with 50 mM HEPES buffer (pH 7.4), 50 mM NaCl, 5 mM MgCl2, 0.1% Triton X-100, and 0.5% SDS. Radiolabeled GDP and GTP were eluted in 2 mM EDTA, 2 mM DTT, 0.2% SDS, 0.5 mM cold GDP, and 0.5 mM cold GTP at 68°C for 20 min and separated on polyethyleneimine-cellulose plates developed in 0.75 M KH2PO4 (pH 3.5). Plates were autoradiographed, and the GDP:GTP ratio was determined by densitometry, as described above.

Mitogen-activated Protein Kinase Assays.
293T cells at 60–80% confluence were transfected with 1 µg of pcDNA-HA-Erk1 and 3 µg of pcDNA3-PCPH or pcDNA3-mt-PCPH. At 12 h after transfection, the cells were washed with PBS and incubated for another 12 h in serum-free medium. Then the cells were lysed in 20 mM HEPES, 10 mM EGTA, 40 mM ß-glycerophosphate, 1% NP40, 2.5 mM MgCl2, 2 mM sodium orthovanadate, 1 mM DTT, 1 mM phenylmethylsulfonyl fluoride, 20 µg/ml aprotinin, and 20 µg/ml leupeptin. Lysates were centrifuged, and supernatants were incubated with 2 µg of anti-HA monoclonal antibody 12CA5 (Babco, Richmond, CA) for 1 h at 4°C. Protein G-Sepharose (20 µl; Amersham Pharmacia Biotech) was used to recover the immunoprecipitates by centrifugation. Pellets were washed three times with PBS containing 1% NP40 and 2 mM sodium orthovanadate, once with 100 mM Tris (pH 7.5) and 0.5 M LiCl, and once with kinase reaction buffer [12.5 mM MOPS (pH 7.5), 12.5 mM ß-glycerophosphate, 7.5 mM MgCl2, 0.5 mM EGTA, 0.5 mM sodium orthovanadate, and 0.5 mM NaF]. Beads were resuspended in a 30-µl final volume containing, per reaction, 1 µCi of [{gamma}-32P]ATP, 20 µM cold ATP, 3.3 mM DTT, and 1.5 mg/ml of MBP (Sigma Chemical Co.) and incubated for 30 min at 30°C. Samples were run in 4–15% SDS-PAGE, blotted, and exposed. As a loading control, blots were incubated with the anti-HA 12CA5 monoclonal antibody and developed.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Increased GDPase Activity in Extracts of Cells Expressing mt-PCPH.
In addition to their partial homology to GTP/GDP exchange factors (6) , the PCPH and mt-PCPH proteins share some homology with members of the family of nucleotide phosphohydrolases, a rather heterogeneous group of proteins with diverse substrate specificities and cleavage patterns (23 , 24) . On the basis of this homology and of our previous finding that the PCPH and mt-PCPH proteins showed particularly high affinity for GDP in nucleotide binding assays (6) , it seemed possible that the PCPH and mt-PCPH proteins may have GDPase activity. This notion was strengthened by the existence of a 54.7% homology (32.4% identity) between the PCPH protein and Gda1p, the S. cerevisiae GDPase (Fig. 1A)Citation . As a first approximation to explore whether the PCPH and mt-PCPH proteins had GDPase activity, we used in-gel activity assays (13) to determine the levels of GDPase activity in total cell extracts of normal Syrian hamster embryo 84-3 cells and immortal mouse NIH3T3 fibroblasts and compared them with Syrian hamster and murine cells that expressed a mutated PCPH allele, either as a result of chemical carcinogenic treatment (81C39 cells) or by gene transfer. Results (Fig. 1B)Citation showed that: (a) the GDPase activity detected in total extracts of 81C39 cells was greater than that detectable in extracts of normal 84-3 cells; (b) comigrated with phosphohydrolase activities on UDP and IDP, also known as possible GDPase (EC 3.6.1.42) substrates; and (c) could be further increased when both 84-3, and especially 81C39 cells, were incubated in serum-free medium (Fig. 1B)Citation , conditions described previously (6) to cause an increase of mt-PCPH mRNA in these cells. Similar results were obtained on determinations of the relative hydrolytic activity on GDP, UDP, and IDP (Fig. 1C)Citation in extracts from stable mt-PCPH-transformed NIH3T3 cells (379) and from NIH3T3 cells stably transfected with PCPH (389), relative to cells transfected with empty vector DNA (391). These data represented an excellent correlation between the expression of the mt-PCPH and PCPH proteins and the detection of GDPase activity in extracts of two different cell types, further strengthening our notion that the PCPH and mt-PCPH proteins may have GDPase activity.



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  		Fig. 1. Increased GDPase activity in extracts of cells expressing PCPH or mt-PCPH. A, homology between PCPH and the yeast GDPase, Gda1p. B, in-gel detection of GDPase activity in neoplastic, mt-PCPH-expressing Syrian hamster 81C39 cells relative to normal 84-3 cells; S and SF, medium with serum or serum free, respectively; experimental details are given in the text. C, nucleotide diphosphatase (NPase) activity on UDP, GDP, and IDP in extracts of mouse NIH3T3 fibroblasts transformed by mt-PCPH (379) and transfected with PCPH (389) or with empty pCDNA3 vector DNA (391).

 
Increased GDPase Activity after in Vitro Transcription/Translation of PCPH and mt-PCPH.
To rule out the possibility that the increased GDPase activity detected in mt-PCPH- or PCPH-expressing cells was not attributable to the PCPH or mt-PCPH intrinsic GDPase activity but resulted as a secondary effect of the transformation process or the overexpression of the PCPH protein on a distinct endogenous gene product, we next synthesized the PCPH and mt-PCPH proteins in vitro using coupled transcription/translation assays and determined the levels of GDPase activity in the extracts before and after translation. Plasmids containing the full-length coding regions of PCPH or mt-PCPH under the transcriptional control of the bacteriophage T7 RNA polymerase promoter and empty vector DNAs were used as templates. There was a significant increase in GDPase activity (Fig. 2A)Citation after translation of both PCPH and mt-PCPH proteins (Fig. 2B)Citation . Although these assays are not quantitative, we consistently observed that the GDPase activity in the lysates used to translate mt-PCPH was greater than the GDPase activity in lysates used to synthesize the PCPH protein. Similar results were observed with rabbit reticulocyte and wheat-germ lysates (Fig. 2A)Citation . No GDPase activity could be detected when the extracts containing the in vitro translated proteins were boiled prior to the GDPase assays. These results strongly suggested that the increased GDPase activity in rabbit reticulocyte and wheat-germ cell lysates was attributable to the translation of the PCPH and mt-PCPH proteins.



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  		Fig. 2. Increased GDPase activity after in vitro translation of PCPH and mt-PCPH. A, relative GDPase activity in rabbit reticulocyte (RR) and wheat-germ (WG) lysates used for the coupled transcription and translation of PCPH and mt-PCPH. GDPase was measured by the colorimetric determination of inorganic phosphate released from GDP. B, autoradiographic confirmation of the synthesis of PCPH and mt-PCPH in vitro. The data shown here correspond to the wheat-germ lysate presented in A. Reactions were carried out with linearized DNA of either empty pcDNA3 vector (Lane 1), the PCPH construct (Lane 2), or the mt-PCPH construct (Lane 3) in the presence of L-[35S]methionine.

 
Expression of the PCPH and mt-PCPH Proteins in Bacteria.
To rule out the possibility that the PCPH proteins had no intrinsic GDPase activity but were activating a latent GDPase preexisting in the rabbit reticulocyte and wheat-germ lysates and to generate high yields of the PCPH and mt-PCPH proteins for further characterization, we expressed them in E. coli. Inducible bacterial expression vectors contained most of the coding regions of the PCPH and mt-PCPH cDNAs (from amino acid 13 to the end of the ORFs), fused for purification purposes to a poly-His tag at their NH2 termini under the control of the bacteriophage T7 promoter and lac operator (Fig. 3A)Citation . The addition of IPTG to the culture media resulted in the accumulation in the cells of polypeptides with sizes corresponding to those expected for the proteins encoded by the ORFs in the expression vectors (Fig. 3B)Citation . Subcellular fractionation of bacteria containing PCPH or mt-PCPH revealed that both proteins were associated with inclusion bodies (data not shown). Recombinant His-tagged proteins were solubilized with 6 M urea, purified by metal chelation chromatography, and refolded by slow, step-wise dialysis in solutions of progressively decreasing urea concentrations. The purified recombinant proteins showed the expected mobility in SDS-PAGE (Fig. 3B)Citation . Aliquots (20 µg) of the purified recombinant proteins were used for GDPase assays in which the GDP phosphohydrolase activity was monitored by the conversion of [3H]GDP into GMP. Results (Fig. 3C)Citation demonstrated that both PCPH and mt-PCPH proteins have intrinsic GDPase activity, and that the activity is lost when the purified proteins were subject to heat denaturation prior to incubation with radiolabeled GDP. No difference was observed between the specific activities of purified recombinant PCPH and mt-PCPH.

PCPH and mt-PCPH Proteins Do Not Complement S. cerevisiae {Delta}gda1 Stains.
Given the existence of significant sequence identity between the PCPH protein and the yeast GDPase, the product encoded by the GDA1 gene of S. cerevisiae (Fig. 1A)Citation , and our observation that the PCPH and mt-PCPH proteins have GDPase activity (Fig. 3)Citation , we examined whether the PCPH proteins shared functional mechanisms with the yeast Gda1p by expressing the mammalian proteins in {Delta}gda1 disruptant strains and testing whether they could complement their reported glycosylation defects (17) . As a glycosylation-dependent end point, we studied the relative electrophoretic mobility of chitinase in {Delta}gda1 yeast cells transformed with the PCPH or mt-PCPH cDNAs in comparison with untransformed {Delta}gda1 cells. Results (data not shown) indicated that the expression of the PCPH or mt-PCPH proteins did not recover the mobility shift of chitinase observed in {Delta}gda1 cells (G2-7) when compared with wild-type cells (G2-5). However, expression of the wild-type GDA1 gene resulted in an almost complete recovery of the mobility shift. This lack of complementation by the PCPH and mt-PCPH proteins was observed regardless of whether high or low copy number plasmid vectors (15) were used for their expression. These results strongly suggested that the primary function of the PCPH and mt-PCPH proteins in mammalian cells is likely to be unrelated to glycosylation, the well-established function of the yeast GDPase (17) .

Transient Expression of mt-PCPH Modulates Ras GDP/GTP Levels in Mammalian cells.
Previous results from our laboratory demonstrated that, in stable cotransfection experiments, mt-PCPH synergized with the human H-ras oncogene to transform NIH3T3 fibroblasts (2) . More recently, we also showed that the PCPH and mt-PCPH products are nucleotide-binding proteins with special affinity for GDP in vitro (6) . These data strongly suggested that PCPH and mt-PCPH proteins may interact directly or indirectly with Ras signaling when expressed in mammalian cells. To test this possibility, we examined whether the expression of mt-PCPH would alter the ratio of GDP:GTP bound to the endogenous Ras protein in NIH3T3 and 293T cells after EGF stimulation. Results were similar for the two cell lines and showed (Fig. 4A)Citation that, as expected, EGF treatment caused an increase in the amount of GTP bound to Ras relative to unstimulated controls, whereas the relative amount of Ras-bound GDP did not change significantly. Accordingly, the GDP:GTP ratio cells went from 7.8 to 1.8 (Fig. 4B)Citation . However, in EGF-stimulated cells expressing mt-PCPH, the GDP:GTP ratio was 2.7. These results indicated that, in the presence of mt-PCPH, a greater proportion of Ras molecules remained in the inactive state in response to EGF. Thus, mt-PCPH appeared to act as a negative regulator of Ras activity.



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  		Fig. 4. Effect of mt-PCPH on the GDP:GTP bound to Ras. Untransfected and mt-PCPH transfected human 293T cells were radiolabeled with [32P]Pi for 4 h, stimulated with EGF for 5 min, and the GDP and GTP bound to p21ras were extracted and resolved by TLC (A). Untransfected, unstimulated cells were used as control. Densitometric analysis (B) was used to determine the GDP:GTP ratios shown at the bottom.

 
PCPH and mt-PCPH Behave as Negative Regulators of the Ras Signaling Pathway.
To assess whether the negative effect on Ras activation had any consequences on the Ras signaling pathway as a whole, we transiently cotransfected the PCPH or mt-PCPH cDNAs into NIH3T3 (Fig. 5ACitation and Fig. 5BCitation ) and COS-7 cells (Fig. 5, C and D)Citation with reporter plasmids in which luciferase expression is regulated by the presence of the AP-1 or SRE transcriptional enhancer elements. Transactivation of the AP-1 and SRE binding sites in the reporter vectors was induced by including vectors for the expression of oncogenic Ras (RasV12; Fig. 5, A and CCitation ) or Raf (v-raf; Fig. 5, B and DCitation ) in some of the cotransfections. Results (Fig. 5)Citation demonstrated that neither PCPH nor mt-PCPH by themselves had any significant effect on the transactivation of AP-1 or SRE elements in either cell type. As expected, expression of either RasV12 or Raf by themselves resulted in considerable increases in AP-1 and SRE transactivation in NIH3T3 and COS-7 cells. However, when the cells were cotransfected with RasV12 or Raf and PCPH or mt-PCPH, the levels of transactivation of both AP-1 and SRE elements were clearly reduced (between 27.4 and 62.5% for AP-1, and between 34.3 and 86.5% for SRE) in both cell types. These data suggested that both PCPH and mt-PCPH were acting as general negative regulators of the Ras/Raf signaling pathway.



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  		Fig. 5. Effect of PCPH and mt-PCPH on AP-1 and SRE transactivation by Ras (A and C) or Raf (B and D). NIH3T3 (A and B) and COS-7 (C and D) cells were cotransfected with RasV12 or v-raf plus either PCPH or mt-PCPH, and their effect on AP-1 and SRE transactivation was assessed by comparison with cells transfected with Ras, Raf, PCPH, or mt-PCPH individually. Background AP-1 and SRE activities were established from cells transfected with empty pcDNA3 vector DNA alone. The extent of transactivation was determined by the expression of luciferase activity from AP-1- and SRE-luciferase reporter constructs introduced in the various cellular backgrounds. Bars, SD.

 
To further characterize the effect of PCPH and mt-PCPH on Ras signaling, we next examined the level and phosphorylation status of other downstream components of the pathway in 293T cells transiently cotransfected to express RasV12 and PCPH or mt-PCPH. As shown in Fig. 6ACitation , expression of RasV12 by itself resulted in a dramatic increase in the level of phosphorylation of MEK1/2, but this increase was reduced by the coexpression of either PCPH (nearly 20% inhibition) or mt-PCPH (~37% inhibition). However, neither PCPH nor mt-PCPH altered the total level of MEK1/2 protein significantly, indicating that the changes observed in MEK1/2 phosphorylation were not attributable to protein down-regulation. These results supported a negative regulatory role of the PCPH and mt-PCPH proteins on the Ras/MEK signaling pathway.



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  		Fig. 6. Effect of PCPH and mt-PCPH on the activation of MEK1/2 and ERK1/2 by oncogenic Ras. 293T cells (A and B) or NIH3T3 (C) cells were cotransfected with RasV12 plus PCPH or mt-PCPH, and their effect on MEK1/2 (A) or ERK1/2 (B and C) activation was assessed by Western blot analysis in comparison with cells transfected with Ras, PCPH, or mt-PCPH individually and relative to background control transfections with pcDNA3 vector DNA alone. PCPH and mt-PCPH expression was confirmed using an antibacterial recombinant PCPH rabbit antiserum. MEK1/2 and ERK1/2 activation was assessed with antibodies specific for their phosphorylated forms. Loading was controlled with anti-MEK1/2 (A) or anti-GAPDH (B) antibodies. Changes in activated ERK1 are shown at the bottom and were determined by densitometry. ERK1 kinase activity in NIH3T3 cells (C) was determined on MBP using immunoprecipitates of HA-tagged ERK1 protein, relative to the total content of ERK1 as determined by Western analysis with an anti-HA antibody. Quantitative data indicated below each lane were normalized for loading and transfection efficiency.

 
Differential Effect of PCPH and mt-PCPH on ERK1 Activation.
Because MEK1/2 phosphorylation was down-regulated in the presence of PCPH or mt-PCPH, we examined the phosphorylation status of ERK1 and ERK2, which are MEK1/2 substrates. To this end, 293T cells were transiently cotransfected with pRasV12 and either pcDNA3-PCPH or pcDNA3-mt-PCPH, and the results were compared with those from transfections with pRasV12, pcDNA3-PCPH, or pcDNA3-mt-PCPH alone. Western analyses with anti-phospho-ERK1/2 showed (Fig. 6B)Citation that neither PCPH nor mt-PCPH alone had any significant effect on ERK1/2 phosphorylation, whereas RasV12 increased ERK1 phosphorylation by ~2-fold. However, when RasV12 was coexpressed with PCPH, the level of phosphorylated ERK1 decreased by almost 20%, relative to cells expressing RasV12 alone. Most surprisingly, when RasV12 was coexpressed with mt-PCPH, the level of phosphorylated ERK1 was almost doubled relative to the induction caused by RasV12 alone, whereas the phosphorylation levels of ERK2 did not change under our experimental conditions. These results suggested that there was a synergism between Ras and mt-PCPH converging specifically on ERK1 and represented a clear difference between the mechanisms of action of PCPH and mt-PCPH.

To confirm that the increased phosphorylation of ERK1 was indeed reflected on its kinase activity, we transiently cotransfected 293T cells to express either HA-ERK1 and RasV12, or HA-ERK1 and RasV12 plus either PCPH or mt-PCPH. Transfected cells were lysed 48 h after transfection, the ERK1 protein was immunoprecipitated with an anti-HA monoclonal antibody, and immune-complex kinase assays were performed using MBP as the substrate. Results (Fig. 6C)Citation showed that, when normalized by the amount of immunoprecipitable ERK1 in each lysate, the maximum ERK1 kinase activity was detected in cells coexpressing RasV12 and the mt-PCPH protein (~33% greater than in the case of RasV12 alone). On the contrary, coexpression of PCPH resulted in an almost 20% decrease of the ERK1 kinase activity stimulation caused by RasV12 alone. These results further supported the notion that the ras and mt-PCPH oncogenes cooperate for the activation of ERK1, despite the general down-regulatory role of mt-PCPH on the Ras-signaling pathway. In addition, these data suggest that mt-PCPH may induce ERK1 activation by a mechanism involving a kinase different from MEK1/2.


   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
This paper reports that the PCPH and mt-PCPH proteins are GDPases. Four lines of evidence support this conclusion: (a) the amino acid sequences of PCPH and mt-PCPH are significantly homologous to Gda1p, the yeast GDPase (Ref. 17 ; Fig. 1ACitation ); b) there was a clear correlation between the expression of mt-PCPH and GDPase activity in carcinogen-initiated cells and cells transformed by mt-PCPH transfection (Fig. 1, B and C)Citation ; (c) GDPase activity was detected in rabbit reticulocyte and wheat-germ extracts after in vitro translation of PCPH or mt-PCPH (Fig. 2)Citation ; and (d) highly purified bacterial recombinant PCPH and mt-PCPH proteins had GDPase activity (Fig. 3)Citation .

The lack of complementation by PCPH or mt-PCPH of the glycosylation-deficient phenotype of S. cerevisiae strains lacking the GDA1-encoded GDPase does not detract from our definition of PCPH and mt-PCPH as GDPases. There are well-documented cases of lack of complementation in S. cerevisiae by the expression of highly conserved mammalian proteins (25) . In some cases, proteins that are extremely well conserved from a structural point of view between yeasts and humans do not have identical functions in S. cerevisiae and mammalian cells. For instance, the Ras proteins function in yeasts to integrate metabolism and control of the cell cycle by modulating adenylate cyclase (26 , 27) , whereas in mammalian cells Ras activity seems to be largely independent of adenylate cyclase (28) . Thus, it is very likely that the primary function of PCPH and mt-PCPH in mammalian cells may not be related to the control of glycosylation. Although our preliminary results seem to confirm this notion, the possibility that PCPH and mt-PCPH may be minor contributors to the glycosylation processes cannot be ruled out. In this regard, PCPH and mt-PCPH might still be functionally comparable in mammalian cells to the yeast Ynd1p, a second protein with GDPase activity described recently (29) as being responsible for the residual GDPase activity detectable in {Delta}Gda1 strains, which is required for O-glycosylation.

Our finding of the involvement of PCPH and mt-PCPH in the regulation of the Ras-signaling pathway provides additional evidence indicating that glycosylation is probably not their primary function. Our results conclusively indicate that both PCPH and mt-PCPH act as negative regulators of Ras signaling by interfering with the activation of several key early and late components of the pathway. The generality of this effect is evidenced by the fact that similar results were obtained with different cell types. Down-regulation of the Ras signaling pathway (Figs. 5Citation and 6)Citation may be an important mechanism by which PCPH contributes to the regulation of normal cell proliferation. Our laboratory has reported (5 , 7) and continues to accumulate data indicating that PCPH is frequently lost in various types of human tumor cells. In agreement with these observations, the loss of a negative regulator of Ras signaling, such as normal PCPH, would result in an up-regulation of the Ras pathway and, in turn, contribute to the development of the neoplastic phenotype.

The overall negative regulatory effect of mt-PCPH on Ras signaling (Figs. 4Citation , 5Citation , and Fig. 6ACitation ) may seem to contradict our previous finding that mt-PCPH and the human H-ras oncogene had a synergistic effect in the transformation of NIH3T3 cells (2) . However, the cooperation between mt-PCPH and Ras may be explained by their convergence on ERK1 activation to a level greater than the addition of the increases induced by Ras and mt-PCPH individually (Fig. 6, B and C)Citation . Nevertheless, the simultaneous down-regulation of upstream components of the Ras-signaling pathway and the activation of ERK1 by mt-PCPH raise the question of what is the MEK1/2-independent mechanism of ERK1 activation in cells coexpressing Ras and mt-PCPH. There are numerous reports in the literature describing various degrees of involvement of Ras, Raf, or MEK on ERK1/2 activation in different experimental systems: from Ras dependent (30 , 31) to Ras independent (31, 32, 33) ; or Ras and Raf independent (34, 35, 36) ; and MEK dependent (32) or independent (37) . The conclusion from all of these data is that EKR1/2 activation can be accomplished via mechanisms not involving components of the Ras-signaling pathway.

Several mechanisms have been proposed as alternatives to the Ras/MEK pathway for ERK1/2 activation, including the inhibition of the VHR or MKP-1 phosphatases for which ERK1/2 are substrates (38 , 39) , tyrosine kinases, phosphatidylinositol 3-kinase, PKC isoforms, phospholipase C (40) , or Ca2+ store depletion (41) . But in the majority of the cases, ERK1/2 activation has been reported to be mediated by PKC (31 , 32 , 42, 43, 44) . This is important, because PKC has been found to be responsible for a slow and sustained activation of ERK1/2 that lasts for days, whereas Ras has been implicated in an immediate, transient activation of ERK that lasts only minutes (44) . In our case, we detected the sustained type of ERK1 activation, at least up to 72 h after transfection, in cells coexpressing Ras and mt-PCPH. Therefore, these results would suggest that, under our experimental conditions, ERK1 activation may have been mediated by a PKC-dependent mechanism. However, the possible involvement of PKC in the activation of ERK1 in cells coexpressing RasV12 and mt-PCPH remains to be elucidated.

The ability to cause a sustained activation of ERK1 may be an important determinant of the transforming activity of mt-PCPH. This suggestion is based on two lines of evidence: (a) the nontransforming PCPH lacks the ability to induce ERK1 activation; and (b) it has been shown in several systems that sustained ERK1/2 activation is necessary for transformation (35 , 45 , 46) through alterations in cell cycle (45 , 47 , 48) , cytoskeleton (49) , and the transcriptional machinery of the cells (47 , 50) . Therefore, ongoing studies are designed to establish the mechanism by which mt-PCPH expression results in ERK1 activation and its contribution to the mt-PCPH transforming activity.


   ACKNOWLEDGMENTS
 
Densitometry was performed at the Lombardi Cancer Center’s Macromolecular Synthesis and Sequencing Shared Resource. We thank Dr. Carlos B. Hirschberg for the S. cerevisiae strains, recombinant plasmids, and anti-Gda1p antiserum.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported by USPHS Grant CA64472 from the National Cancer Institute and by USPHS Grant P30-CA51008. Back

2 To whom requests for reprints should be addressed, at Department of Radiation Medicine, Georgetown University Medical Center, Research Building, Room E215, 3970 Reservoir Road, NW, Washington, DC 20007. Phone: (202) 687-2102; Fax: (202) 687-2221; E-mail: notariov{at}gunet.georgetown.edu Back

3 The abbreviations used are: ORF, open reading frame; ERK, extracellular signal-regulated kinase; MEK, mitogen-activated protein kinase/ERK; EGF, epidermal growth factor; GDPase, guanosine diphosphatase; IPTG, isopropyl-ß-D-thiogalactopyranoside; MBP, myelin basic protein; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HA, hemagglutinin; AP-1, activating protein-1; SRE, serum response element; PKC, protein kinase C. Back

Received 9/22/99. Accepted 1/18/00.


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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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