JOURNAL OF EXPERIMENTAL ZOOLOGY 286:441–449 (2000) Disrupting the Geranylgeranylation at the C-Termini of the Shrimp Ras by Depriving Guanine Nucleotide Binding at the N-Terminal CHEIN-FUANG HUANG AND NIN-NIN CHUANG* Department of Zoology, National Taiwan University and Institute of Zoology, Academia Sinica, Nankang, Taipei, Taiwan 11529 ABSTRACT In order to assess the effects of guanine nucleotide binding on the geranylgeranylation at the CAAX box of the shrimp Ras, we experimented with the shrimp Penaeus japonicus Ras (S-Ras) which is geranylgeranylated at the C-termini, shares 85% homology with mammalian KB-Ras protein and demonstrates identity in the guanine nucleotide binding domains (Huang C-F, Chuang N-N. 1999. J Exp Zool 283:510–521). Several point mutations in the S-ras gene were generated at codons 12 (G12V), 61 (Q61K), and 116 (N116I). The bacterially expressed mutant SRas proteins, G12V and Q61K, were bound with GTP without hydrolysis. In contrast, the mutant S-Ras N116I was defective in its ability to bind any guanine nucleotides. Autoradiography studies showed that the purified shrimp protein geranylgeranyltransferase I (Lin R-S, Chuang N-N. 1998. J Exp Zool 281:565–573) was unable to catalyze the transfer of [3H]-geranylgeranylpyrophosphate to this mutant N116I but very competently caused the geranylgeranylation of GTP-locked mutants, G12V and Q61K. These results demonstrate that the geranylgeranylation at the CAAX box of the shrimp Ras protein requires the proper binding of guanine nucleotide at its N-terminal region. J. Exp. Zool. 286:441–449, 2000. © 2000 Wiley-Liss, Inc. Ras proteins are membrane-associated small guanine nucleotide binding proteins that play critical roles in cellular differentiation (Bar-Sagi and Feramisco, ’85; Swanson et al., ’86; Ngsee et al., ’91), proliferation (Mulcahy et al., ’85; Barbacid, ’87; Daar et al., ’91) and apoptosis (Downward, ’98; Lloyd, ’98). Ras cycles between the active, GTPbound and the inactive, GDP-bound state (Mineo et al., ’96). In many human tumors, Ras is GTPlocked (Boylan et al., ’91). Three most prominent conserved motifs, GXXXXGKS/T, DXXG, and NKXD, localized to the N-terminal domain, form the GDP/GTP binding pocket of Ras (Jurnak, ’85; La Cour et al., ’85; Pai et al., ’89; Tong et al., ’91). The extreme C terminus of Ras is required for lipid binding and membrane localization of the protein (Zhang et al., ’97). The addition of isoprenoid groups, such as geranylgeranylpyrophosphates (GGPP) and farnesylpyrophosphates (FPP), is determined by the X residue of the carboxyl terminal CAAX (C, cysteine; A, an aliphatic amino acid) of proteins. If X is leucine, isoleucine or phenylalanine, the protein is geranylgeranylated (Yokoyama et al., ’91); if X is methionine, serine, alanine, or glutamine, the protein is farnesylated (Hancock et al., ’89; Reiss et al., ’91). Abolishing prenylation disrupts the association of © 2000 WILEY-LISS, INC. Ras with membranes, and thereby disrupts its function (Der and Cox, ’91; Kato et al., ’92). Therefore, inhibitors of prenylation are effective at suppressing the growth of tumor cells possessing oncogenic Ras (Hancock et al., ’89; Seabra et al., ’91; Sun et al., ’95; Lerner et al., ’97). In mammals, four isoforms of Ras exist: H-Ras, KA-Ras, KB-Ras, and N-Ras (Lowy and Willumsen, ’93). All of them are farnesylated (James et al., ’95), except KB-Ras being geranylgeranylated additionally (Armstrong et al., ’95; Lerner et al., ’95). Geranylgeranylation of KB-Ras becomes a potential mechanism of resistance to protein farnesyltransferase (PFTase) inhibitors. Potent and selective inhibitors of protein geranylgeranyltransferase I (PGGTase I) were developed in due course (Lerner et al., ’95; Miquel et al., ’97; Vogt et al., ’97). However, the observation that the number of geranylgeranylated proteins in the cell exceeds overwhelmingly that of farnesylated pro- Grant sponsor: National Science Council, Taiwan. *Correspondence to: Nin-Nin Chuang, Division of Biochemistry and Molecular Science, Institute of Zoology, Academia Sinica, Nankang, Taipei, Taiwan 11529. E-mail: firstname.lastname@example.org Received 26 April 1999; Accepted 3 August 1999 442 C.-F. HUANG AND N.-N. CHUANG teins (Epstein et al., ’90; Farnsworth et al., ’90), plus the critical functional role of geranylgeranylation to process cells from G1 to S phase (Olson et al., ’95; Vogt et al., ’97), indicate that the inhibition of geranylgeranylation stresses cellular vitality. On the other hand, the high incidence of mutated K-Ras in human colon carcinoma (50%) and pancreatic carcinoma (90%) (Bos, ’89) prompts us to examine whether an alternative mechanism exists to regulate geranylgeranylation at the C-termini of Ras, such as evidenced in Rap (Shirataki et al., ’91) and ERM (Ezrin-RadixinMoesin; Gary and Bretscher, ’95), by intramolecular interference of the N-terminal. In this context, we experimented with the Ras of shrimp Penaeus japonicus (S-Ras) which is geranylgeranylated at the C-termini and shares 85% homology with mammalian KB-Ras protein with identity in the guanine nucleotide binding domains (Huang and Chuang, ’99). Several point mutations in the Shrimp Ras gene were generated at the GDP/GTP binding pocket region and then these mutant proteins were bacterially expressed. Our results showed that PGGTase I was unable to catalyze the prenylation at the mutant Ras proteins with defects in binding any guanine nucleotides but very competently caused the geranylgeranylation of GTP-locked mutants. These findings indicate that to block the growth of ras-dependent tumorigenic cells by inhibitors of geranylgeranylation, such as CAAX peptidomimetics, is necessary to have a combined and cooperative inhibition in binding with guanine nucleotide. MATERIALS AND METHODS Materials All reagents used were of the highest grade available commercially. [1(n)-3H]-geranylgeranylpyrophosphate and [α-32P]-guanosine 5′-triphosphate were from New England Nuclear (Boston, MA). Experimental animals Shrimps (Penaeus japonicus), collected off the coast of Taiwan, were kept at 18°C for less than 3 days in a recirculating seawater system. Hepatopancreases were dissected out immediately after shrimps had been killed, frozen in liquid nitrogen, and stored at –80°C. Production and characterization of rasencoded fusion protein in bacteria The open reading frame of shrimp ras cDNA was amplified by PCR and two primers (5′GACGACGACAAGATGACGGAATACAAGCTC- GT-3′, 5′-GGAACAAGACCCGTCTAGAACACAATACACTTCC-3′) with the Ligation-IndependentCloning (LIC) overhang were applied as specified previously (Huang and Chuang, ’99). The PCR was performed in 100 µl of 20 mM Tris-HCl, pH 8.0, 10 mM KCl, 2.0 mM Mg2SO4, 10 mM (NH4)2SO4, 0.1% Triton X-100, and 0.1 mg/ml BSA using 0.5 µM of each primer, 200 µM of each deoxynucleotide triphosphate, 2.5 units of pfu DNA polymerase (Stratagene, La Jolla, CA), and 200 ng of shrimp ras cDNA as template. The template DNA was amplified for 30 cycles consisting of 1 min of template denaturation at 94°C, 1 min of primers annealing at 55°C, and 1 min of primer extension 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 as specified previously (Huang and Chuang, ’99). The enterokinase (EK) site-specific cleavage of CBP-tagged fusion protein could then be rapidly purified by calmodulin affinity resin chromatography (Zheng et al., ’97). Mutation of S-ras expressing plasmids Site-directed mutagenesis by the overlap extension PCR method was used (Ho et al., ’89; Horton et al., ’89) to change codon Gly12 of S-ras to Val (G12V), codon Gln61 to Lys (Q61K) and codon Asn116 to Ile (N116I). Both 5′-3′ and 3′-5′ direction primers used were as follows: 5′-GTCGGAGCTGTAGGCGTTGG-3′ and 5′-CCAACGCCTACAGCTCCGAC-3′ for the mutant G12V; 5′-CAGCCGGGAAAGAAGAATAC-3′ and 5′-GTATTCTTCTTTCCCGGCTG-3′ for the mutant Q61K; 5′GGTGGGCATCAAATGCGAC-3′ and 5′-GTCGCATTTGATGCCCACC-3′ for the mutant N116I. The PCR products were gel-purified by QIAEX II Gel Extraction Kit (QIAGEN, Duesseldorf) from a 1% (w/v) agarose gel and constructed into the pCAL-n-EK expression vector. Both strands of the insert sequences were confirmed by the dideoxynucleotide chain terminator method by applying an ABI PRISM Dye Terminator Cycle Sequencing Kit and ABI Autosequencer377 (Perkin Elmer; Branchburg, New Jersey). The data was reconfirmed with Sequenase Kit (U.S. Biochemical Co., Cleveland, OH). The samples were analyzed by 5% polyacrylamide gel electrophoresis and the gels were exposed to Kodak BioMax-MR film for 16 hr at room temperature. GERANYLGERANYLATION AT C-TERMINI OF SHRIMP RAS Restriction enzyme analysis of S-ras mutants 443 32 GTPase assay Bacterial expression of the shrimp ras-encoding fusion protein was pre-incubated with a buffer containing 50 mM Tris-HCl, pH 8.5, 100 mM NaCl, 2 mM DTT, 80 µg/ml BSA and 50 nM [α- P]-GTP (3,000 Ci/mmol, New England Nuclear) for 30 min at 30°C. At the indicated times, aliquots were filtered on 0.45 µM nitrocellulose filters (MultiScreen-HA; Millipore, France) and washed with 2.5 ml of ice-cold buffer containing 50 mM Tris-HCl, pH 8.5, 100 mM NaCl, and 2 mM DTT. The [α-32P]-GTP bound ras-encoded fusion protein recovered in the lysis buffer containing 10 mM EDTA, 10 mM EGTA, and 0.5% (w/v) SDS was spotted onto thin-layer polyethyleneimine (PEI)cellulose F plates (Merck; Darmstadt, Germany) and chromatographed in 1.6 M LiCl. Radiolabelled spots on autoradiogram were quantified with phosphor-Imager analyzer (Molecular Dynamics; Sunnyvale, CA). Fig. 1. DNA sequences of the shrimp ras mutants. Point mutations in the shrimp ras gene were individually generated at codons G12V (A), Q61K (B), and N116I (C). The primer used for sequencing was an antisense primer, so that the sequence shown is the complementary strand. N denotes normal Ras; M denotes mutant Ras (D). The bacterially expressed Ras mutant proteins (2 µg), G12V, Q61K, and N116I were denatured, analyzed by Tricine-SDS-PAGE on a 10% gel and stained with Coomassie Blue R250. For comparison, E. coli expressing mammalian K-Ras (2 µg) was included. Restriction enzymes were obtained from New England BioLab (MA). The normal S-ras and mutants PCR products (0.25 µg) were treated with 2 units restriction enzyme (Sfc-I specific for G12V mutant, SfaN-I specific for N116I mutant) at 37°C for 3 hr and analyzed by 3.5% agarose gel in 0.5× TBE buffer. 444 C.-F. HUANG AND N.-N. CHUANG Purification of protein GGTase-I Protein GGTase-I was purified from the hepatopancreas of shrimp Penaeus japonicus by essentially the same procedures as those described by Lin and Chuang (’98). All manipulations were carried out at 4°C. Enzyme purification results in a yield relative to ammonium sulfate precipitate of 20% and a specific activity of 376 units per mg of protein (2,506-fold purification). Polyacrylamide gel electrophoresis Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis (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, ’87). Samples were reduced and alkylated (Lane, ’78) before application to the gels. Gels were stained with Coomassie Brilliant Blue R250. Radiolabelled proteins were detected by exposure of the dried gel to BioMaxMS film (Kodak) at –70°C under an intensifying screen (BioMax TranScreen LE, Kodak). Radiolabelled bands were quantified by fluorogram with GEL-PDMS ANALYZER (Silver Spring, MD) using Amplify (Amersham) as specified previously (Tseng and Chuang, ’94). 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., ’51) or by the Micro BCA* Protein Assay (Pierce; Rockford, IL). RESULTS AND DISCUSSIONS Characterization of the shrimp Ras mutants Artificially created substitutions as restriction sites after polymerase chain reaction (PCR) are produced in shrimp ras genes localized to codon G12V (Fig. 1A), Q61K (Fig. 1B), and N116I (Fig. 1C). The results were confirmed by direct sequencing of the expression plasmids. The bacterially expressed SRas and mutant proteins yielded a band at 25 kDa with Tricine-SDS-PAGE, being smaller than 27 kDa of E. coli expressed rat K-Ras (Fig. 1D). By using the nitrocellulose filtration assay, the bacterially expressed S-Ras and mutant proteins, G12V and Q61K, were functional to bind guanine nucleotides (Fig. 2A). On the contrary, some mutation confers the dominant negative activity. We have observed that one S-Ras mutant, N116I, is incapable of binding any guanine nucleotides. Asparagine 116 of Ras is part of the conserved motif NKXD, the domain interacting with guanine base and acting as the foundation for cellular mechanisms of GTP-induced conformational changes, GTP hydrolysis, and guanine nucleotide exchange, as evidenced in mammals (Walter et al., ’86). To measure hydrolysis of the bound [α-32P]GTP, samples of the reactions were analyzed by TLC on PEI cellulose plates (Fig. 2A). The rat K-Ras protein did hydrolyze GTP to GDP with an estimated t1/2 of 2 hr (Fig. 2B), agreeing with the data of Sweet et al. (’84). In contrast, a much slower rate of hydrolysis (t1/2 of 7 hr) was seen with the S-Ras (Fig. 2B). The intrinsic GTPase activity of S-Ras is decreased by a valine at residue 12 (G12V) in the conserved GXXXXGKS/T domain or a lysine at residue 61 (Q61K) next to the conserved DXXG domain (Fig. 2B). In other words, both mutants, G12V and Q61K, are GTP-locked. GTP locking at the N-terminal potentiates the ability of PGGTase-I to prenylate at the C-termini of Ras The purified shrimp PGGTase I effectively catalyzed the transfer [3H]-GGPP to S-Ras, being optimal at pH values around 8.0 (Fig. 3) which is best for the binding of S-Ras with guanine nucleotide (Huang and Chuang, ’99). We have consistently observed that a given amount of S-Ras bound twofold and threefold fewer [3H]-GGPP than the GTP-locked mutants G12V and Q61K (Fig. 4A). That is, the purified shrimp PGGTase I efficiently catalyzed the geranylgeranylation of GTP-locked mutants. Deprived binding of guanine nucleotide at the N-terminal lessens the prenylation by PGGTase I at the C-termini of Ras The purified shrimp PGGTase I poorly carry out the transfer of [3H]-GGPP to the mutant N116I (Fig. 5). We have observed that a given amount of S-Ras bound twofold more [3H]-GGPP than the mutant N116I. The defects of the mutant N116I in binding [3H]-GGPP and any guanine nucleotide in vitro (Fig. 2A) suggest that the overall conformation of this mutant protein had been drastically altered by the single substitution that was introduced into the effector region. We have tried to correct the structure abnormality by conducting refolding. Unfortunately, the purified shrimp PGGTase I was still incompetent to cause the geranylgeranylation to this refolded mutant N116I (Fig. 5). GERANYLGERANYLATION AT C-TERMINI OF SHRIMP RAS Fig. 2. Binding and hydrolysis of [α-32P]-GTP in E. coli expressing shrimp ras-encoding fusion protein. Bacterially expressed shrimp ras-encoding fusion protein (S-Ras, G12V, Q61K, and N116I; 2 µg each) was pre-incubated with a buffer (200 µl) containing 50 mM Tris-HCl, pH 8.5, 100 mM NaCl, 2 mM DTT, 80 µg/ml BSA and 50 nM [α-32P]-GTP (3,000 Ci/ m mol, New England Nuclear; 1 Ci = 37 GBq) for 30 min at 30°C. At the indicated times, aliquots (30 µl) were filtered on 0.45 µM nitrocellulose filters (MultiScreen-HA, Millipore, France) and washed at once with 2.5 ml of ice-cold buffer 445 containing 50 mM Tris-HCl, pH 8.5, 100 mM NaCl, and 2 mM DTT. The ras-encoded fusion protein recovered in the lysis buffer containing 10 mM EDTA, 10 mM EGTA, and 0.5% (w/v) SDS was spotted onto thin-layer polyethyleneimine (PEI)-cellulose F plates (Merck; Darmstadt, Germany) and chromatographed in 1.6 M LiCl (A). (B) Radiolabelled spots on autoradiogram were quantified and analyzed with phosphor-imager analyzer (Molecular Dynamics; Sunnyvale, CA). For comparison, K-Ras of rat was included. 446 C.-F. HUANG AND N.-N. CHUANG Fig. 3. Effect of pH on the prenylation by shrimp PGGTase-I. Purified shrimp ras-encoded fusion protein (5 µg) was assayed in a reaction mixture that consisted of the shrimp PGGTase-I (1 unit), 4.4 µM [3H]-GGPP and 2 mM DTT in 100 mM buffer [citric acid (pH 6.0), Tris-HCl (pH 7.1, 7.5, 8.0, 8.5, and 8.9)], 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. In the present study, we utilized mutants of the shrimp Ras protein to demonstrate that the guanine nucleotide binding at the N-terminal region regulates the geranylgeranylation at the C-terminal. Ras of shrimp is a specific and interesting regulation target for the applications in aquaculture, as suggested by the fact that microinjection of oncogenic mammalian Ras proteins into Xenopus laevis oocytes would induce cellular divisions (Sagata et al., ’88, ’89; Pomerance et al., ’92). Ras kept in the GTP bound form is required to function as a mitogen to induce maturation and release the M-phase arrest in oocytes, similar to the role of Mos in vertebrates (Rhodes et al., ’94, ’97). We intend to use the simplest GTP-locked mutant S-Ras for further investigations of the transformation of shrimp cells to set up cell lines and the precise mechanism by which the geranylgeranylation coordinates ras oncogenes to alter the regulation of signal transduction events in the G0/ G1 phase of the cell cycle (Vogt et al., ’96) for the control of programmed cell death and cell growth (Miquel et al., ’97). Studies would otherwise be rather difficult with Ras available from mammals in more complicated isoforms and alternative choices between farnesylation and geranylgeranylation (Zhang et al., ’97). In addition, we attempt to apply a potent geranylgeranyltransferase I inhibitor to stop the processing of S-Ras in meiotic Fig. 4. GTP-locked Ras mutant proteins are prenylated prominently by PGGTase-I in the presence of [3H]-GGPP. Purified shrimp mutant ras-encoded fusion protein (5 µg; G12V or Q61K) was incubated with a buffer (200 µl) containing 50 mM Tris-HCl, pH 8.5, 100 mM NaCl, 2 mM DTT, 80 µg/ml BSA and 2 µM GTP for 30 min at 30°C before the reaction with the shrimp PGGTase-I (1 unit) in 4 µM [3H]-GGPP, 100 mM Tris-HCl, pH 8.0, and 2 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 (A). For comparison, the Tricine-SDS-PAGE gel (10%) stained with Coomassie Blue R250 was included (B). maturation and probe the parallel pathways in shrimp, which does not possess Mos to coordinate maturation-promoting factor and cytostatic factor. 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