(Received for publication, June 27, 1996, and in revised form, November 19, 1996)
From the Departments of Cardiovascular Biology,
¶ Physiology, and § Internal Medicine, Faculty of
Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113, Japan
and the ** Howard Hughes Medical Institute, Department of Molecular
Physiology and Biophysics, Vanderbilt University School of Medicine,
Nashville, Tennessee 37232-0295
We evaluated the roles of mitogen-activated
protein kinase (MAPK) and Jun N-terminal kinase (JNK) signaling
cascades in G12-induced G1 to S phase cell cycle
progression in NIH3T3(M17) fibroblasts. Transient expression of a
constitutively active mutant of G
12, G
12(R203C), resulted in a
2-fold increase in the number of bromodeoxyuridine-positive S phase
cells over vector control level under serum-deprived conditions. Consistent with the ability of G
12(R203C) to induce G1/S
transition, its expression led to a 2-fold increase in cyclin A
promoter activity, which showed a marked synergism with a low
concentration of serum, resulting in up to a 15-fold elevation over the
basal level. In addition, G
12(R203C) caused a 2-fold stimulation in
E2F-mediated transactivation. Wild type G
12 showed similar
stimulatory effects on cyclin A promoter activity and E2F-mediated
transactivation, although of lesser magnitude. We observed a modest but
constitutive activation of MAPK in cells transfected with
G
12(R203C), which was abolished by a dominant negative form of Ras.
G
12(R203C) also induced a 3-fold increase in JNK activity, which was
abolished by dominant negative forms of either Rac1 or Ras. The
expression of dominant negative forms of Ras, MAPK, Rac1, or JNK
inhibited G
12(R203C)-induced increases in bromodeoxyuridine-positive
cells. Also, the dominant negative forms of Ras, MAPK, and JNK strongly inhibited G
12(R203C)-induced stimulation of cyclin A promoter activity. These results demonstrate that both the Ras/MAPK and Ras/Rac1/JNK pathways convey necessary, if not sufficient, mitogenic signals induced by G
12 activation.
Heterotrimeric G proteins are critical components in transmembrane
signaling via heptahelical receptors and regulate the activities of a
variety of effector enzymes and ion channels. Heterotrimeric G proteins
are composed of three polypeptides, an subunit, a
subunit, and
a
subunit, the latter two of which form a dimer (1). The G
subunits are a family of over 20 different proteins that share 45-95%
amino acid identity. They have been divided into four classes:
Gs
, Gi
, Gq
, and G
12
(1). The G
12 class includes G
12 and G
13 (2, 3) and is
ubiquitously expressed in mammalian tissues and cells (4). In contrast
to Gs, Gi, and Gq class members for
which functional roles have been well established (1), the biological
activity of the G
12 class is not fully clarified. However,
accumulating evidence suggests that G
12 is involved in cell growth
and transformation (5). First, mutationally activated G
12,
G
12(Q229L), stimulated cell proliferation and induced neoplastic
transformation in NIH3T3 cells (3, 6, 7) and Rat-1 cells (8). Second,
expression cloning of a transforming gene from a human sarcoma cDNA
library resulted in isolation of wild type human G
12, suggesting
that overexpression of wild type G
12 was sufficient to cause
neoplastic transformation in the presence of serum growth factors (3). Third, DNA synthesis stimulated by thrombin and serum, but not basic
fibroblast growth factor, was abrogated by microinjection of
anti-G
12 antibody in 1321N1 astrocytoma cells (9).
The molecular mechanisms by which activated G12 promotes cell growth
and causes transformation have not yet been clearly understood. The
expression of G
12(Q229L) was shown to increase the activity of
Na+/H+ exchanger in COS-1 cells (10). It was
demonstrated for Rat-1 cells stably expressing G
12(Q229L) that the
basal activity of MAPK1 (extracellular
signal-regulated kinase) under serum-starved conditions was not
elevated, but MAPK activation induced by epidermal growth factor or
serum was potentiated, as compared with cells transfected with an empty
vector (8). Very recently, constitutive activation of JNK
(stress-activated protein kinase), a new member of the MAPK family, has
been demonstrated in G
12(Q229L)-transformed NIH3T3 cells (11).
However, it remains unknown whether activation of either of these
signaling pathways contributes to G
12-elicited cell growth or
transformation.
Recent progress in the understanding of mammalian cell cycle regulation
has elucidated the principal molecular mechanism of G1 to S
phase progression (12, 13). A number of studies demonstrate that the
E2F family transcription factors play pivotal roles for entry into the
S phase (14). It was reported that microinjection of E2F1 expression
plasmid into quiescent REF-52 fibroblasts is sufficient to induce DNA
synthesis (15). In G0 and early G1 phases, E2F
is complexed with pRb, the product of the retinoblastoma tumor
suppressor gene, and the pRb-related proteins, p107 and p130. The E2F
complex found during G0 and early G1 phases is
inactive as a transcription activator for E2F target genes (12-14) or
may have transcriptional repressor function (16). In middle to late G1 phase, activation of cyclin-dependent
kinases occurs in a temporally ordered manner, and pRb and the
pRb-related proteins become progressively phosphorylated by activated
cyclin-dependent kinases, resulting in inactivation of
their growth-suppressive function (12, 13). Consequently, E2F is
liberated and transactivates a number of the late G1/S
phase genes, including cyclin E, cyclin A, B-myb, cdc2, ribonucleotide reductase, thymidylate synthase, DNA
polymerase , and E2F1 itself, through consensus E2F
binding sites in their promoter regions (14, 17). Cyclin A thus induced
at the G1/S border forms complexes with its catalytic
partner Cdk2. Accumulated evidence indicates that the cyclin A-Cdk2
complex mediates initiation and progression of the S phase (12, 13). It
has also been demonstrated that MAPK is necessary for growth
factor-stimulated G1/S progression (18). However, it is
poorly understood how MAPK activation leads to activation of the
critical events in late G1, i.e. E2F activation
and induction of the E2F target genes that are necessary for
G1 to S progression. Information about how JNK is involved
in G1 to S progression is even more scanty.
In the present study, we observed that the expression of the G12
mutant, G
12(R203C), caused activation of both MAPK and JNK and
stimulation of S phase entry in NIH3T3 fibroblasts. The mutation
corresponding to G
12(R203C) in Gs
,
Gi
2, and Gq
renders them constitutively
active and oncogenic (19-22). We evaluated the role of the MAPK and
the JNK signaling cascades in G
12(R203C)-induced G1 to S
phase progression. We further explored the involvement of the Ras and
Rho subfamilies of low molecular weight G proteins in
G
12(R203C)-induced activation of MAPK and JNK and tried to elucidate
whether E2F-mediated transactivation and cyclin A gene expression, two
major events occurring at the G1/S boundary, are downstream
targets of these signaling molecules. The present results demonstrate
that the expression of G
12(R203C) leads to constitutive activation
of MAPK and JNK in a small G protein-dependent manner and
that both Ras/MAPK and Ras/Rac1/JNK pathways are indispensable for
G
12(R203C)-evoked activation of G1/S gene expression and S phase entry.
A 219-bp fragment
(679-891 when the A residue of the initiation codon ATG is numbered as
1) of rat G12 was obtained from the cDNAs reverse-transcribed
from rat liver poly(A)+ RNA by PCR amplification using two
sets of degenerate oligonucleotide primers corresponding to the two
six-amino acid sequences (182DVGGQR and
290FLNKQD in Gs
) conserved between the
Gs
and Gi
class members that were
designed by Strathmann et al. (24). The sense primer was
GTCTAGAGA(C/T)GTC(A/C/G/T)GG(A/C/G/T)GG(A/G)(A/C)G, and the antisense
primer was GGAATTC(A/G)TC(C/T)TT(C/T)TT(A/G)TT(A/C/G/T)AG(A/G)AA. The
conditions for the PCR were 1 min at 94 °C, 1.5 min at 37 °C, and
2 min at 72 °C. A full-length G
12 cDNA was isolated form a
rat brain
ZAPII cDNA library (Stratagene) by hybridization screening using the 219-bp PCR fragment as a probe under high stringency condition (at 42 °C in the presence of 50% formamide and
0.9 M NaCl). The nucleotide sequence of the cloned cDNA
insert was sequenced by the dideoxy chain termination method with
Sequenase (U.S. Biochemical Corp.). The nucleotide sequence of rat
G
12 has been deposited in the GenBankTM/EMBL Data Bank
with accession number D85760[GenBank]. A constitutively active mutant of G
12,
G
12(R203C), was created with a site-directed mutagenesis kit
(Muta-Gene M13, Bio-Rad) according to the manufacturer's instructions.
The mutagenic primer was CATCCTGTTGGCATGCAAGGACACCAAG. The created
mutation was confirmed by DNA sequencing. It was previously demonstrated for Gs
, Gi
2, and
Gq
that substitution of the corresponding arginine with
cysteine makes the G
proteins constitutively active (19-23). Wild
type G
12 and G
12(R203C) were subcloned into the EcoRI
site of pMT2 expression vector (a generous gift from Dr. Kaufman at
Genetics Institute, Cambridge, MA).
NIH3T3(M17) (a gift from Dr. G. M. Cooper at Harvard Medical School, Boston, MA) is a subclone of NIH3T3 fibroblasts in which the expression of the dominant negative mutant of Ras, Ras(N17), is induced by dexamethasone treatment (25). The cells were maintained in DMEM supplemented with 5% iron-enriched calf serum (25) and 200 µg/ml Geneticin at 37 °C in subconfluent states.
pSV-gal, the expression plasmid for
-galactosidase, was purchased
from Promega. pactEF-MAPK and pactEF-MAPK-DN, the expression vectors of
Xenopus MAPK and its dominant negative form with
Asp170 to Ala substitution, respectively, were kindly
provided by Dr. Okazaki (26) (Kurume University Institute of Life
Science, Kurume, Japan). The expression vector for a Myc epitope
(EQKLISEEDL)-tagged MAPK (pME18S-Myc MAPK) was created by a PCR-based
method (27). The cDNAs of human JNK1 with a Myc epitope tag at its
N terminus and human Rac1 were obtained from a human WI-38 fibroblast
cDNA library by PCR amplification. The cDNA of dominant
negative JNK1 (JNK-DN) with Thr183 to Ala and
Tyr185 to Phe substitutions (28) was generated by a
PCR-based method (27). The cDNAs of Myc epitope-tagged MAPK and
dominant negative Rac1 (Rac1(N17)) were also prepared by a PCR-based
method (27). The nucleotide sequences of the cDNAs obtained by the
PCR method were confirmed by sequencing with an ALFred DNA sequencer
(Pharmacia Biotech Inc.). The cDNAs were subcloned into the
EcoRI site or the BstX1 site of a mammalian
expression vector, pME18S (generously provided by Dr. Maruyama at the
Institute of Medical Science, University of Tokyo, Tokyo, Japan). A
luciferase reporter vector, E2F-Luc, was created as described
previously by Slansky et al. (29). The oligonucleotide
5
-CTAGCAGCTGCTGCGATTTCGCGCCAAACTTGACG-3
, which contains a
20 to +9
from the dihydrofolate reductase promoter plus a PvuII site
for screening and XhoI and NheI sites at the 5
-
and 3
-ends, was inserted into the vector pGL3basic (Promega). A
2.5-kilobase pair BglII fragment of the cyclin A genomic DNA was isolated from a human leukocyte genomic library (Clontech). A
luciferase reporter vector, CycA-Luc, was constructed by ligating the
blunted 1.3-kilobase pair HindIII-SmaI fragment
of cyclin A genomic DNA (30) to pGL2 basic vector (Promega) at the
SmaI site. The bacterial expression plasmid of truncated
c-Jun (amino acids 5-89) fused to glutathione
S-transferase, pGEX-2T-c-Jun-(5-89), was kindly provided by
Dr. A. Kraft (University of Alabama School of Medicine, Birmingham,
AL). The plasmids were purified by two cycles of CsCl2
density gradient centrifugation and introduced into cells by the
calcium phosphate precipitation procedure. The day after transfection,
the cells were serum-deprived by incubating in DMEM containing 0.2%
bovine serum albumin for 24 h. To induce Ras(N17), dexamethasone
(5 × 10
7 M) was added into media at
least 8 h prior to each experiment (25).
Two or three days after transfection,
as indicated in the figure legends, the cells were washed twice with
Ca2+- and Mg2+-free Dulbecco's
phosphate-buffered saline and lysed in 2 × SDS sample buffer.
Gel-loaded sample volumes were adjusted on the basis of protein
concentration (5) determined on parallel dishes so that an equal amount
of total cellular protein was loaded onto gels per well. Proteins were
separated on SDS-10% polyacrylamide gel electrophoresis and
transferred onto Immobilon P membranes (Millipore Corp.). Western blot
analysis was performed by probing with anti-G12 antibody (Santa
Cruz) or a mouse monoclonal anti-Myc epitope antibody (9E10), which
recognized the N-terminal amino acid sequence (EQKLISEEDL) of c-Myc,
and respective alkaline phosphatase-conjugated secondary antibody
(Zymed).
NIH3T3 cells, seeded at 1 × 105 cells/35-mm dish, were co-transfected with pSV-gal
and various expression plasmids at a total of 3 µg of DNA/dish as
indicated in the figure legends. The cells were serum-deprived in the
presence or absence of dexamethasone. Then 10 mM of
BrdUrd (Boehringer Mannheim) was added and further incubated for
17 h in the presence or absence of dexamethasone. The cells
were washed with Ca2+- and Mg2+-free
Dulbecco's phosphate-buffered saline, fixed in 3.7% formaldehyde, and
permeabilized in 0.25% Triton X-100. Cells were first incubated with a
rabbit polyclonal anti-
-galactosidase antibody (Cappel) and then
with a rhodamine-conjugated goat anti-rabbit IgG antibody (Cappel).
After fixation in 3.7% formalin and treatment in 1.5 N HCl
(31), BrdUrd was probed sequentially with a mouse monoclonal anti-BrdUrd antibody (Sigma) and a fluorescein
isothiocyanate-conjugated rabbit anti-mouse IgG antibody (Zymed). Each
incubation was performed according to the manufacturers'
recommendations. More than 200
-galactosidase-positive (transfected)
cells were examined, and BrdUrd-positive cells were counted under a
fluorescent microscope (Olympus, Tokyo, Japan).
NIH3T3(M17) cells (seeded at 5 × 104/well in 12-well plates) were co-transfected with expression plasmids and either CycA-Luc or E2F-Luc (a total of 2 µg of DNA/well), serum-deprived for 24 h, and then incubated in DMEM containing 0.2% bovine serum albumin with or without calf serum (0.5%) and dexamethasone for 17 h. Cell lysates were prepared, and luciferase activity was measured with a Lumat LB95001 luminometer (Berthold) using the luciferase assay system (Promega). Protein concentrations of the same samples were determined using Bio-Rad protein assay reagent, and luciferase activity was normalized for protein content.
Measurement of MAPK ActivityNIH3T3(M17) cells in 35-mm
dishes were co-transfected with pME18S-Myc-MAPK and either
pMT2-G12(R203C) or pMT2 empty vector (a total of 3 µg of
DNA/dish). After incubation for 24 h in DMEM containing 0.2%
bovine serum albumin in the presence or absence of dexamethasone
(5 × 10
7 M), the cells were lysed in a
lysis buffer containing 50 mM Tris (pH 8.0), 1 mM EDTA, 150 mM NaCl, 1 mM
Na3VO4, 1 mM phenylmethylsulfonyl fluoride, 0.5% sodium deoxycholate, 0.1% SDS, and 1% NP-40.
Myc-tagged MAPK was immunoprecipitated by using 9E10 anti-Myc epitope
antibody. MAPK activity associated with the immune complex was assayed
in vitro at 30 °C for 10 min using myelin basic protein
(Sigma) as a substrate as described (32). The reaction was terminated
by adding 4 × SDS sample buffer, and samples were analyzed on
SDS-15% polyacrylamide gel electrophoresis followed by
autoradiography. The radioactivity in the band corresponding to myelin
basic protein was determined with a Fuji BAS 2000 Bio-Image Analyzer
(Fuji Film Co., Tokyo, Japan).
NIH3T3(M17) cells in 35-mm
dishes were co-transfected with pME18S-Myc-JNK and indicated expression
plasmids (a total of 3 µg/dish). The cells were deprived of serum for
24 h and then an immune complex JNK assay was performed as
described by Derijard et al. (33) with a slight
modification. Briefly, cells were lysed in an ice-cold lysis buffer (25 mM HEPES (pH 7.5), 1% Triton X-100, 20 mM Tris
(pH 7.6), 0.5% Nonidet P-40, 500 mM NaCl, 50 mM NaF, 5 mM EDTA, 3 mM EGTA, 1 mM Na3VO4, 10 µg/ml each of
leupeptin and aprotinin, and 1 mM phenylmethylsulfonyl
fluoride). A soluble fraction was obtained by centrifugation and
precleared with protein A-conjugated Sepharose 4B beads (Pharmacia).
The supernatant was incubated with 9E10 antibody and subsequently with
rabbit anti-mouse IgG (Zymed)-bound protein A-Sepharose beads. The
immunoprecipitates were washed three times with the lysis buffer and
twice with a kinase assay buffer (20 mM HEPES (pH 7.6), 20 mM MgCl2, 20 mM -glycerophosphate, 20 mM p-nitrophenyl
phosphate, 2 mM dithiothreitol, 0.1 mM
Na3VO4). The pelleted beads were incubated with
30 µl of the kinase assay buffer containing 3 µg of
GST-c-Jun-(5-89), 20 µM ATP, and 5 µCi of
[
-32P]ATP at 30 °C for 20 min. The reaction was
terminated by adding 10 µl of 4 × Laemmli's SDS-sample buffer
and boiled. The samples were analyzed by SDS-12% polyacrylamide gel
electrophoresis, and the radioactivity in the band corresponding to
GST-c-Jun-(5-89) was measured.
A
2.1-kilobase pair cDNA clone containing the entire coding region of
rat G12 was isolated. Sequence analysis of this clone revealed that
the predicted amino acid sequence was identical to that of mouse G
12
except for one residue (341), where serine in mouse G
12 is changed
to glycine in rat G
12. NIH3T3(M17) cells were transiently
transfected with expression plasmids carrying either wild type G
12
or the constitutively active mutant G
12(R203C). The expression of
either form was confirmed by Western blot analysis using anti-G
12
antibody (Fig. 1), which detected expression of 45-kDa
proteins corresponding to G
12 and G
12(R203C) proteins. A trace
level of endogenous G
12 was detected in empty vector-transfected control cells.
G
Previous studies demonstrated that expression of
G12(Q229L), another constitutively active mutant with
deficient GTPase activity, resulted in an increased thymidine
incorporation into DNA (8). To evaluate whether G
12(R203C) has a
similar stimulatory effect on cell cycle progression, cells were
transfected with pMT2-G
12(R203C), pMT2-G
12, or pMT2 empty vector
together with a
-galactosidase expression plasmid, and BrdUrd
incorporation into nuclei in a
-galactosidase-positive cell
population was determined (Fig. 2). Under serum-deprived
conditions, the expression of G
12(R203C) resulted in an
approximately 2-fold increase in BrdUrd-positive cells over the vector
control level. The stimulatory effect of G
12(R203C) on S phase
progression was also evident in the presence of a low concentration of
serum (0.5%). The expression of wild type G
12 tended to increase
BrdUrd-positive cells in the presence and absence of serum but without
statistical significance.
Recent investigations have elucidated that G1 to S phase
progression requires activation of E2F family transcription factors, a
process dependent on cyclin-dependent kinase-mediated
phosphorylation and inactivation of pRb family proteins (12-14). It is
also known that S phase entry is associated with transcriptional
activation of the cyclin A gene (12, 13). To investigate whether or not activation of G12 elicits these events, we performed a series of
co-transfection experiments using luciferase-reporter plasmids. As
shown in Fig. 3 (left), under serum-deprived
conditions, transfection of G
12(R203C) resulted in a 2-fold increase
in transactivation of a luciferase gene that is under the control of a
consensus E2F binding site in the 5
-upstream region. It was of note
that transfection of wild type G
12 also resulted in stimulation of E2F activity, although to a smaller extent. In the presence of a low
concentration of serum, the basal luciferase activity was slightly
stimulated as compared with serum-deprived cells. Under this
condition, wild type G
12 and G
12(R203C) also caused
stimulation of luciferase activity over the vector control level.
Shown in Fig. 3 (right) is G
12-induced stimulation of
cyclin A promoter activity. Transfection of G
12(R203C) resulted in a
weak but significant increase in luciferase activity in the absence of
serum stimulation, which was potentiated by a low concentration of
serum in a synergistic manner. Again, the wild type G
12 showed
stimulatory effects, although to a lesser extent. These results
demonstrate that G
12(R203)-induced G1/S transition is
associated with stimulation of E2F activity and up-regulation of cyclin
A gene transcription.
G
We evaluated whether G12(R203C) activates MAPK in a
transient co-transfection assay with a Myc epitope-tagged MAPK (Fig. 4). In cells transfected with G
12(R203C), MAPK
activity showed a 1.5-fold increase over the vector control in the
absence of external growth factors. This increase was statistically
significant (p < 0.01) and reproducible. By taking
advantage of inducible expression of the dominant negative mutant of
Ras(N17) by dexamethasone treatment, we examined whether
G
12(R203C)-induced activation of MAPK is dependent on Ras. When
Ras(N17) was induced, MAPK activity in G
12(R203C)-transfected cells
was not significantly different from the vector control level. Western
blot analysis using anti-Myc epitope antibody revealed that the
expression level of a Myc epitope-tagged MAPK was not changed by
co-expression of Ras(N17) and/or G
12(R203C). The results demonstrate
that G
12(R203C) induces stimulation of MAPK in a
Ras-dependent manner.
G
Very recently Prasad et
al. (11) have reported that G12(Q229L) activates JNK. We
examined whether G
12(R203C) activated JNK and, if so, whether
G
12(R203C)-induced JNK activation was dependent on the small G
proteins, Ras and Rac. As shown in Fig. 5A,
co-transfection of G
12(R203C) and a Myc epitope-tagged JNK resulted
in an approximately 2.5-fold increase in JNK activity over the vector
control level. G
12(R203C)-induced activation of JNK was completely
abolished when Ras(N17) was expressed. In addition,
G
12(R203C)-induced JNK activation was entirely dependent on Rac1, as
demonstrated by the inhibition of JNK activation by the dominant
negative Rac1(N17). Western blot analysis using anti-Myc epitope
antibody revealed that the expression level of a Myc epitope-tagged JNK
was not changed by co-expression of either Rac1(N17) or Ras(N17) (Fig.
5B).
G
We investigated whether Ras, MAPK, and JNK are involved in
G1/S cell cycle progression evoked by G12. To this end,
we studied the effects of expression of dominant negative forms of
these signaling molecules on G
12(R203C)-induced activation of
cyclin A promoter and BrdUrd incorporation into DNA. As shown in
Fig. 6A, stimulation of cyclin A promoter
activity by activated G
12 was completely abolished by induced
expression of Ras(N17), both in the presence and absence of a low
concentration of serum. The dominant negative MAPK also strongly
inhibited the stimulatory effect of G
12(R203C) down to the
serum-starved vector control level (Fig. 6B). Similarly,
co-expression of the dominant negative JNK potently inhibited
G
12(R203C)-induced cyclin A promoter activation (Fig.
6C).
G
As described in Fig.
2, BrdUrd-positive cells among the -galactosidase-positive
population were monitored (Table I). The expression of
either dominant negative MAPK or Ras(N17) strongly inhibited basal and
G
12(R203C)-induced BrdUrd incorporation into nuclei. Both dominant
negative JNK and Rac1(N17) also inhibited basal and
G
12(R203C)-induced BrdUrd incorporation; however, they were
less potent as compared with dominant negative MAPK and Ras(N17). We
confirmed the expression of the dominant negative proteins by Western
blot analysis of samples from parallel cultures using specific
antibodies (data not shown). These results clearly indicate that
activation of Ras/MAPK and Ras/Rac1/JNK pathways are both necessary for
G
12(R203C)-induced G1 to S phase progression.
|
In the present work we studied the mitogenic signaling in
G12(R203C)-induced G1 to S phase cell cycle progression.
The mutation corresponding to G
12(R203C) in Gs
renders it constitutively active and oncogenic (gsp) (20).
It is also the site of ADP-ribosylation by cholera toxin, which
activates it (19). The corresponding mutation in Gi
2
also activates it (21) and creates the gip2 oncogene (22).
Finally, the corresponding mutation in Gq
renders it
constitutively active (23). As a general mechanism, the mutation causes
activation by decreasing the GTPase activity (19, 20). In view of these
findings, we think it is reasonable to infer that the R203C mutation in
G
12 would also render it constitutively active, particularly since
the data are consistent with this conclusion. The present study
demonstrated that G
12(R203C) stimulated G1 to S phase
cell cycle progression in NIH3T3 cells in a manner dependent upon both
MAPK and JNK cascades. Consistent with the ability of G
12(R203C) to
promote G1 to S progression, we observed that the
expression of G
12(R203C) led to stimulations of E2F-mediated transactivation and the cyclin A promoter activity, the latter of which
was also dependent upon both MAPK and JNK. In addition, we found that
the small G protein Ras was required for G
12(R203C)-induced activation of MAPK, while both Ras and Rac were required for JNK activation and S phase entry. The results clearly indicate the critically important roles of both Ras/MAPK and Ras/Rac/JNK cascades in
activated G
12-induced G1/S cell cycle progression.
Besides well studied growth factor ligands for receptor
protein-tyrosine kinases (34), certain agonists for G protein-coupled receptors, including thrombin, lysophosphatidic acid, and
gastrin-releasing peptide/bombesin, are capable of stimulating
mitogenesis (35-37). Several previous studies demonstrated that
pertussis toxin caused substantial inhibition of thrombin- and
lysophosphatidic acid-induced mitogenesis in fibroblasts, indicating a
role for a heterotrimeric G protein of the Gi or
Go classes (36-38). Subsequent studies demonstrated that
activation of pertussis toxin-sensitive G proteins by these ligands led
to activation of Ras, which was shown to mediate stimulation of DNA
synthesis (39-41). On the other hand, there are also many reports
describing that agonists for G protein-coupled receptors stimulate DNA
synthesis through pertussis toxin-insensitive G proteins (1, 42). In
addition, more recent studies demonstrated that thrombin and
angiotensin II can activate Ras through a pertussis toxin-insensitive G
protein in astroglial cells (9, 43) and cardiac myocytes (44). A very
recent study by Aragay et al. (9) using microinjection of
anti-G12 antibody indicated that G
12 functions as a pertussis
toxin-insensitive G protein that mediates thrombin-induced,
Ras-dependent mitogenesis in human astroglial cells.
Consistent with this report, the present results directly document that
G
12 causes stimulation of DNA synthesis in a
Ras-dependent manner (Table I). In view of the ubiquitous expression of G
12 in various tissues and cell types (4), it is a
likely possibility that G
12 is involved in the pertussis toxin-insensitive, Ras-dependent mitogenesis evoked by G
protein-coupled receptor agonists in a variety of cell types.
There is now much evidence for the notion that Ras activates multiple
signaling pathways for cell proliferation and differentiation. The
Raf/MEK/MAPK cascade is the best characterized example of Ras-dependent signaling. The activation of the MAPK cascade
is critically important for growth factor-induced G1/S
progression, because expression of dominant negative forms of MEK (18)
and a synthetic inhibitor of MEK (45) were shown to inhibit growth factor-induced DNA synthesis, and, conversely, expression of a constitutively active MEK induced cell proliferation and transformation (18, 46). It is known that activation of MAPK can be brought about by
both Ras-dependent and -independent mechanisms in response to various external stimuli (18, 39-41, 43, 44, 47). The present study
demonstrated that the expression of G12(R203C) led to activation of
MAPK via a Ras-dependent mechanisms (Fig. 4).
G
12(R203C)-induced activation of MAPK is not as potent as that
observed with acute growth factor stimulation but is very likely
sustained, i.e. still evident 2 days after transfection. A
previous study demonstrated that in fibroblasts stably expressing activated G
12, MAPK activity was not increased under serum-deprived conditions as compared with cells transfected with an empty vector, although epidermal growth factor-stimulated MAPK activation was enhanced in G
12-transformed cells (8). The reason for the discrepancy between the previous report and the present result is not
clear. However, one reason might be that we adopted a transient transfection method with the G
12(R203C) expression vector and the
Myc-tagged MAPK expression vector. This may have allowed for the
detection of a small increase in MAPK in the present study. The
activation of MAPK in G
12(R203C)-transfected cells appears to be
critically important for G1/S cell cycle progression,
because the dominant negative MAPK totally abolished
G
12(R203C)-induced DNA synthesis in the present study (Table I). It
is poorly understood how the MAPK functions in mitogenic signaling,
although the MAPK cascade was shown to be associated with activation of
several different transcription factors including the Ets family
proteins (48, 49). In the present study we demonstrated for the first time that MAPK is involved in activation of cyclin A promoter. It is
now established that E2F functions as a crucial cell cycle-specific transcription factor to activate a number of genes (including cyclin A)
that control cell cycle progression (14, 17). Therefore, G
12(R203C)-induced activation of E2F is likely an important
mechanism underlying G
12(R203C)-evoked G1/S progression.
Accumulated evidence indicates that E2F activation is brought about by
phosphorylation of pRb and its related proteins by
cyclin-dependent kinases (12-14). In middle to late
G1, cyclin D-Cdk4 or cyclin D-Cdk6 complex is first
activated to initiate phosphorylation of pRb family proteins. With
regard to the activation mechanisms of Cdk4 or Cdk6, a very recent
study demonstrated that a dominant negative MAPK inhibited epidermal
growth factor-induced activation of cyclin D1 promoter (50). Since the
induction of cyclin D1 in middle to late G1 is a
prerequisite for activation of Cdk4 and/or Cdk6 (12-14), which in turn
activates E2F, MAPK-mediated cyclin D1 induction may be an upstream
mechanism for MAPK-mediated cyclin A induction at the G1/S
boundary.
Recent evidence suggests that Ras may activate a second signaling
pathway for cell proliferation and transformation, which involves Rac,
a member of the Rho subfamily of small G proteins. Several studies
demonstrated that expression of a dominant negative form of Rac1
blocked transformation by oncogenic Ras, suggesting that Rac functions
downstream of Ras (51, 52). It was also shown that an activated form of
Rac1 markedly synergized with activated Raf to enhance transformation
(52). Further, it was demonstrated that microinjection of activated
forms of Rac into quiescent fibroblasts stimulated G1/S
cell cycle progression, whereas expression of a dominant negative form
of Rac1 blocked serum-induced DNA synthesis (53). These observations
suggest that in addition to the Raf/MEK/MAPK cascade, the Rac1
signaling cascade is required for Ras-mediated cell proliferation and
transformation. In the present study we have shown that
G12(R203C)-induced G1 to S progression is also dependent
upon Rac (Table I). Recent studies identified JNK as a downstream
effector of Rac (54, 55). JNK phosphorylates and activates the
transcription factors c-Jun and ATF-2, which results in induction of
c-Jun itself via two AP-1 sites in its promoter region (28, 54, 55). We
demonstrated by employing a dominant negative form of JNK (Figs. 4 and
5, Table I) that JNK is actually involved in the
Rac-dependent mitogenic signaling. Previous studies
demonstrated that c-Jun, in conjunction with several related AP-1
proteins, promotes G1 phase progression and S phase entry
(56, 57). The important AP-1 target genes implicated in G1
to S progression include cyclin D1 (50, 56). Therefore,
G
12(R203C)-induced, JNK-mediated cyclin A promoter activation (Fig.
4) may be mediated partly by cyclin D1 induction via the AP-1 site.
Thus, both MAPK and JNK appear to be involved in sequential induction
of cyclins and resultant activation of cyclin-dependent
kinases at G1 and S.
Recent data demonstrate that subunits of heterotrimeric G
proteins can mediate activation of both MAPK and JNK in a
Ras-dependent manner (58, 59). However, the present study
demonstrated that the
subunit of G
12 alone is able to activate
MAPK and JNK in Ras- and Ras/Rac-dependent manners,
respectively. The Ras dependence of G
12- or G
13-induced JNK
activation was also recently shown by another group (11). These
observations provide compelling evidence that there is a
Ras-dependent pathway activated by the
subunits of G12
and G13. The direct effector of G
12 in terms of Ras activation is
presently unknown. Recent studies have demonstrated that
Gq-coupled receptor agonists activate Ras via activation of
nonreceptor tyrosine kinases through a mechanism involving tyrosyl
phosphorylation of Shc, a SH2 domain-containing adaptor protein (44,
60). Shc thus phosphorylated at specific tyrosyl residues mediates
recruitment of the Ras GDP/GTP exchange factor mSOS to the plasma
membrane (61). Rac1 activation that appears to be required for
G
12-induced JNK activation is probably caused by recruitment and
activation of a GDP/GTP exchange factor for Rac1. The activation of Ras
might be necessary for the activation of the Rac1 GDP/GTP exchange
factor. Further work will be required to understand the G
12 effector
pathway more thoroughly.
We thank R. Nakanishi, F. Iwase, and W. Zhou for technical and secretarial assistance. We also thank Drs. G. M. Cooper, A. Kraft, and K. Okazaki for giving us NIH3T3 cells (M17), pGEX-2T-c-Jun-(5-89), and pactEF-MAPK and pactEF-MAPK-DN, respectively.