(Received for publication, March 30, 1995; and in revised form, May 19, 1995)
From the
Signal transduction pathways regulated by G
The Activation of the extracellular response kinase/mitogen-activated
protein kinase (ERK/MAPK) pathway has been strongly correlated with
growth and transforming activities in fibroblasts(11) .
Expression of The JNK/SAPKs phosphorylate and activate c-Jun, the basal
transcription factor involved in the regulation of different phases of
cell growth, differentiation, and other physiological
responses(18) . Previously, it was demonstrated that
Expression of GTPase-inhibited mutants of
Figure 1:
Constitutive activation of JNK/SAPK in
Figure 2:
Constitutive activation of JNK/SAPK in
multiple clones of
In contrast to the constitutive activation of
JNK/SAPK, the ERK/MAPK pathway was not activated. Our results indicated
that the ERK1 and ERK2 activities of NIH 3T3 cells expressing
Figure 3:
Effect of
Further demonstrating that the activation of JNK/SAPK is mediated by
the expression of the activated
Figure 4:
Activation of JNK/SAPK by
In
addition to the inducible vector system, we also transiently expressed
Figure 5:
Effect of transient expression of
Figure 6:
Effect of constitutively activated
Diverse stimuli appear to activate ERKs and JNKs through different
signaling
pathways(11, 14, 15, 16, 17, 20, 21, 23) .
Ras appears to play a pivotal role in the activation of both ERK/MAPK
and JNK/SAPKs in response to specific growth factors and
hormones(13, 15, 21) . Ras is capable of
activating JNK/SAPK through its activation of MAPK/ERK kinase kinase
1(21) . MAPK/ERK kinase kinase 1 (MEKK1) in turn activates a
dual specificity kinase MAP kinase kinase 4 (MKK4)/SAPK/ERK kinase 1
(SEK1) that consequently activates JNK(24, 25) .
Cytokines such as TNF
Figure 7:
It has been proposed that JNK/SAPKs may be more involved in
the regulation of transmitting growth inhibition rather than mediating
mitogenic responses(24, 25) . However, our studies
reported here present an apparent paradox to this paradigm. The
GTPase-deficient mutants of
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
and
G
heterotrimeric G proteins are largely unknown.
Expression of activated, GTPase-deficient mutants of
and
alter physiological responses such as
Na
/H
exchanger activity, but the
effector pathways controlling these responses have not been defined. We
have found that the expression of GTPase-deficient mutants of
(
Q229L) or
(
Q226L) leads to robust activation of the Jun
kinase/stress-activated protein kinase (JNK/SAPK) pathway. Inducible
Q229L and
Q226L expression vectors
stably transfected in NIH 3T3 cells demonstrated JNK/SAPK activation
but not extracellular response/mitogen-activated protein kinase
activation. Transient transfection of
Q229L and
Q226L also activated the JNK/SAPK pathway in COS-1
cells. Expression of the GTPase-deficient mutant of
(
Q209L) but not
(
Q205L) or
(
Q227L) was also able to activate the JNK/SAPK
pathway. Functional Ras signaling was required for
Q229L and
Q226L activation of the
JNK/SAPK pathway; expression of competitive inhibitory
N
Ras inhibited JNK/SAPK activation in response to both
Q229L and
Q226L. The results
describe for the first time a Ras-dependent signal transduction pathway
involving JNK/SAPK regulated by
and
.
- and the
-subunits of heterotrimeric G
proteins (
)regulate the activity of diverse effectors in
response to the activation of seven transmembrane
receptors(1, 2, 3) . G
-subunits are
grouped based on amino acid homology into four sub-families:
G
, G
, G
, and
G
(3) . Cellular signal transduction pathways
regulated by the G
members,
and
, remain most poorly defined. Several studies suggest
and
regulate signal pathways
involved in controlling cell growth and differentiation (4, 5, 6, 7, 8, 9, 10) .
For example, mutation of cta, a Drosophila homologue
of
disrupts the ventral furrow formation in Drosophila embryos(4) ;
has been
identified as a putative oncogene of soft tissue sarcomas(5) ;
the overexpression of wild type
and
or the expression of activated GTPase-deficient mutants of
(
Q229L) and
(
Q226L) transform fibroblast cell
lines(6, 7, 8, 9, 10) .
Q229L or
Q226L did
not activate ERK/MAPK pathway but may enhance epidermal growth
factor-stimulated ERK/MAPK activity in Rat1 cells(10) .
Expression of an activated mutant of
also does not
activate the ERK/MAPK pathway in COS-1 cells(12) . More
recently, proline-directed protein kinases related to ERK/MAPK referred
to as Jun kinase/stress-activated protein kinases (JNK/SAPK) have been
identified(13, 14, 15, 16, 17) .
Although the role of JNK/SAPKs in the control of cell growth and
differentiation remains unclear at this time, they do appear to be
activated in response to cellular stress and osmotic
imbalance(13, 14, 15, 16, 17) .
Q229L- and
Q226L-transformed NIH
3T3 cells had increased c-Jun expression(9) . Here, we
demonstrate that the expression of
Q229L and
Q226L lead to the activation of JNK/SAPK. The
results demonstrate a Ras-dependent signal transduction pathway
controlled by
and
subunit
proteins.
Plasmids, Cell Culture, and
Transfections
COS-1 and NIH 3T3 cells (ATCC) were
maintained by serial passage in Dulbecco's modified Eagle's
medium (DMEM, Life Technologies, Inc.) containing 10% newborn calf
serum (Life Technologies, Inc.), 50 units/ml penicillin, and 50
µg/ml streptomycin at 37 °C in a 5% CO incubator.
pcDNA3-
Q229L plasmid was constructed by ligating the EcoRI-XbaI (1.8 kilobase) fragment from
pcDNA1-
Q229L into the EcoRI-XbaI
site of pcDNA3 vector (Invitrogen). pcDNA3-
Q226L was
constructed by ligating the BamHI-XbaI fragment (2.4
kilobases) into the BamHI-XbaI site of pcDNA3.
Plasmids were purified using a cesium chloride gradient. The procedures
for the transfection and transformation of NIH 3T3 cells have been
previously described(9) . NIH 3T3 cell lines expressing
inducible
Q229L and
Q226L were
established using the Lac Switch inducible expression system
(Stratagene). Blunt-ended 1.8-kilobase HindIII-XbaI
fragment of
Q229L or the 2.4-kilobase BamHI-XbaI fragment of
Q226L
excised from the respective pcDNA3 vectors was ligated into the blunted NotI site of pOPRSVI plasmid following the published
procedures(19) . The orientation of the constructs was verified
by restriction analysis, and the plasmids were purified. NIH 3T3 cells
(0.75
10
) were cotransfected with p3`SS plasmid
vector expressing the Lac repressor and pOPRSVI-
Q229L
or pOPRSVI-
Q226L vector (5 µg each) by
electroporation. A control NIH 3T3 cell line was established by
cotransfecting p3`SS and pOPRSVI vectors alone. Electroporation was
carried out using a Bio-Rad gene pulser as follows: 4-mm
electroporation cuvette containing 0.75
10
cells in
0.5 ml of DMEM with 10% FBS and 5 µg of plasmid DNA was placed in a
gene pulser chamber, and a pulse was delivered at 0.2 kV and 960
microfarads. After the pulse, the cells were incubated at room
temperature for 5 min, resuspended in 10 ml of DMEM containing 10% FBS,
and plated in a 100-mm culture dish. After 48 h, the cells were split
into 1:4 ratio, and hygromycin (50 µg/ml, Calbiochem) and G-418
(400 µg/ml, Life Technologies, Inc.) were added to select the
transformed cells. The selection was carried out for 3 weeks with
biweekly feedings, and individual clones were isolated and propagated.
The expression of
Q229L and
Q226L
following induction with 1 mM IPTG was determined by Northern
blot analyses using total RNA from these cells. Clones having the
lowest expression of the mutant G
subunit in the absence of IPTG
were selected and used for further experiments. Transfection of COS-1
cells was carried out using lipofectamine reagent (Life Technologies,
Inc.) following the manufacturer's protocol. COS-1 cells (2
10
) were seeded in a 35-mm dish with 2 ml of DMEM
containing 10% FBS. 24 h later, the cells were washed with 2 ml of
serum-free medium. Meanwhile, the respective plasmids (0.5 µg each)
along with 0.5 µg of the reporter plasmid pSV
Gal (Promega)
were taken in 100 µl of DMEM and mixed with 100 µl of DMEM
containing 6 µl of lipofectamine (Life Technologies, Inc.). The
DNA-lipid complexes were allowed to form at room temperature for 30
min. The complex was supplemented with 0.8 ml of DMEM and was added to
the cells. The dishes were incubated at 37 °C for 6 h, after which
1 ml of DMEM with 20% FBS was added, and the incubation was continued.
The medium was replaced at 24 h, and the cells were harvested for
analysis at 60 h post-transfection.
Solid Phase JNK/SAPK Assay
Assay of Jun
kinase activity was carried out following previously published
procedures(20, 21) . Actively growing NIH 3T3 cells (1
10
) were seeded in 100-mm dishes containing DMEM
with 10% newborn calf serum. 24 h later, the cells were serum starved
by replacing them in DMEM containing 0.2% bovine serum albumin. The
cells were allowed to reach quiescence for 16-24 h, and they were
stimulated with or without growth factors. After growth factor
stimulation, the cells were rinsed with ice-cold phosphate-buffered
saline and harvested in 400 µl of lysis buffer containing 25 mM HEPES (pH 7.6), 0.1% Triton X-100, 300 mM NaCl, 1.5
mM MgCl
, 20 mM
-glycerophosphate,
100 µM Na
VO
, 0.2 mM EDTA,
0.5 mM dithiothreitol, 2 µg/ml leupeptin, 2 µg/ml
aprotinin, 100 µM phenylmethylsulfonyl fluoride, and 1
mM benzamidine. The cell lysates were centrifuged for 10 min
in a microcentrifuge (16,000
g), and the supernatants
were normalized for protein content. The solid phase kinase assay was
carried out using GST-c-Jun(1-79) bound glutathione-Sepharose
(Pharmacia Biotech Inc.) beads as substrate.
GST-c-Jun(1-79)-bound glutathione-Sepharose beads were prepared
as previously described(20) . 10 µl of 50%
GST-c-Jun(1-79)-bound glutathione-Sepharose beads were washed
with JNK lysis buffer and incubated with 100 µg of lysate protein
(in 100 µl) for 2 h at 4 °C in a rotator. At the end of the
incubation, the beads were washed twice in JNK lysis buffer followed by
two washes in JNK buffer containing 20 mM HEPES, pH 7.6, 20
mM
-glycerophosphate, 10 mM MgCl
,
and 100 µM Na
VO
. The kinase
reaction was carried out by resuspending the beads in 40 µl of JNK
buffer containing 20 µM
[
-
P]ATP (5000 cpm/pmol) and incubating them
for 20 min at 30 °C. After stopping the reaction with the addition
of Laemmli's buffer followed by boiling the samples for 3 min,
the phosphorylated GST-c-Jun was separated on 12% SDS-PAGE. The gel was
dried, and an autoradiogram was developed. The radioactive GST-c-Jun
band was excised out and quantitated in a liquid scintillation counter.
Immune Complex JNK/SAPK Assay
COS-1 cells
transfected with HA-JNK1 along with test plasmids were subjected to
immune complex JNK assay according to the previously published
procedures(20, 21) . The transfected COS-1 cells were
lysed in 300 µl of JNK lysis buffer, and the lysate was prepared as
described in the case of NIH 3T3 cells. The cell lysates were
normalized for transfection using the expression of -galactosidase
reporter activity(22) . The HA epitope-tagged JNK1 in 100
µg of cell extract was immunoprecipitated by incubating the lysate
with 1 µg of monoclonal (12CA5) HA antibodies (Boehringer Mannheim)
for 1 h. This was followed by an additional incubation with 20 µl
of protein A-Sepharose (Pharmacia) for 1 h. The protein A-Sepharose
beads were washed twice with JNK lysis buffer followed by two washes
with JNK reaction buffer. The beads were resuspended in 40 µl of
JNK buffer containing 20 µM [
-
P]ATP (5000 cpm/pmol), and the
kinase reaction was carried out for 20 min at 30 °C using 3 µg
of purified GST-c-Jun(1-79) as substrate. The reaction was
stopped, and the radioactivity in the phosphorylated GST-c-Jun was
quantitated as described above.
ERK1/2 Assay
Cell extracts were prepared
as described above using ERK lysis buffer containing 20 mM HEPES (pH 7.4), 50 mM -glycerophosphate, 0.5% Triton
X-100, 2 mM MgCl
, 1 mM EGTA, 1 mM dithiothreitol, 2 µg/ml leupeptin, 2 µg/ml aprotinin, 100
µM phenylmethylsulfonyl fluoride, and 1 mM benzamidine. ERK1 or ERK2 in 100 µg of cell lysate was
immunoprecipitated by incubating 100 µg of lysate protein with 1
µg of the respective polyclonal antibodies (antibodies sc-19 and
sc-154 to ERK1 and ERK2, respectively, are from Santa Cruz
Biotechnology, Inc., Santa Cruz, CA) for 1 h followed by an additional
incubation with 20 µl of protein A-Sepharose (Pharmacia) for 1 h.
The immune complex-bound protein A-Sepharose beads were washed twice
with lysis buffer and twice with ERK/MAPK buffer containing 20 mM HEPES (pH 7.4), 50 mM
-glycerophosphate, 10 mM MgCl
, 1 mM EGTA, 1 mM
dithiothreitol, and 100 µM Na
VO
.
The beads were resuspended in 40 µl of MAPK buffer containing 100
µM [
-
P]ATP (5000 cpm/pmol),
and the kinase assay was carried out for 20 min at 30 °C using 5
µg of myelin basic protein as substrate. The reaction was stopped
by the addition of Laemmli's sample buffer followed by the
boiling of the samples for 3 min. The proteins were resolved on 12%
SDS-PAGE; the gel was dried, and an autoradiogram was developed.
Northern Blot Analysis
Northern analysis
was performed following previously published
methods(9, 22) . Total RNA from NIH 3T3 cells was
prepared using 10 cells, and the
and
probes (50 ng) were labeled with
[
P]dCTP as previously
described(9, 22) . Total RNA (20 µg) was resolved
on a denaturing 1% agarose and 2.2 M formaldehyde gel. The RNA
was blotted onto a zeta probe-GT membrane using a vacuum blotter and
cross-linked to the membrane by UV; the blot was then probed with the
cDNA probes. Other general analytical procedures were carried out as
previously described (9, 22) .
-subunit
results in the constitutive activation of the signaling pathway
regulated by the respective
-subunit. Hence, the cells expressing
the GTPase-deficient mutants of
(
Q229L) and
(
Q226L) were analyzed for the constitutive
activation of any of the known G protein-coupled signaling pathways.
Stable expression of either of these mutant
-subunits readily
confers transforming ability to fibroblast cell lines such as NIH 3T3
or Rat1. However, these studies failed to identify a constitutively
activated
- or
-specific signaling
pathway(6, 7, 8, 9, 10) .
To investigate whether the expression of
Q229L or
Q226L activates JNK/SAPK pathway, we determined
JNK/SAPK activity in vector-transfected and
Q229L-
and
Q226L-transformed NIH 3T3 cells. JNK/SAPK
activities were determined in the lysates from these cells by a solid
phase kinase assay using GST-c-Jun(1-79) fusion protein
immobilized on glutathione-Sepharose beads(21) . Our results
using this assay indicated that the JNKs were constitutively activated
in both
- and
-transformed 3T3
cells (Fig. 1). JNK responses to growth factors in
Q229L and
Q226L transformants were
analyzed by stimulating cells with 10% FCS or FGF (5 ng/ml). FGF or
serum stimulation of JNK/SAPK was only weak in control NIH 3T3 cells.
FGF and FCS only modestly or not at all enhanced JNK/SAPK activation
over that observed with the expression of
Q229L or
Q226L alone (Fig. 1). JNK/SAPK activation was
found in eight of eight
Q229L- and six of six
Q226L-transformed clones, indicating this is a
response reproducibly correlated with expression of activated
and
(Fig. 2, A and B). The differences in JNK/SAPK activation between
Q229L and
Q226L clones in the
presence of FCS may be indicative of the subtle differences in the
signaling pathways regulated by these
-subunits ( Fig. 1and Fig. 2).
Q229L- and
Q226L-transformed NIH
3T3 cells. The cell lysates from vector control,
Q229L- and
Q226L-transformed NIH
3T3 cells, were subjected to a solid phase JNK assay using
glutathione-Sepharose-bound GST-c-Jun(1-79) as described under
``Experimental Procedures.'' Similar sets of cells were
either stimulated with 10% FBS or 5 ng/ml basic FGF. The phosphorylated
proteins were resolved on SDS-PAGE and visualized by autoradiography (upper panel). The radioactive bands were cut and counted to
quantify the JNK activity. JNK activity is presented as the cpm in the
radioactive GST-c-Jun(1-79) band (lower panel). The
experiment was repeated a minimum of six times, and the results are
from a typical experiment.
Q229L- and
Q226L-transformed NIH 3T3 cells. Different clonal
isolates of
Q229L (A) and
Q226L transformants (B) were analyzed for
JNK activities. Cells were processed for JNK solid phase JNK assay as
described under ``Experimental Procedures.'' Following 24 h
serum starvation, these cells were challenged with (openbars) or without (hatchedbars) 10%
FCS. The phosphorylated protein was separated by SDS-PAGE, an
autoradiogram was developed, and the radioactive bands were cut and
counted. The JNK activity was presented as fold stimulation over
non-FCS-stimulated control groups. This experiment was repeated three
times.
Q229L or
Q226L were not altered (Fig. 3). Their activities determined in
Q229L
or
Q226L transformants were comparable to the control
cells. Both serum and FGF stimulated the basal ERK/MAPK activities in
all the cells, suggesting that the
or
pathway does not have a significant role in stimulating or
inhibiting the ERK/MAPK pathway in NIH 3T3 cells (Fig. 3).
Q229L and
Q226L expression on ERK/MAPK activity. The pcDNA3
transfected NIH 3T3 cells along with
Q229L and
Q226L transformed cells were processed for ERK/MAPK
immune complex kinase assay as described under ``Experimental
Procedures'' using myelin basic protein (MBP) as a substrate. The
basal and serum- and FGF-stimulated activities were assayed as
indicated. The phosphorylated myelin basic protein was separated on 12%
SDS-PAGE and visualized by autoradiography.
and
polypeptides, NIH 3T3 cells stably transfected with
IPTG-inducible
Q229L and
Q226L
expression vectors were utilized. Addition of 1 mM IPTG to the
culture medium induced the transcription of
Q229L or
Q226L within 30 min (Fig. 4). Analysis of
JNK/SAPK activity in cell lysates at different time points following
IPTG induction demonstrated that JNK/SAPK was activated by 1 h
following the induction of either
Q229L or
Q226L (Fig. 4). These results indicate that
and
activation of JNK/SAPK is an
acute response and not due to an adaptive response produced by the long
term expression of the respective mutant
or
polypeptides in NIH 3T3 cells. Reproducibly,
induction of
Q226L gave an earlier activation of
JNK/SAPK relative to
Q229L. This was most likely due
to higher levels of
Q226L expression compared to
Q229L. Alternatively, this could be due to the
differences in the potency of
Q229L and
Q226L in activating the JNK/SAPK pathway.
Q229L and
Q226L in NIH 3T3 cells. 1
mM IPTG was added to the culture dishes, and the cells were
harvested at various times after IPTG induction as indicated. Cells
were harvested for RNA isolation to monitor the respective
-subunit expression. Northern blots were developed using the
respective cDNA probes. Cells were also harvested at these time points
and processed for JNK assay. JNK was assayed using solid phase kinase
assay using GST-c-Jun(1-79)-bound glutathione-Sepharose beads.
Protein was separated by SDS-PAGE, and a 1-h autoradiogram was
developed. GAPDH, glyceraldehyde-3-phosphate
dehydrogenase.
Q229L or
Q226L in COS-1 cells to
demonstrate the ability of
and
to
activate the JNK/SAPK pathway. The expression vectors containing
Q229L or
Q226L inserts were
cotransfected with an expression vector containing HA epitope-tagged
JNK1(21) . The cells were lysed after 60 h, and HA-JNK1 was
immunoprecipitated using the monoclonal antibodies to HA epitope tag.
An immune complex JNK/SAPK assay was carried out using GST-c-Jun as
substrate(21) . The results clearly indicated that the
expression of
Q229L or
Q226L
stimulated the activity of JNK/SAPK (Fig. 5). It is worth noting
that the expression of wild type
or
failed to activate JNK/SAPK in contrast to the GTPase-deficient
mutants (data not shown). Although the fold increases in JNK activity
mediated by
Q229L and
Q226L in
COS-1 cells were less than those observed with NIH 3T3 cells, the
results suggested that the transient expression system can be
effectively used to dissect the signaling components involved in the
activation of JNK/SAPK by
. To examine whether the
activation of JNK is specific to
and
alone, we tested the ability of GTPase-deficient
,
, and
to
stimulate the activity of JNK in COS cells. Constitutively activated
mutants of these
-subunits were cotransfected with HA-JNK1, and 60
h later, JNK activities of the transfectants were determined by immune
complex JNK assay. The results indicated that only
Q209L activated JNK/SAPK in the COS cell transient
expression system with equal potency as
(Fig. 6). Expression of
Q227L and
Q205L was largely without effect on JNK activity.
Q229L and
Q226L on JNK/SAPK
activity of COS-1 cells. Respective
-subunits (0.5 µg)
contained in pcDNA3 expression vector were cotransfected with HA-JNK1
(0.5 µg) using lipofectamine (Life Technologies, Inc.). After 48 h,
the HA-tagged JNK was immunoprecipitated with anti-HA antibodies
(Boehringer Mannheim), and an immune complex kinase assay was carried
out using GST-c-Jun(1-79) as a substrate. The phosphorylated
proteins were separated by SDS-PAGE and visualized by autoradiography.
The experiment was repeated at least four times with similar results. Upperpanel, lane 1, control; lane
2,
Q229L; and lane 3,
Q226L. Lowerpanel, the radioactive
bands were excised and counted in a scintillation counter. JNK activity
was expressed as % of control.
-subunits on JNK/SAPK activity. Respective
-subunits
contained in pcDNA3 expression vector (0.5 µg each) were
cotransfected with HA-JNK1 (0.5 µg) into COS-1 cells using
lipofectamine reagent. After 48 h, the HA-tagged JNK was
immunoprecipitated, and an immune complex kinase assay was carried out
using GST-c-Jun(1-79) as a substrate. The phosphorylated proteins
were separated by 12% SDS-PAGE and visualized by autoradiography. The
experiment was repeated at least four times with similar
results.
and IL1 activate JNK/SAPK pathways
independent of Ras (21, 26) . Thus,
activation of JNK may involve a Ras-dependent or -independent
mechanism. To investigate whether
activation of
JNK/SAPK is dependent or independent of Ras, inhibitory Ras
(N
Ras) was coexpressed with
Q229L and
Q226L. N
Ras blocked the ability of
Q229L and
Q226L to stimulate
JNK/SAPK activity (Fig. 7). The results demonstrate
Q229L and
Q226L require Ras to
activate JNK/SAPK. While our results presented here provide evidence
that
Q229L and
Q226L communicate to
Ras in activating JNK/SAPK in the COS cells, it is likely that
and
may also interact with
additional signaling pathways in different cell types. In fact, we have
evidence indicating that
Q229L and
Q226L activation of JNK/SAPK may involve a
Rho-dependent signaling cascade in Swiss 3T3 cells. (
)At
present, the nature of the molecular communication between
and Ras as well as other Ras-like GTPases is
unknown.
Q229L and
Q226L require functional Ras for the activation of
JNK/SAPK. JNK activities in the respective COS cell transfectants were
determined using an immune complex kinase assay with immunoprecipitated
HA epitope-tagged JNK1. Recombinant GST-c-Jun(1-79) was used as a
substrate. The phosphorylated GST-c-Jun was separated by 12% SDS-PAGE,
and an autoradiogram was developed (upper panel). The
radioactive bands were cut and quantitated by liquid scintillation
counting. JNK activity is expressed as percent of control (lower
panel). Lane 1, vector-transfected COS cells; lane
2,
Q229L transfectant; lane 3,
Q226L transfectant; lane 4,
N
ras transfectants; lane 5,
Q229L plus N
ras transfectant; lane 6,
Q226L plus N
ras transfectant. The experiment was repeated four times with similar
results.
and
activate a mitogenic
pathway(6, 7, 8, 9, 10) .
However, they also constitutively activate JNK/SAPKs, whose activation
has been proposed to be associated with growth arrest (24, 25) . It is likely that the role of JNK/SAPK in
cell proliferation or growth arrest is cell-type specific, and thus the
activation of JNK/SAPK by
and
may
have different consequences in different cell types. Consistent with
this notion is the observation that c-Jun is essential for the
transformation of 3T3 cells by Ha-Ras (27) and the
overexpression of c-Jun itself can transform rat
fibroblasts(28, 29) . Presumably, the opposing effects
of c-Jun in different cells (and sometimes in the same cell in a
different context) may be due to its interaction with different
transactivators. c-Jun can form heterodimers with at least nine
different proteins(18, 20, 30) . Each of
these heterodimers along with c-Jun homodimers can potentially
transactivate different sets of genes by binding to distinct DNA sites
leading to diverse functional responses(18) . In this context,
it is interesting to note that E1A transformation of 3T3 cells is
stimulated by Jun-ATF heterodimer while inhibited by Jun-Jun
homodimers(31) . The relative abundance of transcription
factors capable of dimerizing to c-Jun can thus dictate cell responses.
Alternatively,
and
activation of
JNK/SAPK may be indicative of a signaling cascade independent of cell
proliferation. It has been shown that JNK/SAPK can be activated upon
hyperosmolar shock(16) . In addition, JNK1 has been
demonstrated to complement yeast cells lacking the hyper osmolarity
glycerol response gene 1 (HOG1) in regulating cell volume in
response to hyperosmotic shock(16) . Based on these
observations, it has been proposed that JNK1 may be involved in the
cell volume regulation of higher eukaryotes. However, the upstream
``osmotic sensor,'' the transducer that stimulates JNK/SAPK,
and the effector that is activated by JNK/SAPK activity are not known.
It is significant to note that the regulation of
Na
/H
exchanger activity is considered
to play an important role in the regulatory volume increase following
hyperosmotic shock in eukaryotic cells(32) . Our observation
that
and
acutely activate
JNK/SAPK pathway in NIH 3T3 and COS-1 cells becomes more significant in
this context since the G
family of G proteins has been
shown to stimulate Na
/H
exchanger(22, 33, 34) . The findings
are suggestive that the regulation of the JNK/SAPK pathway and
Na
/H
exchanger may be an integrated
response controlled by G
and G
. This
observation associates volume regulation with specific gene
transcription, a question that has long eluded an explanation.
Experiments are now possible to directly test the integration of
Na
/H
exchanger activity with the
JNK/SAPK pathway.
-D-galactopyranoside.
We thank Drs. Scott K. Shore and E. Premkumar Reddy
for valuable suggestions and encouragement.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.