Departments of Pharmacology and Anesthesiology, University of Illinois, Chicago, Illinois 60612
Submitted 25 March 2003 ; accepted in final form 1 May 2003
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ABSTRACT |
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mitogen-activated kinase; effector mutant; Src kinase
Investigation of the signaling properties of mutant proteins in which
effector-protein binding is defective has provided a useful approach to the
functional study of Rho and Ras GTPases
(3,
55). Moreover, effector
mutants of Gs, G
q, and
G
i proteins were used intensively to analyze the activation
of specific signaling pathways
(5,
30). So far, effector mutants
of G
13 have not been identified. While screening multiple
samples obtained from human tumors and tumor cell lines for the mutations in
the
-chain of G13 protein, we found a mutation-encoding
substitution of Arg260 to a stop codon in G
13 mRNA obtained
from the human adenocarcinoma cell line LoVo. We found that
G
13-T lost its ability to promote proliferation and
transformation but retained its ability to induce apoptosis. Here, we analyzed
which signaling pathways are required for G
13-induced
mitogenesis or apoptosis using a novel mutant of G
13 as a
specific tool to dissect G
13-mediated signals.
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MATERIALS AND METHODS |
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Reverse transcription. Reverse transcription (RT) was performed
with 1 µg of total RNA, 10 µlof5x first-strand buffer, 10 mM
dithiothreitol, 0.5 mM of each dNTP, 10 ng/µl oligo-(dT)12-18, 200 U
Moloney murine leukemia virus reverse transcriptase; final volume was adjusted
with water to 20 µl. After incubation for 60 min at 37°C, the reaction
was stopped by heating for 3 min at 100°C. Samples were kept at -20°C.
To amplify the human G13 gene, oligonucleotide primers were
designed on the basis of published sequence
(22).
Polymerase chain reaction. Polymerase chain reaction (PCR) was
performed with 2 µl of RT mixture, 2.5 µl of 10x PCR buffer, 1.5
mM MgCl2, 0.2 mM of each dNTP, 1 µl [32P]dCTP
5000 Ci/mmol, 1 µM of each primer, 2.5 U TaqI, and 0.16 U
PfuI. PCR was performed using DNA thermal cycler and Perkin Elmer
Cetus for 35 cycles as follows: 1 min at 94°C, 1 min at 61°C, and 2
min at 72°C, followed by 10 min of elongation at 72°C. PCR products
were examined by 1.0% agarose gel electrophoresis after ethidium bromide
staining.
Single-strand conformation polymorphism analysis. Single-strand conformation polymorphism analysis (SSCP) of PCR products was performed essentially as described (37a). To denature DNA before electrophoresis, 3 µl of samples were mixed with 17 µl of 95% formamide, 20 mM EDTA, 0.05% bromophenol blue, and 0.05% xylene cyanol and heated at 97°C for 5 min. Then, samples were immediately chilled on ice and loaded onto 8% polyacrylamide gel with N,N'-methylene bisacrylamide/acrylamide ratio 1:29, wt/wt. Electrophoresis was performed in 90 mM Tris-borate, pH 8.3, and 4 mM EDTA at 10 V/cm for 20 h at room temperature. After electrophoresis, the gels were transferred to Whatman paper and dried on a gel dryer (Bio-Rad). Transferred products were analyzed with PhosphorImager (Bio-Rad).
Site-directed mutagenesis. To introduce Q226L activation mutation
into GTPase domain of truncated and full-version human G13,
we used a PCR-based site-directed mutagenesis kit provided by Stratagene (La
Jolla, CA). Two synthetic oligonucleotide primers, direct and reverse,
containing the mutation that converts CAG codon for Gln226 to CTT
codon for Leu226 were extended during temperature cycling with Pfu
polymerase, and methylated, nonmutated parental DNA template was with DpnI.
DNA fragments that incorporated the desired mutation were transfected into
HB101 competent cells. Plasmids containing the mutated gene were recovered by
screening with restriction endonuclease, and the presence of the mutation was
determined by sequencing. Hemagglutinin (HA)-epitope tagging of
G
13-T was performed with double-stranded linker containing
sequences for HA epitope (YPYDVPDYAS), followed by stop codon and flanking
sites for restriction endonucleases to introduce the linker into plasmid.
Primary structure was confirmed by sequencing.
Cell culture, transfection, foci formation, and colony-forming efficiency. Rat-1 murine fibroblasts were grown in high-glucose DMEM supplemented with 10% calf serum, 100 IU/ml penicillin G, 100 IU/ml streptomycin, and 0.25 mg/l amphofericine B until 80% confluence and passage every 2-4 days. For foci formation assay, Rat-1 cells were transfected on 60-mm dishes using 15 µl LipofectAMINE 2000 and 5 µg of DNA constructs and maintained in growth medium reduced to 5% heat-inactivated calf serum. The appearance of foci was scored in 2-3 weeks upon cells being fixed with paraformaldehyde and being stained with Giemsa stain.
Colony-survival assay. Colony-survival assay was performed by transfection of fibroblasts as described above. After transfection, cells were reseeded into 6-cm-diameter dishes. G418 selection (0.15 mg/ml) was applied the following day, and individual colonies (comprising >100 cells) were scored after a further 2 wk.
Luciferase assays. Elk-dependent gene expression was assayed by
ELK-1 "PathDetect" trans-reporter system (Stratagene).
Briefly, cells at 90% confluency grown on 24-well plates were transfected with
the following plasmids (per well): 200 ng pFR-Luciferase (reporter plasmid),
12.5 ng pFA2-ELK1 (fusion trans-activator plasmid) (for ELK-dependent
gene expression) with 50 ng pCMV-gal (transfection efficiency control
plasmid), and 50 ng G
13Q229L, balanced with pCMV vector alone. The day
before stimulation, cells were incubated in either 0.2 or 0% serum to achieve
serum starvation. Activation of Elk1-dependent gene expression was similar in
the cells exposed to either 0 or 0.2% serum. Cells were washed twice with PBS
and lysed in protein extraction reagent, and the cleared lysates were assayed
for luciferase and
-galactosidase activity using the corresponding assay
kits (Promega, Madison, WI). In order to account for differences in
transfection efficiency, the Luciferase activity of each sample was normalized
to
-galactosidase activity and expressed as percent of the maximal
response to G
-subunit stimulation.
JNK, ERK1, and ASK1 activity assays. JNK, ERK-1, and ASK1 activities were determined as described previously (4, 19). Briefly, HA-JNK or HA-ASK1 were transfected in the presence of various cDNA constructs as described in RESULTS. The kinase activity of HA-JNK using recombinant c-Jun and the kinase activity of HA-ERK or HA-ASK1 were measured or myelin basic protein (MBP) as a substrate, respectively. Aliquots of the whole cell lysates from the same experiment were subjected to immunoblotting analysis to confirm the appropriate expression of transfected proteins. In some experiments, phosphospecific ERK1/2 antibody (Cell Signaling Technology) was used.
Src kinase assay. Src family kinase assay was performed after the
immunoprecipitation of endogenous c-Src (Src2, Santa Cruz) from NIH3T3 cells
transfected with G13Q229L. The immunocomplexes were
collected with protein-A-agarose (GIBCO) and washed three times with lysis
buffer. Kinase activity of c-Src was evaluated by its ability to phosphorylate
the acid-denatured enolase
(36). The radioactivity
incorporated into enolase was measured by a BioImaging System (Bio-Rad).
Cell death assays. DNA fragmentation was determined as previously
described using the terminal deoxynucleotidyl transferase (TdT)-mediated dUTP
nick end labeling (TUNEL method) (Apoptag Plus, Intergen) according to
manufacturer's instructions
(4). In this assay, residues of
digoxigenin-nucleotide are catalytically added to the DNA by TdT. The
incorporated digoxigenin is then recognized by fluorescein-coupled digoxigenin
antibody. For apoptosis analysis, cells were cotransfected with cDNA-encoding
-galactosidase. Twenty-four hours after transfection, cells were serum
starved for an additional 16 h and then fixed with 2% paraformaldehyde for 15
min. Cells were permeabilized with 0.1% Triton X-100 for 10 min, and
fragmented genomic DNA was stained as described
(4).
-Galactosidase was
stained with polyclonal
-galactosidase antibody, followed by a rabbit
rhodamine-conjugated IgG. The percentage of
-galactosidase-positive
cells with fragmented nuclei was assessed by fluorescence microscopy. In each
experiment, 300-400 cells were counted.
Caspase 3/7 activity assay. Caspase 3/7 activity was determined using Apo-ONE kit (Promega) according to the manufacturer's instructions. Briefly, Rat-1 cells seeded onto 24-well plates were transfected with indicated constructs for 24 h. Cells were washed with phosphate-buffered saline (PBS). Caspase 3/7 reagent (200 µl) was added to each well and incubated at 30°C for 4 h. The fluorescence was measured using spectrofluorimeter with an excitation wavelength of 485 nm and an emission wavelength of 530 nm using CytoFluor II. In some experiments, caspase activity was blocked by pretreatment with 10 µM Z-VAD-FMK caspase inhibitor for 16 h before experiment.
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RESULTS |
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Thereafter, the whole coding region of G13 from human
adenocarcinoma was cloned into mammalian expression vector by PCR approach
using primers flanking the gene. DNA sequencing showed mutation at codon 260
(Arg260
Stop) that resulted in the introduction of the premature stop
codon. The predicted molecular mass of the truncated protein was 30 kDa.
Immunoblotting analysis using antibody against the NH2 terminus
(Santa Cruz Biotechnology) of G
13 showed that both
full-length and truncated copies of G
13 were expressed
endogenously in LoVo cells (Fig.
1B). To control the specificity of staining,
G13-specific antibody was preabsorbed with immunizing peptide; the
right panel depicts the absence of staining with
G13-specific antibody that was preabsorbed with immunizing peptide
(Fig. 1B). Similarly,
in NIH3T3 cells transfected with truncated G
13,
immunoblotting analysis of total cell lysates detected a single band with an
apparent molecular mass of 30 kDa (Fig.
1B).
These data suggest that G13-T forms a stable polypeptide
that both exists as endogenous protein and can be transiently expressed in the
cells.
G13-mediated transformation of
Rat-1 cells is permissively dependent on basal ERK activity. We have
previously shown that mutationally activated G
13 induces
neoplastic transformation of Rat-1 and NIH3T3 fibroblasts
(52). As truncated
G
13 protein was cloned from a LoVo cell line that derived
from metastatic tumor node from a patient with adenocarcinoma of the colon, we
have tested whether the G
13-T could induce neoplastic
transformation. Focus formation assay in Rat-1 fibroblasts was used as a test
for cell transformation. Rat-1 cells were transfected with corresponding
constructs and maintained after transfection in the medium supplemented with
5% calf serum for 2 wk. Only constitutively activated mutant of human
full-length G
13 (G
13Q226L) induced focus
formation with transformation-specific morphology as a 0.1 to 1-mm area of
multilayer cell growth (Fig.
2A). These data were consistent with previously published
results obtained with constitutively activated mouse G
13
(52). Both wild-type
G
13 and G
13-T did not induce focus
formation in Rat-1 fibroblasts (Fig.
2A). To model the presence of two distinct
G
13 polypeptides in LoVo cells, we determined whether
equimolar amounts of wild-type G
13 and
G
13-T could induce focus formation in Rat-1 fibroblasts.
G
13-T did not enhance the transformation by wild-type
G
13 (Fig.
2A). Finally, G
13Q226L-T (Q226L
mutation was introduced in a GTPase region of G
13-T) also
did not induce focus formation in Rat-1 fibroblasts
(Figure 2A). Similar
data were obtained using NIH3T3 fibroblasts (data not shown). Thus
G
13-T failed to induce focus formation in Rat-1 fibroblasts
and probably cannot be considered as a reason for the neoplastic phenotype of
LoVo adenocarcinoma.
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Interestingly, both G13-T and
G
13Q226L-T inhibited G
13Q226L-induced foci
formation by 65% (data not shown). Because transformation inhibition seen with
G
13-T may not be specific but instead may be a consequence
of nonspecific growth inhibition, we determined whether
G
13-T could cause inhibition of cell growth by employing a
colony survival assay by drug selection of transfected Rat-1 cells. As shown
in Fig. 2B, both
G
13-T and G
13Q226L-T significantly
inhibited the number of surviving colonies. We also noted that many of the
colonies generated from G
13-T or
G
13Q226L-T transfected Rat-1 cells grew poorly and had a
propensity to detach from the culture dish, with widespread cell death,
suggesting that these two mutants may induce apoptosis in Rat-1 cells.
We have previously shown that in Rat-1 cell lines stably transfected with
G13, EGF-stimulated ERK1 activity was potentiated
(52); however, significance of
this phenomenon is not understood. Involvement of the ERK signaling pathway in
cell transformation induced by a variety of oncogenes is now well documented
(for review, see Ref. 29);
however, the involvement of this pathway in G
13-dependent
transformation has not been investigated. Interestingly, we could not detect
G
13-dependent activation of ERK1 using kinase assay
(52) or using or phospho-ERK1
antibody (data not shown).
Signaling pathways such as the MAP kinases, ERK1 and 2, p38, and JNK induce
phosphorylation of the transcription factor Elk-1, resulting in its activation
(46). Therefore, Elk-1-induced
expression of the reporter gene was used as a readout of
G13-induced gene transcription. In transient transfection,
full-length G
13Q226L stimulated Elk-1 by fivefold
(Fig. 3). The constitutively
active mutant form of MEK1 (S218E/S222D)
(33) stimulated Elk-1 by
50-fold as compared with vector-transfected cells
(Fig. 3). To determine whether
G
13-induced activation of Elk-1 occurs via simulation of the
MEK-ERK signaling pathway, we measured Elk-1 activation by
G
13 in the presence of the dominant-negative (K79M) form of
MEK1 (33) or the
dominant-negative (K432A) form of MEKK1
(51). In some experiments,
cells were pretreated with the pharmacological inhibitor of MEK PD-98059 or
the pharmacological inhibitor of p38MAPK and JNK SB-202190. SB-202190 was
initially described as an inhibitor of p38 MAPK
(27); however, it was recently
shown that at higher concentration, it also inhibits JNK activity
(20). Because our unpublished
data confirmed that SB-203580 inhibited p38 at 4 µM and inhibited JNK at 40
µM, we used SB-203580 at 40 µM, a concentration that was capable of
inhibiting both pathways.
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Our data showed that Elk-1 activation by G13 was
suppressed by dominant-negative MEK1 but not by dominant-negative MEKK1
(Fig. 3). In addition, PD-98059
but not SB-202190 inhibited G
13-induced Elk1 activation,
suggesting that G
13 can activate the ERK signaling pathway.
Interestingly, G
13wt, G
13-T, and
G
13Q226L-T did not stimulate Elk-1 activation
(Fig. 3).
We then determined whether the MEK-ERK pathway is involved in the
G13-dependent regulation of cell growth and transformation.
To characterize growth rate, Rat-1 cells stably transfected with
constitutively activated G
13Q226L were plated in medium
containing 5% calf serum, and cells were counted every day. In the presence of
5% calf serum, G
13Q226L-expressing cells proliferated faster
than vector only-expressing cells and reached a confluent state faster, and
maximal cell density was higher as compared to cells transfected with vector
alone (Fig. 3B). The
overexpression of G
13Q226L was about twofold when compared
with endogenously expressed G
13
(Fig. 3B,
inset). Proliferation of the Rat-1 cell line expressing wild-type
G
13 was similar to that of the vector only-expressing cell
line (data not shown). Specific MEK inhibitor PD-98059 inhibited
G
13Q226L-induced cell growth
(Fig. 3B), whereas
inhibitor of p38MAPK and JNK SB-202190 did not affect
G
13-induced proliferation (data not shown). Similarly,
PD-98059 and dominant-negative MEK inhibited
G
13Q226L-induced focus formation
(Fig. 3C), whereas
SB-202190 and dominant-negative MEKK1 did not affect the foci-forming activity
of G
13Q226L. Thus, although G
13 did not
activate ERK, the basal ERK activity may be essential for transformation,
whereas p38MAPK and JNK pathways did not seem to be involved in
G
13-induced transformation.
Finally, we determined whether activation of ERK pathway could salvage
G13-T deficiency to induce neoplastic transformation in
Rat-1 cells. For these experiments, we cotransfected Rat-1 cells with a
constitutively active mutant form of MEK1 (S218E/S222D)
(33). Increased activity of
ERK in the cells transfected with constitutively active MEK1 was confirmed by
Luciferase-based determination of ERK activity (data not shown).
Constitutively active MEK1 alone induced a small but significant increase of
foci formation (Fig.
3C) that was consistent with previously published data
(33). Cotransfection of MEK1
(S218E/S222D) with G
13Q226L-T resulted in a synergistic
increase of foci formation (Fig.
3C). These results indicate that MEK-ERK pathway is
required for G
13-induced transformation of Rat-1 cells.
These results also indicate that failure to induce transformation by truncated
G
13 may be explained by its failure to activate the MEK-ERK
pathway.
To further examine the mechanism of G13-induced cell
transformation, we investigated the possible role of Src, whose involvement in
the regulation of cell proliferation and transformation is well established.
To determine the activity of the endogenous c-Src kinase, we used Rat-1 cells
transfected with G
13Q226L
(Fig. 4A). Equal
quantities of endogenous Src family kinases were immunoprecipitated from Rat-1
cells, and Src kinase activity was estimated by phosphorylation of
acid-denatured enolase. G
13Q226L increased the activity of
endogenous Src kinase by
5.4-fold. Pretreatment of the cells with
tyrosine kinase inhibitor genistein inhibited G
-induced activation of
Src kinase (Fig. 4A).
Importantly, truncated G
13 did not induce Src
phosphorylation (Fig.
4A).
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Finally, we determined how inhibition of Src affected
G13-induced mitogenic signaling. Data showed that both
dominant-negative c-Src (c-Src K295M/Y527F, SrcDN) and genistein completely
inhibited G
13 Q226L-dependent activation of Elk-1
(Fig. 4B). Similarly,
genistein inhibited G
13-dependent cell growth
(Fig. 4C), whereas
SrcDN inhibited G
13Q226L-induced focus formation
(Fig. 4D). To rule out
nonspecific growth inhibition, we determined whether SrcDN could cause
inhibition of cell growth by employing a colony-survival assay by drug
selection of transfected Rat-1 cells. No difference was observed between the
vector and dominant-negative Src with respect to the number of colonies (data
not shown). Thus the inhibition of focus-forming activity that we detected
could not be the result of a nonspecific growth inhibition by mutant protein.
Therefore, the data suggest that activation of Src pathway is required for
G
13 subunit-dependent transformation.
Mitogen-activated signaling is required for
G13-induced apoptosis in Rat-1
fibroblasts. As we have shown previously that G
13
activates the JNK pathway
(51), we have now tested
whether G
13-T and G
13Q226L-T could
activate the JNK pathway. Consistent with previously published data,
G
13Q226L-induced phosphorylation of c-Jun by JNK was
inhibited by dominant-negative MEKK1 and SB-202190
(51), whereas
dominant-negative MEK1 and PD-98059 did not affect JNK activity
(Fig 5A).
Interestingly, two truncated mutants of G
13 also induced JNK
activation that was inhibited by both dominant-negative MEKK1 and SB-202190
(Fig. 5B).
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We have recently described that although G13 induces
neoplastic transformation in Rat-1 and NIH3T3 cells
(52), it induces apoptosis in
other cell lines such as COS-7 and CHO cells
(1,
4); the reason for these
cell-specific effects remains unknown. Because two truncated mutants of
G
13 had decreased colony-forming efficiency
(Fig. 2B), we tested
whether they could induce apoptosis in Rat-1 cells using in situ detection of
DNA fragmentation using the TUNEL method
(4). Different
-subunit
constructs were transiently transfected (at a ratio of 1 µg of
cDNA:106 cells), and 24 h after transfection, cell death was
estimated measuring the number of cells with fragmented DNA labeled with TdT
(Apoptag Plus kit) (Fig.
6A) in cells expressing G
13 constructs.
Under these conditions, 2 ± 1.1% of vector-transfected cells underwent
apoptosis (Fig. 6A).
Neither wild-type G
13 nor activated
G
13Q226L mutant induced apoptosis in these cells; these data
were consistent with our previous observations
(4,
52). However, both
G
13-T and G
13Q226L-T induced apoptosis in
46 ± 7% of transfected cells (Fig.
6A). Dilution of G
13-T or
G
13Q226L-T cDNA 10-fold with the vector cDNA during
transfection resulted in a reduced percentage of apoptotic cells (13 ±
5.5%, data not shown), suggesting that G
13-T-induced
apoptosis is a function of G
13-T expression levels.
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Because mitogenic stimuli through the ERK cascade have been shown to have
inhibitory effect on the induction of apoptosis
(56), and our data showed that
G13-T could not stimulate ERK-dependent Elk-1 activation, we
determined whether ERK-mediated signaling could be involved in protection of
Rat-1 cells from G
13-induced apoptosis. Therefore, we
determined whether activation of the ERK pathway could inhibit
G
13-T-induced apoptosis. For these experiments, we
cotransfected Rat-1 cells with a constitutively active mutant form of MEK1
(S218E/S222D). Under these conditions, Rat-1 cells were efficiently protected
against G
13-T-induced apoptosis
(Fig. 6A). Similarly,
inhibition of JNK and p38 kinase by SB-202190 effectively protected Rat-1
cells from apoptosis (Fig.
6A).
Because caspase 3 and 7 are activated during the earliest phases of
apoptotic induction (41), we
determined the activity of caspase-3/7 using rhodamineconjugated caspase 3/7
substrate,
bis-(N-CBZ-L-aspartyl-L-glutamyl-L-valyl-L-aspartic
acid amide, Z-DEVDR110) according to the manufacturer's instruction (Apo-ONE
Promega). Consistently, only the truncated construct of G13
induced caspase 3/7 activation (Fig.
6B). Pretreatment of the cells with cell-permeable
caspase inhibitor Z-VAD-FMK blocked G
13-T-induced caspase
activation.
We have previously shown that G13-induced apoptosis in
COS-7 cells is mediated by apoptosis signal-regulating kinase (ASK1)
(4). To examine whether
truncated G
13 could affect the same signaling pathway, ASK-1
activity was determined in the Rat-1 cells expressing full-length and
truncated G
13 and ASK1
(Fig. 7A). Consistent
with previously published data, mutationally activated G
13
induced ASK1 activity (4). In
addition, truncated G
13 also stimulated ASK1
(Fig. 7A). Finally, we
determined whether dominant-negative ASK1 could inhibit apoptosis induced by
truncated G
13 in Rat-1 cells. Data showed that
cotransfection of truncated G13 with dominant-negative ASK1
resulted in significant inhibition of apoptosis
(Fig. 7B). These
results indicate that apoptosis induced by truncated G
13 can
be mediated via ASK1. Besides, both full-length and truncated
G
13 stimulated ASK1; however, only truncated
G
13 induced apoptosis in Rat-1 cells, suggesting that
full-length G
13 may stimulate additional signaling pathways
that protect cells from apoptosis.
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Figure 8 shows the model 3-D
structures of truncated G13
(Fig. 8A) and
full-length protein (Fig.
8B). The very first 19 amino acids from
G
13 NH2 terminus are unique and not homologous to
any known sequence. Therefore, they were excluded from our 3-D model. The
region of deletion, amino acids 260-377 of G
13, is shown in
purple (Fig. 8B).
Major putative sites for interaction with G
-subunits,
sites for lipid modification, regulators of G protein signaling
(RGS)-interacting domains (shown as three blue loops), and
G1-G3 sites, ensuring chelating of guanine nucleotide,
are present in G
13-T. G1, G2, and
G3 domains could theoretically coordinate
,
, and
phosphates, as well as provide coordinated molecule of water and
Mg2+ in the catalytic site of the protein for GTP/GDP turnover.
However, two extra sites responsible for the interaction of
-subunit
with guanine nucleotide are lost. The G4 region is responsible for
coordination of the guanine ring and, by this means, secures GTP/GDP
interaction. The function of G5 region that is conserved among G
proteins is unknown.
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Thus, from the analysis of 3-D crystal models for G13, we
can predict that guanine nucleotide binding and GTP hydrolysis will be
seriously impaired in G
13-T mutant. Interestingly, mitogenic
response that required a GTP-bound form of G
13 (at least, in
Rat-1 cells Ref. 52) was
absent in G
13-T. However, G
13-T has
maintained its ability to induce apoptosis, a response that did not seem to
depend on the guanine nucleotide-binding state of the mutant protein. We
conclude that this mutant protein will be a useful tool for identifying
G
13-interacting protein(s) that define the ability of
G
13 to induce mitogenesis or apoptosis. Yeast two-hybrid
studies and DNA array displays using truncated mutant of G
13
are currently in progress.
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DISCUSSION |
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G12 and
G
13 are localized in chromosomal regions
showing association with colon, pancreas, and brain tumors. In this
study, extensive screening of tumors from different tissues did not reveal
mutations that could increase mitogenic potential of G
13.
Comparing this result with the level of polymorphism for G
s,
it is necessary to underscore that all activated mutations of
G
s were restricted to three types of tumor: pituitary
adrenocorticotropic growth hormone-secreting adenomas, thyroid tumors, and
adrenocortical adenomas. The probability of finding a mutated form of
G
s in these tumors was from 10 to 40%
(32). Mutated
G
i2 is also tissue specific and can be found mainly in
ovarian sex cord stromal tumors and adrenal cortical tumor with a probability
of about 30% (32). An
extensive search for activated mutations in G
11,
G
14, and G
q did not detect mutations of
GTPase domain in neoplasias from different origins
(14). Because
G
12 was identified as a putative oncogene in soft tissue
sarcomas (8), we cannot exclude
the possibility that both G
12 and G
13
could be putative oncogenes in tissues that we did not test in this study.
Here, we report the identification and characterization of the novel mutant
of G13 protein. The mutation encoding the substitution of
Arg260 to stop the codon in mRNA of the G
13 subunit produced
a mutant protein (G
13-T) that lacks a COOH terminus and is
endogenously expressed in LoVo cells as a polypeptide of 30 kDa. The LoVo cell
line was initiated in 1971 from a metastatic tumor nodule in the left
ventricular region of a patient with adenocarcinoma of the colon
(15,
16). The established cell line
retains many features associated with malignant cells; for example, it
secretes carcinoembryonic antigen and induces tumors in nude mice with 100%
frequency (15,
16). Although
G
13-T failed to induce focus formation in Rat-1 fibroblasts
and probably cannot be considered as a reason for the neoplastic phenotype of
LoVo adenocarcinoma, it may contribute to the sensitivity of this cell line to
apoptosis-inducing agents
(42).
Three-dimensional model structure of
G13. The crystal structure of
G
13 has not been solved yet. However, 3-D structures for a
number of G proteins
-subunits in a complex with
-subunits, different guanine nucleotide analogs, and RGS were
successfully solved (11,
24,
44,
45). Alignment of amino acid
sequence for the protein against G
s, G
t,
G
i1, p21ras, and other homologous
proteins reveals a high level of identity and similarity
(24). Therefore, using
Swiss-Pdb Viewer v. 3.5 and Geneva Biomedical Research Center homology
modeling server "Swiss-Model"
(39), we threaded a
G
13 protein primary sequence onto a 3-D template of
homologous protein (Fig. 6).
Human G
13 contains several conservative amino acid regions
responsible for chelating and hydrolysis of guanine nucleotide
(6,
22). These regions are G1,
G2, G3, G4, and G5 according to Bourne nomenclature
(6).
Studies of crystal structures for
p21ras,Gt, and G
i1
proteins complexed with guanine nucleotide analogs showed that the first
region (A or G1) is responsible for chelating
-,
-,
and
-phosphates, the second (C or G2) interacts with
-phosphate, the third (G or G3) is a catalytic domain and
interacts with
-phosphate, and the fourth (I or G4) is
responsible for interaction with the guanine ring
(6).
RGS proteins, which accelerate the rate of GTP hydrolysis, can interact
with the surface of protein globule that is formed by amino acids from switch
I, II, and III the Ras-like domains and do not make significant contact with
the -helical domain of G
, as was shown for RGS4 bound to
AlF- 4 -activated G
i1. RGS4 could
enhance catalysis by binding a molecule of water in the active site through
the Asn128 residue that is in close vicinity with Gln204 and Ser206 of the
G
i1 switch II domain
(11,
45). Guanine
nucleotide-releasing proteins (GNRP) interact with the very C-end of G
protein (6).
Figure 6 shows the model 3-D
structures of truncated G13
(Fig. 8A) and
full-length protein (Fig.
8B). The very first 19 amino acids from the
G
13 NH2 terminus are unique and not homologous to
any known sequence. Therefore, they were excluded from our 3-D model. The
region of deletion, amino acids 260-377 of G
13, is shown in
purple (Fig. 8B).
Major putative sites for interaction with G
-subunits, sites for
lipid modification, RGS interacting domains (shown as a three blue loops), and
G1-G3 sites ensuring chelating of guanine nucleotide are
present in G
13-T. G1, G2, and
G3 domains could theoretically coordinate
-,
-, and
-phosphates, as well as provide a coordinated molecule of water and
Mg2+ in the catalytic site of the protein for GTP/GDP turnover.
However, two extra sites responsible for the interaction of the
-subunit with the guanine nucleotide are lost. The G4 region
is responsible for coordination of the guanine ring and, by this means,
secures GTP/GDP interaction. The function of the G5 region that is
conserved among G proteins is unknown.
Thus, from the analysis of 3-D crystal models for G13, we
can predict that guanine nucleotide binding and GTP hydrolysis will be
seriously impaired in G
13-T mutant. Interestingly, the
mitogenic response that required a GTP-bound form of G
13 (at
least, in Rat-1 cells, Ref.
52), was absent in
G
13-T. However, G
13-T has maintained its
ability to induce apoptosis, a response that did not seem to depend on the
guanine nucleotide-binding state of the mutant protein. We conclude that this
mutant protein will be a useful tool for identifying
G
13-interacting protein(s) that define the ability of
G
13 to induce mitogenesis or apoptosis. Yeast two-hybrid
studies and DNA array displays using the truncated mutant of
G
13 are currently in progress.
Regulation of mitogenesis and apoptosis by MAPK pathways. With the
recognition that G13 protein is functionally coupled to both
mitogenic and apoptotic responses, we hypothesized that G
13
regulates multiple signaling pathways that produce bifurcating signals leading
to mitogenic or apoptotic response. The variable effects of
G
13 on mitogenic and apoptotic responses could reflect the
use of these multiple pathways under different circumstances and in different
cell types.
MAPK are a family of Ser/Thr kinases involved in the regulation of a wide range of cellular responses, including cell proliferation and survival (43). To date, several distinct MAPK have been identified that act independently on signaling pathways. These include ERK, JNK, and p38MAPK; these kinases represent the terminal stages of growth/survival factor or death receptors (54). The MAPK pathways act as key regulators of mitogenic and apoptotic pathways. Deregulation of ERK pathway leads to cell proliferation and neoplastic transformation (23, 33). The role of JNK pathway in mitogenesis and neoplastic transformation is poorly understood; however, several reports suggest that the JNK pathway also participates in neoplastic transformation (40).
The role of G13 in ERK activation is complex and seems to
be cell dependent. Several studies have not detected ERK activation or even
detected ERK inhibition by G
12 and G
13
(51), whereas other studies
have shown these proteins can either activate ERK
(13,
35) or enhance the extent and
persistence of the agonist-dependent ERK activation
(52). Our data showed that
basal activity of ERK (but not JNK pathway) is involved in
G
13-mediated proliferation and transformation. Although we
could not detect G
13-induced ERK activation using kinase
assay and phospho-ERK antibody, pharmacological and genetic inhibition of
MEK-ERK pathway inhibited G
13-induced proliferation and
transformation. Similar inhibition of JNK-p38 MAPK pathway did not have any
effect on G
13-induced proliferation and transformation.
Moreover, constitutively active MEK partially salvaged the ability of
G
13-T to transform Rat-1 cells. It is not clear how
G
13 could modulate basal level of ERK activity. One
possibility is that G
13 could affect the expression level of
some of the components of ERK signaling pathway, thus modulating the basal ERK
activity. This possibility is currently under investigation.
Another interesting possibility is in a recently described interaction
between G13 and E-cadherin
(34). E-cadherin is a long
recognized tumor suppressor
(50), and inhibition of the
ERK pathway induces synthesis of E-cadherin
(10). It is tempting to
speculate that the expression of G
13-T may result in an
increased expression of E-cadherin due to inhibition of the ERK pathway, which
may lead to the enhanced tumor suppressor function of E-cadherin. This
hypothesis is currently under investigation.
Our data suggest that the Src pathway mediates
G13-dependent gene expression, cell proliferation, and
transformation. Thus G
13-induced cell proliferation and foci
formation were abolished by either tyrosine kinase inhibitor or by the
dominant-negative mutant of Src. In addition, G
13Q226L could
stimulate Src activity.
The MAPK pathways, in particular the JNK pathway, participate in the induction of apoptosis (56). The activation of JNK and p38 MAPK is generally associated with the promotion of apoptosis, whereas ERK activity inhibits apoptosis. Thus, in PC-12 cell during apoptosis induced by growth factor withdrawal, JNK and p38 MAPK are activated, whereas ERK is inhibited (56). In contrast, the activation of ERK has been shown to inhibit apoptosis induced by hypoxia (7), growth factor withdrawal (17), and H2O2 (53).
The striking difference between G13 and
G
13-T revealed in the present work is the ability of
G
13-T to induce apoptosis in a cell line that is unaffected
by full-length G
13. We propose that modulation of basal ERK
activity by G
13 is one of the mechanisms that protect the
cells from apoptosis on the basis of the following observations. We have shown
that G
13 inhibits the ERK pathway in COS-7 cells
(51); at the same time,
G
13 induces apoptosis in COS-7 cells
(4). However, in Rat-1 cells in
which G
13 seems to potentiate the ERK pathway, it did not
induce apoptosis but instead promotes proliferation and transformation.
Finally, G
13-T that lacks the ability to modulate the ERK
pathway also causes apoptosis in Rat-1 cells. Interestingly, the cells were
partially rescued from G
13-T-induced apoptosis by
stimulation of ERK with constitutively active MEK. The mechanism of
G
13-induced apoptosis is not understood. Our published and
unpublished data suggest that signaling proteins that can contribute to the
G
13-dependent signaling interact with G
13
in both guanine nucleotide-dependent and -independent ways
(37,
47,
48). Due to the low level of
expression, it was not possible to address the guanine nucleotide-binding
properties of G
13-T. Because both G
13-T
and G
13-T harboring putative GTPase-inhibiting mutation
(G
13Q226L-T) were capable of stimulating the JNK pathway and
inducing apoptosis, it is conceivable that G
13-T either
exists in the GTP-bound state or constitutively interacts with molecules that
signal apoptosis. This important question is a target of our further
investigation.
In conclusion, we have shown that 1) mitogen-activated signaling
is involved in the regulation of mitogenic and apoptotic responses induced by
G13, and 2) the ability of G
13 to
transform cells could be dissociated from its propensity to promote apoptotic
response. We conclude that this mutant protein will be a useful tool for
identifying G
13-interacting protein(s) that define the
ability of G
13 to induce mitogenesis or apoptosis.
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DISCLOSURES |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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