From the Program in Molecular Signal Transduction,
Division of Basic Sciences, National Jewish Medical and Research
Center, Denver, Colorado 80206 and the ¶ Department of
Pharmacology, University of Colorado Medical School,
Denver, Colorado 80262
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ABSTRACT |
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Cell shape change and the restructuring of the
cytoskeleton are important regulatory responses that influence the
growth, differentiation, and commitment to apoptosis of different cell types. MEK kinase 1 (MEKK1) activates the c-Jun
NH2-terminal kinase (JNK) pathway in response to
exposure of cells to microtubule toxins, including taxol. MEKK1
expression is elevated 3-fold in mitosis and microtubule toxin-treated
cells accumulated at G2/M of the cell cycle. Targeted
disruption of MEKK1 expression in embryonic stem cells resulted in the
loss of JNK activation and increased apoptosis in response to taxol.
Targeted disruption of the MEK kinase 2 gene had no effect on
activation of the JNK pathway in response to microtubule toxins
demonstrating a specific role of MEKK1 in this response. Cytochalasin
D-mediated disruption of actin fibers activates JNK and stimulates
apoptosis similarly in MEKK1 Restructuring of the microtubule cytoskeleton occurs in response
to a variety of events such as immune cell infiltration and invasion,
interaction of cells with the extracellular matrix, and during the
mitotic phase of the cell cycle. Cell shape change and the underlying
restructuring of the cytoskeleton regulates gene expression and
contributes to the commitment of cells to grow, undergo apoptosis, or
differentiate. The importance of the cytoarchitecture and its
regulation was realized when it was described that a reduction in cell
spreading resulting in a spherical versus flat cell shape
had an inhibitory effect on DNA synthesis (1, 2). More recently, it was
demonstrated that cytoarchitecture determines whether a cell will grow
or undergo apoptosis, as decreased cell spreading, which results in
cell rounding using micropatterned substrates of various dimensions,
induced an apoptotic response whereas cell spreading allowed survival
and proliferation (3). The underlying restructuring of the cytoskeleton
during these responses has been shown to influence gene expression. For
example, microtubule disruption induces expression of the
urokinase-type plasminogen activator and interleukin 1- Reorganization of the cytoarchitecture regulates signaling pathways
including the mobilization of intracellular calcium, activation of
tyrosine kinases, Ras, extracellular signal-regulated kinase (ERK),1 and c-Jun
NH2-terminal kinase (JNK) (9-12). Consistent with the activation of signal pathways, specific transcription factors are
activated by cytoskeletal restructuring (8, 13). In this report, we
show that targeted disruption of MEKK1 expression selectively inhibits
JNK activation in response to microtubule toxins. Functionally, MEKK1
is the transducer for the specific regulation of the JNK pathway and
promotes cell survival during changes in microtubule integrity.
Cell Culture--
Cells were maintained in a humidified 7.0%
CO2 environment in Dulbecco's modified Eagle's medium
supplemented with 100 units/ml penicillin, 100 µg/ml streptomycin
(Life Technologies, Inc.). Medium for T47D human breast adenocarcinoma
cells was supplemented with 10% fetal bovine serum (Life Technologies,
Inc.). Medium for MEKK1 Analysis of Kinase Activity--
MEKK1 and JNK activies were
measured as described previously (14), except 1 µg of recombinant
purified kinase-inactive glutathione S-transferase-c-Jun
NH2-terminal kinase kinase (GST-JNKKk-r) was used as a substrate to assay MEKK1 activity. To determine ERK and p38
activity, cell lysates were Western blotted with either an
anti-phospho-ERK or anti-phospho-p38 antibody (New England Biolabs, MA)
as a measure of kinase activity.
Apoptosis Assay--
Apoptosis was measured using acridine
orange/ethidium bromide staining. Apoptotic cell death was verified
using DNA ladder formation assays.
Microscopy--
Cells were plated onto uncoated glass coverslips
2 days before being fixed in a solution containing 3%
paraformaldehyde and 3% sucrose in phosphate-buffered saline (pH 7.4).
Cells were permeabilized with 0.2% Triton X-100 and incubated with an
anti-tubulin antibody, followed by an incubation with 1.5 mg/ml
Cy3-conjugated affinity-purified donkey anti-rabbit Ig
(Jackson ImmunoResearch Laboratories, West Grove, PA). Coverslips were
mounted and analyzed as described previously (14).
JNK Is the Dominant MAPKActivated by Microtubule
Toxins--
Exposure of cells to conditions and drugs that alter the
cytoarchitecture strongly activate the JNK pathway. Fig.
1, A and B, shows
that with T47D human breast carcinoma cells and ES cells, respectively,
microtubule toxins (taxol and nocodazole), actin fiber disruption
(cytochalasin D), and hyperosmolarity (sorbitol) strongly activate the
JNK pathway. In these studies modest concentrations of these stimuli
were used for short times where the cells were not adversely affected,
demonstrating that activation of the JNK pathway is an early major
response to these stimuli.
Analysis of the ERK and p38 pathways indicates that the different
treatments differentially activate MAPK pathways, even though each
stimulus significantly activates JNK. As predicted, hyperosmolarity achieved with sorbitol addition to the growth medium activated p38 in
both cell types. Cytochalasin D and nocodazole activated p38 similarly
in T47D cells but not in ES cells. ERK activation is weak in ES cells
in response to most stimuli2
and not measurably activated in response
to the compounds used to alter cell shape. In contrast, for T47D cells,
cytochalasin D and nocodazole modestly activated ERK, whereas taxol was
a very weak activator of the ERK pathway. Sorbitol-induced
hyperosmolarity stimulated significant ERK activation. Interestingly,
taxol did not significantly activate p38 in either cell type; JNK
activation is the primary response to taxol treatment. This finding
indicates microtubule poisoning by taxol is sensed differently by the
cell than nocodazole-induced depolymerization of microtubules.
MEKK1 Is Activated in Response to Stimuli That Alter
Microtubules--
MEKKs regulate the JNK pathway (15-17), suggesting
that a specific MEKK could mediate the JNK activation in response to
microtubule reorganization of the cytoarchitecture. We found that MEKK1
is activated by stimuli that alter microtubule dynamics. Nocodazole disrupts microtubules in T47D cells (Fig.
2A) and activates MEKK1 in a
concentration- and time-dependent manner (Fig. 2,
B and C). Taxol treatment of T47D cells, which
stabilizes microtubule structures (Fig. 2A), activates MEKK1
(Fig. 2D). Thus, the microtubule toxins, nocodazole and
taxol, activate MEKK1.
MEKK1 Protein Expression Is Increased during the M Phase of the
Cell Cycle--
During the cell cycle the greatest change in
microtubule structure occurs in mitosis. Analysis of T47D breast
carcinoma cells proliferating in normal growth conditions demonstrated
that the expression of MEKK1 is increased in the M phase of the cell
cycle. Fig. 3A shows T47D
cells, co-stained using an antibody recognizing MEKK1 and the DNA
stain, DAPI. Immunofluorescence microscopy readily demonstrated that
mitotic cells have significantly higher levels of MEKK1 expression than
non-mitotic cells. To quantitate differences in anti-MEKK1
immunofluorescence in mitotic versus non-mitotic cells,
deconvolved confocal three-dimensional images were constructed. Images
of mitotic and non-mitotic cells were quantitated for anti-MEKK1 immunofluorescence. Fig. 3B shows that mitotic T47D cells
express approximately 3-fold higher MEKK1 protein levels than
non-mitotic cells. Similar results have been observed in other cell
types (not shown).
To biochemically confirm the immunofluorescence analysis, T47D cells
were treated with microtubule toxins to arrest cells at
G2/M in the cell cycle. The G2/M block of
treated cells was confirmed by cell cycle analysis using flow cytometry
(not shown). Immunoblotting with the anti-MEKK1 antibody used for
immunofluorescence demonstrated that MEKK1 protein levels were
increased in drug-treated cells accumulated at G2/M,
compared with untreated cell populations having cells randomly in all
phases of the cell cycle (Fig. 3C, upper left
panel). As a control, treatment of cells with etoposide, which
induces DNA damage in S phase and blocks cells in G1/S of the cell cycle, did not increase MEKK1 expression (Fig. 3C,
upper right panel). In fact, etoposide, which acutely
activates MEKK1, induces a loss of MEKK1 expression in T47D cells after
several hours of drug exposure, resulting from the induction of caspase 3 cleavage of the 196-kDa MEKK1 protein (18, 19). We have found that
taxol and nocodazole treatment of cells does not induce the cleavage of
MEKK1 like that observed for DNA-damaging drugs. Thus, the regulation
of MEKK1 protein levels and caspase cleavage in response to microtubule
toxins and DNA-damaging drugs is different. Consistent with the
increased levels of MEKK1 in T47D cells blocked at G2/M,
the total MEKK1 activity is also increased by the microtubule toxin-induced block at G2/M (Fig. 3C,
lower panel). In contrast, hydroxyurea and etoposide, which
act in S phase of the cell cycle do not cause an increase in MEKK1
activity following prolonged cellular exposure to these drugs.
Cumulatively, the findings demonstrate increased MEKK1 expression at
the G2/M phase of the cell cycle and that microtubule
toxins activate MEKK1. Thus, MEKK1 regulation is responsive to changes
in microtubule organization in the cell.
Targeted Disruption of MEKK1 Expression Causes Loss of JNK
Activation in Response to Microtubule Reorganization--
We have
targeted the disruption of MEKK1 expression by homologous recombination
(20). MEKK1
We have also disrupted the expression of MEKK2 by homologous
recombination in ES cells (Fig. 5C).3
Both microtubule and actin fiber
disruption by nocodazole and cytochalasin D, respectively, activated
the JNK pathway similarly in MEKK2+/+ and
MEKK2 Targeted Disruption of MEKK1 Expression Increases Apoptosis in
Response to Microtubule Toxins--
We have shown that
MEKK1
Cytochalasin D disrupts the actin cytoskeleton but has little effect on
microtubule integrity. Cytochalasin D treatment of T47D cells strongly
stimulates MEKK1 and JNK activities (Fig. 7A). Similarly, cytochalasin D
treatment of wild type ES cells activates MEKK1 (Fig. 7B)
and strongly activates JNK in both MEKK1+/+ and
MEKK1 Changes in cytoskeletal dynamics stimulate signal transduction
pathways. Several studies indicate that the microtubules play an
integral part in regulating signaling pathways. We have shown that JNK
is the dominant MAPK activated by microtubule toxins, including taxol
and nocodazole. MEKK1 is absolutely required for JNK activation when
cells are exposed to nocodazole and taxol. MEKK1 is similarly required
for JNK activation in response to mild hyperosmolarity and cold stress,
both of which alter the integrity of the microtubule cytoskeleton and
cell shape. Targeted disruption of MEKK1 expression unequivocally
defined its role by being the MAPK kinase kinase regulating the JNK
pathway in response to changes in microtubule integrity. How might
microtubule restructuring activate MEKK1? One hypothesis would involve
the GTP-binding protein Rac1 (21). Rac1 has been shown to bind
The targeted gene disruption of MEKK1 expression unequivocally defined
the function of MEKK1 in responding to changes in microtubule dynamics
in mouse ES cells, namely the protection of ES cells from committing to
apoptosis. The role of MEKK1 is specific in that loss of its expression
causes complete loss of JNK activation in response to microtubule
disruption. Targeted disruption of MEKK2 expression had no effect on
the JNK response to microtubule disruption in ES cells. Thus, each MEKK
will be predictably found to respond to very specific upstream stimuli.
Support for this prediction is observed with the loss of specific
receptor activation of the JNK pathway in MEKK2 knockouts that is
not observed with MEKK1
knockouts.3
The role of MEKK1 in cell survival is contrary to its pro-apoptotic
functions when cleaved by caspases (18, 25). MEKK1 is a substrate for
caspase 3 and is cleaved at Asp874. The cleavage of MEKK1
at Asp874 releases a 91-kDa activated kinase domain that
amplifies the activation of caspases. The activated full-length 196-kDa
MEKK1 does not activate caspases or apoptosis (18, 19, 25). Thus, there
is a dual role of MEKK1 that is controlled by caspases. Activation of
MEKK1 promotes cell survival; targeted disruption of MEKK1 expression
unequivocally defines this function of MEKK1. Caspase cleavage of MEKK1
causes loss of the survival response and conversion to a pro-apoptotic
response of the newly generated 91-kDa MEKK1 COOH-terminal kinase
domain. If the survival response mediated by MEKK1 could be abrogated,
then microtubule toxins like taxol would have greater efficacy as
chemotherapeutic drugs. From a practical standpoint, defining proteins
and genes specifically regulated by the MEKK1-activated JNK pathway
might define new drug targets for the treatment of cancer and other diseases.
/
and wild type cells. The
results show that MEKK1 is required for JNK activation in response to
microtubule but not actin fiber toxins in embryonic stem cells. MEKK1
activation can protect cells from apoptosis in response to change in
the integrity of the microtubule cytoskeleton.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
but inhibits
tubulin synthesis (4-6). The promoter region for the B chain of
platelet-derived growth factor was shown to contain a cis-acting
response element that was regulated by shear stress induced by changes
in cytoarchitecture (7). The collagenase-1 gene, which encodes a matrix
metalloproteinase important for cell migration and invasion, is also
regulated in response to cytochalasin D disruption of the actin
cytoskeleton (8).
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/
and MEKK1+/+ mouse
embryonic stem (ES) cells was supplemented with 15% heat-inactivated fetal bovine serum (Summit Biotechnology, CO), 144 µM
monothioglyerol (Sigma), and 1% leukemia inhibitory factor Chinese
hamster ovary cell-conditioned medium.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Regulation of JNK, ERK and p38 pathways by
cytoskeletal toxins and hyperosmolarity. T47D human breast
carcinoma cells (A) or mouse ES cells (B) were
incubated with 2 µg/ml cytochalasin D (Cyto D), 5 µM taxol, 0.5 µg/ml nocodazole (Nocod), or
400 mM sorbitol for the indicated times. JNK was assayed
using GST-c-Jun as a substrate. For analysis of ERK and p38 activity
cell lysates were prepared, resolved by SDS-polyacrylamide gel
electrophoresis, and immunoblotted with either anti-phospho-ERK or
anti-phospho-p38 antibodies. The experiment shown is representative of
several experiments analyzing time courses and activation of MAPK
pathways in response to these stimuli.
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Fig. 2.
MEKK1 is activated in response to treatment
of cells with nocodazole or taxol. A, T47D cells
exposed to 0.5 µg/ml nocodazole for 30 min or 10 µM
taxol for 16 h have an altered microtubule cytoskeleton. Shown are
immunofluorescence images of representative control, and nocodazole-
and taxol-treated cells stained with anti-tubulin antibodies. T47D
cells were treated with the indicated concentrations of nocodazole
(B) or taxol (D) for 30 min or for different
times with 0.5 µg/ml nocodazole (C). Cells were lysed and
MEKK1 immunoprecipitated and assayed measuring in vitro
kinase activity using JNKKk-r as substrate. The experiment
is representative of 2-4 independent determinations for each
condition.
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Fig. 3.
MEKK1 expression is increased in the M phase
of the cell cycle. A, proliferating T47D breast
adenocarcinoma cells were fixed and fluorescently stained with an
anti-MEKK1 antibody followed by a Cy3-tagged secondary
antibody. DNA was stained with DAPI. Metaphase cells, evident by
chromosome condensation, were stained more intensely with the
anti-MEKK1 antibody relative to G1/S phase T47D cells.
Shown are 0.5-µm deconvolved confocal images of the thickest region
of each the cell. B, for quantitation, 10 M phase and 10 G1/S phase cells were analyzed by three-dimensional
reconstruction of 0.5-µm confocal sections of the cell. The
fluorescence signal over the entire thickness of the cell was summed.
Shown is the mean ± S.D. of the summed MEKK1 immunofluorescence of G1/S
versus M phase T47D cells. The difference in
immunofluorescence between G1/S and M phase cells is
significant at a p < 0.05 using the Student's
t test. C, upper panels, T47D cells
were incubated for 20 h in 10% serum without (control) or with
0.05 µg/ml nocodazole, 1 µM taxol, 1 µM
colchimide, or 0.1 µM etoposide. Flow cytometry analysis
verified that taxol, nocodazole, and colchimide arrested cells at
G2/M and etoposide-treated cells were blocked in
G1/S. Cells were harvested and analyzed by immunoblotting
for MEKK1 expression. The blot showing control and etoposide
(right) was exposed longer than the blot showing MEKK1
expression (left) in cells exposed to microtubule toxins.
Lower panel, MEKK1 was immunoprecipitated and assayed from
lysates prepared from T47D cells treated with 1 mM
hydroxyurea, 0.1 µM etoposide, 1 µM taxol,
0.05 µg/ml nocodazole, or no drug for 20 h. MEKK1 activity was
assayed using JNKKk-r in an in vitro kinase
assay.
/
ES cells do not express the MEKK1 protein.
Treatment of wild type ES cells with either taxol or nocodazole
activates MEKK1 measured by immunoprecipitation and in vitro
kinase assay (Fig. 4, A and
B). Importantly, no kinase activity is observed in
immunoprecipitates from lysates of MEKK1
/
ES cells
treated with nocodazole (Fig. 4B). This finding
unequivocally demonstrates that our antibodies selectively
immunoprecipitate MEKK1 and that MEKK1 is responsible for the JNKK
phosphorylation in the in vitro kinase assay. As predicted
from this result, JNK activation in response to the microtubule toxins
nocodazole and taxol is lost in two independent MEKK1
/
clones (Fig. 5A).
Interestingly, the disruption of the actin cytoskeleton with
cytochalasin D activates the JNK pathway in MEKK1
/
and
MEKK1+/+ ES cells (Fig. 5A). Thus, MEKK1 is
absolutely required for JNK activation in response to microtubule but
not actin fiber disruption. Re-expression of the 196-kDa MEKK1 protein
by stable transfection of MEKK1
/
with a plasmid
encoding the full-length MEKK1 cDNA reconstituted the regulation of
the JNK pathway by taxol (Fig. 5B). The reconstitution of
JNK activation in response to taxol demonstrates that this response
is specifically mediated by MEKK1.
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Fig. 4.
Specificity of the MEKK1 in vitro
kinase assay from microtubule poison-treated cells. ES
(MEKK1+/+ or MEKK1 /
) cells were
treated with 5 µM taxol for the indicated times or with
different concentrations of nocodazole for 30 min. Cells were lysed and
MEKK1-immunoprecipitated. JNKKk-r was added with
[
-32P]-ATP to the immunoprecipitate.
MEKK1-dependent phosphorylation of JNKKk-r was
determined by resolving the proteins in the assay using
SDS-polyacrylamide gel electrophoresis followed by
autoradiography.
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Fig. 5.
MEKK1 but not MEKK2 is required for JNK
activation in response to exposure of ES cells to microtubule
poisons. A, wild type ES cells (MEKK1+/+)
and two ES cell clones having targeted disruption of MEKK1 expression
(MEKK1 /
#4 and #15) were exposed to 4 µg/ml cytochalasin D (Cyto D), 5 µM taxol,
0.5 µg/ml nocodazole (Nocod) or growth medium only
(Control) for 2 h. Cells were lysed and JNK activity
was measured using GST-c-Jun as substrate. B, the cDNA
encoding the full-length 196-kDa MEKK1 in pCEP4 was stably transfected
into MEKK1
/
cells. Hygromycin-resistant,
MEKK1-expressing clones were characterized by immunoblotting. Two
re-expression clones (AB1 and AB2) and
MEKK1
/
and MEKK1+/+ ES cells were
challenged with 5 µM taxol for 1 h, lysed, and
assayed for JNK activity. C, immunoblot of
MEKK2+/+, MEKK2+/
, and MEKK2
/
cells, showing the loss of MEKK2 expression in ES cells having targeted
disruption of the MEKK2 gene. D, cytochalasin D (2 µg/ml)
and nocodazole (0.5 µg/ml) were incubated with wild type (+/+) and
MEKK2
/
(
/
) ES cells for 2 h. Cells were lysed
and assayed for JNK activity using GST-c-Jun as substrate.
/
ES cells (Fig. 5D). Thus, MEKK2 is
not involved in the regulation of the JNK pathway in response to
changes in the microtubule and actin cytoskeleton; the microtubule
response involves MEKK1. This finding demonstrates the selectivity of
different MEKKs for regulation by specific stimuli.
/
ES cells do not activate JNK following treatment
with nocodazole or taxol. Prolonged shape change and microtubule
disruption induce apoptosis in many cell types (3, 20). Fig.
6A shows that
MEKK1
/
ES cells have a significantly greater apoptotic
index in response to taxol, relative to MEKK1+/+ ES cells.
Fig. 6B shows that temporally MEKK1
/
cells
become apoptotic more rapidly than MEKK1+/+ cells. At a
significantly slower rate, MEKK1+/+ cells will reach the
same apoptotic index as taxol-treated MEKK1
/
cells.
Thus, MEKK1 activation has a protective function in response to
microtubule poisoning. To prove this fact, the two
MEKK1
/
ES cell lines having MEKK1 expression
re-established by stable transfection of a MEKK1 expression plasmid
were tested for their sensitivity to taxol-induced apoptosis (Fig.
6A). Just as in the reconstitution of JNK activation, the
re-expression of MEKK1 expression rescued the survival of cells exposed
to taxol similar to wild type ES cells.
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Fig. 6.
MEKK1 expression protects cells from
taxol-induced apoptosis. A, ES cells, wild type (+/+),
MEKK1 /
(
/
), and MEKK1 re-expression (AB)
clones were incubated in growth medium without (control) or
with 100 nM taxol for 12 h. B, the
designated ES cell clones were incubated with 100 nM taxol
for 4, 8, or 12 h. Apoptotic cells were determined by quantitating
acridine orange staining of condensed nuclei. The results represent the
mean ± S.E. and are representative of three independent
experiments.
/
ES cells (Fig. 5A). The activation of
JNK, independent of MEKK1 in ES cells, allowed the question of whether
MEKK1 or JNK mediates the cell survival response to actin cytoskeleton
poisoning. Fig. 7, C and D, shows that the
sensitivity of ES cells to undergo cytochalasin D-induced apoptosis is
virtually identical in a MEKK1
/
or MEKK1+/+
background. The increased apoptotic response of MEKK1
/
cells is specific to microtubule toxins and not actin cytoskeleton toxins. Thus, the enhanced sensitivity of MEKK1
/
cells
to undergo apoptosis in response to microtubule toxins may involve the
loss of JNK activation regulated by MEKK1.
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Fig. 7.
MEKK1 is activated but is not required for
JNK stimulation following exposure of ES cells to cytochalasin D. A, T47D cells were exposed to 8 µg/ml cytochalasin D for
the indicated times, 0.5 µg/ml nocodazole (N) for 2 h, or buffer with Me2SO for 2 h ( ). Cells were lysed
and MEKK1-immunoprecipitated with anti-MEKK1 antibody.
Immunoprecipitates were incubated with recombinant JNKKk-r
and [
-32P]-ATP, resolved by SDS-polyacrylamide gel
electrophoresis, and analyzed by autoradiography. Autophosphorylation
of MEKK1 and phosphorylation of JNKKk-r is shown by
arrows. For assaying JNK activity, GST-c-Jun was added to
aliquots of the same cell lysates used for immunoprecipitation.
B, wild type ES cells were incubated with 8 µg/ml
cytochalasin D for the indicated times. Cells were lysed, MEKK1
antibody was added to lysates for immunoprecipitation, and MEKK1
activity was assayed as described in A. C and
D, wild type (+/+) and MEKK1
/
(
/
) ES
cells were incubated without or with 2 µg/ml cytochalasin D
(CD) for 24 h (C) or 20 or 24 h
(D). Apoptotic cells were quantitated by acridine orange
staining. The results represent the mean ± S.E. of triplicate
determinations and are representative of three independent
experiments.
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-tubulin in a GTP-dependent manner (22). The binding of
Rac1·GTP did not influence tubulin polymerization, and it was
proposed that tubulin-Rac1·GTP complexes would control Rac1
signaling. MEKK1 binds Rac1 in a GTP-dependent manner (14).
Rac1 activates the JNK pathway as does MEKK1 (23, 24), and inhibitory
MEKK1 mutants block Rac1 activation of JNK (14). If Rac1 functions as a
sensor for cytoskeletal changes it could stimulate MEKK1, resulting in JNK activation and protection of cells from apoptosis. The increased expression of MEKK1 during the G2/M phase of the cell cycle
is consistent with its involvement in sensing the dramatic microtubule changes that occur during mitosis. The protective properties of MEKK1
would prevent cells from defaulting into apoptosis during progression
through the G2/M phase. The increased apoptotic response of
MEKK1
/
ES cells to microtubule toxins is consistent
with this hypothesis. Confirmation of this hypothesis will require the
use of an inducible dominant-negative Rac1 construct to determine the
role of Rac1 in microtubule disruption-stimulated JNK activity. In the
future, cell lines expressing an inducible dominant-negative Rac1 will be generated to test this hypothesis.
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ACKNOWLEDGEMENT |
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We thank Nancy Lassignal Johnson for expert assistance in digital confocal immunofluorescence imaging and figure preparation.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants DK37871, DK48845, GM30324, and CA58157.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.
§ Leukemia Society Fellow.
To whom correspondence should be addressed: Program in
Molecular Signal Transduction, Division of Basic Sciences, National Jewish Medical and Research Center, 1400 Jackson St., Denver, CO 80206. Tel.: 303-398-1504; Fax: 303-398-1225; E-mail: johnsong{at}njc.org.
2 T. Yujiri and G. L. Johnson, unpublished observations.
3 T. P. Garrington, T. Yujiri, S. Gibson, and G. L. Johnson, manuscript in preparation.
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ABBREVIATIONS |
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The abbreviations used are: ERK, extracellular signal-regulated kinase; MEKK, MEK kinase; JNK, c-Jun NH2-terminal kinase; MAPK, mitogen-activated protein kinase; ES, embryonic stem; JNKK, JNK kinase; GST, glutathione S-transferase; JNKKk-r, recombinant kinase-inactive JNKK; DAPI, 4',6-diamidino-2-phenylindole.
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