close

Вход

Забыли?

вход по аккаунту

?

72

код для вставкиСкачать
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: zonnc@sinica.edu.tw
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.
In the meantime, it is anticipated that a retrospective reaction from guanine nucleotide bind-
GERANYLGERANYLATION AT C-TERMINI OF SHRIMP RAS
447
ACKNOWLEDGMENT
C.-F. Huang is a recipient of a National Science
Council Graduate Fellowship, Taiwan.
LITERATURE CITED
Fig. 5. Guanine nucleotide deprived mutant of S-Ras attenuates the geranylgeranylation at the C-termini. Purified
E. coli expressing Ras proteins of mutant N116I-1 (5 µg) 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 reversibly denatured and refolded mutant Ras was included (N116I-2) (B).
ing protein, such as tubulin (Hertzler and Clark,
’92), will be shown to coordinate S-Ras to process
cell rearrangements during gastrulation (Ettensohn, ’84; Hardin and Cheng, ’86; Stephens et al.,
’86; Burker et al., ’91), as indicated by the findings in mammals (Daar et al., ’91).
Armstrong SA, Hannah VC, Goldstein JL, Brown MS. 1995.
CAAX geranylgeranyltransferase transfers farnesyl as efficiently as geranylgeranyl to RhoB. J Biol Chem 270:7864–
7868.
Barbacid M. 1987. ras genes. Annu Rev Biochem 56:779–827.
Bar-Sagi D, Feramisco JR. 1985. Microinjection of the ras
oncogene protein into PC12 cells induces morphological differentiation. Cell 42:841–848.
Bos JL. 1989. ras oncogenes in human cancer: a review. Cancer Res 49:4682–4689.
Boylan JF, Jackson J, Steiner MR, Shih TY, Duigou GJ,
Roszman T, Casey PJ, Thissen JA, Moomaw JF. 1991. Enzymatic modification of proteins with a geranylgeranyl isoprenoid. Proc Natl Acad Sci USA 89:8313–8316.
Burker RD, Myers RL, Sexton TL, Jackson C. 1991. Cell
movements during the initial phase of gastrulation in the
sea urchin embryo. Dev Biol 146:542–547.
Daar I, Nebreda AR, Yew N, Sass P, Paules R, Santos E,
Wigler M, Woude GFV. 1991. The ras oncoprotein and Mphase activity. Science 253:74–76.
Der CJ, Cox AD. 1991. Isoprenoid modification and plasma
membrane association: critical factors for ras oncogenicity.
Cancer Cells 3:331–340.
Downward J. 1998. Ras signalling and apoptosis. Curr Opin
Genet Dev 8:49–54.
Epstein WW, Lever DC, Rilling HC. 1990. Prenylated proteins: synthesis of geranylgeranylcysteine and identification
of this thioether amino acid as a component of proteins in
CHO cells. Proc Natl Acad Sci USA 87:7352–7354.
Ettensohn CA. 1984. Primary invagination of the vegetal plate
during sea urchin gastrulation. Am Zool 24:571–588.
Farnsworth CC, Gelb MH, Glomset JA. 1990. Identification
of geranylgeranyl-modified proteins in HeLa cells. Science
247:320–322.
Gary R, Bretscher A. 1995. Ezrin self-association involves
binding of an N-terminal domain to a normally masked Cterminal domain that includes the F-actin binding site. Mol
Biol Cell 6:1061–1075.
Hancock JF, Magee AI, Childs JE, Marshall CJ. 1989. All ras
proteins are polyisoprenylated but only some are palmitoylated. Cell 57:1167–1177.
Hardin JD, Cheng LY. 1986. The mechanisms and mechanics
of archenteron elongation during sea urchin gastrulation.
Dev Biol 115:490–501.
Hertzler PL, Clark WH. 1992. Cleavage and gastrulation in
the shrimp Sicyonia ingentis: invagination is accompanied
by oriented cell division. Development 116:127–140.
Ho SN, Hunt HD, Horton RM, Pullen JK, Pease LR. 1989.
Site-directed mutagenesis by overlap extension using the
polymerase chain reaction. Gene 77:51–59.
Horton RM, Hunt HD, Ho SN, Pullen JK, Pease LR. 1989.
Engineering hybrid genes without the use of restriction
enzymes: gene splicing by overlap extension. Gene
77:61–68.
Huang C-F, Chuang N-N. 1999. Facilitated geranylgeranylation of shrimp ras-encoded p25 fusion protein by the
binding with guanosine diphosphate. J Exp Zool 283:
510–521.
448
C.-F. HUANG AND N.-N. CHUANG
James GL, Goldstein JL, Brown MS. 1995. Polylysine and
CVIM sequences of K-RasB dictate specificity of prenylation
and confer resistance to benzodiazepine peptidomimetic in
vitro. J Biol Chem 270:6221–6226.
Jurnak F. 1985. Structure of the GDP domain of EF-Tu and
location of the amino acids homologous to ras oncogene proteins. Science 230:32–36.
Kato K, Cox AD, Hisaka MM, Graham SM, Buss JE, Der
CJ.1992. Isoprenoid addition to Ras protein is the critical
modification for its membrane association and transforming activity. Proc Natl Acad Sci USA 89:6403–6407.
La Cour TFM, Nyborg J, Thirup S, Clark BF. 1985. Structure
details of the binding of guanosine diphosphate to elongation factor Tu from E. coli as studied by x-ray crystallography.
EMBO J 4:2385–2388.
Lane LC. 1978. A simple method for stabilizing protein-sulfhydryl groups during SDS-gel electrophoresis. Anal Biochem
86:655–664.
Lerner EC, Qian Y, Hamilton AD, Sebti SM. 1995. Disruption of oncogenic K-Ras4B processing and signaling by a
potent geranylgeranyltransferase I inhibitor. J Biol Chem
270:26770–26773.
Lerner EC, Zhang TT, Knowles DB, Qian Y, Hamilton AD,
Sebti SM. 1997. Inhibition of prenylation of K-Ras, but not
H- or N-Ras, is highly resistant to CAAX peptidomimetics
and requires both a farnesyltransferase and a geranylgeranyltransferase I inhibitor in human tumor cell lines.
Oncogene 15:1283–1288.
Lin R-S, Chuang N-N. 1998. Carboxyl-terminal CFFL-sequence-specific monomeric protein geranylgeranyltransferase I from the eyes of the shrimp Penaeus japonicus. J
Exp Zool 281:565–573.
Lloyd AC. 1998. Ras versus cyclin-dependent kinase inhibitors. Curr Opin Genet Dev 8:43–48.
Lowry OH, Rosebrough NJ, Farr AE, Randall RJ. 1951. Protein measurement with the Folin pheno reagent. J Biol
Chem 193:265–275.
Lowy DR, Willumsen BM. 1993. Function and regulation of
ras. Annu Rev Biochem 62:851–891.
Mineo C, James GL, Smart EJ. 1996. Localization of epidermal growth factor-stimulated Ras/Raf-1 interaction to
caveolae membrane. J Biol Chem 271:11930–11935.
Miquel K, Pradines A, Sun J, Qian Y, Hamilton AD, Sebti
SM, Favre G. 1997. GGTI-298 induces G0-G1 block and
apoptosis whereas FTI-277 causes G2-M enrichment in A549
cells. Cancer Res 57:1846–1850.
Mulcahy LS, Smith MR, Stacey DW. 1985. Requirement for
ras proto-oncogene function during serum-stimulated
growth of NIH3T3 Cells. Nature 313:241–243.
Ngsee JK, Elferink LA, Scheller RH. 1991. A family of raslike GTP-binding proteins expressed in electromotor neurons. J Biol Chem 266:2675–2680.
Olson MF, Ashworth A, Favre G. 1995. An essential role for
Rho, Rac, and Cdc42 GTPases in cell cycle progression
through G1. Biochem Biophys Res Commun 225:869–876.
Pai EF, Kabsch W, Krengel U, Holmes KC, John J, Wittinghofer A. 1989. Structure of the guanine-nucleotide-binding
domain of the Ha-ras oncogene product p21 in the triphosphate conformation. Nature 341:209–214.
Pomerance M, Schweighoffer F, Tocque B, Pierre M. 1992.
Stimulation of mitogen-activated protein kinase by oncogenic Ras p21 in Xenopus oocytes. J Biol Chem 267:16155–
16160.
Reiss Y, Seabra MC, Armstrong SA, Slaughter CA, Goldstein JL, Brown MS. 1991. Nonidentical subunits of
p21H-ras farnesyltransferase: peptide binding and
farnesyl pyrophosphate carrier functions. J Biol Chem
266:10672–10677.
Rhodes N, Hicks R, Kasenally AB, Innes CL, Paules RS,
Propst F. 1994. V-mos-transformed cells fail to enter quiescence but growth arrest in G1 following serum withdrawal.
Exp Cell Res 213:210–217.
Rhodes N, Innes CL, Propst F, Paules RS. 1997. Serum
starved v-mos-transformed cells are unable to appropriately downregulate cyclins and CDKs. Oncogene 14:
3017–3027.
Sagata N, Oskarsson M, Copeland T, Brumbaugh J, Vande
Woude GF. 1988. Function of c-mos proto-oncogene product
in meiotic maturation in Xenopus oocytes. Nature (London)
335:519–525.
Sagata N, Daar I, Oskarsson M, Showalter SD, Vande Woude
GF. 1989. The product of the mos proto-oncogene as a candidate “initiator” for oocyte maturation. Science 245:643–646.
Schagger H, von Jagow G. 1987. Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Anal Biochem
166:368–379.
Seabra MC, Reiss Y, Casey PJ, Brown MS, Goldstein JL. 1991.
Protein farnesyltransferase and geranylgeranyltransferase
share a common alpha subunit. Cell 65:429–434.
Shirataki H, Kaibuchi K, Hiroyoshi M, Isomura M, Araki S,
Sasaki T, Takai Y. 1991. Inhibition of the action of the stimulatory GDP/GTP exchange protein for smg p21 by the
geranylgeranylated synthetic peptides designed from its Cterminal region. J Biol Chem 266:20672–20677.
Stephens L, Hardin J, Keller R, Wilt F. 1986. The effects of
aphidicolin on morphogenesis and differentiation in the sea
urchin embryo. Dev Biol 118:64–69.
Sun J, Qian Y, Hamilton AD, Sebti SM. 1995. Ras CAAX
peptidomimetic FTI 276 selectively blocks tumor growth in
nude mice of a human lung carcinoma with K-Ras mutation and p53 deletion. Cancer Res 55:4243–4247.
Swanson ME, Elste AM, Green berg SM, Schwartz JH, Aldrich
TH, Furth ME. 1986. Abundant expression of ras proteins
in Aplysia neurons. J Cell Biol 103:485–492.
Sweet RW, Yokoyama S, Kamata T, Feramisco JR, Rosenberg
M, Mitchell G. 1984. The product of ras is a GTPase and
the T24 oncogenic mutant is deficient in this activity. Nature 311:273–275.
Tong LA, De Vos AM, Millburn MV, Kim S-H. 1991. Crystal
structures at 2.2 A resolution of the catalytic domains of
normal ras protein and an oncogenic mutant complexed with
GDP. J Mol Biol 217:503–516.
Tseng S-F, Chuang N-N. 1994. The binding of corticosterone
to the class-theta glutathione S-transferase from the eyes
of the shrimp Penaeus japonicus (Crustacea: Decapoda).
Comp Biochem Physiol 108B:215–219.
Vogt A, Qian Y, McGuire TF, Hamilton AD, Sebti SM. 1996.
Protein geranylgeranylation, not farnesylation, is required
for the G1 to S phase transition in mouse fibroblasts.
Oncogene 13:991–999.
Vogt A, Sun J, Qian Y, Hamilton AD, Sebti SM. 1997. The
geranylgeranyltransferase-I inhibitor GGTI-298 arrests human tumor cells in G0/G1 and induces p21 (WAF1/CIP1/
SDI1) in a p53-independent manner. J Biol Chem 272:
27224–27229.
Walter M, Clark SG, Levinson AD. 1986. The oncogenic activation of human p21ras by a novel mechanism. Science 233:
649-652.
Yokoyama K, Goodwin GW, Ghomashci F, Glomset JA, Gelb
MH. 1991. A protein geranylgernayltransferase from bovine
GERANYLGERANYLATION AT C-TERMINI OF SHRIMP RAS
brain: implications for protein prenylation specificity. Proc
Natl Acad Sci USA 88:5302–5306.
Zhang FL, Kirschmeier P, Carr D, James L, Bond RW, Wang
L, Patton R, Windsor WT, Syto R, Zhang R, Bishop WR.
1997. Characterization of Ha-Ras, N-Ras, Ki-Ras4A, and
Ki-Ras4B as in vitro sybstrates for farnesyl protein trans-
449
ferase and geranylgeranyl protein transferase type I. J Biol
Chem 272:10232–10239.
Zheng CF, Simcox T, Xu L, Vaillancourt P. 1997. A new expression vector for high level protein production, one step
purification and direct isotopic labeling of calmodulin-binding peptide fusion proteins. Gene 186:55–60.
Документ
Категория
Без категории
Просмотров
23
Размер файла
466 Кб
Теги
1/--страниц
Пожаловаться на содержимое документа