(Received for publication, November 30, 1994)
From the
The expression of human muscarinic acetylcholine receptors
(mAChRs) in NIH 3T3 cells has been used as a model for studying
proliferative signaling through G protein-coupled receptors. In this
biological system, the m1 class of mAChRs can effectively transduce
mitogenic signals (Stephens, E. V., Kalinec, G., Brann, M. R., and
Gutkind, J. S.(1993) Oncogene 8, 19-26) and induce
[Medline]
malignant transformation if persistently activated (Gutkind, J. S.,
Novotny, E. A., Brann, M. R., and Robbins, K. C.(1991) Proc. Natl.
Acad. Sci. U. S. A. 88, 4703-4708). Moreover, available
evidence suggests that the m1-signaling pathway converges at the level
of p21 with that emerging from tyrosine kinase
receptors (Crespo, P., Xu, N., Simonds, W. F., and Gutkind, J. S.(1994) Nature 369, 418-420). To explore nuclear events involved
in growth regulation by G protein-coupled receptors in this setting, we
compared the effect of platelet-derived growth factor (PDGF) and the
cholinergic agonist, carbachol, on the expression of mRNA for members
of the jun and fos family of nuclear proto-oncogenes.
We found that activation of m1 receptors by carbachol induces the
expression of a distinct set of nuclear transcription factors. In
particular, carbachol caused a much greater induction of c-jun mRNA and AP-1 activity. These responses did not correlate with
protein kinase C stimulation nor with the activation of
mitogen-activated protein (MAP) kinases. Recently, it has been shown
that a novel family of kinases structurally related to MAP kinases,
stress-activated protein kinases, or Jun kinases (JNKs), phosphorylate in vivo the amino-terminal transactivating domain of the c-Jun
protein, thereby increasing its transcriptional activity. In view of
our results, this observation prompted us to ask whether m1 and PDGF
can differentially activate JNKs. Here, we show that m1 mAChRs can
induce a remarkable increase in JNK activity, which was temporally
distinct from that of MAP kinase and was entirely protein kinase C
independent. In contrast, PDGF failed to activate JNK in these cells,
although it stimulated MAP kinase to an extent even greater than that
for carbachol. These findings demonstrate that G protein-coupled
receptors can signal through pathways leading to the activation of JNK,
thus diverging at this level with those signaling routes utilized by
tyrosine kinase receptors.
Critical molecules participating in the transduction of
proliferative signals have just begun to be identified. One such
example is the family of extracellular signal-regulated kinases (ERKs) ()or MAP kinases(7) . The enzymatic activity of
these kinases increases in response to most mitogens, such as those
acting on receptor-protein tyrosine kinases or on receptors coupled to
heterotrimeric guanine nucleotide binding proteins (G
proteins)(3, 4, 7) . Activation of the
tyrosine kinase class of receptors transmits signals to MAP kinases in
a multistep process. For the EGF receptor, essential components of this
process include the adaptor protein GRB2/SEM-5, a guanine nucleotide
exchange protein such as SOS, p21
, and a cascade
of protein kinases defined sequentially as MAP kinase kinase kinase,
represented by c-Raf-1 and MEKK, and MAP kinase kinase, such as MEK1
and MEK2(7, 8, 9) . MEKs ultimately
phosphorylate MAP kinases in both threonine and tyrosine residues,
thereby increasing their enzymatic
activity(7, 8, 9) . In turn, MAP kinases
phosphorylate and regulate the activity of key enzymes including the
EGF receptor, phospholipase A
, p90
,
and nuclear proteins such as c-Myc and p62
Elk1(10) , which ultimately regulate the expression of
genes essential for proliferation.
Little is known about the nature
of proliferative pathways that participate in growth stimulation by G
protein-coupled receptors. We have used the expression of human
muscarinic receptors for acetylcholine (mAChRs) in NIH 3T3 cells as a
model for studying proliferative signaling through this class of
receptors(1) . The mAChR family consists of five distinct but
highly related subtypes (m1-m5) (11, 12) . In
this biological system, mAChR subtypes coupled to phosphatidylinositol
biphosphate catabolism (m1, m3, and m5) can effectively transduce
mitogenic signals (1) and, when persistently activated, can
induce malignant transformation(2) . In contrast, those mAChRs
coupled to the inhibition of adenylyl cyclase (m2 and m4) fail to
induce the transformed phenotype(2) . Using this system, we
have recently shown that triggering m1 receptors with the cholinergic
agonist carbachol induces the activation of c-Raf and MAP
kinases(3) . Moreover, using transient expression in COS-7
cells, we have found that activation of MAP kinases by muscarinic
receptors involves subunits of G proteins acting on a
Ras-dependent pathway(4) . These findings strongly suggest that
the G protein-coupled receptor signaling pathway converges at the level
of Ras with that emerging from receptors of the tyrosine kinase class.
Thus, activation of either type of receptor would be expected to elicit
a similar response at the level of nuclear transcription factors. Here,
we present evidence that activation of m1 receptors in NIH 3T3 cells
induces a distinct pattern of expression of immediate early genes of
the jun and fos family and a much greater expression
from an AP-1-responsive reporter construct. These responses did not
correlate with the activation of MAP kinases. We found that triggering
m1 mAChRs potently stimulate the activity of a novel family of enzymes
closely related to MAP kinases, known as Jun kinases (JNKs) or stress-activated protein kinases
(SAPKs)(5, 6) . In contrast, PDGF failed to activate
JNK in these cells, although it stimulated MAP kinase to an even
greater extent and for a more prolonged period of time than carbachol.
These findings demonstrate that G protein-coupled receptors can signal
through pathways leading to the activation of JNK, thus diverging at
this level with those pathways utilized by receptors of the tyrosine
kinase class.
Confluent plates of transfected NIH
3T3 cells were incubated overnight in serum-free medium. Cells were
then stimulated with agonists for different periods of time at 37
°C, washed with cold PBS, and lysed at 4 °C in a buffer
containing 25 mM HEPES, pH 7.5, 0.3 M NaCl, 1.5
mM MgCl, 0.2 mM EDTA, 0.5 mM dithiothreitol, 20 mM
-glycerophosphate, 1 mM vanadate, 0.1% Triton X-100, 1 mM phenylmethylsulfonyl
fluoride, 20 µg/ml aprotinin, and 20 µg/ml leupeptin. Cleared
lysates were rocked for 3 h at 4 °C in the presence of 1 µg of
GST-cjun79 fusion protein bound to glutathione-agarose beads (5 mg of
GST-cjun79 protein/25 ml of beads). Beads were washed three times with
PBS containing 1% Nonidet P-40 and 2 mM vanadate, once with
100 mM Tris, pH 7.5, 0.5 M LiCl, and once in kinase
reaction buffer (12.5 mM MOPS, pH 7.5, 12.5 mM
-glycerophosphate, 7.5 mM MgCl
, 0.5
mM EGTA, 0.5 mM sodium fluoride, 0.5 mM vanadate). Samples were then resuspended in 30 µl of kinase
reaction buffer containing 1 µCi
[
-
P]ATP per reaction and 20 µM of unlabeled ATP. After 20 min at 30 °C, the reactions were
terminated by addition of 10 µl of 5
Laemmli buffer.
Samples were heated at 95 °C for 5 min and analyzed by SDS-gel
electrophoresis on 12% acrylamide gels. Autoradiography was performed
with the aid of an intensifying screen.
Immunocomplex JNK activity was determined upon immunoprecipitation with a JNK-specific polyclonal antibody(5) . Cleared lysates were incubated with 2 µl of antisera for 4 h at 4 °C. Immunocomplexes were recovered with the aid of protein G-Sepharose, and precipitates were processed as above. Kinase reaction was performed using 1 µg of purified GST-cjun79 protein as a substrate.
One of the earliest nuclear events resulting from exposure of quiescent cells to mitogens is the induction of expression of genes of the jun and fos family(18) . Homodimers of the Jun protein product or Jun/Fos heterodimeric complexes form the AP-1 transcription factor, which binds the palindromic TRE sequence(19) , thereby controlling the expression of genes possessing this regulatory element. The Jun family consists of c-jun, junB, and junD while members of the Fos family include c-fos, fosB, fra-1, and fra-2(15) . To assess whether triggering m1 receptors in NIH 3T3 cells affects transcriptional activation mediated by AP-1, we used the transient expression of a reporter plasmid containing multiple TRE elements fused to the CAT gene (3XTRE-CAT)(16) . As shown in Fig. 1, carbachol addition to transfected NIH-m1.2 cells dramatically increased expression from the reporter gene construct. Interestingly, the AP-1-dependent response elicited by carbachol (6-fold increase) was much greater than that induced by activation of PDGF receptors or by direct stimulation of PKC by TPA (20) (approximately 2-fold increase for both cases).
Figure 1:
Induction of AP-1
activity in NIH 3T3 cells expressing m1 mAChRs (effect of PKC
blockade). NIH-m1.2 cells were cotransfected with 3xTRE-CAT and
pCMV--gal plasmids. 48 h later, cells were serum starved for 18 h
and left untreated or treated for 30 min with 1 µM of the
PKC inhibitor GF 109203X. Cells were then exposed for 4 h to 100
µM carbachol, 10 ng/ml PDGF or 50 ng/ml TPA, as indicated.
CAT activity was measured and data normalized by
-galactosidase
activity present in each sample. Data represent the average ±
S.E. of triplicate samples from a typical experiment expressed as -fold
induction with respect to unstimulated cells (control). Similar results
were obtained in four independent
experiments.
Activation of m1 receptors induces the
hydrolysis of PIP and consequently activates
PKC(1, 3, 20) . To explore whether PKC
mediates the AP-1 response to carbachol, we pretreated cells with the
non-toxic PKC-specific inhibitor GF 109203X and then challenged these
cells with the various agonists. Under these conditions, we have
previously shown that this PKC inhibitor effectively blocks
phosphorylation of endogenous PKC substrates as well as biological
responses induced by phorbol esters(3) . Accordingly, GF
109203X pretreatment abolished the CAT activity elicited by phorbol
esters. However, PKC inhibition reduced only about 40% of the AP-1
response to carbachol (Fig. 1). In contrast, stimulation of CAT
activity by PDGF was not affected by GF 109203X. Thus, m1 signaling
pathways induce AP-1-dependent transcriptional activation more
effectively than those pathways triggered by tyrosine kinases or
phorbol esters. In addition, m1 receptors appear to induce AP-1
activity through both PKC-dependent as well as PKC-independent
pathways.
The activity of AP-1 is regulated at the level of jun and fos gene transcription and by post-translational modification of their protein products(18, 19, 21) . Because of the remarkable effect of m1 on AP-1 activity, we next examined the ability of carbachol, PDGF, and TPA to induce expression of jun and fos family members. As shown in Fig. 2, all agonists rapidly affected the expression of mRNA for these immediately early genes but to different extents. Carbachol induced a much greater expression of c-jun, junD, c-fos, and fosB mRNA, whereas PDGF preferentially induced junB and egr-1 (another immediate early gene unrelated to jun and fos)(16) . TPA was less effective in most cases, although it potently induced expression of egr-1 (Fig. 2). PDGF and TPA not only failed to increase levels of junD mRNA but instead they decreased its expression (Fig. 2). All agonists induced fra-1 messages much later than for the other genes (Fig. 2).
Figure 2:
Expression of immediate early genes in
response to agonists. Quiescent NIH-m1.2 cells were stimulated with 100
µM carbachol, 10 ng/ml PDGF, or 50 ng/ml TPA for the
indicated period of time. Cells were lysed, and total RNAs were
extracted as described under ``Experimental Procedures.''
Samples containing 10 µg of total RNA per lane were fractionated
and analyzed by Northern blotting, using the indicated P-labeled DNA probes. Material present in each lane was determined to be equivalent by ethidium bromide staining of
ribosomal RNAs and by hybridization with a radiolabeled
glyceraldehyde-3-phosphate dehydrogenase probe (data not shown).
Similar results were obtained in at least three independent
experiments.
We next explored which of these responses were mediated by PKC. Treatment of cells with the PKC inhibitor abolished all responses induced by TPA, demonstrating the effectiveness of this procedure (Table 1). In contrast, responses induced by PDGF were not affected by this treatment except for c-fos, which was slightly reduced. For carbachol, the picture appears to be more complex. The induction of egr-1 was completely abolished, and the expression of c-fos and fosB was reduced by 50 and 25%, respectively. In contrast, PKC blockade did not have any demonstrable effect on m1-mediated induction of c-jun and junD. Thus, the most striking difference between carbachol and the other agonists is the potent induction of c-jun and junD mRNA, in both cases involving PKC-independent pathways.
The activity of c-Jun appears to
be controlled by a novel family of enzymes structurally related but
clearly distinct from MAP kinases. These enzymes, named JNKs (6) or SAPKs(5) , selectively phosphorylate the
amino-terminal transactivating domain of the c-Jun protein, thereby
increasing its transcriptional activity(5, 6) . In
light of our results, we decided to compare the ability of carbachol,
PDGF, and TPA to induce MAP kinase and JNK (SAPK) activity. The former
was determined in NIH-m1.2 cells expressing an epitope-tagged ERK2
cDNA(3) . The latter was assayed by first retaining activated
JNK using bacterially expressed recombinant GST-c-jun79 protein bound
to glutathione-agarose and then assessing its phosphorylating activity
on GST-c-jun79. Alternatively, JNK activity was measured in an
immunocomplex kinase assay upon immunoprecipitation of SAPKs with a
specific antiserum, using purified GST-c-jun79 as a
substrate(5) . As previously reported (3) , carbachol
potently induced MAP kinase activation, which peaked after 3-5
min, and remained slightly above unstimulated levels for more than 2 h.
PDGF and TPA also induced a marked increase in MAP kinase activity;
however, its activity remained higher for a more prolonged period of
time (Fig. 3). In contrast, only carbachol induced JNK
activation and, to an extent, similar to that caused by cycloheximide,
which was used as a control (5) (Fig. 3). It is
noticeable that m1-mediated activation of JNK was delayed with respect
to MAP kinase. It peaked between 10-15 min upon carbachol
addition and remained higher than unstimulated cells for approximately
40 min. Furthermore, JNK activity in response to carbachol in NIH-m1.2
cells was similar or even greater than that induced by known JNK
activators, such as tumor necrosis factor-, interleukin-1, or heat
shock (5) (data not shown). On the other hand, TPA or agonists
acting on tyrosine kinase receptors, such as epidermal growth factor or
fibroblast growth factor, have been shown to poorly induce JNK
activity, and only in a few cell types(5, 22) . The
lack of JNK activation by PDGF and TPA in NIH 3T3 cells is in line with
those observations. We next determined whether PKC activation plays a
role in JNK activation through m1 G protein-coupled receptors. We found
that neither blockade of PKC nor depletion of this enzyme by prolonged
treatment with TPA (1, 3) affect the JNK response to
carbachol in m1-expressing cells (Fig. 4), thus demonstrating
that signaling from m1 to JNK involves PKC-independent pathways.
Figure 3: The cholinergic agonist carbachol induces MAP kinase and JNK (SAPK) activity in NIH 3T3 cells expressing m1 mAChRs. Confluent plates of NIH-m1.2 cells were serum starved for 18 h and treated with 100 µM carbachol, 10 ng/ml PDGF, or 50 ng/ml TPA for the indicated times. Treatment with 180 µM cycloheximide (Cx) for 1 h was used as a control. Cells were lysed, and kinase activity present in cell extracts was recovered by immunoprecipitation using the monoclonal antibody 12CA5 for MAP kinase (MAPK) or affinity precipitated using recombinant GST-cjun79 fusion protein bound to glutathione beads for JNK. Kinase reactions were performed as described under ``Experimental Procedures.'' The products of kinase reactions were fractionated in 12% SDS-polyacrylamide gel electrophoresis gels. Position of labeled myelin basic protein (MBP) or GST-jun79 are indicated. Similar results were obtained in four independent experiments. For JNK, almost identical results were obtained by immunoprecipitation of endogenous JNK using a specific antibody (data not shown and Fig. 4).
Figure 4: Effect of PKC depletion or blockade on JNK activity. NIH-m1.2 cells were serum starved for 18 h(-), depleted of PKC by treatment with 1 µg/ml TPA in serum-free conditions (TPA O/N), or serum starved for 18 h and treated for the last 30 min with 1 µM of the specific PKC inhibitor GF 109203X, as indicated. Cells were treated with 100 µM carbachol, 10 ng/ml PDGF, or 50 ng/ml TPA for 20 min and lysed. Cell extracts were processed as described under ``Experimental Procedures'' for immunocomplex JNK assay. Results are representative from three independent experiments. Near identical results were obtained using the GST-jun79 affinity precipitation technique (data not shown).
Recent findings have demonstrated that mitogens acting on a large
variety of cell surface receptors converge at the level of Ras to
induce a cascade of serine-threonine kinases leading to the activation
of MAP kinases. In turn, MAP kinases phosphorylate a number of
intracellular substrates that are important for cell proliferation (10) . For example, MAP kinases phosphorylate the transcription
factor ternary complex factor, known as p62 or
Elk-1(23) , which represents a critical event in controlling
the expression of c-fos(24) . Interestingly, this
observation might account for the decreased c-fos and AP-1
response to carbachol upon blockade of PKC, as we have recently shown
that MAP kinase activation through m1 mAChRs is partially dependent on
PKC(3) . However, other nuclear events in response to
environmental stimuli might involve additional members of the MAP
kinase family, such as JNK and the mammalian homolog of HOG
kinase(25) . In particular, JNK is thought to be responsible
for phosphorylating in vivo the transactivating domain of
c-Jun protein (and probably Jun
D)(
)(5, 6, 22) . Phosphorylated
c-Jun homodimers have potent AP-1 activity, and this complex appears to
control the expression of c-jun mRNA(27, 28) . Consistent with this idea,
activation of JNK by m1 mAChRs correlated well with both the potent
induction of AP-1 activity and the remarkable expression of c-jun mRNA in response to carbachol. Conversely, induction of AP-1 and
c-jun expression by PDGF and TPA might involve alternate
mechanisms, including MAP kinase-dependent events or as the result of
dephosphorylation of inhibitory phosphorylated sites in the c-Jun
protein, as previously suggested(28) .
Our findings also
have implications with regard to activating pathways for JNK. Although
it was initially suggested that these enzymes were located downstream
from Ras, this hypothesis is in conflict with recently available
data(26) , including the lack of activation of JNK by PDGF
(this study) or by other agonists acting on receptors that are known to
couple to the ras pathway(5, 22) .
Furthermore, agonists such as tumor necrosis factor- and
interleukin-1 potently induce JNK, but they activate MAP kinase poorly
( (5) and data not shown). Taken together, these observations
suggest the existence of parallel pathways leading to the activation of
either MAP kinase or JNK. Based upon our results, both pathways can be
effectively stimulated by transforming G protein-coupled receptors.
Although the precise role of JNK activation by m1 mAChRs is not known,
it correlates well with agonist-induced expression of genes important
for cell growth. Thus, JNK is a likely candidate to be an integral part
of the mitogenic signaling pathway utilized by these G protein-coupled
receptors. Whether that is the case warrants further investigation.