From the Department of Medicine and
§ Herbert Irving Comprehensive Cancer Center, College of
Physicians & Surgeons, Columbia University, New York, New York 10032 and ¶ Cell Pathways, Inc., Horsham, Pennsylvania 19044
Received for publication, February 12, 2001, and in revised form, March 12, 2001
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ABSTRACT |
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We recently obtained evidence that treatment of
human colon cancer cells with exisulind (sulindac sulfone) and related
compounds induces apoptosis by activation of protein kinase G (PKG) and c-Jun kinase (JNK1). The present study further explores this mechanism. We demonstrate that in NIH3T3 cells a constitutively active mutant of
PKG causes a dose-dependent activation of JNK1 and thereby transactivates c-Jun and stimulates transcription from the AP-1 enhancer element. The activation of JNK1 and the transactivation of
c-Jun by this mutant of PKG were inhibited by a dominant negative MEKK1. In vitro assays showed that a purified PKG directly
phosphorylated the N-terminal domain of MEKK1. PKG also directly
phosphorylated a full-length MEKK1, and this was associated with
enhanced MEKK1 phosphorylation. Thus, it appears that PKG activates
JNK1 through a novel PKG-MEKK1-SEK1-JNK1 pathway, by directly
phosphorylating and activating MEKK1.
Cyclic GMP (cGMP) is an important second messenger that mediates
several signal transduction pathways in mammalian cells (1). It is
involved in the regulation of various physiological functions, including neurotransmission, cell differentiation, proliferation, and
platelet aggregation (2). Cyclic GMP also modulates intracellular calcium levels in vascular smooth muscle cells and thereby modulates smooth muscle tone (3). Intracellular levels of cGMP are tightly regulated through synthesis by guanylate cyclases and hydrolysis by specific phosphodiesterases
(PDEs)1 (4, 5). cGMP has
several intracellular targets, including gated ion channels,
cGMP-dependent protein kinases (PKG), cGMP-activated phosphodiesterases, and cGMP-inhibited phosphodiesterases (6, 7).
PDE2 and PDE5 are cGMP phosphodiesterases (5). In recent studies we
obtained evidence that in human colon cancer cell lines, novel PDE2/5
inhibitors, including exisulind (sulindac sulfone) and the high
affinity derivatives CP248 and CP461, increase cellular levels of cGMP
and that this leads to activation of PKG and c-Jun kinase (JNK1) and
induction of apoptosis (8, 9). However, the biochemical details by
which cGMP leads to activation of JNK1 were not definitely established.
The present study presents evidence that the activation of PKG by cGMP
leads to direct phosphorylation and activation of MEKK1. This then
leads to activation of SEK1 and subsequently JNK1. This
PKG-MEKK1-SEK1-JNK1 pathway represents a novel signal transduction
pathway that may be important in understanding the roles of cGMP and
PKG in cell proliferation and apoptosis.
Cell Cultures and Transfection and Reporter Assays--
NIH3T3
mouse fibroblasts were routinely grown in Dulbecco's minimal essential
medium (DMEM) containing 10% calf serum. For reporter assays,
triplicate samples of 1 × 105 cells in 35-mm plates
were transfected using Lipofectin (Life Technologies, Inc.) with 1 µg
of the reporter plasmid, 0.05-5 µg of various expression vectors,
and 1 µg of the control plasmid pCMV- JNK1 Kinase Assays--
The cells were lysed in a lysis buffer
(20 mM Tris-HCl, pH 7.5, 0.5% Nonidet P-40, 250 mM NaCl, 3 mM EDTA, 3 mM EGTA, 2 mM DTT, 0.5 mM phenylmethylsulfonyl
fluoride, 1 µg/ml leupeptin, 20 mM
For transient transfection experiments, subconfluent cultures of cells
in 10-cm plates were transfected, using Lipofectin (Life Technologies,
Inc.), with 2.5 µg of the pCMV5-M2-JNK1 plasmid and either 5 µg of
pRc/CMV-
The intensities of the bands on gels were determined with a
PhosphorImager (Molecular Dynamics), and the ratio of the treated sample to the control untreated sample was expressed as "relative kinase activity" or "fold activation." The experiments were
repeated three times with similar results.
PKG Assays--
Purified recombinant PKG I MEKK1 Assays--
COS-7 cells were grown in DMEM containing 10%
fetal bovine serum, and subconfluent cultures in 10-cm plates were
transiently transfected with either a HA epitope-tagged MEKK1 WT (wild
type) or the D1369A mutant plasmid, using Lipofectin (Life
Technologies, Inc.), as described above. After 24 h, the cells
were lysed in the above-mentioned lysis buffer. MEKK1 was then
immunoprecipitated with an anti-HA antibody (Berkeley Antibody
Company), and in vitro kinase assays were performed in the
absence or presence of 100 units of purified PKG I PKG Activates JNK1--
To directly address the role of PKG, we
examined the effects of a constitutively active mutant of PKG on the
JNK1 pathway in NIH3T3 mouse fibroblasts. For this purpose, we utilized
the pRc/CMV-
We first established that in NIH3T3 cells, as in SW480 human colon
cancer cells (8), increased levels of cGMP lead to activation of JNK1.
Exponentially growing NIH3T3 cells were treated with various cGMP
modulators for 1 h, and protein extracts were then collected for
JNK1 assays (Fig. 1A).
Anisomycin, a known activator of JNK1 (14), was used as a positive
control. Treatment with 8-bromo-3':5' cyclic GMP (8-Br-cGMP, 500 µM), a cell permeable cGMP analog, led to rapid
activation of JNK1. YC-1 (100 µM), a guanylate cyclase
activator, and CP248 (1 µM), a PDE2/5 inhibitor (8, 9),
also rapidly activated JNK1. Western blot analysis using a JNK1
antibody (Fig. 1A) ensured that equal amounts of the
immunoprecipitated JNK1 protein were added to the kinase reactions. Therefore, increase in cellular levels of cGMP does lead to activation of JNK1 in NIH3T3 cells.
We then examined whether the constitutively active mutant of PKG causes
JNK1 activation in NIH3T3 cells. The cells were transiently transfected
with the pCMV5-M2-JNK1 plasmid and increasing amounts of the
constitutively active PKG plasmid MEKK1 Is Required for PKG-mediated JNK1 Activation--
We
previously reported that in SW480 cells submicromolar concentrations of
the exisulind analog CP248, a potent PDE2/5 inhibitor (8, 9), causes
rapid (within 30 min) activation of MEKK1 (8). To determine whether
MEKK1 is actually required for PKG-mediated JNK1 activation, we studied
the effects of a dominant negative mutant of MEKK1, encoded by the
plasmid pCEP4-HA-MEKK1-D1369A, on PKG-mediated activation of JNK1. The
MEKK1-D1369A mutant encodes a kinase-inactive protein, due to an
Asp1369 to Ala point mutation in its activation loop (12).
NIH3T3 cells were transiently transfected with the pCMV5-M2-JNK1
plasmid and either PKG Directly Phosphorylates MEKK1 in Vitro--
In view of the
above results we investigated whether MEKK1 is a direct downstream
target of PKG in the PKG-JNK1 pathway by examining whether MEKK1 is an
in vitro substrate for PKG. Purified wild type PKG I
Phosphorylation of MEKK1 activates its kinase activity, and this, in
turn, leads to autophosphorylation of the MEKK1 protein (15).
Therefore, we sought evidence that the in vitro
phosphorylation of MEKK1 by PKG also activates the kinase activity of
MEKK1, thereby resulting in its autophosphorylation. COS-7 cells were
transiently transfected with either a HA epitope-tagged WT MEKK1 or the
D1369A (dominant negative) mutant of MEKK1. After 24 h, the WT and
mutant MEKK1 proteins were immunoprecipitated from cell extracts with an anti-HA antibody. The immunoprecipitated MEKK1-WT or MEKK1-D1369A proteins were incubated in [
These data suggest that phosphorylation of MEKK1-D1369A is due to
phosphorylation by PKG and that phosphorylation of MEKK1-WT is due to
both phosphorylation by PKG and MEKK1 autophosphorylation. The high
level of phosphorylation of MEKK1-WT was dependent upon the addition of
both PKG and cGMP (Fig. 2B), indicating that this was
mediated by activated PKG. These results indicate that cGMP-activated PKG can directly phosphorylate MEKK1 and suggest that this activates MEKK1.
PKG Activation Also Leads to Transactivation of c-Jun and Increased
AP-1 Activity--
Since activated PKG resulted in the activation of
JNK1 in NIH3T3 cells, it was of interest to look at immediate events
downstream of JNK1. Activation of JNK1 leads to phosphorylation and
thereby transactivation of the transcription factor c-Jun. Therefore, we examined whether transfection of NIH3T3 cells with the
constitutively active mutant of PKG led to c-Jun transactivation, using
transient transfection reporter assays (Fig.
3). The pG5-luciferase reporter plasmid
has five copies of GAL4 binding sites, and the pGAL4-c-Jun plasmid
encodes the GAL4 DNA binding domain protein fused to the transactivation domain of c-Jun (16). Activation of c-Jun by phosphorylation of its transactivation domain leads to activation of
transcription of the pG5-luciferase reporter. NIH3T3 cells were
transfected with the pG5-luciferase reporter plasmid together with the
pGAL4-c-Jun plasmid, and with increasing amounts of the
In additional studies, NIH3T3 cells were transfected with the
pG5-luciferase reporter plasmid and the pGAL4-c-Jun plasmid, together
with either
We then examined the ability of the
We reported previously that sulindac sulfone (exisulind) and related
compounds that inhibit PDEs 2 and 5 and, therefore, increase cellular
levels of cGMP, can lead to activation of the MEKK1-SEK1-JNK1 pathway
in SW480 human colon cancer cells (8). The present studies provide
evidence that PKG can directly phosphorylate and activate MEKK1. A
hypothetical signaling pathway based on these results is shown in Fig.
4. Intracellular levels of cGMP can
increase, either through activation of guanylate cyclase or through
inhibition of PDEs 2 and 5. This leads to activation of PKG. Activated
PKG can then directly phosphorylate and activate MEKK1. The activated MEKK1 then phosphorylates and activates SEK1, which in turn
phosphorylates and activates JNK1. Previous studies indicate that JNK1
activation can play a critical role in the activation of c-Jun, gene
transcription, and the induction of apoptosis (8, 19).
It is known that MEKK1 can play an important role in various stress
responses and in apoptosis, through activation of the downstream
kinases SEK1 and JNK1 (20). However, the precise proteins that act
upstream of MEKK1 to cause its activation are not clearly defined. Deak
et al. (15) identified a major site of autophosphorylation
(Thr575) within the "activation loop" of MEKK1.
Phosphatase treatment of a constitutively active MEKK1 or mutation of
Thr575 to alanine decreased the kinase activity of MEKK1
(15). PAK3 and PKC were reported to result in activation of MEKK1
in vivo (21). However, this interaction was indirect, since
there was no direct phosphorylation of MEKK1 by PAK3 or PKC or of PAK3
by PKC. Our evidence that PKG can directly phosphorylate and activate MEKK1 suggests a novel signaling pathway that can cause apoptosis.
Although the present studies were done mainly with NIH3T3 cells, in
unpublished studies we found that transfection of SW480 human
colon cancer cells with the constitutively active mutant of PKG also
leads to JNK1 activation. Therefore, this signaling pathway probably
applies to a variety of mammalian cells and may have more general
relevance with respect to growth control and apoptosis.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
-gal. The pcDNA3 plasmid
DNA was added to the transfections, as needed, to achieve the same
total amount of plasmid DNA per transfection. Twenty-four hours after
transfection, cell extracts were prepared, and luciferase assays were
done using the Luciferase Assay System (Promega). Luciferase activities
were normalized with respect to parallel
-gal activities, to correct
for differences in transfection efficiency.
-gal assays were
performed using the
-Galactosidase Enzyme Assay System (Promega).
The plasmid pRc/CMV-
93GK was kindly provided by R. Pilz (University
of California, San Diego) (10), the pAP-1-luciferase plasmid by J. Pierce (NCI) (11), the pGAL4DB-c-Jun and pG5-luciferase plasmids by A. Minden (Columbia University), and the pCEP4-HA-MEKK1-WT and
pCEP4-HA-MEKK1-D1369A plasmids by M. H. Cobb (University of Texas
Southwestern Medical Center) (12). The pCMV-
-gal plasmid was
purchased from Stratagene.
-glycerophosphate, 25% glycerol) and then JNK1 was
immunoprecipitated for 2 h with an anti-JNK1 antibody (Santa Cruz)
and assayed for in vitro kinase activity with
GST-c-Jun-(1-79) (New England Biolabs) as the substrate, in a
kinase reaction buffer (20 mM HEPES, pH 7.5, 10 mM MgCl2, 1 mM DTT, 20 µM ATP, 20 mM
-glycerophosphate, 1 µCi
of [
-32P]ATP), for 20 min, as described previously
(8). The reaction mixture was then subjected to 10% SDS-PAGE.
93GK or pCEP4-HA-MEKK1 plasmid DNA. The pcDNA3 plasmid
DNA was used as a control. Twenty-four hours after transfection, the
cells were lysed in the lysis buffer described above and then JNK1 was
immunoprecipitated with an anti-FLAG antibody (Sigma) for 2 h and
assayed for in vitro kinase activity, as described above.
(100 units,
Calbiochem) was incubated with 2 µg of recombinant GST-MEKK1-(1-301)
(Santa Cruz) in a reaction buffer (20 mM Tris-HCl, pH 7.5, 10 mM MgCl2, 10 µM dipyridamole,
10 mM DTT, 200 µM ATP, 10 mM NaF,
1 µCi of [
-32P]ATP), for 20 min, in the absence or
presence of 100 µM cGMP. The reaction mixture was
subjected to 10% SDS-PAGE, and the intensities of the bands were
determined as described above. The ratio of the treated sample to the
control untreated sample was expressed as fold activation.
(Calbiochem) in a
kinase reaction buffer (20 mM HEPES, pH 7.5, 10 mM MgCl2, 1 mM DTT, 20 µM ATP, 20 mM
-glycerophosphate, 1 µCi
of [
-32P]ATP), for 20 min, as described previously
(13). The pCEP4-HA-MEKK1-WT and pCEP4-HA-MEKK1-D1369A plasmids encode a
wild type MEKK1 and a kinase-inactive mutant, respectively (12).
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
93GK plasmid, which encodes a mutant PKG I
sequence with an N-terminal truncation. Deletion of the N-terminal 93 amino acids renders this PKG independent of cGMP, and therefore, it is
constitutively active (10).
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Fig. 1.
cGMP and a constitutively active PKG activate
JNK1. A, NIH3T3 cells were grown in DMEM medium with
10% calf serum and during exponential growth they were treated with
either 0.1% Me2SO ( ) or 8-Br-cGMP, 500 µM; YC-1, 100 µM; CP248 1, µM, for 1 h. Anisomycin (0.1 mg/ml) was used as a
positive control. The cells were lysed and then JNK1 was
immunoprecipitated with an anti-JNK1 antibody and assayed for in
vitro kinase activity with GST-c-Jun-(1-79) as the substrate, as
described under "Experimental Procedures" (8). The experiments were
repeated three times with similar results. Fold activation was measured
with a PhosphorImager. Western blot analysis using a JNK1 antibody
(Santa Cruz) ensured that there were equal amounts of the
immunoprecipitated JNK1 protein in the kinase reactions. B,
NIH3T3 cells were transiently transfected with 2.5 µg of the
pCMV5-M2-JNK1 plasmid and either 5 µg of the pRc/CMV-
93GK or the
pCEP4-HA-MEKK1 plasmids. These plasmids encode the wild type JNK1 with
a FLAG epitope tag, a constitutively active PKG, and wild type MEKK1
proteins, respectively. The pcDNA3 plasmid DNA was used as a
control (
). Twenty-four hours after transfection, cells were lysed in
a lysis buffer and then JNK1 was immunoprecipitated for 2 h with
an anti-FLAG antibody and assayed for in vitro kinase
activity. Western blot analysis using an HA antibody ensured that there
were equal amounts of the immunoprecipitated HA-JNK1 protein in the
kinase reactions. C, NIH3T3 cells were transiently
transfected with the pCMV5-M2-JNK1 plasmid (2.5 µg) and either the
pRc/CMV-
93GK (5 µg) or the pCEP4-HA-MEKK-D1369A (+, 5 µg; ++, 10 µg) plasmid or with both of the latter two plasmids as indicated. The
pcDNA3 plasmid DNA was used as a control (
). Twenty-four hours
after transfection, cells were lysed in a lysis buffer and then JNK1
was immunoprecipitated for 2 h with an anti-FLAG antibody and
assayed for in vitro kinase activity. Western blot analysis
using an HA antibody ensured that equal amounts of the
immunoprecipitated HA-JNK1 proteins were present in the kinase
reactions.
93GK. The pCMV5-M2-JNK1 plasmid
encodes a FLAG-tagged JNK1. The pCEP4-HA-MEKK1 plasmid, which encodes
the wild type MEKK1, was used as a positive control, since when
overexpressed it leads to JNK1 activation (12). Twenty-four hours after
transfection, FLAG-tagged JNK1 was immunoprecipitated with an anti-FLAG
antibody and assayed for JNK1 in vitro kinase activity,
using GST-c-Jun as a substrate. As shown in Fig. 1B, transfection with
93GK activated JNK1, in a
dose-dependent manner. Western blot analysis indicated that
the kinase assays contained equal amounts of the FLAG-JNK1 protein
(Fig. 1B). These data demonstrate that a constitutively
active PKG mutant is sufficient to induce JNK1 activity in NIH3T3 cells.
93GK or pCEP4-HA-MEKK1-D1369A, or both of the
latter two plasmids. Twenty-four hours after transfection, the
FLAG-tagged JNK1 protein was immunoprecipitated with an anti-FLAG
antibody and assayed for JNK1 kinase activity. Fig. 1C shows
that the dominant negative MEKK1 construct strongly inhibited
PKG-mediated JNK1 activation. These results indicate that in NIH3T3
cells PKG-mediated activation of JNK1 requires MEKK1, thus placing
MEKK1 upstream of JNK1 in the pathway by which PKG leads to JNK1 activation.
(Calbiochem) was incubated with a recombinant GST-MEKK1-(1-301)
protein, which contains the N-terminal domain of MEKK1, in a reaction
buffer containing [
-32P]ATP, in the absence or
presence of 100 µM cGMP. We found that PKG did
phosphorylate the N-terminal domain of MEKK1 and that this
phosphorylation was stimulated by cGMP (Fig.
2A).
View larger version (20K):
[in a new window]
Fig. 2.
Purified PKG phosphorylates and activates
MEKK1 in vitro. A, purified
recombinant PKG I (100 units, Calbiochem) was incubated with 2 µg
of recombinant GST-MEKK1-(1-301) in a reaction buffer containing
[
-32P]ATP, for 20 min, in the absence or presence of
100 µM cGMP. The reaction mixture was then subjected to
SDS-PAGE. The intensities of the bands were determined with a
PhosphorImager, and the ratio of the treated sample to the control
untreated sample was expressed as fold activation. B, NIH3T3
cells were transiently transfected with the HA epitope-tagged MEKK1 (WT
or D1369A mutant) plasmids, using Lipofectin. After 24 h, the
cells were lysed in a lysis buffer and then the HA-MEKK1-WT and
HA-MEKK1-D1369A mutant proteins were immunoprecipitated with an anti-HA
antibody. In vitro kinase assays were then performed for 20 min in assays, containing [
-32P]ATP, in the absence or
presence of 100 units of purified PKG, and in the absence or presence
of 100 µM cGMP, as described under "Experimental
Procedures." Western blot analysis using an HA antibody ensured that
equal amounts of the immunoprecipitated HA-MEKK1 WT and mutant proteins
were present in the kinase reactions. The intensities of the bands were
quantitated with a PhosphorImager. The panel on the
right was exposed for a longer time than the
panel on the left because the bands in the
panel on the right would otherwise not be well
displayed.
-32P]ATP-phosphorylation
assays, with or without purified wild type PKG, and in the absence or
presence of cGMP. Fig. 2B shows that in the presence of cGMP
and PKG the MEKK1-WT protein underwent more extensive phosphorylation
than the MEKK1-D1369A mutant protein, even though similar amounts of
both proteins were immunoprecipitated. The panel on the right was
exposed for a longer time than the panel on the left, because the bands
in the panel on the right would otherwise not be well displayed. The
more extensive phosphorylation of MEKK1-WT suggests that
phosphorylation of this protein by cGMP-activated PKG leads to
activation of the kinase activity of MEKK1, and subsequent MEKK1
autophosphorylation. This is consistent with evidence that when MEKK1
is activated in vivo it undergoes autophosphorylation (15),
but this requires further studies.
93GK plasmid
or the pCEP4-HA-MEKK1 plasmid. Luciferase assays (Fig. 3A)
indicated that transfection with
93GK led to c-Jun transactivation,
in a dose-dependent manner. Again, MEKK1 served as a
positive control.
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Fig. 3.
PKG transactivates c-Jun and also activates
AP-1 transcription in NIH3T3 cells. A, NIH3T3 cells
were transfected with the pG5-luciferase reporter plasmid (1 µg) and
pGAL4-c-Jun plasmid (50 ng), together with increasing amounts (0, 0.1, 0.5, or 2 µg) of the pRc/CMV- 93GK plasmid or the pCEP4-HA-MEKK1
plasmid. The pG5-luciferase reporter plasmid has five copies of GAL4
DNA binding sites, and the pGAL4-c-Jun plasmid encodes the GAL4 DNA
binding domain fused to the transactivation domain of c-Jun. The cells
were also cotransfected with the pCMV-
-gal plasmid. The pcDNA3
plasmid DNA was used as a control (
). Twenty-four hours after
transfection, cell extracts were prepared, and luciferase activities
were measured and normalized with respect to
-galactosidase
activities. For all experiments, the data shown are representative of
at least three independent experiments with each assay done in
triplicate. The error bars indicate the S.D. values.
Luciferase activities are expressed as fold induction, after correction
for
-galactosidase activities. B, NIH3T3 cells were
transfected with the pG5-luciferase reporter plasmid (1 µg) and the
pGAL4-c-Jun plasmid (50 ng), together with either pRc/CMV-
93GK (2 µg), pCEP4-HA-MEKK1-D1369A (+, 2 µg; ++, 5 µg), or both of the
latter plasmids, as indicated. The MEKK1-D1369A plasmid encodes a
dominant negative mutant of MEKK1. The pcDNA3 plasmid DNA was used
as a control (
). Twenty-four hours after transfection, cell extracts
were prepared, and luciferase activities were measured and normalized
with respect to
-galactosidase activities. C, NIH3T3
cells were transfected with the pAP-1-luciferase reporter plasmid (1 µg), which has three copies of AP-1 sites, together with increasing
amounts (0, 0.1, 0.5, or 2 µg) of the pRc/CMV-
93GK plasmid or the
pCEP4-HA-MEKK1 plasmid. The cells were also cotransfected with the
pCMV-
-gal plasmid. The pcDNA3 plasmid DNA was used as a control
(
). Twenty-four hours after transfection, cell extracts were
prepared, and luciferase activities were measured and normalized with
respect to
-galactosidase activities.
93GK, pCEP4-HA-MEKK1-D1369A, or both of the latter two
plasmids (Fig. 3B). The constitutively active mutant of PKG
again stimulated c-Jun activation, and this activation was markedly
inhibited by the dominant negative MEKK1 construct (D1369A), in a
dose-dependent manner (Fig. 3B). Therefore, the ability of PKG to cause transactivation of c-Jun is mediated through MEKK1, which then, presumably, activates SEK1 and then JNK1.
93GK mutant of PKG to mediate
the activation of an AP-1 enhancer element. The AP-1 enhancer element
is responsive to various stimuli that activate the c-Jun/c-Fos heterodimer (17, 18). NIH3T3 cells were transfected with a pAP-1-luciferase reporter plasmid, together with increasing amounts of
the
93GK plasmid (Fig. 3C). Again the pCEP4-HA-MEKK1
plasmid was used as a positive control. Twenty-four hours after
transfection, cell extracts were prepared and assayed for luciferase
activity. We found that
93GK also activated the AP-1 reporter, in a
dose-dependent manner (Fig. 3C). Therefore, a
constitutively active mutant of PKG is sufficient to lead to activation
of the AP-1 enhancer element.
View larger version (11K):
[in a new window]
Fig. 4.
JNK1 signal transduction pathway activated by
PKG. Intracellular levels of cGMP can increase through either
activation of guanylate cyclase or through inhibition of PDE2/5. This
activates PKG and then PKG directly phosphorylates and activates MEKK1.
MEKK1 then phosphorylates and activates SEK1, which in turn
phosphorylates and activates JNK1. Activation of JNK1 then plays a
critical role, together with other signals, in activation of c-Jun,
gene transcription, and the induction of apoptosis by mechanisms yet to
be determined.
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ACKNOWLEDGEMENT |
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We are grateful to Wang-Qui Xing for valuable technical assistance.
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FOOTNOTES |
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* This work was supported by grants from Cell Pathways, Inc., the T. J. Martell Foundation, and the National Foundation for Cancer Research (to I. B. W.).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.
To whom correspondence should be addressed: Herbert
Irving Comprehensive Cancer Center, College of Physicians & Surgeons, Columbia University, HHSC-1509, 701 West, 168th St., New York, NY
10032. Tel.: 212-305-6921; Fax: 212-305-6889; E-mail:
weinstein@cuccfa.ccc.columbia.edu.
Published, JBC Papers in Press, March 14, 2001, DOI 10.1074/jbc.C100079200
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ABBREVIATIONS |
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The abbreviations used are:
PDE, phosphodiesterase;
PKG, protein kinase G;
PKC, protein kinase C;
DMEM, Dulbecco's minimal essential medium;
-gal,
-galactosidase;
DTT, dithiothreitol;
GST, glutathione S-transferase;
PAGE, polyacrylamide gel electrophoresis;
HA, hemagglutinin;
WT, wild type;
8-Br-cGMP, 8-bromo-3':5' cyclic GMP.
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