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Modulatory effects of amino acids on neuromuscular transmission on the crayfish fast flexor muscle

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EXPERIMENTAL ZOOLOGY 283:510–521 (1999)
Facilitated Geranylgeranylation of Shrimp rasEncoded p25 Fusion Protein by the Binding With
Guanosine Diphosphate
Department of Zoology, National Taiwan University and Institute of Zoology,
Academia Sinica, Nankang 11529, Taipei, Taiwan
A cDNA was isolated from the shrimp Penaeus japonicus by homology cloning. Similar to the mammalian Ras proteins, this shrimp hepatopancreas cDNA encodes a 187-residue polypeptide whose predicted amino acid sequence shares 85% homology with mammalian KB-Ras proteins
and demonstrates identity in the guanine nucleotide binding domains. Expression of the cDNA of
shrimp in Escherichia coli yielded a 25-kDa polypeptide with positive reactivity toward the monoclonal antibodies against Ras of mammals. As judged by nitrocellulose filtration assay, the specific
GTP binding activity of ras-encoded p25 fusion protein was approximately 30,000 units/mg of protein, whereas that of GDP was 5,000 units/mg of protein. In other words, the GTP bound form of rasencoded p25 fusion protein prevails. Fluorography analysis demonstrated that the prenylation of
both shrimp Ras-GDP and shrimp Ras-GTP by protein geranylgeranyltransferase I of shrimp Penaeus
japonicus exceeded that of nucleotide-free form of Ras by 10-fold and four-fold, respectively. That is,
the protein geranylgeranyl transferase I prefers to react with ras-encoded p25 fusion protein in the
GDP bound form. J. Exp. Zool. 283:510–521, 1999. © 1999 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 signaling is activated when bound
to GTP and is inactivated when GDP is bound
(Mineo et al., ’96). At least two proteins appear to
regulate Ras activity. One, guanine-nucleotide-exchange factor (GEF), promotes GDP to GTP exchange (Wolfman and Macara, ’90; Ueda et al., ’91).
The other, GTPase-activating protein (GAP), stimulates the conversion of Ras GTP to Ras GDP by
increasing its intrinsic GTPase activity over 100fold (Trahey and McCormick, ’87). Therefore, the
ratio of GTP-bound to GDP-bound Ras is crucial
and this ratio has to be controlled precisely (Bourne
et al., ’90; Bourne et al., ’91). In many human tumors, Ras is GTP-locked (Boylan et al., ’90) and
not confined in caveolae (Song et al., ’96). In mammals, four isoforms of Ras exist: H-Ras, N-Ras, KARas, and KB-Ras (Lowy and Willumsen, ’93). These
are products of three genes. KA-Ras and KB-Ras
are 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 motifs of prenylation (Del Villar
et al., ’96).
Lipid modification of Ras results in an increase
in its intrinsic affinity for the plasma membranes
at which they can participate in signal transduction (Zhang et al., ’97). The addition of isoprenoid
groups, such as geranylgeranyl (20-carbon) and
farnesyl groups (15-carbon), is determined by the
X residue of the carboxyl terminal CAAX (C, cysteine; A, an aliphatic amino acid) of proteins. If X
is leucine or phenylalanine, the protein is geranylgeranylated (Casey et al., ’91; Moores et al., ’91;
Yokoyama et al., ’91); if X is methionine, serine, alanine, or glutamine, the protein is farnesylated
(Hancock et al., ’89; Schaber et al., ’90; Reiss et al.,
’91). Ras is preferentially farnesylated in mammals
(James et al., ’95). Abolishing prenylation disrupts
the association of 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).
The cultivation of penaeid shrimp is a world-
Grant sponsors: National Science Council and Agriculture Council,
*Correspondence to: Nin-Nin Chuang, Division of Biochemistry and
Molecular Science, Institute of Zoology, Academia Sinica, Nankang
11529, Taipei, Taiwan. E-mail:
Received 20 May 1998; Accepted 11 August 1998.
wide economically important industry. However,
diseases reduce the production of shrimp, which
results in vast economic losses. In this context,
the provision of a molecular index for growth has
become a priority to insure the long-term survival
of shrimp aquaculture. Therefore, we have undertaken the cloning and characterization of Ras in
the tropical shrimp Penaeus japonicus.
We report here, for the first time in shrimp, the
biochemical characterization and the cDNA cloning of genes specifying Ras protein. We intend to
experiment by using the shrimp cDNA probe of
ras as an in situ field monitor and indicator for
environmental pro-carcinogen hazards.
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., U.K.).
Mouse monoclonal antibody against Ras of mammals was from Santa Cruz Biotechnology, Inc.
(Santa Cruz, CA). The prepackaged SuperdexTM75,
SuperdexTM200 column, and HiTrap Phenyl Sepharose 6 Fast Flow (high substitution) were obtained from Pharmacia (Uppsala, Sweden), and
those for polyacrylamide gel electrophoresis were
obtained as described previously (Chuang and
Shih, ’90).
Experimental animals
Shrimps (Penaeus japonicus), collected off the
coast of Taiwan, were kept at 18°C for less than
3 days before experiments in a recirculating seawater system. Hepatopancreas were dissected out
immediately after shrimps had been killed, frozen in liquid nitrogen, and stored at –80°C.
Ras cDNA cloning
A lambda ZAP cDNA library for shrimp hepatopancreas was constructed 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 Inc., MA) and screened with
a 239-bp fragment cDNA as a probe covering nt
214 to nt 452. The duplicate membranes were prehybridized 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 ci-
trate (SSC), 2% (w/v) blocking reagent (Boehringer
Mannheim, Germany) for 2 hr at 42°C. For hybridization, the membranes were incubated in the
same solution containing the denatured Dig-11dUTP labeled ras cDNA probe for 17 hr at the
same temperature. The membranes were washed
twice in 2× SSC and 0.1% (w/v) SDS for 5 min at
room temperature, and then twice in 0.1× SSC
containing 0.1% (w/v) SDS at 55°C for 15 min.
The nylon membranes were then incubated in 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
CSPD® (Boehringer Mannheim) by exposure to
Kodak BioMax-MR film at room temperature for
16 hr. The pBluescript phagemids containing the
target inserts were in vivo excision from the UniZAP vector of positive plaques. PCR and sequencing were used to confirm and select the correct
colony. DNA was sequenced following the dideoxynucleotide method with modifications 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.
Production and characterization of shrimp
ras-encoded p25 fusion protein
The open reading frame of shrimp ras was amplified by PCR with two primers containing the
Ligation-Independent-Cloning (LIC) overhangs.
The ATG initiation codon was designed in front
of the exon I, and the stop codon was connected
after the final sequence of the exon IV of shrimp
ras. The polymerase chain reaction was performed
in 100 µl of 20 mM Tris-HCl, pH 8.4, 50 mM KCl,
and 1.5 mM MgCl2 using 0.5 µM of each primer,
200 µM of each deoxynucleotide triphosphate, 2.5
units of Taq DNA polymerase (Gibco-BRL, MD),
and 200 ng of shrimp ras cDNA as template. The
template DNA was amplified for 30 cycles consisting of 1 min denaturation at 94°C, 1 min renaturation at 55°C, and 1 min polymerization at
72°C. The PCR product was cloned into expression vector (pCAL-n-EK) from the CalmodulinBinding-Peptide (CBP)-tagged fusion system
(Stratagene). We chose the CBP-tagged fusion system (pCAL-n-EK) to express ras of shrimp as a
fusion protein because CBP-tagged fusion protein
would tend to be soluble so that it is not necessary to isolate them from inclusion bodies. Moreover, the Ras fusion protein was produced in
sufficient amounts, and could be rapidly purified
by calmodulin affinity resin chromatography
(Zheng et al., ’97). The expression vector was
transformed into BL21 (DE3) pLysS Escherichia
coli cells and selected by ampicillin.
A single colony of BL21(DE3) pLysS Escherichia
coli cells containing a recombinant pCAL-ras plasmid was inoculated in 5 ml of LB-A-C medium
(10 g/liter peptone; 5 g/liter yeast extract; 5 g/liter NaCl; 50 µg/ml ampicillin; 34 µg/ml chloramphenicol) and incubated with shaking at 37°C
until an OD600 of 0.6–1.0 was reached. The culture was then transferred into 500 ml of LB-A-C
medium and incubated at 37°C with shaking at
300 rpm until an OD600 of 0.6 was reached. For
induction of the recombinant protein expression,
Isopropyl-thio-D-galactoside (IPTG) was added to
the culture to reach a final concentration of 1 mM.
After further incubation with shaking at 37°C for
5 hr, the culture was harvested by centrifuging
at 7,000g for 15 min at 4°C. The resulting pellets
were resuspended in 30 ml of ice-cold buffer A (50
mM Tris-HCl, pH 8.0, 150 mM NaCl, 10 mM βmercaptoethanol, 1 mM magnesium acetate, 1 mM
imidazole, and 2 mM CaCl2). The cells were disrupted by sonication and 0.1% Triton X-100 (final concentration) was added. The sonicated cell
extract was centrifuged for 15 min at 20,000g to
remove unbroken cells and cell debris. The supernatant was mixed with 5 ml of calmodulin affinity resin and incubated at 4°C overnight. The
calmodulin affinity resin was washed with 10× resin
volume of buffer A and then eluted with 15× resin
volume elution buffer (50 mM Tris-HCl, pH 8.0, 2
mM EGTA, and 10 mM β-mercaptoethanol). The
ras-encoded p25 fusion protein was further purified by chromatography on a SuperdexTM75 column.
The pooled fractions containing ras-encoded p25 fusion protein was stored at –80°C in aliquots.
RNA extractions and northern blotting
Total RNA was isolated from 1 g fresh weight
individual hepatopancreas using the rapid RNA
extraction kit from TEL-TEST (Friendswood, TX)
following the guanidine thiocyanate method
(Chirgwin et al., ’79) after disruption of hepatopancreas in liquid nitrogen and quantified by spectrophotometry. Poly(A)+ RNAs were purified by
affinity chromatography on oligo dT-oligotex column (Qiagen; Chatsworth, CA). A 3′-end 223 bp
Dig-11-dUTP labeled fragment of the shrimp ras
cloned cDNA (nt 339-561) was used to probe RNA
which was fractionated by denaturing electrophoresis on 1.2% agarose-formaldehyde gel in 20
mM MOPS, pH 7.0, 8 mM sodium acetate, and 1
mM EDTA. Transfer to nylon membrane (Hybond
N+, Amersham) was performed as described by the
Purification of protein
geranylgeranyltransferase I
Protein geranylgeranyltransferase I was purified
by essentially the same procedures as those described by Lin and Chuang (’98) but with modifications. All manipulations were carried out at 4°C.
Hepatopancreas from the shrimp Penaeus japonicus (21 g) were homogenized with a Polytron
tissue grinder (three 10-sec bursts; Kinematica
GmbH, Brinkmann Instruments, Westbury, NY)
in 20 ml of ice-cold buffer that contained 50 mM
citric acid, pH 6.0, 1 mM EDTA, 1 mM EGTA, 1
mM DTT, 0.2 mM PMSF, 1 µM pepstatin, 5 µM
E64, and 0.02 mM leupeptin, and the extract was
centrifuged at 6,000g (Beckman, JA 17) for 20 min.
The supernatant was brought to 20% saturation
with ammonium sulfate, stirred for 30 min on ice,
and centrifuged at 6,000g (Beckman, JA 17) for
20 min to remove precipitated proteins. The resulting supernatant was adjusted to 40% saturation with ammonium sulfate and centrifuged at
6,000g (Beckman, JA 17) for 20 min. The pellets
were then dissolved in 5 ml of buffer A (20% ammonium sulfate in 50 mM citric acid, pH 6.0) and
was loaded on a HiTrap Phenyl Sepharose 6 Fast
Flow (high substitution) column that had been
equilibrated with buffer A. The unbound protein
was washed out with 10 volumes of the equilibration buffer, and the column was eluted in gradient with decreasing concentrations of ammonium
sulfate. The fraction eluted in 3.5% ammonium
sulfate from the Sepharose 6 Fast Flow (high substitution) column, which contained protein geranylgeranyltransferase I, was dialyzed for 12–16
hr at 4°C against 50 mM citric acid, pH 6.0, and
1 mM DTT. The dialyzed solution was concentrated on a YM 10 membrane (Amicon, Danvers,
MA) then subjected to affinity chromatography on
a 0.5 ml affinity column prepared with ras-encoded p25 fusion protein bound to agarose that
had been equilibrated in 50 mM citric acid, pH
6.0, 50 mM NaCl, and 1 mM DTT (buffer B). The
column was washed with 20 ml of buffer B that
contained 0.01% (w/v) Triton X-100 (buffer C). The
enzyme was then eluted with 5 ml of 50 mM citric acid, pH 6.0, that contained 1 mM DTT, 0.2%
(w/v) Triton X-100. The eluent was concentrated
and chromatographed on a SuperdexTM200 FPLC
column that had been equilibrated with 50 mM
citric acid, pH 6.0, 150 mM NaCl and 1 mM DTT.
The pooled fractions with highest specific activity
were dialyzed for 12–16 hr at 4°C against 50 mM
citric acid, pH 6.0, and concentrated in an ultrafiltration cone (CF25 Centriflo; Amicon, MA). Enzyme purification results in a yield relative to
ammonium sulfate precipitate of 20% and a specific activity of 376 units/mg of protein (2,506-fold
GNP binding assay
Reaction mixtures (50 µl) contained 20 mM TrisHCl, pH 8.5, 2 mM DTT, 100 mM NaCl, 2 µg of
bovine serum albumin (Bethesda Research Laboratories, Bethesda, MD), and 2 µM [3H]GNP (10.8
Ci/m mol, Amersham; 1 Ci = 37 GBq) and were
incubated at 30°C for 30 min. Aliquots (40 µl) were
filtered on 0.45 µm nitrocellulose filters (MultiScreen-HA, Millipore, France) and washed at once
with 3 ml of ice-cold buffer containing 20 mM TrisHCl, 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
Polyacrylamide gel electrophoresis
Tricine-sodium dodecyl sulfate-polyacrylamide
gel electrophoresis (Tricine-SDS-PAGE) was conducted on slab gels that contained 10 % (w/v)
acrylamide and 0.61% (w/v) N, N′-methylenebisacrylamide (Laemmli, ’70; Schagger and von
Jagow, ’87). Samples were reduced and alkylated
(Lane, ’78) before application to the gels. Gels were
Coomassie brilliant Blue R250-stained or silverstained by the method of Merril et al. (’81). 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 fluorography by use of Amplify (Amersham) as
specified before (Tseng and Chuang, ’94).
Western immunoblotting
Proteins that had been separated by TricineSDS-PAGE were transferred electrophoretically to
nitrocellulose membranes (Bio-Rad, Richmond,
CA) and probed by Western immunoblotting with
antibody. In brief, the nitrocellulose membranes
were incubated in a quenching solution (3% BSA
in PBS buffer), and then they were incubated overnight at room temperature in a solution of antibodies (1 µg/ml) in quenching solution. The
nitrocellulose membrane was washed and incu-
bated with second antibodies, namely goat antibodies against mouse IgG, conjugated with biotin
that had been diluted 1:1,000 in the same quenching solution. Immunoreactive proteins were detected by exposure of the nitrocellulose membrane
to avidin-conjugated alkaline phosphatase, and
visualization of bands was achieved at room temperature in the presence of 5-bromo-4-chloro-3indolyl phosphate in nitroblue tetrazolium salt
(Zymed, San Francisco, CA).
Quantitation of protein
Bovine serum albumin served as the standard
in the measurement of levels of protein. Amounts
of protein were determined by Lowry’s method
(Lowry et al., ’51) or by the Micro BCA* Protein
Assay (Pierce, Rockford, IL).
Isolation of a Ras cDNA from
hepatopancreas of shrimp
Penaeus japonicus
The most conserved sequences from the Ras
were used to design degenerate oligonucleotide
primers, and a first strand cDNA pool from shrimp
hepatopancreas was used as the template for polymerase chain amplification. The 239-bp probe (nt
214-452) was used to isolate five positive plaques
from shrimp hepatopancreas cDNA library. The
1,294-bp insert contained a 128-bp 5´-untranslated
sequence preceding an initiation site consensus. A
561-bp open reading frame was followed by 3′-untranslated sequence containing a polyadenylation
consensus (Fig. 1A). 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,
KA-Ras and KB-Ras, the shrimp Ras shared 80–
85% sequence similarity and 75–80% sequence
identity respectively. The shrimp Ras shared the
highest similarity with mammalian KB-Ras that
is 85% similar in sequence.
Tissue distribution of Ras mRNA in the
shrimp Penaeus japonicus
Total RNA or poly(A)+RNA from shrimp hepatopancreas showed a prominent band of approximately 3.5 kb, and analyses of RNA from heart,
and eye were similar. In contrast, when comparable amounts of RNA from muscle were probed,
only very faint signals were detected and analyzed
(Fig. 2).
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. (B) Alignment of the amino acid sequence
of the shrimp Ras protein (S-Ras), H-Ras, N-Ras, KARas and KB-Ras. The five conserved regions involved
in GTP metabolism are boxed. Identical positions are
marked with dots.
Fig. 2. Northern analysis of Ras
mRNA in shrimp tissues. Total RNA (20
µg) from heart, muscle, hepatopancreas,
and eye, and poly(A)+ RNA (10 µg) from
shrimp hepatopancreas (*) were hybridized with Dig-11-dUTP labeled probe (nt
339-561) corresponding to carboxyl terminal of Ras cDNA (A). Equal amounts
of RNA were loaded as verified by the
abundance of 18 S RNA (B).
Fig. 3. Tricine-SDS-polyacrylamide electrophoresis of recombinant ras-encoded fusion protein. Ras-encoded p25 fusion protein (R, 0.5 µg) and CBP-tag removed fusion protein
(R′, 1.5 µg) were 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: 1,000 dilution (B). For comparison, E. coli lysates
Characterization of the shrimp ras protein
Bacterial expression of the cDNA encoding Ras
yielded a protein that migrated at 25 kDa in
Tricine-SDS-polyacrylamide gel electrophoresis
(Fig. 3A, lane R). After the removal of 41 amino
acid residues of the calmodulin binding peptide
from the ras-encoded fusion protein, a corresponding protein that migrates at 21 kDa (Fig. 3A, lane
R′) was recovered. The protein could be detected
with the monoclonal antibodies against mammalian Ras (Fig. 3B), in agreement with the previous amino acid alignment results, indicating that
the highest levels of homology lie in the guanine
nucleotide binding domains (Fig. 1B).
Functional analysis of recombinant ras
proteins: guanine nucleotide binding
[3H]GTP binding to ras-encoded p25 fusion protein was optimal at pH values around 8.5 (Fig. 4A)
(OD600 = 1) before (O) and after (I) induction with IPTG (1
mM) were included. Biotinylated phosphorylase b (Mr 97,400),
biotinylated bovine serum albumin (Mr 68,000), biotinylated
ovalbumin (Mr 46,000), biotinylated carbonic anhydrase (Mr
31,000), biotinylated soybean trypsin inhibitor (Mr 20,100)
and biotinylated lysozyme (Mr 14,400) were applied as molecular weight markers.
Fig. 4. Components required for stable and maximal
binding of [3H]GTP to ras-encoded p25 fusion protein. (A)
[ 3H]GTP binding to 1 µg of ras-encoded p25 fusion protein was determined in the presence of 20 mM Tris-HCl
adjusted to the indicated pH. (B) [3H]GTP binding to 1
µg of ras-encoded p25 fusion protein was determined under standard assay conditions except that the concentrations of dithiothreitol were varied as indicated. (C)
[ 3H]GTP binding to 1 µg of ras-encoded p25 fusion protein was determined as a function of MgCl 2 concentration. (D) [3H]GTP binding to 1 µg of ras-encoded p25 fusion
protein was determined as a function of incubation time
at 30°C. A reaction mixture (600 µl) containing 20 mM
Tris-HCl, pH 8.5, 2 mM dithiothreitol, 100 mM NaCl, 12
µg of ras-encoded p25 fusion protein, and 2 µM [3H]GTP
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. At the indicated intervals, 50 µl aliquots of the reaction mixture were removed
and immediately filtered through 0.45 µm nitrocellulose filters. Filters were washed at once and processed as described
for the determination of the bound radioactivity.
Figure 4.
and was markedly increased by dithiothreitol (Fig.
4B) but inhibited by MgCl2 (Fig. 4C). The [3H]GTP
binding activity was a linear function of the amount
of ras-encoded p25 fusion protein present in the assay. The specific GTP binding activity of ras encoded
p25 fusion protein was approximately 30,000 units/
mg of protein, whereas that of GDP was 5,000 units/
mg of protein. That is, the nucleotide binding site
on ras-encoded p25 fusion protein shows preference
for GTP, with an affinity approximately 6-fold
greater than that for GDP (Fig. 4D). In parallel
studies applying the nitrocellulose assay, [γ-32P]-labeled dATP, dCTP and ATP were discovered to be
unable to compete with binding of [3H]GTP to rasencoded p25 fusion protein.
Prominent geranylgeranylation of GDP
bound recombinant ras proteins
The purified protein geranylgeranyltransferase
I from the hepatopancreas of the shrimp Penaeus
japonicus effectively catalyzed the transfer of
[3H]geranylgeranyl pyrophosphate to ras-encoded
p25 fusion protein (Fig. 5a). Fluorography analysis demonstrated that the prenylation of both GDP
and GTP bound forms of Ras by protein geranylgeranyltransferase I exceeded that of nucleotidefree form of Ras by 10-fold (Fig. 5c) and four-fold
(Fig. 5b) respectively. In other words, the protein
Fig. 5. Fluorography of the ras-encoded p25 fusion protein
after acylation by protein geranylgeranyltransferase I in the
presence of [3H]geranylgeranyl pyrophosphate. After incubation with GTP (b), GDP (c) or without (a), purified ras-encoded
p25 fusion protein (2 µg) was prenylated by the protein
geranylgeranyltransferase I in 4 µM [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-polyacrylamide gel (10%). The fluorogram of the processed gel is shown.
geranylgeranyltransferase I selectively reacted
with ras-encoded p25 fusion protein in the GDP
bound form.
Using the nitrocellulose filtration assay, the recombinant ras-encoded p25 fusion protein of shrimp
was found to be functional in binding guanine nucleotides, in agreement with the previous report
(Manne et al., ’84). We consistently observed that a
given amount of ras-encoded p25 fusion protein
bound six-fold fewer GDP than GTP at their respective saturating concentration. That is, ras-encoded p25 fusion protein prevails in its GTP bound
form, suggesting that large amounts of GTPase-activating proteins (GAPs) may be required to release
shrimp cells from oncogenicity, since GAPs promote
GTP hydrolysis and thereby favor the GDP bound
inactive form of Ras. Evidence for the excessive expression of GAP-related genes in cytosol (Halenbeck
et al., ’90; LeebLaudberg and Song, ’93; Zhang et
al., ’93; Baba et al., ’95) were previously evidenced
in Drosophila melanogaster (Gaul et al., ’92) and
mammals (Maekawa et al., ’94). However, the possibility of other mechanisms to decrease the availability of Ras to bind with GTP, such as binding to
GEFs in the nucleotide-free form of Ras (Fam et
al., ’97) or binding to caveolin (Song et al., ’96), cannot be ignored.
The recombinant ras-encoded p25 fusion protein
of shrimp was prenylated by the purified protein
geranylgeranyltransferase I from the hepatopancreas of shrimp, but not by the protein farnesyltransferase, in agreement with previous results
from studies in Drosophila (Therrien et al., ’95).
The ras encoded p25 fusion protein favorably reacted with the protein geranylgeranyltransferase
I of shrimp Penaeus japonicus in its GDP bound
form. We cannot rule out the possibility that carboxyl terminal geranylgeranylcysteine residue
bearing this modification cannot by itself confer
efficient membrane binding to proteins, as advised
by Silvius and L’Heureux (’94). However, the observation that microsomal membranes contain a
high-affinity binding site for prenylated peptides
(Thissen and Casey, ’93), plus the present data of
prevalent prenylation of Ras in the GDP bound
form with protein geranylgeranyltransferase I,
suggest that prenylation might be a crucial step
for GDP bound Ras to become segregated from
bulky cytosolic GTP bound Ras to receive the message at the membrane. In other words, protein
geranylgeranyl-transferase I might play a prelude
here for Ras signaling.
Ras produces more than one species of transcript
in shrimp tissues and is ubiquitously expressed.
Based on an analysis of the 5′ and 3′ ends of ras
transcripts, we believe that the cDNA of 1,294 bp
that we have isolated is a full length representative of the largest ras transcript. The finding that
our cDNA of ras seems to be shorter than the prominent ras transcript (3.5 kb) on the northern blot is
likely due to differences in the length of the poly(A)+
tail that is present on the ras transcript (De Leon
et al., ’83; Jeffers et al., ’90; Proudfoot, ’94; Wahle
and Keller, ’96; Staton and Leedman, ’98) and/or
differences in the 3′ UTR sequences that contain
the information required for the correct spatial and/
or temporal interactions of translational factors
(Bonifer et al., ’97).
The report (Schweins et al., ’97) that the GTPhydrolysis rate of Ras GTP is significantly faster
when Mg++ is present cannot be confirmed in our
experiments. However, it is not inconceivable that
Mg++, instead of Ca++, which transmits fertilization signal in oocytes of shrimp (Goudeau and
Goudeau, ’96) cannot facilitate the GTP-hydrolysis rate of Ras in due course. 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). Thus, if to
increase the rate of GTP-hydrolysis is necessary
afterward for feedback regulation, an additional
factor, such as Mn++ (Schweins et al., ’97), might
be required to replace Mg++ for functions.
Data presented here provide evidence that Ras
of shrimp will prove as 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).
C.-F.H. is a recipient of a National Science Council Graduate Fellowship, Taiwan.
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