From the Lineberger Comprehensive Cancer Center and
the ¶ Department of Biology and Curriculum in Genetics and
Molecular Biology, University of North Carolina School of Medicine,
Chapel Hill, North Carolina 27599
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ABSTRACT |
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Tumors frequently contain mutations in
ras genes, resulting in constitutive activation of
Ras-activated signaling pathways. The ultimate targets of these signal
transduction cascades are transcription factors required for cellular
proliferation. Understanding how constitutive activation of Ras
contributes to tumorigenesis requires an understanding of both the
signaling pathways that Ras activates and how these pathways in turn
regulate gene expression. Gene expression from The small GTP-binding protein Ras functions as an inducer of
intracellular signaling pathways that are responsible for regulating cellular functions including proliferation (1, 2). Mutations in Ras
alleles are found in approximately 30% of human cancers, leading to
chronic GTP binding and constitutive activation of signal transduction
cascades. Thus, it has been shown that oncogenic Ras stimulates
mitogen-activated protein
(MAP)1 kinase signaling
cascades and that these and other signal transduction pathways are
critical for Ras to initiate proliferative and oncogenic functions
(3-10). Three MAP kinases have been identified, the extracellular
signal-regulated kinases (ERKs), the c-Jun N-terminal/stress-activated protein kinases (JNK/SAPKs), and the p38 kinases. The MAP kinases are
activated in response to a wide variety of stimuli including growth
factors, cytokines, and cellular stress. The ERKs are primarily responsible for responding to cellular proliferation signals, while
JNKs and p38 respond to cellular stress signals (11, 12, 13). Each MAP
kinase is regulated by different upstream signaling pathways that
consist of similar elements. ERK is activated by a MAP kinase kinase,
MEK, which is activated by a MAP kinase kinase kinase, Raf. JNK and p38
are activated by the MAP kinase kinase MKK4/SEK1, which is activated by
the MAP kinase kinase kinase MEKK. Once activated, the MAP kinases
translocate to the nucleus, where they activate transcription factors
(4). Therefore, Ras regulates cell growth through activation of
transcription factors that regulate the expression of genes involved in
cellular proliferation and oncogenic transformation.
The mammalian transcription factor NF- NF- Several lines of evidence suggest that NF- Cell Culture and Reagents--
Parental Ras-transformed, and
Raf-transformed NIH-3T3 cells were maintained in Dulbecco's modified
Eagle's medium (high glucose) supplemented with 10% fetal calf serum,
penicillin, and streptomycin. For experiments using conditioned medium,
the conditioned medium was made by taking medium that had been on
parental Ras-transformed, or Raf-transformed cells for 48 h and
adding fresh medium at a 3:1 ratio. Conditioned medium was added to
cells 16-24 h after they were transfected (see below), and cells were
harvested 24 h after the addition of conditioned medium. The
PD98059, SB202190, and SB203580 compounds were obtained from Calbiochem
and were used at a final concentration of 4 µM, 700 nM, and 1.2 µM, respectively. The compounds
were added to cells at the indicated concentrations in fresh medium
16-24 h after the cells had been transfected (see below), and cells
were harvested 24 h after the addition of the compounds.
Plasmid Constructs--
The 3X Cell Transfections and Luciferase Assays--
Cells were
transfected by the calcium phosphate precipitation method essentially
as described previously (24). Briefly, semiconfluent cells were
transfected with 10.0-15.0 µg of DNA. Unless otherwise indicated,
the DNA precipitate contained 1.0 µg of luciferase reporter
construct, 1.0 µg of CMV-lacZ plasmid as an internal control for
transfection efficiency, and 5.0-7.0 µg of expression vector. For
Gal4-p65, 0.4 µg of the plasmid was used. The pGEM plasmid was used
as carrier DNA to bring the final DNA concentration to 10.0-15.0 µg.
The DNA precipitate was added to cells, and 16 h later the cells
were washed and fresh medium was added. Cells were harvested
approximately 24 h later, washed twice with phosphate-buffered
saline, and resuspended in 0.25 M Tris (pH 7.8). Cell
lysates were made by freeze-thawing three times. Protein concentrations
were determined, and 100 µg of protein was assayed for luciferase
activity as described previously (28). Blocking the Raf/MEK/ERK Pathway Does Not Inhibit Ras Activation of
NF-
In addition to using dominant negative Raf to block MEK/ERK signaling,
we have also used a commercially available MEK/ERK-inhibitory compound,
PD98059, which inhibits MEK activation. To test the effect of this
compound in our system, cells were cotransfected with the constructs
shown in Fig. 1B. The medium on these cells was changed
16-24 h after transfection, and fresh medium containing 4 µM PD98059 was added to the cells. The cells were
harvested 24 h later, and luciferase assays were performed. These
results, shown in Fig. 1B, demonstrate that blocking the
Raf/MEK/ERK pathway does not inhibit Ras activation of
NF- Ras Does Not Require an Interaction with Raf to Activate
NF- Raf Activates NF-
First, we wanted to determine if there was a difference in the time it
takes for Ras and Raf to activate NK-
To determine if the delayed activation of NF- Activation of the Rac Pathway Enhances NF- Blocking the JNK/p38 Signaling Pathway Inhibits Ras as Well as Raf
Activation of NF-
The Pak family of protein kinases is regulated by Rac and Cdc42, and
they are an early step in the signaling cascade that leads to JNK and
p38 activation (37). PAK is required for Ras transformation, since
kinase-deficient mutants of PAK inhibit Ras transformation of Rat-1
fibroblasts (38). To determine if PAK is involved in Ras activation of
NF- Blocking p38 Activity Inhibits Ras Activation of NF- We have presented evidence, summarized in Fig.
7, that oncogenic Ras enhances NF-B sites is enhanced
in cells transformed with activated Ras and NF-
B activity is
required for oncogenic Ras to transform NIH-3T3 and Rat-1 fibroblasts.
Both dominant negative and constitutively active components of
signaling pathways have been tested for their ability to regulate
NF-
B. These experiments show that Ras utilizes
Raf-dependent and Raf-independent pathways to activate
NF-
B transcriptional activity, both of which require the
stress-activated kinase p38 or a related kinase. In the case of Raf,
activation of NF-
B by an autocrine factor stimulates
B-dependent transcriptional activity.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B is involved in regulating
the expression of genes required for inflammatory responses, for
suppression of apoptosis, and for controlling cell growth (14, 15).
NF-
B is a member of the Rel family of transcription factors, which
consists of five members, c-Rel, p50 (NF-
B1), p65 (RELA), p52
(NF-
B2), and RELB. The different family members can form homodimers
or heterodimers, and different subunit combinations have different
functions in regulating transcription (14). The classic NF-
B
activator of transcription is a heterodimer composed of a p50 and p65
subunit. This heterodimer is a potent activator of gene expression from
B sites due to the presence of at least two transactivation domains
in the C-terminal region of p65 (16, 17). The carboxyl-terminal 30 amino acids comprise a transactivation domain (TA1) that belongs to the
class of acidic activators and can activate transcription from GAL4
binding sites when fused to the GAL4 DNA-binding domain.
B activity is regulated, at least in part, by its subcellular
localization. NF-
B is held in the cytoplasm, where it is inactive,
through an interaction of the p65 or c-Rel subunits with the inhibitor
protein, I
B (14). A variety of extracellular stimuli activate signal
transduction pathways that target the NF-
B·I
B complex for
disruption (15). These pathways target I
B for degradation by the
proteasome by leading to the phosphorylation and ubiquitination of
I
B (14, 15). Degradation of I
B results in the disruption of the
NF-
B·I
B complex, allowing NF-
B to be transported into the
nucleus, where it can activate gene expression (14, 15). Serine
residues 32 and 36 are required for the inducible phosphorylation of
I
B through the recently identified I
B kinases (18-22).
B plays an important role
in cellular transformation. NF-
B is activated by a variety of
cellular oncogenes including Her2/Neu (23), and two members of the
NF-
B family, v-rel and p52/lyt-10, and the
I
B family member Bcl-3 are potentially oncogenic (14). Transient
transfection experiments have shown that oncogenic forms of H-Ras and
Raf-1 can activate a reporter driven by NF-
B binding sites (24, 25). Importantly, expression of a modified, superrepressor form of I
B
blocked the ability of oncogenic Ras alleles to induce focus formation
in 3T3 cells (26). The requirement of NF-
B for Ras transformation is
based partly on the ability of NF-
B to suppress transformation-associated apoptosis (27). Interestingly, it was
observed that oncogenic Ras activates NF-
B through the ability to
stimulate transcriptional function dependent on the transactivation domains of the p65/RelA subunit (26). We show here that oncogenic Ras
activates NF-
B transcriptional activity largely through a Raf-independent mechanism that utilizes a SEK- and
p38-dependent pathway. The ability of Raf-1 to activate
NF-
B is MEK/ERK-dependent but appears to require an
autocrine pathway that ultimately utilizes the same
SEK/p38-dependent mechanisms as oncogenic Ras.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B-luc, Gal4-p65, Gal5-luc,
Ras12V, RafBXB, and Raf130 plasmids have been described previously (24,
26). PAK299R was provided by Jonathan Chernoff. SEK(KR) was provided by
Dennis Templeton. Ras12V37G, Ras12V40C, Ras12V35S, and Rac115 were
provided by Channing Der.
-Galactosidase assays were
also performed to correct for variations in transfection efficiency.
All transfections were performed in duplicate, and all were repeated at
least three times.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B--
Since previous work had shown that NF-
B
transcriptional activity was enhanced by expression of either oncogenic
Ras or Raf (24-26), we assumed that the Ras/Raf/MEK/ERK signaling
pathway was responsible for NF-
B activation in response to oncogenic Ras. To determine if oncogenic Ras required activation of Raf to
activate NF-
B, we used a Raf construct that expresses only the first
130 amino-terminal amino acids or Raf, Raf130. This construct expresses
the regulatory but not the catalytic domain of Raf and functions as a
dominant negative by blocking signaling through Raf (25). Parental
NIH-3T3 cells were transiently cotransfected with an activated Ha-Ras
expression vector, HRas12V, as described previously (24), and the
Raf130 expression vector. NF-
B activity was measured in two ways.
First, NF-
B-dependent gene expression was measured by
using a luciferase reporter plasmid, 3X
B-luc, where luciferase gene
expression is under control of a promoter that contains three NF-
B
DNA binding sites. Second, enhancement of NF-
B transcriptional
activity was measured by using a Gal4-p65 fusion expression vector.
This fusion protein contains the carboxyl-terminal 30 amino acids of
p65/RelA (TA1) fused to the DNA binding domain of the yeast
transcription factor Gal4 (26). For cotransfections that contained
Gal4-p65, a reporter plasmid, Gal5-luc, that puts luciferase gene
expression under control of a promoter that contains five Gal4
DNA-binding sites was included. The results of these transfections are
shown in Fig. 1A. These
experiments show that blocking Raf does not have a significant effect
on the ability of oncogenic Ras to enhance
NF-
B-dependent gene expression or transcriptional
activity. Possibly, Raf130 cannot compete for an endogenous effector
that activates NF-
B, or the region required for NF-
B activation
is found outside of the Ras effector domain. To show that the dominant
negative Raf construct was producing a functional protein, cells were
transfected with HRas12V, Raf130, and a Gal4-Elk1 construct. Elk1 is a
transcription factor that is activated in response to activation of MAP
kinases (29, 30). The Gal4-Elk1 construct contains the Gal4 DNA binding
domain fused to the carboxyl-terminal transactivation domain of Elk1.
Raf130 was able to inhibit the Ras activation of Gal4-Elk1 (see Fig. 1A), demonstrating that functional Raf130 was produced in
these cells.
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Fig. 1.
Blocking either Raf or MEK does not inhibit
oncogenic Ras activation of NF- B.
A, NIH-3T3 cells were cotransfected with the indicated
expression vectors and either 1.0 µg of the 3X
B-luc reporter or
1.0 µg of the Gal4 reporter, Gal5-luc, for cotransfections containing
Gal4 expression vectors. Transfections containing vector only used 5.0 µg of empty expression vector with the 3X
B-luc reporter or 0.4 µg of the empty Gal4 expression vector with the Gal5-luc reporter.
The concentrations of other expression vectors were as follows: 2.0 µg of the oncogenic Ras expression vector (HRas12V), 3.0 µg of the
dominant negative Raf expression vector (Raf130), 0.4 µg of the Gal4
expression vector containing the TA1 region of p65 (Gal4-p65), 1.0 µg
of the Gal4 expression vector containing Elk1. Luciferase activity was
determined as described under "Experimental Procedures."
B, NIH-3T3 cells were cotransfected as described for
A except that 24 h after transfection medium containing
either PD98059 or Me2SO (DMSO) was added to the
cells. Cells were harvested 24 h later, and luciferase activity
was determined as described under "Experimental Procedures." -Fold
luciferase activation is presented relative to the transfection that
contained empty expression vector whose value was placed at 1.0. Bars represent S.D. values determined from at least three
independent transfections.
B-dependent gene expression or transcriptional
activity. As a control, PD98059 was found to block Ras activation of
Gal4-Elk1 (see Fig. 1B). In addition, PD98059 inhibited the
activation of NF-
B by an activated Raf construct (RafBXB), showing
that Raf activation of MEK/ERK is required for Raf to activate NF-
B.
These data indicate that oncogenic Ras can activate NF-
B
transcriptional activity independently of Raf.
B--
Ras has been shown to interact with several different
effectors in addition to Raf (31, 32). Selected mutations within the
Ras effector domain (amino acids 32-40) result in a Ras protein that
retains the ability to interact with some effectors while losing the
ability to interact with others. For example, Ras40C and Ras37G
interact with PI-3 kinase and Ral-GDS, respectively, but they no longer
interact with Raf (7-9). An additional effector mutant, Ras35S, can
interact with Raf, but it no longer interacts with PI-3 kinase or
Ral-GDS (9). We have tested each of these Ras effector mutants for
their ability to activate NF-
B-dependent gene
expression. Each of the Ras effector mutants is a double mutant that
contains the activating 12V mutation in addition to the effector loop
mutation (9). NIH-3T3 cells were cotransfected with the effector
mutants and the 3X
B-luc reporter plasmid, and the results are shown
in Fig. 2. Each effector mutant activates NF-
B, showing that Ras utilizes effectors other than Raf to activate NF-
B. Similar results were obtained for activation of Gal4-p65 (data
not shown).
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Fig. 2.
Ras effector mutants retain the ability to
activate NF- B. NIH-3T3 cells were
cotransfected with the indicated Ras effector mutants (2.0 µg) and
1.0 µg of 3X
B-luc reporter plasmid. Luciferase activity was
determined as described under "Experimental Procedures." -Fold
luciferase activation is presented relative to the transfection that
contained empty expression vector, whose value was placed at 1.0. Bars represent S.D. values determined from at least three
independent transfections performed in duplicate.
B through an Autocrine Feedback
Loop--
Since oncogenic Ras does not require activation of Raf to
activate NF-
B, we wanted to determine how oncogenic Raf is able to
activate NF-
B. Activation of Raf has been shown to result in JNK
activation in addition to ERK activation. However, activation of JNK
occurs within 16-24 h of Raf activation, while ERK activation occurs
after a few minutes (31). Experiments using conditioned medium from
Raf-transformed cells have suggested that the delayed JNK activation is
due to the release of autocrine factors (31, 32). Since our results
suggested that Ras does not require the activation of the Raf/MEK/ERK
pathway to activate NF-
B, we wanted to determine if activation of
NF-
B by Raf could be due to its delayed ability to activate JNK or a
related kinase through the release of an autocrine factor.
B. NIH-3T3 cells were
cotransfected with either HRas12V or RafBXB and 3X
B-luc. The cells
were harvested at different time points after transfection as shown in
Fig. 3A. These transient
transfections show that Ras can activate NF-
B as early as 12 h
after transfection, while Raf takes 24 h to give the same level of
NF-
B activity. The same results are obtained when cells are
cotransfected with Gal4-p65 (data not shown), indicating that the
effect on NF-
B activity occurs at the transcriptional activity
level. This result suggested, but did not prove, that the ability of
oncogenic Raf to activate NF-
B required an autocrine function.
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Fig. 3.
Raf activates NF- B
through production of an autocrine factor. A, NIH-3T3
cells were cotransfected with either the oncogenic Ras expression
vector, HRas12V (2.0 µg), or the oncogenic Raf expression vector,
RafBXB (2.0 µg), and 1.0 µg of 3X
B-luc reporter plasmid. Plates
of each were harvested at 6, 12, 24, and 48 h after transfection,
and luciferase activity was measured as described under "Experimental
Procedures." The results are presented as -fold luciferase activity
over time, with circles representing luciferase activity
from cells transfected with HRas12V and squares representing
luciferase activity from cells transfected with RafBXB. B,
NIH-3T3 cells were transfected with either 2.0 µg of HRas12V or 2.0 µg of RafBXB and 1.0 µg of 3X
B-luc reporter plasmid. Medium was
changed 8 h after transfection and then changed every 8-10 h
after that for 48 h. Cells were harvested, and luciferase activity
was determined. -Fold luciferase activity is shown relative to the
activity from a transfection containing empty expression vector, whose
value was placed at 1.0. Bars represent S.D. values
determined for three independent transfections performed in duplicate.
C, parental NIH-3T3 cells were transfected with either 1.0 µg of 3X
B-luc reporter plasmid or 0.4 µg of Gal4-p65 expression
vector and 1.0 µg of G5-luc. Conditioned medium (see "Experimental
Procedures") from parental Ras-transformed, or Raf-transformed cells
(as indicated) was added 24 h after transfection, and cells were
harvested 24 h later. Luciferase activity was determined, and
-fold luciferase activation is presented relative to the activity
obtained from using parental medium, which was placed at 1.0. Bars represent S.D. values determined from three independent
transfections performed in duplicate.
B by Raf could be due
to the production of an autocrine factor, two different approaches were
used. First, the medium on cells that had been transfected with HRas12V
or RafBXB was changed every 8-10 h for 48 h after transfection.
Changing the medium on RafBXB-transfected cells reduced NF-
B
activation by approximately 3-4-fold, while changing the medium had no
significant effect on the ability of oncogenic Ras to activate NF-
B
(Fig. 3B). Next, medium from Ras- or Raf-transformed NIH-3T3
cells was collected and added to parental NIH-3T3 cells that had been
transfected with 3X
B-luc (data not shown) or Gal4-p65. The cells
were harvested 24 h later, and luciferase assays were performed.
The cells that received medium from parental NIH-3T3 cells had little
Gal4-p65 activity, while those that received medium from Ras- or
Raf-transformed cells had approximately 6-fold higher Gal4-p65 activity
(Fig. 3C). These results suggest that Raf does not activate
NF-
B directly but that activation of Raf results in the production
of an autocrine factor that can function to activate Ras or a signaling
pathway utilized by Ras.
B Activity--
The
preceding results suggest that JNK or a JNK-related kinase may be
required for Ras to activate NF-
B. Ras activates the JNK signaling
pathway by activating the small GTP-binding proteins Rac and Cdc42.
Activation of Rac leads to the activation of both JNK and p38 MAP
kinases (11-13). To determine if activated Rac could activate NF-
B,
NIH-3T3 cells were cotransfected with a constitutively active form of
Rac, Rac115I, and 3X
B-luc. Fig. 4
shows that Rac115I can enhance NF-
B-dependent gene
expression as well or better than oncogenic Ras. Similar results were
obtained with the Gal4-p65 assay (data not shown), indicating that
Rac-stimulated signals target the transcriptional activation function
of NF-
B. These results are consistent with a previous report (33),
where it was shown that Rac as well as Cdc42 can activate a
B-dependent reporter. Since Rac does not activate ERK,
these data provide more support for the utilization of downstream
kinases, such as JNK and/or p38, in the activation of NF-
B by
oncogenic Ras.
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Fig. 4.
Activated Rac activates
NF- B. NIH-3T3 cells were cotransfected
with either oncogenic Ras expression vector, HRas12V (2.0 µg), or
oncogenic Rac expression vector, Rac115I (2.0 µg), and 1.0 µg of
3X
B-luc reporter plasmid. Luciferase activity was determined as
described under "Experimental Procedures." -Fold luciferase
activity is presented relative to the activity obtained from the
transfection containing empty expression vector, whose value was placed
as 1.0. Bars represent S.D. values obtained from at least
three independent transfections performed in duplicate.
B--
To investigate the potential involvement of
the JNK and p38 pathways in regulation of NF-
B activity, we wanted
to determine the effect that blocking the activation of these MAP
kinases would have on Ras activation of NF-
B. The MAP kinase kinase
SEK1/MKK4 is responsible for phosphorylating and activating JNK and p38 (34-36). To test the effect of inhibiting JNK and p38, we used a
dominant negative, kinase-inactive form of SEK1, SEK(KR). SEK(KR) has
the lysine residue at position 129 in the ATP binding domain changed to
arginine (34). NIH-3T3 cells were cotransfected with HRas12V;
3X
B-luc; or Gal4-p65, Gal5-luc, and SEK(KR). The results of these
transfections are shown in Fig.
5A. These results show that
Ras activation of NF-
B can be blocked by blocking the activation of
JNK and p38. To show that SEK(KR) was not blocking the activation of
ERK, cells were cotransfected with HRas12V, Gal4-Elk1, Gal5-luc, and
SEK(KR). Expression of SEK(KR) did not affect Ras activation of
Gal4-Elk1, showing that the SEK(KR) is not exhibiting nonspecific effects (see Fig. 5A). To show that Raf requires the
activation of JNK and p38 to activate NF-
B, cotransfections with
RafBXB, 3X
B-luc, and SEK(KR) were also performed. Coexpression of
SEK(KR) was able to block Raf activation of NF-
B, indicating that
Raf activates NF-
B because of its ability to activate non-ERK MAP kinases (see Fig. 5A).
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Fig. 5.
Blocking JNK/p38 signaling inhibits Ras
activation of NF- B. A, NIH-3T3
cells were cotransfected with the indicated expression vectors at the
following concentrations: 1.0 µg of 3X
B-luc, 1.0 µg of Gal5-luc,
0.4 µg of Gal4-p65, 1.0 µg of Gal4-Elk1, 2.0 µg of HRas12V, 2.0 µg of RafBXB, 3.0 µg SEK(KR). Cells were harvested 48 h after
transfection, and luciferase activity was determined as described under
"Experimental Procedures." -Fold luciferase activation is presented
relative to the activity obtained from the transfection containing
empty expression vector, whose value was placed at 1.0. Bars
represent S.D. values obtained from at least three independent
transfections performed in duplicate. B, cells were
transfected as described for A except that 3.0 µg of
PAK299R was used instead of SEK(KR).
B, we tested a kinase-deficient mutant of PAK that contains a
lysine to arginine mutation at residue 299, PAK299R. NIH-3T3 cells were
cotransfected with HRas12V, 3X
B-luc, or Gal4-p65 and Gal5-luc, and
PAK299R, and the results are shown in Fig. 5B. Like SEK(KR),
PAK299R blocks Ras activation of NF-
B-dependent gene
expression and transcriptional activity. This result shows that
blocking the upstream JNK and p38 signaling pathways at multiple control points inhibits Ras activation of NF-
B.
B--
The
preceding results demonstrated that oncogenic Ras utilizes signaling
components that can activate both JNK and p38 MAK kinases to activate
NF-
B, but they have not shown whether JNK or p38 or both are
required for Ras to activate NF-
B. p38 is phosphorylated, indicating
that it is activated, in Ras-transformed cells2; therefore, we wanted
to determine if p38 was required for NF-
B activation in these cells.
To do this, we used the commercially available p38 inhibitor compounds
SB202190 and SB203580. These compounds were added to Ras-transformed
cells that had been transfected with 3X
B-luc or Gal4-p65 and
Gal5-luc. The results of these transfections are shown in Fig.
6. These experiments show that blocking
p38 inhibits Ras activation of NF-
B-dependent gene
expression and transcriptional activity. Ras-transformed cells were
also transfected with Gal4-Elk1, which we have shown to be activated by
the Raf/MEK/ERK pathway in our system. The addition of the p38
inhibitors had no effect on activation of Gal4-Elk1 (data not shown),
while the addition of the MEK inhibitor PD98059 blocked Gal4-Elk1
activity in these cells. Additionally, dominant negative forms of JNK
did not inhibit the ability of oncogenic Ras to activate
B-dependent gene expression (data not shown). These
results demonstrate that p38 or a related kinase is involved in the
activation of the transcription function of the p65/RelA NF-
B
subunit in response to signals initiated by oncogenic Ras.
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Fig. 6.
Blocking p38 inhibits Ras activation of
NF- B. A, parental or
Ras-transformed NIH-3T3 cells were transfected with 1.0 µg of the
3X
B-luc reporter plasmid. Me2SO (DMSO) or the
indicated inhibitor compounds were added 24 h after transfection
at the following concentrations: 4.0 µM PD98059
(PD), 1.2 µM SB203580 (SB80), 0.7 µM SB202190 (SB90). Cells were harvested
24 h later, and luciferase activity was determined as described
under "Experimental Procedures." -Fold luciferase activation is
presented relative to the activity obtained from the transfection of
3X
B-luc into parental NIH-3T3 cells, whose value was placed at 1.0. Bars represent S.D. values determined from three independent
transfections performed in duplicate. B, Ras-transformed
NIH-3T3 cells were cotransfected with either 0.4 µg of Gal4 (vector)
or 0.4 µg of Gal4-p65 and 1.0 µg of the Gal4 reporter, Gal5-luc.
Me2SO and inhibitor compounds were added as described for
A. -Fold luciferase activation is presented relative to the
activity obtained from the transfection containing empty expression
vector, whose value was placed at 1.0. Bars represent
S.D. values determined from three independent transfections
performed in duplicate.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B
transcriptional activity through a signaling pathway that utilizes the
p38 MAP kinase. Ras activates a MAP kinase signaling cascade that most
likely includes the small GTP-binding protein Rac and the MEKK, PAK,
and MKK4/SEK1 kinases. MKK4/SEK1 can activate both the JNK kinases and
p38. However, none of the dominant negative JNK constructs we tested
blocked Ras activation of NF-
B, while compounds that blocked p38
blocked Ras activation of NF-
B. Therefore, we conclude that p38 or a
closely related kinase and/or the downstream kinases it activates are
responsible for oncogenic Ras activation of NF-
B.
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Fig. 7.
Model for Ras and Raf activation of
NF- B.
We have also shown that oncogenic Raf enhances NF-B transcriptional
activity apparently through the same pathway as Ras although by a
different initiating mechanism. Raf activation of NF-
B most likely
occurs through an autocrine feedback loop, since conditioned medium
from Raf-transformed cells activated NF-
B transcriptional activity
when added to parental cells. Raf has been shown to induce the
expression of hb epidermal growth factor and transforming growth
factor-
, and Raf's ability to activate the SAPKs, to which JNK and
p38 belong, is assumed to require the production of these or related
factors (31). We do not know the identity of the autocrine factor that
Raf utilizes to activate NF-
B in our system, but it is possible that
it is hb epidermal growth factor or transforming growth factor-
.
Recent work has shown that oncogenic Raf activates NF-
B in HEK 293 cells through an autocrine loop that activates the SAPKs (39). In that
study, blocking the epidermal growth factor receptor, which binds both
hb epidermal growth factor and transforming growth factor-
,
inhibited the ability of Raf to activate NF-
B-dependent
gene expression. Future studies will address the identity of this
factor in our system.
The p38 family of MAP kinases consists of four members, p38, p38
,
p38
, and p38
(e.g. see Ref. 40). These kinases are activated both by stress-inducing signals including osmotic shock and
by UV irradiation and inflammatory cytokines like interleukin-1 and
tumor necrosis factor-
(11-13). NF-
B is activated by many of
same stimuli that activate p38, and recent evidence indicates that p38
plays a role in the ability of certain stimuli to activate NF-
B
transcriptional activity (e.g. see Ref. 41). The exact mechanism that p38 uses to activate NF-
B is not understood. For example, we do not know if p38 phosphorylates NF-
B itself or if one
of the p38-regulated kinases can phosphorylate NF-
B. Phosphorylation of the p65 NF-
B subunit has been shown to stimulate NF-
B
transcriptional function (42, 43). Another possibility is that NF-
B
is not the direct target of one of these kinases but that p38 targets a
transcriptional coactivator such as CBP to stimulate transcriptional function. Additional studies will be required to determine if p38 acts
directly on NF-
B or if it targets other components of the
transcription machinery.
Ras utilizes multiple signaling components to transform cells. These
include Raf, Pak, Rac, and PI3-K (1-3, 7, 38, 44), and our data
demonstrate that the transcription factor NF-B is activated by Raf
(Refs. 24 and 26; Fig. 1B), Rac (Fig. 4), and
phosphatidylinositol 3-kinase2 and is inhibited by blocking
PAK (Fig. 5B). Based on our data with oncogenic Ras and Raf,
the p38 stress-activated kinase pathway appears to control this
response. Since NF-
B has been found to be required for the ability
of Ras to transform cells (26, 27), it will be important to determine
whether the p38 pathway is a necessary component in Ras-induced
cellular transformation.
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ACKNOWLEDGEMENTS |
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We thank Channing Der for providing the Ras- and Raf-transformed NIH3T3 cells, the Ras effector mutants, and the activated Rac expression vector. We also thank Jonathan Chernoff for providing the dominant negative PAK construct and Dennis Templeton for providing the dominant negative SEK/MKK4 construct.
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FOOTNOTES |
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* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Supported by National Institutes of Health (NIH) National Research Service Award Fellowship 5F32 CA73487.
Supported by NIH Grant CA73756. To whom correspondence should
be addressed: Lineberger Comprehensive Cancer Center, CB# 7295, University of North Carolina, Chapel Hill, NC 27599. Tel.:
919-966-3652; Fax: 919-966-0444.
2 J. L. Norris and A. S. Baldwin, Jr., unpublished results.
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ABBREVIATIONS |
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The abbreviations used are:
MAP kinase, mitogen-activated protein kinase;
IB, inhibitor of NF-
B;
SAPK, stress-activated protein kinase;
ERK, extracellular signal-regulated
kinase;
MEK, MAP/ERK kinase;
JNK, Jun N-terminal kinase;
SEK, SAPK-activating kinase.
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REFERENCES |
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