Molecular cloning of ras cDNA from Penaeus japonicus (Crustacea, Decapoda): geranylgeranylation and guanine nucleotide binding

Gene 224 (1998) 117–122

Molecular cloning of ras cDNA from Penaeus japonicus (Crustacea, Decapoda): geranylgeranylation and guanine nucleotide binding Chein-Fuang Huang, Nin-Nin Chuang * Department of Zoology, National Taiwan University and Institute of Zoology, Academia Sinica, Nankang 11529, Taipei, Taiwan Received 26 June 1998; received in revised form 10 September 1998; accepted 16 September 1998; Received by M. Schartl

Abstract A cDNA was isolated from the shrimp Penaeus japonicus by homology cloning. The shrimp hepatopancreas cDNA encodes a 187-residue polypeptide whose predicted amino acid sequence shares 85% homology with mammalian K-Ras 4B protein and demonstrates identity in the guanine nucleotide binding domains. Expression of the shrimp cDNA in Escherichia coli yielded a 21-kDa polypeptide with a positive reactivity towards the monoclonal antibodies against mammalian Ras. The GTP binding of the shrimp ras-encoded fusion protein was approximated to be 30 000 units/mg of protein, whereas the binding for GDP was 5000 units/mg of protein. Fluorography analysis demonstrated that the prenylation of both shrimp Ras GDP and shrimp Ras GTP by protein geranylgeranyltransferase I of shrimp Penaeus japonicus exceeded the shrimp Ras nucleotide-free form by 10-fold, and fourfold, respectively; that is, the shrimp protein geranylgeranyltransferase I prefers to react with the shrimp ras-encoded p25 fusion protein in the GDP-bound form. © 1998 Elsevier Science B.V. All rights reserved. Keywords: Ras; Protein geranylgeranyltransferase I; GTP; GDP; Invertebrate; Crustacea

1. Introduction Ras proteins are membrane-associated small guanine nucleotide binding proteins that play critical roles in cellular differentiation (Bar-Sagi and Feramisco, 1985), proliferation (Daar et al., 1991) and apoptosis (Downward, 1998). Ras signaling is activated when bound to GTP and is inactivated when GDP is bound (Mineo et al., 1996). At least two proteins appear to regulate Ras activity. One, guanine-nucleotide-exchange factor (GEF ), promotes GDP to GTP exchange ( Wolfman and Macara, 1990; Fam et al., 1997). The other, GTPase activating protein (GAP), stimulates the conversion over 100-fold of Ras GTP to Ras GDP by increasing Ras intrinsic GTPase activity ( Trahey and McCormick, 1987). Therefore, the ratio of Ras GTP to Ras GDP is crucial, and this ratio has to be controlled * Corresponding author. Tel: +886 2 2789 9531; Fax: +886 2 27858059; e-mail: [email protected] Abbreviations: CBP, calmodulin-binding-peptide; Dig-11-dUTP, digoxigenin-11-2∞-deoxy-uridine-5∞-triphosphate; GAP, GTPase activating protein; GDP, guanosine 5∞-diphosphate; GEF, guanine-nucleotide-exchange factor; GTP, guanosine 5∞-triphosphate; PGGT-I, protein geranylgeranyltransferase I.

precisely (Bourne et al., 1990). In many human tumors, Ras is GTP-locked (Boylan et al., 1990) and not confined in caveolae (Song et al., 1996). In mammals, four isoforms of Ras exist: H-Ras, N-Ras, K-Ras 4A, and K-Ras 4B (Lowy and Willumsen, 1993). They are products of three genes, with K-Ras 4A and K-Ras 4B being splice variants of the same gene. Among them, the protein sequences are 80% identical with major differences residing in their carboxyl termini, including the CAAX motif of prenylation (Del Villar et al., 1996). Lipid modification of Ras results in an increase in its intrinsic affinity for the plasma membrane where it can participate in signal transduction (Zhang et al., 1997). The addition of isoprenoid residues, such as geranylgeranyl (20-carbon) and farnesyl (15-carbon) residues, is determined by the X residue of the carboxyl terminal CAAX motif (C, cysteine; A, an aliphatic amino acid ) of proteins. If X is leucine or phenylalanine, the protein is geranylgeranylated (Moores et al., 1991); if X is methionine, serine, alanine, or glutamine, the protein is farnesylated (Reiss et al., 1991). Ras is preferentially farnesylated in mammals (James et al., 1995). Abolishing prenylation disrupts the association of Ras with membranes, thereby disrupting its function ( Kato

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et al., 1992). Inhibitors of prenylation are effective at suppressing the growth of tumor cells transformed by oncogenic Ras (Sun et al., 1995). The cultivation of penaeid shrimp is a world-wide economically important industry. However, diseases cause a decline in the shrimp production. We report here, for the first time in shrimp, the biochemical characterization and the cDNA cloning of genes specifying Ras protein. We intend to utilize the shrimp cDNA probe of ras as an in-situ field monitor and indicator for environmental pro-carcinogen hazards.

2. Materials and methods All reagents used were of the highest grade available commercially. [1(n)-3H]-Geranylgeranyl pyrophosphate, [8-3H]-guanosine 5∞-diphosphate, [8-3H]-guanosine 5∞-triphosphate and biotinylated goat polyclonal antibodies against mouse IgG were from Amersham Corp. (Amersham, Bucks, UK ). The mouse monoclonal antibody against Ras of mammals was from Santa Cruz Biotechnology (Santa Cruz, CA). The prepacked Superdex@75, Superdex@200 column, and HiTrap Phenyl Sepharose 6 Fast Flow (high substitution) were obtained from Pharmacia ( Uppsala, Sweden). Shrimps (Penaeus japonicus), collected off the coast of Taiwan, were kept at 18°C for less than 3 days in a recirculating seawater system before the experiments. Hepatopancreases were dissected out immediately after shrimps had been killed and frozen in liquid nitrogen and stored at −80°C. 2.1. cDNA cloning of ras The most conserved sequences of the Ras were used to design degenerate oligonucleotide primers: 5∞-ATG(A/C )G(A/T/G/C )GA(C/T )CA(A/G)TA(C/T )ATG3∞, 5∞-GT(A/G)TA(A/G)AA(A/T/G/C )GC(A/G)TC(C/T )TC(A/T/G/C )AC-3∞, and a first-strand cDNA pool was synthesized from shrimp (Penaeus japonicus) hepatopancreas as the template for PCR to find the specific shrimp ras cDNA fragment. A lambda ZAP cDNA library for shrimp hepatopancreas was established following the manufacturer’s protocol (Stratagene, La Jolla, CA). The cDNA library contains 1.95×106 phages per ml, and plaques were transferred to Magna Graph (Micron Separations) and screened using a 239-bp Dig-11-dUTP labeled probe (primers: 5∞-ATGCGAACAGGGGAAGG-3∞, 5∞-CCCATGCGGGTCTTGGC-3∞) covering nt 214 and 452. Prehybridization of the duplicate membranes was achieved in a 50% (w/v) formamide solution containing 0.02% (w/v) sodium dodecyl sulfate (SDS), 0.1% (w/v) N-laurylsarcosine, 5× sodium saline citrate (SSC ), 2% (w/v) blocking reagent (Boehringer Mannheim,

Germany) for 2 h at 42°C. For hybridization, the denatured Dig-11-dUTP labeled ras cDNA probe was incubated with the membranes for 17 h at 42°C. The filters were washed twice in 2× SSC and 0.1% (w/v) SDS for 5 min at room temperature, then twice in 0.1× SSC containing 0.1% (w/v) SDS at 55°C for 15 min. The nylon membranes were then incubated with antibodies against digoxigenin, conjugated with alkaline phosphatase that had been diluted 1:10 000. Visualization of bands was achieved at room temperature in the presence of 1:100 diluted CSPDA (Boehringer Mannheim) by exposure to Kodak BioMax-MR film at room temperature for 16 h. PCR (with primers of shrimp Penaeus japonicus for the synthesis of a 239-bp probe) and sequencing were used to confirm and select the correct colony. DNA was sequenced following the dideoxynucleotide method with modification for extended DNA sequencing. Sequence alignment of the various ras sequences was performed at Intelligenetics (Genbank Online Service, Mountain View, CA), using the GENALIGN program. 2.2. Production and characterization of shrimp rasencoded p25 fusion protein The open reading frame of shrimp ras cDNA was amplified by PCR with two primers of the LigationIndependent-Cloning (LIC ) overhang. The ATG initiation codon was designed in the front of the exon I, and the stop codon was connected after the final sequence of the exon IV of shrimp ras. The PCR was performed in 100 ml of 20 mM Tris–HCl, pH 8.4, 50 mM KCl, and 1.5 mM MgCl using 0.5 mM of each primer, 200 mM of 2 each deoxynucleotide triphosphate, 2.5 units of Taq DNA polymerase (Gibco-BRL), and 200 ng of shrimp ras cDNA as template. The template DNA was amplified for 30 cycles consisting of 1 min of denaturation at 94°C, 1 min of renaturation at 55°C, and 1 min of polymerization at 72°C. The PCR products were constructed with calmodulin-binding-peptide (CBP)-tagged fusion system (Stratagene) of pCAL-n-EK expression vector. The expression vector was transformed into BL21(DE3) pLysS Escherichia. coli cells and selected by ampicillin. The enterokinase ( EK ) site-specific cleavage of CBP-tagged fusion protein could then be rapidly purified by clamodulin affinity resin chromatography ( Zheng et al., 1997). 2.3. Purification of protein geranylgeranyltransferase I Protein geranylgeranyltransferase I (PGGT-I ) was purified from the hepatopancreas of shrimp Penaeus japonicus by essentially the same procedures as those described by Lin and Chuang (1998). All manipulations were carried out at 4°C. Enzyme purification results in a yield relative to

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Fig. 1. Sequence analysis of Ras from the hepatopancreas of shrimp Penaeus japonicus. Open reading frame nucleotide sequence (A) of the clone isolated from a shrimp hepatopancreas cDNA library is shown. Alignment of the amino acid sequence (B) of the shrimp Ras protein (S-Ras), H-Ras, N-Ras, K-Ras 4A and K-Ras 4B. The five conserved regions involved in GTP metabolism are boxed. Identical positions are marked by dashes.

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ammonium sulfate precipitate of 20% and a specific activity of 376 units per mg of protein (2506-fold purification). 2.4. GNP binding assay Reaction mixtures (50 ml ) contained 20 mM Tris–HCl, pH 8.5, 2 mM DTT, 100 mM NaCl, 2 mg of bovine serum albumin (Bethesda Research Laboratories), and 2 mM [3H]GNP (10.8 Ci/mmol, Amersham; 1 Ci=37 GBq) and were incubated at 30°C for 30 min. Aliquots (40 ml ) were filtered on 0.45-mm nitrocellulose filters (MultiScreen-HA, Millipore, France) and washed immediately with 3 ml of ice-cold buffer containing 20 mM Tris–HCl, pH 8.5, 2 mM DTT, and 100 mM NaCl. In all of the experiments, 1 pmol of [3H]GNP represents 6 000 cpm. One unit of ras-encoded p25 fusion protein is taken as the amount of protein that binds 1 pmol of [3H]GNP under standard assay conditions. 2.5. Polyacrylamide gel electrophoresis Tricine–sodium dodecyl sulphate–polyacrylamide gel electrophoresis ( Tricine–SDS–PAGE ) was conducted on slab gels containing 10% (w/v) acrylamide and 0.61% (w/v) N,N∞-methylenebis-acrylamide (Schagger and von Jagow, 1987). Samples were reduced and alkylated (Lane, 1978) before application to the gels. Gels were stained with Coomassie Brilliant Blue R250. Radiolabelled proteins were detected by exposure of the dried gel to BioMax-MS film ( Kodak) at −70°C under

an intensifying screen (BioMax TranScreen LE, Kodak). Radiolabelled bands were quantified by fluorogram with GEL-PDMS ANALYZERA (Silver Spring, MD) using Amplify (Amersham) as specified previously ( Tseng and Chuang, 1994). 2.6. Western immunoblotting Proteins that had been separated by Tricine– SDS–PAGE were transferred electrophoretically to nitrocellulose membranes (Bio-Rad, Richmond, CA) and incubated in a quenching solution (3% BSA in PBS buffer) and then incubated overnight at room temperature in a solution of antibodies (1 mg/ml ) in quenching solution. The nitrocellulose membrane was washed and incubated with goat antibodies against mouse IgG, conjugated with biotin that had been diluted 1:1000 in the same quenching solution. Immunoreactive proteins were detected by exposure of the nitrocellulose membrane to avidin-conjugated alkaline phosphatase, and the bands were visualized at room temperature in the presence of 5-bromo-4-chloro-3-indolyl phosphate (BCIP) in nitroblue tetrazolium (NBT ) salt ( Zymed, San Francisco, CA). 2.7. Quantitation of protein Bovine serum albumin served as the standard in the measurement of levels of protein. The amounts of protein were determined by Lowry’s method (Lowry et al., 1951) or by the Micro BCA* Protein Assay (Pierce, Rockford, IL).

Fig. 2. Tricine–SDS–PAGE of bacterially expressed shrimp ras-encoded fusion protein. Shrimp ras-encoded p25 fusion protein (R, 0.5 mg) and vector CBP-tag removed Ras protein (R∞, 1.5 mg) was denatured, analyzed by Tricine–SDS–PAGE on a 10% gel and stained with Coomassie Blue R250 (A). After electrophoresis, proteins were Western-blotted with monoclonal antibody against Ras of mammals (Pan, Santa Cruz) at 1:1000 dilution (B). For comparison, E. coli lysates (OD =1) before (O) or after (I ) induction with final 1 mM Isopropyl-b--thiogalactopyranoside 600 (IPTG) were included. Biotinylated phosphorylase b (M 97,400), biotinylated bovine serum albumin (M =68 000), biotinylated ovalbumin r r (M =46 000), biotinylated carbonic anhydrase (M =31 000), biotinylated soybean trypsin inhibitor (M =20 100) and biotinylated lysozyme r r r (M =14 400) were applied as molecular weight markers. r

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3. Results and discussion 3.1. Isolation of a ras cDNA from hepatopancreas of shrimp Penaeus japonicus The Dig-11-dUTP labeled 239-bp probe (nt 214–452) was applied to isolate five positive plaques from a cDNA library from shrimp hepatopancreas. The 1294-bp insert contained a 128-bp 5∞- untranslated region preceding an initiation site consensus. A 561-bp open reading frame (Fig. 1A) was followed by 3∞- untranslated region containing a polyadenylation consensus. The open reading frame encodes a polypeptide of 187 amino acids. A detailed comparison of the shrimp Ras protein with all entries of the Swissprot databank revealed substantial similarities with mammalian members of the Ras-like superfamily (Fig. 1B). With H-Ras, N-Ras, K-Ras 4A and K-Ras 4B, the shrimp Ras shared 80–85% sequence similarity and 75–80% sequence identity, respectively. The similarity reaches 85% with K-Ras 4B of mammals.

Fig. 4. Fluorography of the shrimp ras-encoded p25 fusion protein after acylation by shrimp protein geranylgeranyltransferase I in the presence of [3H]-geranylgeranyl pyrophosphate. Purified shrimp rasencoded p25 fusion protein (2 mg) was prenylated after reaction with GTP (b), GDP (c) or without (a) by the shrimp protein geranylgeranyltransferase I in 4 mM [3H]-geranylgeranyl pyrophosphate in 50 mM citric acid, pH 6.0, and 1 mM DTT at 30°C for 60 min. The mixture was precipitated with trichloroacetic acid (10%) and treated with SDS, reduced, alkylated, and subjected to electrophoresis on a Tricine–SDS–PAGE gel (10%). The fluorogram of the processed gel is shown.

3.2. Characterization of the shrimp Ras protein Bacterial expression of the shrimp ras-encoding fusion protein yielded a band at 25 kDa with Tricine– SDS–PAGE ( Fig. 2A-R). After the removal of vector CBP-tag (41 amino acid residues), the shrimp rasencoding fusion protein was resolved into a corresponding band at 21 kDa (Fig. 2A-R∞). The protein was positively reacted with monoclonal antibodies against Ras of mammals ( Fig. 2B), in agreement with the previous amino acid alignment, with the highest levels of homology in the guanine nucleotide binding domains ( Fig. 1B). 3.3. Functional analysis of bacterially expressed Ras proteins

Fig. 3. Binding of shrimp ras-encoded p25 fusion protein with guanine nucleotides. [3H]GTP and [3H]GDP binding to shrimp ras-encoded p25 fusion protein was determined as a function of incubation time at 30°C. A reaction mixture (600 ml ) containing 20 mM Tris–HCl, pH 8.5, 2 mM dithiothreitol, 100 mM NaCl, 12 mg of ras-encoded p25 fusion protein, and 2 mM [3H]GTP or [3H]GDP was incubated at 30°C. All of the components were preincubated at the indicated temperature for 5 min prior to initiation of the reaction with [3H]GTP or [3H]GDP. At the indicated intervals, 50-ml aliquots of the reaction mixture were removed and immediately filtered through 0.45-mm nitrocellulose filters. Filters were washed at once and processed as described for the determination of the bound radioactivity.

By using the nitrocellulose filtration assay, the bacterially expressed shrimp ras-encoded p25 fusion protein was found to be functional to bind guanine nucleotides, agreeing with the previous report (Manne et al., 1984). [3H]GTP binding was a linear function of the amount of shrimp ras-encoded p25 fusion protein present in the assay. We have consistently observed that a given amount of shrimp ras-encoded p25 fusion protein bound sixfold fewer GDP than GTP at their respective saturating concentration ( Fig. 3). The binding of shrimp rasencoded p25 fusion protein for GTP was approximated to be 30 000 units per mg of protein, whereas the binding for GDP was 5000 units per mg of protein. That is, shrimp ras-encoded p25 fusion protein prevails in the GTP bound form (data not shown). In parallel studies with shrimp ras-encoded p25 fusion proteins by applying a nitrocellulose assay, [c-32P]labeled dATP or ATP were unable to compete the

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binding of [3H]GTP to shrimp ras-encoded p25 fusion protein (data not shown). 3.4. Geranylgeranylation of bacterially expressed Ras proteins The bacterially expressed shrimp ras-encoded p25 fusion protein was prenylated by the purified PGGT-I from the hepatopancreas of shrimp Penaeus japonicus, not by the protein farnesyltransferase, in agreement with the previous results using Drosophila ( Therrien et al., 1995). The purified shrimp PGGT-I effectively catalyzed the transfer of [3H] geranylgeranyl pyrophosphate to shrimp ras-encoded p25 fusion protein ( Fig. 4a). A fluorography analysis revealed that the prenylation of both Ras GDP and Ras GTP by PGGT-I exceeded that of the nucleotide-free form of Ras by 10-fold ( Fig. 4c), and fourfold (Fig. 4b), respectively. This experiment was repeated seven times by quantitating the flurogram with GEL-PDMS ANALYZERA (Silver Spring, MD); that is, there is a preference for the PGGT-I to react with ras-encoded p25 fusion protein in the GDPbound form. The data presented here provide evidence that Ras of shrimp is interesting in respect of its strong binding with GTP and its enhanced reaction with PGGT-I in the GDP-bound form. Further studies are directed towards the understanding the regulation mechanism of shrimp Ras during embryogenesis.

Acknowledgements We are grateful to the National Science Council and the Agriculture Council for financial support. C.-F.H. is a recipient of a National Science Council Graduate Fellowship.

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