(Received for publication, October 10, 1995; and in revised form, November 28, 1995)
From the
Mitogen-activated/extracellular response kinase kinase (MEK) kinase (MEKK) is a serine-threonine kinase that regulates sequential protein phosphorylation pathways, leading to the activation of mitogen-activated protein kinases (MAPK), including members of the Jun kinase (JNK)/stress-activated protein kinase (SAPK) family. In Swiss 3T3 and REF52 fibroblasts, activated MEKK induces cell death involving cytoplasmic shrinkage, nuclear condensation, and DNA fragmentation characteristic of apoptosis. Expression of activated MEKK enhanced the apoptotic response to ultraviolet irradiation, indicating that MEKK-regulated pathways sensitize cells to apoptotic stimuli. Inducible expression of activated MEKK stimulated the transactivation of c-Myc and Elk-1. Activated Raf, the serine-threonine protein kinase that activates the ERK members of the MAPK family, stimulated Elk-1 transactivation but not c-Myc; expression of activated Raf does not induce any of the cellular changes associated with MEKK-mediated cell death. Thus, MEKK selectively regulates signal transduction pathways that contribute to the apoptotic response.
The c-Jun kinase (JNK)()/stress-activated protein
kinase (SAPK) has been shown to be activated by diverse stimuli,
including growth factors, cytokines, gamma and ultraviolet irradiation,
and protein synthesis inhibitors(1, 2, 3) .
Growth factors activate the extracellular response kinase pathway and
may also activate the JNK/SAPK pathway. In contrast, specific cytokines
and stresses to the cell appear to preferentially activate the JNK/SAPK
pathway(1, 2, 3, 4, 5) .
Several of the cytokines and stresses that activate the JNK/SAPK
pathway also induce cell death characteristic of apoptosis. The
JNK/SAPKs have been shown to phosphorylate and regulate the activity of
several transcription factors including c-Jun, Elk-1, and
ATF-2(1, 2, 4, 6, 7) .
Similar to the ERK members of the MAPK family, JNK/SAPK is a component
of a sequential protein kinase
pathway(1, 4, 8, 9, 10, 11, 12, 13) .
JNK/SAPK is phosphorylated, resulting in its activation, by JNK kinase
(JNKK)/stress-activated Erk kinase
(SEK-1)(9, 10, 14, 15) . JNKK/SEK-1
is itself regulated by phosphorylation by an upstream kinase referred
to as MEK kinase (MEKK)(13) . The MEKK-regulated JNK/SAPK
sequential protein kinase pathway is parallel to the Raf/Erk pathway.
Apoptosis is a regulated cell death process characterized by
cytoplasmic shrinkage, nuclear condensation, and DNA fragmentation (16) . Apoptosis can be induced by signaling from specific cell
surface receptors (i.e. tumor necrosis factor (TNF) and
Fas)(17, 18) , expression of viral proteins such as
E1A(19, 20, 21) , and anticancer agents
including chemotherapeutic drugs and ionizing radiation(22) .
It is known that specific growth factors and cytokines can protect
cells against apoptosis(23, 24, 25) , Bcl-2
and Bcl-x are protective against apoptosis(23, 25) , a
wild-type p53 protein is often involved in the nuclear events mediating
apoptosis(20, 27, 28) , and c-Myc can be
required for apoptosis(24, 30) . Despite the
identification of proteins involved in mediating or protecting against
apoptosis, biochemical pathways involved in apoptosis are poorly
defined.
Receptor-stimulated apoptosis must involve signal transduction pathways that transfer cell surface events to cytoplasmic and nuclear proteins that initiate the cell death program. Signal transduction pathways involved in apoptosis appear to overlap with those that mediate growth and differentiation(31, 32) . The integration of signal transduction pathways regulated by growth factor and cytokine receptors commits a cell either to proliferation or apoptosis(33) . Cellular stress such as DNA damage can stimulate signal transduction pathways and alter their integration so that the cell commits to a pathway of apoptosis(34) . Several checkpoints exist in the pathways leading to apoptosis that involve proteins such as Bcl-2 and p53. Overexpression of Bcl-2 can collaborate with oncoproteins to suppress apoptosis and increase tumor cell growth. Similarly, mutational inactivation of p53 can inhibit apoptosis and contribute to the resistance of tumor cells to chemotherapeutic agents and irradiation(28) .
To date, candidate molecules involved in
signaling apoptosis include ceramide(35) , Ras(31) ,
Rho(36) , c-Myc(36) , c-Jun(30) , and the
proteins associated with the TNF receptor (38) and
Fas(39) . The interleukin converting enzyme-like proteases
appear to be frequent end point mediators of apoptosis(40) .
Members of the MAPK family may be predicted to be involved in apoptotic
signaling because Ras can regulate their activity and may be involved
in ceramide-mediated apoptosis(31) . MAPKs are also involved in
the regulation of transcription factors including c-Myc and c-Jun,
which have been shown to influence apoptosis. The regulation of MAPKs
including the ERKs, JNK/SAPK and p38/Hog1 involves sequential
phosphorylation pathways. For ERKs the pathway is Raf-MEK-ERK, which is
strongly implicated in the regulation of growth and differentiation of
different cell types. For JNK/SAPK and p38/Hog1 the proposed pathway
involves a MEKK, JNKK/SEK, JNK/SAPK, or p38/Hog1. The exact number and
selectivity of members of these kinase pathways is still being defined.
Members of the TNF receptor family including the TNF
receptor(4) , Fas, (
)and CD40 (5) activate
JNK/SAPK pathways. The TNF
receptor and Fas can mediate apoptosis,
but CD40 actually can be protective against apoptosis in B
cells(33, 41, 42) . Even though JNK/SAPK
activation is a common signal response to these receptors, their
influence on cell function can be quite different. Thus, JNK/SAPK
activation per se would not be predicted to be the primary
mediator of apoptotic signaling by TNF receptor family members. Signal
transduction pathways in addition to or other than JNK/SAPK activation
must therefore be involved in mediating receptor-stimulated apoptosis.
In this report, we demonstrate that expression of activated MEKK
mediates a cell death response characterized by cytoplasmic shrinkage,
nuclear condensation, and DNA fragmentation. MEKK-mediated cell death
appears independent of JNK/SAPK activation and has properties
characteristic of apoptosis.
Swiss 3T3 cells were microinjected with 100 ng/µl pCMV-gal
and 20 ng/µl pCMV5MEKK
. To label free DNA ends,
fixed and rehydrated cells were incubated with terminal
deoxytransferase (TDT) and 10 nM biotin-dUTP following the
manufacturer's instructions (Boehringer Mannheim). Cells were
stained with FITC-streptavidin to label DNA fragments.
-Galactosidase (
-gal) was detected using rabbit
anti-
-gal antibody (Cappel Laboratories) and a rhodamine-labeled
goat anti-rabbit antibody (Cappel Laboratories).
Figure 1:
Morphological changes induced by
expression of activated MEKK (MEKK). Swiss 3T3 cells
were microinjected with pCMV5
-gal alone (Control) or in
the presence of pCMV5MEKK
(MEKK). At
17-18 h post-injection, cells were fixed and stained with X-gal
to identify injected cells. The control panel shows six injected cells
denoted by arrows, all of which have a normal flattened
morphology similar to the adjacent non-injected cells. The MEKK panel
shows six injected cells denoted by arrows and identified by
X-gal staining, all of which have an altered morphology. Three of the
MEKK
-expressing cells have a highly condensed cytoplasm
and the three cells in the bottom part of the panel are at earlier
stages of cytoplasmic shrinkage and cellular condensation. None of the
uninjected cells in the panel have an altered morphology and are
similar to the control cells. The panels are representative of
10-12 different experiments and 3-4 independent plasmid
preparations.
For further analysis and comparison cells were
microinjected with BxBRaf, a truncated activated form of Raf-1 (51) that selectively activates the ERK pathway(12) .
In microinjected cells, expression of -gal, MEKK
,
or BxBRaf was demonstrated by indirect immunofluorescence using
specific antibodies recognizing each protein (Fig. 2). Swiss 3T3
cells (panels A and B) and REF 52 cells (panels C and D) microinjected with the indicated expression
plasmid were fixed and stained only 8 h post-injection to demonstrate
that each protein was being expressed in the cytoplasm of the cells.
Since the cells shown in Fig. 2were fixed and stained soon
after injection, they were not yet undergoing significant cellular
condensation as observed in Fig. 1. However, it is apparent with
the REF 52 cells expressing MEKK (panel C) that they have
begun to undergo a morphological change relative to
-gal-expressing cells (panel D).
Figure 2:
Expression MEKK and BxBRaf
in Swiss 3T3 and REF52 cells. Swiss 3T3 cells (panels A and B) and REF52 cells (panels C and D) were
micoinjected with pCMV5MEKK
, pCMV5BxBRaf, or
pCMV
-gal as indicated. Eight hours after injection, cells were
fixed and stained by indirect immunofluorescence for expression of each
protein. The MEKK
protein has the 9-amino acid
hemagglutinin tag (YPYDVPDYA) at its NH
terminus and was
detected using the mouse monoclonal 12CA5 antibody and a secondary
FITC-rabbit anti-mouse antibody. BxBRaf was detected using a rabbit
anti-Raf antibody recognizing the COOH terminus of Raf-1, and a
rhodamine-donkey anti-rabbit secondary antibody.
-Gal was detected
using a mouse anti-
-gal monoclonal antibody, and the FITC-rabbit
anti-mouse secondary antibody. At 8 h post-injection, the morphological
changes in the MEKK
-expressing REF52 cells (panel
C) are beginning to be apparent while the changes in Swiss 3T3
cells are not yet apparent (compare panels A and B).
Quantitation of
microinjection experiments demonstrated that expression of
MEKK resulted in significant cell death characterized by
the dramatic morphological condensation (Table 1). In contrast,
BxBRaf expression did not affect cell viability relative to control
cells expressing only
-gal. Approximately 84% of all
MEKK
-injected cells had a highly condensed cellular
morphology 17 h after injection. This count actually underestimates the
number of condensed cells because Swiss 3T3 cells in advanced stages of
the cell death response were often nonadherent to coverslips. Some of
the nonadherent highly condensed cells could be found to be released
from the coverslip into the culture medium, but were not scored in the
quantitation. In contrast, fewer than 3% of BxBRaf and 1% of control
-gal-injected cells had an altered morphology even after
48-72 h post-injection.
Cell death resulting from
MEKK expression required the kinase activity of the
enzyme; the kinase inactive mutant of MEKK
was without
effect (Table 1). The apoptotic-like cell death was also
dependent on the MEKK
concentration as measured by
serial dilution (0-100 ng/µl) of the expression plasmid used
for microinjection. Maintenance of the MEKK
-expressing
cells in 10% serum slightly prolonged the time required for induction
of cytoplasmic shrinkage, nuclear condensation, and cell death,
suggesting that growth factors and cytokines had some influence on the
onset of the response induced by MEKK
but high serum
could not prevent MEKK
induced cell death. Greater than
80% of MEKK
-expressing cells had a cytoplasmic and
nuclear morphology characteristic of apoptosis 18 h post-injection.
Fig. 3A demonstrates in more detail the dramatic
morphological changes in Swiss 3T3 cells resulting from expression of
MEKK. Cytoplasmic shrinkage is evident from the
-gal staining and nuclear condensation is obvious in
MEKK-1-expressing cells stained with propidium iodide. In contrast,
cells expressing BxBRaf do not demonstrate any detectable morphological
difference from control cells expressing only
-gal. A similar
dramatic cytoplasmic shrinkage and nuclear condensation was observed
with MEKK
expression in REF52 cells (Fig. 3B), where BxBRaf again had no effect on
cytoplasmic and nuclear integrity. To assess if DNA fragmentation was
induced by MEKK
expression, TDT was used to covalently
transfer biotin-dUTP to the ends of DNA breaks in situ (Fig. 4). Streptavidin-FITC was then used for detection of
dUTP incorporated into cellular DNA. Even though Swiss 3T3 cells do not
undergo significant DNA degradation and laddering at the nucleosomal
level, they do generate larger DNA fragments when stimulated to undergo
apoptosis(32) . The condensed nuclei of
MEKK
-injected cells were highly fluorescent, indicating
significant DNA fragmentation (Fig. 4, B, D,
and F). It is also apparent that the cytoplasm has become
highly condensed and the condensed chromatin is distinct from the
cytoplasm. Microinjected cells not yet undergoing cytoplasmic and
nuclear condensation in response to MEKK
did not
incorporate dUTP into their DNA. Thus, expression of MEKK
induced all the hallmarks of apoptosis, including cytoplasmic
shrinkage, nuclear condensation, and DNA fragmentation.
Figure 3:
Expression of MEKK induces
cytoplasmic shrinkage and nuclear condensation. Swiss 3T3 cells (A) and REF52 cells (B) were microinjected with the
pCMV5 expression plasmid encoding
-gal in the absence (Control) or the presence of pCMV5 expression plasmids for
MEKK
or BxBRaf. Seventeen h after injection, Swiss 3T3
cells were fixed and stained by indirect fluorescence for
-gal
expression to detect injected cells. REF52 cells were fixed 42 h after
injection. Cells were also stained with propidium iodide for detection
of DNA and nuclear morphology. Expression of MEKK
caused
dramatic cytoplasmic shrinkage and nuclear condensation. One of the two
REF52 cells expressing MEKK
has lost its nucleus, and
only the cytoplasmic blebs remain on the
coverslip.
Figure 4:
Detection of DNA fragments in Swiss 3T3
cells expressing MEKK. Cells were microinjected with the
expression plasmids for
-gal and MEKK
. Eighteen h
after injection, cells were fixed and incubated with TDT and 10 nM biotin-dUTP to label the ends of DNA fragments. The fixed cells
were then washed and incubated with a rabbit anti-
-gal antibody
and detected with a rhodamine-goat anti-rabbit secondary antibody.
Biotin-dUTP was stained with FITC-streptavidin. Panels A-D represent two different fields showing an
MEKK
-expressing cell having a highly condensed cell
morphology and TDT-positive labeling. In the same field are injected
cells that have not yet undergone the morphological changes and are
TDT-negative in their nuclear staining. Panels E and F are 3-fold magnifications of the apoptotic cell in panels C and D. The TDT reaction clearly labels DNA that is highly
condensed in the nucleus, whereas the
-gal staining is in the
condensed cytoplasm. Cells microinjected with pCMV5
-gal alone were
normal, had a flattened morphology, and were
TDT-negative.
Expression
of BxBRaf did not induce a response measured by any of the criteria
mentioned above. BxBRaf-expressing cells displayed a normal flattened
morphology similar to -gal-expressing cells or to uninjected cells (Fig. 3, A and B). Transient BxBRaf expression
in Swiss 3T3 cells stimulated ERK activity (data not shown), and the
transactivation function of the Gal4/Elk-1 chimeric transcription
factor (Fig. 5) whose activation is dependent on phosphorylation
by Erk members of the MAPK
family(45, 52, 53) . Cumulatively, the
results indicate that activation of the Raf/ERK pathway does not induce
the cytoplasmic and nuclear changes observed with MEKK.
Figure 5: Wild-type Swiss 3T3 cells were transfected with pCMV5BXBRaf or pCMV5 without a cDNA insert in the presence of expression plasmids encoding Gal4/Elk-1 and Gal4-TK-luciferase as described under ``Materials and Methods.'' Forty-eight h post-tranfection, cells were lysed and assayed for luciferase activity.
Figure 6:
Induction of MEKK expression in Swiss 3T3 cells increases the number of condensed
cells. A, Swiss 3T3 cells having an inducible MEKK
under the control of the lac repressor were isolated
using the LacSwitch inducible mammalian expression system (Stratagene).
IPTG (5 mM) induced MEKK
expression in two
independent clones (MEKK-1 and MEKK-2). MEKK
protein
expression was detected using an antibody recognizing the extreme COOH
terminus of MEKK(13) . B, parental
LacR
, MEKK-1, and MEKK-2 cells were incubated in the
presence or absence of 5 mM IPTG for 48 h. Cells were stained
with acridine orange and condensed cells quantitated per 1000 cells
counted per coverslip. Results are representative of three independent
experiments with each clone. C, Swiss 3T3 cells having
inducible MEKK
were incubated in the presence or absence
of 5 mM IPTG for 17 h. The indicated cells were then exposed
to UV-C irradiation 39 J/M
). Two h after irradiation, cells
were fixed and stained with propidium iodide. Condensed cells having a
morphology similar to that shown in Fig. 1were quantitated for
each condition. Results are representative of three independent
experiments.
It
was found that IPTG-induced MEKK expression stimulated
signal transduction pathways that made the cells significantly more
sensitive to stresses that induce cell death. For example, cells
expressing MEKK
were highly sensitive to ultraviolet
irradiation (Fig. 6C). Two hours after exposure to
ultraviolet irradiation greater than 30% of the
MEKK
-expressing cells became morphologically highly
condensed and appeared apoptotic. In contrast, the population of
uninduced cells showed no increase in condensed apoptotic-like cells at
this time point. Thus, overnight induction of MEKK
expression modestly increased the basal index of morphologically
condensed cells and primed the cells for apoptosis in response to UV
irradiation. The results indicate that MEKK-regulated signal
transduction pathways enhance apoptotic responses to external stimuli.
Figure 7:
Assay of MEKK-regulated protein kinases in
Swiss 3T3 cells. A, the parental LacR or
MEKK-2 Swiss 3T3 cells were incubated for 17 h in the absence or
presence of 5 mM IPTG and assayed for JNK/SAPK activity. The
LacR
clone lacks the MEKK
expression
plasmid. Induction of MEKK
results in the activation of
JNK/SAPK, which phosphorylates glutathione S-transferase-c-Jun. B, induction of MEKK
does not activate ERK (p42/44 MAPK) or p38/Hog1 activity in the
MEKK-2 Swiss 3T3 clone. Treatment of MEKK-2 Swiss 3T3 cells with PDGF
(10 ng/ml, 10 min) or sorbitol (400 mM, 20 min) activated ERK
and p38/Hog1 activity, respectively, demonstrating these response
pathways were functional. C, regulation of specific
transactivation by MEKK
. Left panel, the MEKK-2
Swiss 3T3 clone was used for measurement of
Gal4/Jun
, Gal4/Myc
and
Gal4/Elk-1
transactivation in response to
IPTG-induced MEKK
expression. Induction of MEKK
expression did not significantly increase Gal4/Jun
transactivation. Right panel, transient transfection of
MEKK
resulted in increased Gal4/Jun transactivation in
the MEKK-2 Swiss 3T3 cell clone. A similar result was seen with or
without IPTG treatment. The condition shown is with IPTG
incubation.
The failure of IPTG-induced MEKK expression to activate Gal4/Jun may be related to the multiple
c-Jun NH
-terminal phosphorylation sites involved in
regulating c-Jun transactivation. Serines 63 and 73 and threonines 91
and 93 are apparent regulatory phosphorylation sites in
c-Jun(1, 54, 55, 56) . Both clusters
are proposed to be sites of phosphorylation for Erks and
JNK/SAPKs(56) . Transient transfection of MEKK
activates JNK/SAPK (9) but also activates
ERKs(13) . In contrast IPTG induction of MEKK
results in the activation of JNK/SAPK but not Erks. The
difference in regulation of c-Jun transactivation may be related to the
differential phosphorylation of these sites by JNK/SAPK and ERKs.
Further studies will be required to address this question.
Expression of activated Raf in Swiss 3T3 cells stimulated Elk-1
transactivation (Fig. 5) but not c-Myc or c-Jun transactivation
(not shown). This result indicates that Elk-1 transactivation alone
does not mediate the cell death response in fibroblasts observed with
MEKK. Cumulatively, the findings demonstrate that
induction of MEKK
expression enhances cell death
independent of ERK, p38/Hog-1, or c-Jun transactivation in Swiss 3T3
cells and may involve c-Myc transactivation.
Figure 8:
Competitive inhibitory JNK/SAPK(APF)
attenuates Gal4/Jun but not Gal4/Myc activation. Swiss 3T3 cells were
transfected using the calcium phosphate procedure with pCMV5 with no
cDNA (3 µg) or pCMV5MEKK (3 µg) and
pSR
JNK/SAPK (9 µg) or pSR
JNK/SAPK (9 µg). All of
the dishes were transfected with the Gal4/Jun
(3 µg) and Gal4-TK-luciferase (9 µg) reporter constructs.
Cells were harvested 42 h post-transfection and assayed for luciferase
activity. The results are representative of three independent
experiments where a 3-fold excess of JNK/SAPK(APF) inhibited
approximately 65% of Gal4/Jun activation with no effect on Gal4/Myc
activation.
The
cell death response to MEKK also appeared to be largely
independent of JNK/SAPK. In several experiments, expression of
JNK/SAPK(APF) alone had no demonstrative effect on Swiss 3T3 cells (Fig. 9, A and B). The expressed JNK/SAPK(APF)
was localized in both the cytoplasm and nucleus, while
-gal
expression was restricted to the cytoplasm. Co-expression of
JNK/SAPK(APF) with MEKK
did not block
MEKK
-induced cytoplasmic shrinkage and cellular
condensation. As shown in Fig. 9C, a 20-fold lower
concentration of MEKK
than that used in Fig. 1Fig. 2Fig. 3still induced the cytoplasmic
shrinkage characteristic of apoptosis in microinjected Swiss 3T3 cells.
Co-microinjection of a 30-fold greater concentration of JNK/SAPK(APF)
plasmid relative to the MEKK
plasmid did not affect the
MEKK
-mediated cell death response (Fig. 9D). Panels C and D show the
cells undergoing a dramatic cytoplasmic shrinkage. Because of the low
amount of MEKK
expression plasmid used, the cell
condensation response was slower in onset than cells in Fig. 1Fig. 2Fig. 3. The percentage of
MEKK
-microinjected cells committed to cytoplasmic
shrinkage and cellular condensation and the timing of this response was
the same in the presence or absence of JNK/SAPK(APF). In addition, the
competitive inhibitory mutant K116RJNKK/SEK-1, the kinase immediately
upstream of JNK/SAPK which phosphorylates and activates
JNK/SAPK(14, 15) , was also unable to attenuate
MEKK
induced cell death. Expression of JNK/SAPK(APF) or
K116RJNKK/SEK-1 alone had no measurable effect on the morphology of
Swiss 3T3 cells (data not shown). Thus, MEKK
induces
cell death via the regulation of signal pathways that appear largely
independent of JNK/SAPK regulation and c-Jun transactivation. Finally,
BxBRaf neither induced cell death nor activated c-Myc (not shown),
indicating that MEKK
-regulated responses were not
mediated by the Erk1 and 2 proteins (p42/p44 MAP kinases), consistent
with the lack of ERK activation in the inducible MEKK
Swiss 3T3 cells.
Figure 9:
Expression of JNK/SAPK(APF) does not
affect the MEKK-induced cellular condensation response.
Swiss 3T3 cells were microinjected with expression plasmid encoding
JNK/SAPK(APF) (150 ng/µl) in the absence (panels A and B) or presence of pCMV5 expression plasmids encoding
MEKK
(5 ng/µl) (panels C and D).
This is 20-fold less MEKK
expression plasmid than that
used in Fig. 1Fig. 2Fig. 3. Each injection
condition also included 50 ng/µl pCMV
-gal expression plasmid. Panels A, C, and D are stained for
-gal
expression. Panel B is stained for JNK(APF) expression where
the construct was tagged at the NH
terminus with the
9-amino acid HA sequence and detected with the 12CA5 monoclonal
antibody. Experiments were also conducted with the competitive
inhibitory K116RJNKK/SEK-1 kinase inactive mutant with similar results
(not shown). Data are representative of five to six experiments each
for JNK(APF) and K116RJNKK/SEK-1 inhibitory
mutants.
Our results demonstrate, for the first time, a role for MEKK
in mediating a cell death response characteristic of apoptosis.
Receptors such as the cytotoxic TNF receptor and Fas must be
capable of regulating signal transduction pathways controlling
cytoplasmic and nuclear events involved in apoptosis. The enhanced
apoptosis to ultraviolet irradiation observed with MEKK
expression in Swiss 3T3 cells indicates that MEKK-regulated
signal transduction pathways integrate with the apoptotic response
system. MEKK
-expressing cells have a higher basal
apoptotic index and are primed to undergo apoptosis in response to a
stress stimulation. The short time required to observe the enhanced
apoptosis (2 h) suggests that cell cycle traverse, DNA synthesis, or
significant transcription/translation is not required for the enhanced
cell death in response to ultraviolet irradiation in cells expressing
MEKK
. This finding is striking and suggests that genetic
or pharmacological manipulation of MEKK activity could be used to
sensitize cells to irradiation-induced death.
The ability to
dissociate c-Jun transactivation from MEKK-stimulated
cell death argues that the JNK/SAPK activity achieved in the inducible
Swiss 3T3 cell clones is insufficient alone to activate c-Jun
transactivation or induce cell death. It is more likely that the
JNK/SAPK activity we have measured is involved in stimulating a
protective program in response to potentially lethal stimuli as
previously proposed(34) . Protective responses could involve
changes in metabolism or alterations in the activity of proteins such
as Bcl-2(23, 25) . This prediction is consistent with
the activation of JNK/SAPK mediated by CD40 ligation in B cells, which
protects against rather than stimulates
apoptosis(5, 33, 41, 42) .
Recently, it was shown that dominant negative c-Jun could protect
neurons from serum deprivation-induced apoptosis(37) . It was
proposed that the dominant negative c-Jun inactivated c-Jun and
prevented an attempt by the post-mitotic neurons to enter an abortive
cell cycle progression that triggered a cell death program. Thus,
dominant negative c-Jun was believed to maintain the neurons in
stringent growth arrest. At first glance, the protective effect of
dominant negative c-Jun seems contradictory to our results that
JNK/SAPK and c-Jun transactivation are not involved in MEKK-induced
cell death. Our results demonstrate that the dramatic cytoplasmic
shrinkage, nuclear condensation, and onset of cell death induced by
MEKK are largely independent of JNK or c-Jun
transactivation. Importantly, MEKK
-induced cell death
occurs in high serum where growth factor and cytokine stimulation of
the cells is normal. We have also determined that expression of
MEKK
in Swiss 3T3 cells does not significantly inhibit
or alter cell cycle progression. Thus, an abnormal cell cycle event
that may occur with serum deprivation does not appear to account for
MEKK-induced cell death.
Expression of MEKK increased
the transactivation of c-Myc and Elk-1 in Swiss 3T3 cells. c-Myc has
been shown to be required for apoptosis in
lymphocytes(57, 58, 59) , to induce apoptosis
when overexpressed in growth factor-deprived
fibroblasts(24, 29, 30) , and to enhance
TNF-mediated apoptosis(60) . The requirement of c-Myc for
apoptosis is not understood mechanistically, but c-Myc is proposed to
transcriptionally activate an apoptotic
pathway(24, 29, 30, 58, 59) .
The activation of Elk-1 by MEKK
induction in Swiss 3T3
cells correlates best with the stimulation of JNK/SAPK. Recently, it
was found that JNK/SAPK in addition to Erks phosphorylated and
activated Elk-1 consistent with our findings(7) . In contrast,
we demonstrate that c-Jun is not significantly activated in
MEKK
-expressing cells. These findings are provocative
because they indicate that MEKK-stimulated JNK/SAPK activation
preferentially regulated Elk-1 and not c-Jun. A second signal in
addition to JNK/SAPK may be required for c-Jun transactivation in cells (56) . We are unaware of any proposed role for Elk-1 in
inducing an apoptotic response, but serum deprivation-induced apoptosis
of Swiss 3T3 cells results in the increased expression of early cell
cycle genes consistent with an increased serum response factor/serum
response element activity associated with elevated Elk-1
activity(32) . The induction of apoptosis in several cell types
does not appear to require transcription, but our inducible cell lines
and plasmid microinjection experiments do not allow us to test whether
MEKK
can induce cell death in the absence of
transcription. We are currently attempting to make active recombinant
MEKK
to test this possibility. In cells where
transcription is not necessary for the induction of apoptosis, it is
likely that proteins required for apoptosis are already expressed and
may be post-translationally regulated by sequential protein kinase
pathways involving MEKK. For example, the phosphorylation of nuclear
proteins could alter their activity independent of transcription and
contribute to a cell death response.
In Jurkat cells, a human T cell line, Fas-induced apoptosis has been proposed to involve a ceramide-stimulated, Ras-dependent signaling pathway(31) . We recently demonstrated that MEKK activity can be stimulated by Ras and that MEKK1 physically binds to Ras in a GTP-dependent manner(61, 62) . The ability of MEKK to regulate an apoptotic-like cell death response suggests it is a candidate component for the ceramide-regulated apoptotic pathway.
The importance of our
observations describing the involvement of MEKK regulated sequential
protein kinase pathways in physiologically relevant signaling leading
to cell death is supported by several findings. First, MEKK induces or enhances a cell death response in the presence of 10%
calf serum, indicating that growth factor deprivation is not a
prerequisite for MEKK-induced cell death. This is similar to TNF
,
Fas, and ceramide-mediated apoptosis, which proceeds in high serum.
Thus, the involvement of MEKK in cell death responses is not simply to
activate a subset of growth factor-stimulated signaling events causing
an aborted cell cycle-induced apoptosis that would normally be
prevented by serum factors. Second, the enhanced cell death to
ultraviolet irradiation indicates that expression of MEKK
may activate signals that potentiate stresses to the cell. This
finding indicates that MEKK-regulated signal transduction pathways
integrate with cellular responses involved in mediating apoptosis, that
ultraviolet irradiation likely activates additional pathways, and that
MEKK
-mediated signaling synergizes with the ultraviolet
response to accelerate apoptosis. Third, MEKK stimulated sequential
protein kinase pathways independent of ERK, JNK/SAPK, p38/Hog1, and
c-Jun transactivation that can stimulate c-Myc transactivation. These
results indicate that MEKK-regulated pathways traverse the cytoplasm to
regulate as yet undefined protein kinases that activate c-Myc in the
nucleus. The regulation of c-Myc activity is a unique function of MEKK
signaling and one that we postulate is likely to contribute to the cell
death response. Serum deprivation significantly induces JNK/SAPK
activation in several cell types including Swiss 3T3 cells.
Similarly, TNF
stimulates a JNK/SAPK pathway (9) ,
and we have recently demonstrated TNF
stimulation of MEKK activity
in mouse macrophages (63). c-Myc overexpression has been shown to
enhance TNF
receptor stimulation of apoptosis(21) . These
findings are consistent with a linkage between TNF
receptor
signaling, MEKK, and c-Myc. Cumulatively, the findings define MEKK as a
potentially important component in the regulation of signal
transduction pathways involved in apoptosis.