(Received for publication, August 3, 1995)
From the
To elucidate cellular pathways involved in
Jun-NH-terminal kinase (JNK) activation by different forms
of stress, we have compared the effects of UV irradiation, heat shock,
and H
O
. Using mouse fibroblast cells (3T3-4A)
we show that while H
O
is ineffective, UV and
heat shock (HS) are potent inducers of JNK. The cellular pathways that
mediate JNK activation after HS or UV exposure are distinctly different
as can be concluded from the following observations: (i)
H
O
is a potent inhibitor of HS-induced but not
of UV-induced JNK activation; (ii) Triton X-100-treated cells abolish
the ability of UV, but not HS, to activate JNK; (iii) the free radical
scavenger N-acetylcysteine inhibits UV- but not HS-mediated
JNK activation; (iv) N-acetylcysteine inhibition is blocked by
H
O
in a dose-dependent manner; (v) a Cockayne
syndrome-derived cell line exhibits JNK activation upon UV exposure,
but not upon HS treatment. The significance of Jun phosphorylation by
JNK after treatment with UV, HS, or H
O
was
evaluated by measuring Jun phosphorylation in vivo and also
its binding activity in gel shifts. HS and UV, which are potent
inducers of JNK, increased the level of c-Jun phosphorylation when this
was measured by [
P]orthophosphate labeling of
3T3-4A cultures. H
O
had no such effect.
Although H
O
failed to activate JNK in vitro and to phosphorylate c-Jun in vivo, all three forms of
stress were found to be potent inducers of binding to the AP1 target
sequence. Overall, our data indicate that both membrane-associated
components and oxidative damage are involved in JNK activation by UV
irradiation, whereas HS-mediated JNK activation, which appears to be
mitochondrial-related, utilizes cellular sensors.
The response of mammalian cells to stress in the form of UV
irradiation or heat shock (HS) ()involves key regulatory
proteins such as p53(1) , GADD45(2, 3) ,
WAF1/p21/cip(4, 5) , NF
B(6, 7) ,
and c-Jun(8, 9, 10) . Changes in the
expression and activities of the stress-modulated cellular proteins
affect cell cycle distribution(11) , rate of repair, and DNA
replication(12) . Such changes also lead to temporal growth
arrest (2, 13) or apoptosis(14) . While the
primary sensors that trigger the stress response are not known, a
subset of ras-dependent protein
kinases(15, 16, 17) , including
Src(18) , Raf(19) , Jun-NH
-terminal
kinase(19, 20) , and mitogen-activated protein
kinase(21) , as well as growth factor receptors(22) ,
were shown to participate.
To understand the nature of the stress
response, one needs to identify the mechanisms involved in the
activation of stress-related protein kinases. Pathways that were thus
far shown to mediate the stress response include cell surface
receptors, such as epidermal growth factor receptor. These are capable
of activating mitogen-activated protein kinase (22) through ras, raf-1, MEK, and ERK, leading to the phosphorylation and
activation of the transcription factors TCF/ELK1 and
c-Fos(23) . A second pathway includes yet unidentified
cytoplasmic components that can activate JNK via its own kinases,
including MKK4,(17) , which leads to the phosphorylation of
c-Jun. Interestingly, JNK is also activated by osmotic shock as was
demonstrated in Chinese hamster ovary cells(24) . Upstream JNK
kinases also phosphorylate p38-MpK2 (25) which is able to
reconstitute osmotic response in yeast strains that lack HOG1, the
yeast homologue of p38 (24) . HOG1 was reported to share
similarity with the mitogen-activated protein kinase activating protein
2-reactivating kinase that mediates the response to heat shock and
phosphorylates small heat shock proteins (26) . Augmented by ras, mitogen-activated protein kinase and JNK activities
contribute to the overall phosphorylation of
c-Jun(20, 27) . JNK phosphorylates c-Jun and ATF2 on
their NH-terminal region, whereby these transcription
factors are enabled to mediate transcription, replication, and
transformation activities(28, 29) . To exert such
activities, JNK requires interaction with the
domain of
c-Jun(30) . This key component is deleted in c-Jun's
oncogenic counterpart v-Jun(31, 32) .
In elucidating mechanisms involved in the cellular response to UV irradiation and HS treatment as two different forms of stress, we have focused on the role of JNK. Although UV and HS are potent inducers of JNK, they are expected to damage cells via alternate pathways.
To
distinguish between the effects of UV and HS, we have evaluated the
role of oxidative stress which is induced by UV to a greater extent
than by HS. Oxidative stress can induce c-Jun, c-Fos, NFB, early
growth response gene (EGR1), and heme oxygenase expression at
the transcriptional level(33, 34) . This induction
correlates with transcriptional activities as demonstrated for Jun, and
NF
B(35, 36) , and it appears to be mediated via
tyrosine kinases in a protein kinase C-dependent
manner(37, 38) . Poly-(ADP-ribosylation) was also
shown to participate in AP1 activation by
H
O
(39) . Hydrogen peroxide-mediated
cellular changes can be suppressed by Bcl-2 through inhibitory effects
on lipid peroxidation at the site of free radical generation within the
mitochondria(40) . Suppression of mitochondrial activities has
been associated with elevated levels of oxidative damage and uncoupling
of electron transport from ATP production. It is also linked to the
induction of apoptosis(39, 41, 42) .
The role of oxygen radicals and membrane components, which are involved in cellular stress response, was evaluated to enable the identification of mechanisms involved in JNK activation by different forms of stress. The degree of JNK activation by either UV or HS was further correlated with c-Jun phosphorylation and DNA binding activities as measures for the functional significance of JNK activity.
An in-gel kinase assay was performed by
embedding the c-Jun NH-terminal region that was purified
from the pGEX-jun within the acrylamide gel (10 µg/ml). Equal
amounts of proteins (50 µg) were separated in a routine 10%
SDS-PAGE. To remove SDS, the gels were washed for 4-12 h in Hepes
(40 mM, pH 7.4) with 0.2 M EDTA. The gel was then
incubated in kinase buffer (see above) in the presence of 1 mCi of
[
-
P]ATP (specific activity, 6000 Ci/mol)
for 30 min. Followed by extensive washings in the presence of Dowex
2-X8 (Bio-Rad), mixed with DEAE-650 (Supelco), and packed within a
dialysis bag (to compete for nonspecific bound
[
-
P]ATP), the gel was silver-stained to
ensure proper distribution and that equal amounts of protein substrate
were loaded, dried, and autoradiographed.
Figure 1:
Cockayne
syndrome and mtDNA cells exhibit JNK activation by UV
but not by HS. A, cells of a Cockayne syndrome patient were
exposed to HS or UV-C treatment, and JNK activity was measured. In all
cases, proteins were prepared 30 min after UV and 60 min after HS
treatment, and JNK assays were performed using pGEX-Jun as a substrate
in the presence of [
-
P]ATP and the
respective kinase buffer. The phosphorylated Jun was identified as a
35-kDa band representing the fusion protein Gst-NH
-terminal
c-Jun after its separation on SDS-PAGE and detection by
autoradiography. B, analysis of kinases present in these
cellular proteins that can phosphorylate c-Jun was performed via an
``in-gel kinase'' assay (see ``Materials and
Methods'') in which NH
-terminal Jun was embedded in
the acrylamide. The arrow points to the position of one of the
JNK isozymes with a molecular mass of 54 kDa, which appears as a
doublet. The upper component of this doublet, seen in HeLa cells, is
probably another JNK isozyme which is also seen in respective
immunoprecipitations (not shown).
To further elucidate this observation, we
have performed in-gel kinase reactions. To this end, Jun (corresponding
to the NH-terminal region which was eluted from the GST-Jun
construct) was fixed in the acrylamide gel prior to separation of
proteins prepared from control and UV- or HS-treated CS cells. As shown
in Fig. 1B, a 54-kDa band appeared as a doublet; it
corresponded to the M
of JNK2 (48, 49) and was thus identified as the kinase that
phosphorylates c-Jun. The lower component of this 54-kDa doublet was
clearly induced, thus exhibiting greater intensity in the proteins of
UV-treated cells, but substantially lower intensity in HS-treated CS
cells. JNK2 was also identified in HeLa cell proteins which we used as
an internal control (Fig. 1B). That equal amounts of
proteins were separated on each lane was verified via silver staining
of the gel before the autoradiography (not shown). The 54-kDa doublet
may represent different JNK isozymes that are expressed in these cells
(see below) and exhibit different kinase activities. A similar pattern
of JNK activation by UV, but not by HS, treatment was observed in cells
that were depleted of their mitochondrial DNA (Fig. 1B). The 54-kDa band was also noticed in
HS-treated HeLa cells, whereas both 54- and 46-kDa bands (46 kDa
corresponding to JNK1) were identified in HS-treated 3T3-4A cells (not
shown).
Figure 2:
JNK isozymes in 3T3-4A cells. Protein
extracts were prepared from sham, UV, or HS-treated 143B205 cells
(depleted of mitochondrial DNA), 3T3-4A (mouse fibroblasts), and JC133
(Cockayne syndrome derived fibroblasts). These proteins were incubated
with pGEX-Jun as in the kinase reaction. After washing, the bound
proteins were eluted and analyzed via Western blotting on SDS-PAGE
using antibodies to JNK. M markers are indicated
on the right panel, and the position of JNK1 and JNK2 is shown
by arrows on the left
panel.
Figure 3:
HO
inhibits HS-
but not UV-mediated JNK activation. A, mouse fibroblasts were
exposed to the indicated doses of H
O
, UV-C (20
J/m
), or sham treatment, either alone or in combination as
indicated. Proteins were then used for JNK assays as detailed under
``Materials and Methods.'' After their incubation with the
pGEX-Jun in the presence of kinase buffer and
[
-
P]ATP, beads were washed and separated on
SDS-PAGE which was autoradiographed. The bands shown reflect the 35-kDa
fusion protein Gst-Jun (NH
-terminal region). B,
mouse fibroblasts were exposed to heat shock (HS) (60 min at
42 °C), H
O
, sodium vanadate (SV),
or sham treatment, either alone or in the indicated combinations. JNK
assays were performed as described in the text. HS in vitro indicates the increase in level of active JNK after exposure of
proteins from control (untreated cells) to HS in vitro. C, the
actual gels were scanned with a radioimaging blot analyzer and values
obtained were normalized per JNK activity in sham-treated cells. Values
are shown as n-fold increase in JNK
activity.
To quantify the degree of JNK activation noticed after HS and UV exposure, we have scanned the respective gels by a radioimaging blot analyzer. The counts/min obtained under each of these experimental conditions were normalized per counts measured in control (untreated) extracts, resulting in values that represent an n-fold increase in JNK activation (Fig. 3C).
While the role of tyrosine phosphorylation in JNK activation was shown previously(48, 49) , we have determined the influence of tyrosine phosphorylation on JNK activities in mouse fibroblast 3T3-4A cells. One of the potent modulators of tyrosine kinases is sodium vanadate. When added to cultures of 3T3-4A cells, sodium vanadate (1 mM) itself caused a significant increase in JNK activities (Fig. 3B), suggesting that the degree of JNK phosphorylation directly contributes to its activity. Although UV is already a potent inducer of JNK activities, an enhanced effect is noted when UV is combined with sodium vanadate. Similar to the UV effect, heat shock treatment of 3T3-4A cells leads to a strong activation of JNK, which is further enhanced by sodium vanadate (not shown). Additional support for the role of tyrosine kinases in JNK activities comes from the use of genistein, a potent inhibitor of tyrosine kinase. When genistein was added to 3T3-4A cells, we have noticed a dose-dependent decrease in JNK activation after UV irradiation (not shown).
Figure 4:
NAC inhibits UV- but not HS-mediated JNK
activation. A, 3T3-4A cells were exposed to NAC (40
mM), UV-C (20 J/m), or H
O
(10-100 µM) as indicated. Proteins (10 µg)
were used as source of JNK which was added to the pGEX-Jun coupled to
GST beads. Analysis of JNK activity was performed as outlined. B, quantitative JNK assays were performed on 3T3-4A cell
proteins prepared from cells treated with UV (20 J/m
), HS
(60` 42 °C), NAC (40 mM), or H
O
(100 µM), alone or combined as
indicated.
Since NAC acts as a potent scavenger of free oxygen radicals, we have tested how glutathione in its oxidized and reduced forms contributes to JNK activation. When tested by itself, neither form induced JNK activation. Yet, when added to UV-treated cells, the oxidized form of glutathione had an additive effect on JNK activity. The reduced form of glutathione had an inhibitory effect on UV-mediated JNK activation. When added to HS-treated cells, the oxidized form of glutathione was a potent inhibitor of JNK, whereas the reduced form increased JNK activation (not shown). These observations further support our former experiments and suggest that HS utilizes a different cellular component than UV in mediating JNK activation.
Figure 5:
Effect of Triton X-100 on UV- and
HS-mediated JNK activation. A, cells were exposed to the
indicated concentrations of Triton X-100 for 5 min before their
exposure to HS (60 min at 42 °C). The control lane represents the
basal level of JNK activity. B, cells were exposed to the
indicated concentrations of Triton X-100 for 5 min before irradiation
with UV (20 J/m). Proteins were prepared 30 min after
treatment and analyzed as indicated. C, quantitative data with
the gel shown in B were obtained through the use of a
radioimaging blot analyzer. Counts/min values of UV-mediated JNK
activation were considered as 100%, and those obtained after
pretreatment with Triton X-100 were normalized to reflect percent
inhibition of JNK activity after UV irradiation. D, 3T3-4A
cells were incubated for 5 min with Triton X-100 (0.008%) 30 min after UV (40 J/m
, UV + T) or after
sodium vanadate (SV, 1 mM; SV + T)
treatment. Protein extracts were prepared and assayed for JNK activity
as indicated. Lanes T + UV and T + SV represent JNK assays performed with proteins prepared from cells
growing in culture that were incubated with Triton X-100 at the same
concentration for 5` before they were treated with UV or
sodium vanadate.
To determine whether c-Jun phosphorylation in vivo reflects
the patterns noticed in the JNK assays, we have incubated 3T3-4A cells
in [P]orthophosphate for 60 min after their
exposure to UV, HS, or H
O
. Following extensive
washing, proteins were prepared and the c-Jun protein was then
immunoprecipitated with the aid of antibodies to c-Jun and with protein
A/G beads. Immunoprecipitated material was separated on SDS-PAGE and
autoradiographed. As shown in Fig. 6A, clear
differences were noticed in the overall level of phosphorylated jun
after these treatments. Both UV and HS were potent inducers of c-Jun
phosphorylation in vivo, whereas H
O
was not. The degree of phosphorylated c-Jun is even lower after
H
O
treatment when compared with that
precipitated from control proteins. The doublet seen in c-Jun after
H
O
treatment indicates a phosphorylated form,
although the overall level of phosphorylation was substantially lower
than that found after UV or HS treatment, as seen in shorter exposures
(not shown). The pattern seen here is in agreement with that observed in vitro after using pGEX-Jun (NH
-terminal region)
as a substrate ( Fig. 3and Fig. 4). Longer incubation
periods did not affect the overall level of phosphorylation (Fig. 6A), suggesting that the immediate response of
JNK is the key determinant in c-Jun phosphorylation. The position of
c-Jun was confirmed by both the molecular mass of 39 kDa and by the use
of in vitro translated c-Jun which was subjected to
phosphorylation by JNK in vitro and separated in parallel on
SDS-PAGE (Fig. 6A).
Figure 6:
A, changes in jun phosphorylation in
vivo after HS, HO
, or UV treatment.
[
P]Orthophosphate was added to 3T3-4A cells that
were grown in 150-mm dishes in phosphate-free medium. After 60-min
incubation (unless otherwise indicated) proteins were prepared,
immunoprecipitated with antibodies to c-Jun, and separated on SDS-PAGE,
which was then subjected to autoradiography. The molecular mass of the
band shown was calculated to be 39 kDa. On the same gel (a few lanes
apart that were spliced out) in vitro translated, and
immunoprecipitated c-Jun was also separated as an internal control. B, binding to AP1 target sequence after HS,
H
O
, or UV treatment. Proteins prepared from
mouse fibroblast 3T3-4A cells 1 h after exposure to HS,
H
O
, or UV irradiation were incubated (5 µg)
with a
P-labeled AP1 target sequence in the presence of
nonspecific DNA and DNA binding buffer (lane marked with -).
Competition experiments were performed using a 50-fold excess of the
respective nonlabeled target sequence and using AP1 or NF
B as
indicated in the second and third lanes of each panel. Upon their
separation on a 5% PAGE, protein complexes were visualized via
autoradiography. The first three lanes from the left represent
free probes; the arrow points to the position of the major
complex.
As a measure for transcriptional
activities that are mediated by proteins which interact with the AP1
target sequence, we have performed EMSA. In these assays we have
compared the binding of proteins prepared after HS, UV, or
HO
treatments. As demonstrated in Fig. 6B, whereas binding activities have increased
after each of these treatments, proteins derived after HS and
H
O
treatments were more potent than those
obtained after UV exposure. The specificity of this reaction was
demonstrated through the use of AP1 and NF
B as specific and
nonspecific competitors, respectively (Fig. 6B). While
AP1 was capable of inhibiting binding to the AP1 target sequence,
NF
B had no effects. A similar pattern was observed when the
NF
B target sequence was used (not shown).
The present study provides direct evidence for the existence
of alternate pathways in JNK activation by different forms of stress,
as shown for UV and HS. The distinction of these alternate pathways
lies in the requirement for membrane-associated components for
UV-mediated, but not for HS-mediated, JNK activation. This can be
concluded on the basis of the ability of Triton X-100 to block JNK
activation by both vanadate and UV, when these are administered before,
but not after, UV irradiation. As vanadate has been shown to mediate
its effects via cell membrane receptors(54) , changes in such
receptors are expected to abolish its activities as was observed in our
experiments (Fig. 5). Moreover, it has been previously
demonstrated that Triton X-100 alters membrane organization and
modulates the ability of p21 to mediate mitogen-activated
protein kinase activation(53) . Similar detergents were shown
to alter the ability of the epidermal growth factor receptor to
dimerize and autophosphorylate(50, 51, 52) .
It is likely, therefore, that due to the effect of Triton X-100 on
receptor organization, the receptor can no longer dimerize to
trans-phosphorylate and thus fails to activate downstream protein
kinases. Furthermore, we have recently shown the association between
p21
and JNK(55) . We, therefore, cannot exclude
the possibility that membrane-anchored components, such as p21
were also altered, so that they could not associate/activate JNK.
Accordingly, HS-mediated JNK activation may utilize ras-independent pathways which were previously documented (25, 56) . We conclude, therefore, that UV requires
cellular membrane signaling, whereas HS does not. It is, therefore,
surmised that different kinases are involved in JNK activation in
response to alternate forms of stress as shown here for UV and HS.
UV-mediated JNK activation also involves oxidative damage, as
demonstrated by the inhibition of this response through the radical
scavenger NAC. In contrast, oxidative damage by HO
inhibits HS-mediated JNK activation, and we have shown that NAC
is unable to inhibit HS-mediated JNK activation. That oxygen radicals,
such as H
O
, inhibit the HS-mediated pathway
suggests that cellular components other than catalase, superoxide
dismutase, or glutathione peroxidase (which are involved in the
response to UV and its oxidative damage) are playing an important role
in the regulation of JNK activation. That there are different cellular
sensors for UV and HS damage can also be deduced from the observation
that in vitro HS treatment of cellular extracts can induce JNK (Fig. 3B), whereas UV-mediated JNK activation requires
a complete and precise in vivo setting of membrane components
as demonstrated in the Triton X-100 experiments. The different
responses to HS and UV are not mediated by different JNK isozymes, as
both the 46-kDa and the 54-kDa proteins representing JNK1 and JNK2,
respectively, were equally capable of binding to the
NH
-terminal region of Jun.
Both UV and HS were potent inducers of JNK activation leading to increased c-Jun phosphorylation in vivo and to a corresponding increase in binding to the AP1 target sequence. The degree of JNK activity and, in turn, c-Jun phosphorylation is expected to be tightly regulated by specific protein phosphatases, which are also induced by external stress, as shown for HS. For example, protein phosphatase 2C is induced by HS in Schizosaccharomyces pombe(57) . Similarly, PAC1, a mitogen-induced nuclear protein with tyrosine phosphatase activities, is also induced by HS(58) .
Interestingly, in the cell
system studied here, HO
was neither capable of
activating JNK, nor was it able to increase in vivo labeling
of c-Jun; however, it was as potent as UV or HS in forming complexes
with AP1 target sequence DNA. It is thus possible that although
complexes of similar size were formed in all cases, the binding
observed in our EMSA may have been mediated not only by c-Jun proteins,
but by other members of the AP1 or ATF families that can interact with
the AP1 target sequence and have a M
similar to
that of c-Jun. However, the overall transcriptional signal is expected
to be different in the case of H
O
than in that
of UV or HS.
A possible clue as to the nature of the different cellular components involved in JNK activation evolved from the reactions with different cell lines. We have shown this for CS cells which mediate selective activation of JNK2 by UV but not by HS. A similar observation was made in cells that lack mitochondrial DNA and exhibit JNK activation after UV exposure but not after HS treatment (Fig. 1B and (2) ). These observations suggest that HS activation of JNK requires mitochondria-related components, which are not available upon deregulation of the mitochondria. Impaired mitochondrial function has been widely documented for aged cells(41) . The latter coincides with our observations that higher levels of oxidative stress inhibit HS-mediated JNK activities and that aged cells abrogate heat shock response(59, 60, 61) .
Overall, that alternate pathways mediate JNK activation by different forms of stress indicates the existence of multiple cellular sensors which are triggered by the respective stimuli. UV-induced JNK activation is dependent on membrane-associated components and free oxygen radicals (our present study) and DNA damage per se(47) , whereas mitochondria-related components appear to be involved in the HS response.