From the
Irradiation of mammalian cells with short wavelength ultraviolet
light (UVC) evokes a cascade of phosphorylation events leading to
altered gene expression. Both the classic mitogen-activated protein
(MAP) kinases and the distantly related c-Jun N-terminal kinases (JNK)
contribute to the response via phosphorylation of transcription factors
including AP-1. These kinases are themselves regulated via reversible
phosphorylation, and several recently identified specific MAP kinase
phosphatases (MKP) have been implicated in down-regulating MAP
kinase-dependent gene expression in response to mitogens. Here, we
provide evidence that MKP-1 plays a role in regulating transcriptional
activation in response to UVC as well as another genotoxic agent,
methyl methanesulfonate (MMS). We further demonstrate that JNK is a
likely target for MKP-1. JNK is shown to be activated by UVC and MMS
treatment, while MAP kinase activation occurs only with UVC. Like JNK
activation, MKP-1 mRNA is induced by both treatments, and elevated
MKP-1 expression coincides with a decline in JNK activity. Constitutive
expression of MKP-1 in vivo inhibits JNK activity and reduces
UVC- and MMS-induced activation of AP-1-dependent reporter genes.
Exposure of cells to genotoxic agents evokes a series of
phosphorylation events leading to the modification of transcription
factors and altered gene expression
(1, 2) . Although
short wavelength ultraviolet light (UVC)
The activity of both the classic MAP
kinases as well as JNKs is regulated via reversible phosphorylation of
tyrosine and threonine residues. They are distinguished by the
tripeptide phosphorylation motif required for their activation:
Thr-Glu-Tyr for MAP kinases and Thr-Pro-Tyr for JNK
(6, 7, 9, 10, 14, 18, 19) .
Several protein phosphatases with high specificity for MAP kinases have
been described. These include the ubiquitously expressed mouse MAP
kinase phosphatase 1 (MKP-1), its human homologue CL100, and the
lymphocyte-specific PAC-1 protein
(20, 21, 22, 23, 24) . MKP-1 and
PAC-1 have been shown to dephosphorylate phosphothreonine and
phosphotyrosine residues of MAP kinase resulting in its inactivation.
MKP-1 exhibits high selectivity for dephosphorylation of MAP kinase
from a spectrum of phosphotyrosine-containing peptides including JNK
(21, 25) . Overexpression of PAC-1 as well as MKP-1
inhibits MAP kinase-regulated reporter gene expression in response to
mitogenic stimulation
(21, 24) . Recent studies have
shown that overexpression of MKP-1 likewise inhibits Ras-induced DNA
synthesis in quiescent cells further establishing its role during
mitogenesis
(21) .
In this study we address the role of MKP-1
in regulating gene activation in response to genotoxic stress using two
different treatments, UVC and MMS. Our findings provide evidence that
MKP-1 serves as an important regulator of gene activation in response
to genotoxic stress, and indicate that JNK can be a target for MKP-1.
For experiments examining the
effect of MKP-1 expression on JNK activity in vivo, the HA-JNK
and MKP-1 expression plasmids were transiently transfected into HeLa
cells at a ratio of 1:0, 1:4, or 1:10 using Lipofectamine (Life
Technologies, Inc.). Total DNA was kept constant using pSG5 as carrier
DNA. Forty-eight h following transfection, cells were treated with
either UVC or MMS and harvested after 60 min (for UVC) or 90 min (for
MMS). HA-JNK was immunoprecipitated using anti-HA tag antiserum (5
µg) (Babco, Berkeley, CA). and assayed for kinase activity.
MAP
kinase was immunoprecipitated using anti-p42
JNK1 has been shown to phosphorylate c-Jun and activate AP-1 in
response to UVC irradiation
(1, 8, 9, 10, 11) . Since, like
UVC, MMS treatment results in enhanced AP-1 activity
(30) , we
sought to examine whether JNK1 might be involved in mediating this
response. Accordingly, we examined the relative JNK1 activity in
extracts of UVC and MMS treated cells using an immunocomplex kinase
assay (Fig. 2). JNK1 was rapidly activated in response to UVC treatment
with maximum activity observed within 30 min post-treatment. JNK1
activity was also markedly increased following MMS treatment, although
the kinetics of induction were slower (maximum activity was seen at 90
min) and the magnitude of activation was less than that seen with UVC.
In other experiments we have observed that JNK2 is similarly activated
in response to both UVC and MMS treatment (data not shown).
We thank J. Luethy-Martindale for technical assistance
and M. Lee for helpful discussions.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
(
)
irradiation has been the most widely studied treatment in
this regard, other agents, including the DNA alkylating agent methyl
methanesulfonate (MMS), result in a similar pattern of gene expression
(3, 4) . The pathway(s) mediating the response to UVC
damage overlap with those mediating the response to proliferative
stimuli, and at least two different phosphorylation cascades appear to
be involved. The first involves activation of membrane-associated
tyrosine kinases followed by the sequential activation of Ras and Raf
(1, 2) . Raf phosphorylates the mitogen-activated
protein (MAP) kinase kinase (MEK), which in turn activates MAP kinase
(5, 6, 7) . MAP kinases, also called
extracellular signal-regulated kinases (ERKs) are members of a
ubiquitous family of serine/threonine kinases that are responsible for
the phosphorylation and activation of various transcription factors,
including c-Myc, ATF2, and p62
(7) . A second
pathway relies on the c-Jun N-terminal kinases (JNK) for gene
activation following UVC treatment
(8, 9, 10) .
JNKs can be activated by a variety of stresses and hence are also
referred to as stress-activated protein kinases (SAPK)
(11) . In
addition to phosphorylating c-Jun protein, JNKs have also recently been
shown to phosphorylate ATF2
(12, 13) . The events
leading to JNK activation are less defined than those involved in MAP
kinase activation. However, recent reports indicate that JNK is
phosphorylated by the MEK homologue, SEK1/MKK4
(14, 15) , which is in turn phosphorylated by MEK kinase
(16, 17) .
Recombinant Plasmids
A rat cDNA homologous to
the mouse MKP-1 gene (rMKP-1) was isolated from a gt11
rat lung cDNA library (data not shown). Plasmids expressing sense and
antisense rMKP-1 were constructed by inserting the cDNA fragment of
rMKP-1 in opposing directions into the EcoRI site of
pSG5 (Stratagene, La Jolla, CA). The GST-c-Jun(1-135) plasmid was
provided by J. Woodgett
(11) . Hemagglutinin (HA)-tagged JNK1,
and jun-LUC (containing c- jun promoter sequences from
-70 to +170 linked to a luciferase reporter) plasmids were
provided by M. Karin
(9) . The coll-CAT construct,
encompassing the human collagenase promoter region from -517 to
+63 linked to the chloramphenicol acetyltransferase (CAT) reporter
gene, was provided by P. Herrlich
(26) .
Cell Culture and Treatment Conditions
HeLa cells
were cultured in Dulbecco's modified Eagle's medium
containing 10% fetal bovine serum (Life Technologies, Inc.). For UVC
treatment, the medium was removed, and the dishes washed twice with
phosphate-buffered saline. Cells were irradiated using a germicidal
lamp at a dose rate of 1.3 J/m/s at 254 nm, after which the
original culture medium was added back to the dishes.
12- O-Tetradecanoylphorbol-13-acetate (TPA) and MMS were added
directly to cells from concentrated stocks.
Northern and Western Blot Analysis
For mRNA
analysis, HeLa cells were harvested at various times after treatment.
Total RNA was extracted using Stat 60 (Tel-test B, Friendswood, TX).
Northern blot analysis was performed with the r MKP-1 cDNA
probe using standard procedures
(27) . Western blot analysis was
performed utilizing monoclonal antibodies against ERK1 and ERK2
(Transduction Laboratories, Lexington, KY). Immune complexes were
detected using the enhanced chemiluminescence detection system
(Amersham Corp.).
JNK and MAP Kinase Assays
JNK1 was
immunoprecipitated and kinase activity was measured using an
immunocomplex kinase assay employing GST-c-Jun as substrate as
described
(11) . Briefly, cells (60-80% confluent) were
washed twice with ice-cold phosphate-buffered saline and broken in
lysis buffer
(28) . One mg of soluble protein was collected and
incubated with protein A-Sepharose and antiserum against p46(2.5 µg) (Santa Cruz Biotechnology; Santa Cruz, CA). After
incubation for 3 h at 4 °C, immunoprecipitates were washed
extensively and assayed for kinase activity at 30 °C for 20 min
using 6 µg of GST-c-Jun(1-135). Proteins in the reaction were
resolved by SDS-polyacrylamide gel electrophoresis (12% gel) and
subjected to autoradiography. The incorporated
P was
quantitated by scintillation counting.
antiserum
(Santa Cruz Biotechnology) and its activity assayed essentially as
described above except that myelin basic protein (Sigma) was used as a
substrate.
Gene Expression
HeLa cells were transfected with 1
µg of either coll-CAT or jun-LUC along with 10
µg of either pSG5, or constructs expressing sense (pSG5-rMKP-1) or
antisense (pSG5-rMKP-1as) rMKP-1 using calcium phosphate precipitation
as described previously
(27) . Twenty h later they were treated
with either TPA (60 ng/ml), UVC (40 J/m), or MMS (100
µg/ml). TPA was left in the medium overnight. MMS was removed after
4 h and the cells given fresh medium. UVC-treated cells were also given
fresh medium after irradiation. The following day cell extracts were
prepared from the various treatment groups and assayed for CAT
(29) or luciferase activity using a luciferase assay system kit
(Promega, Madison, WI)
Differential Activation of JNK and MAP Kinase by UVC
and MMS
The kinetics of MAP kinase activation were examined in
UVC-irradiated (40 J/m) or MMS-treated (100 µg/ml) HeLa
cells by two different methods (Fig. 1). In the first, phosphorylated
forms of the ERK1 and ERK2 MAP kinase isoforms were identified by
Western blot analysis based on their slower electrophoretic mobility
compared to nonphosphorylated forms
(2, 5) . Both ERK1
and ERK2 were rapidly phosphorylated (within 15 min) in response to UVC
irradiation, followed by dephosphorylation by 60 min post-treatment. In
contrast, no phosphorylated forms of either kinase were evident in
MMS-treated cells. Similar results were seen when ERK2 activity was
assessed by phosphorylation of myelin basic protein (Fig. 1 B).
While UVC resulted in a >30-fold increase in ERK2 kinase activity,
MMS showed only a 4-fold increase in phosphorylation of the substrate.
Induction of MKP-1 by UVC and MMS
MKP-1 is highly
induced by mitogen stimulation as well as a variety of stresses
(22, 31, 32) . It can dephosphorylate the
classic MAP kinases and has been implicated in the regulation of gene
expression during mitogenesis
(20, 21) . To explore its
role in regulating gene expression in response to genotoxic stress, we
examined the expression of MKP-1 following treatment of cells with
either UVC or MMS. As shown in Fig. 3, MKP-1 mRNA was induced more than
10-fold by both treatments. Maximum MKP-1 mRNA expression coincided
with a decline in MAP kinase (for UVC treatment; Fig. 1) and JNK
activity (for both UVC and MMS treatment; Fig. 2), consistent
with it playing a role in the inactivation of either or both of these
kinases. Importantly, however, given that MMS treatment does not result
in significant MAP kinase activation, this kinase is unlikely to be the
target for MKP-1 in response to this treatment.
Figure 1:
MAP kinase activation in response to
UVC and MMS treatment. Panel A, Western blot analysis of ERK1
and ERK2 MAP kinase isoforms at various times following treatment of
HeLa cells with UVC (40 J/m) or MMS (100 µg/ml).
Panel B, MAP kinase (ERK2) activity was determined in cell
extracts from HeLa cells treated with UVC (40 J/m
) or MMS
(100 µg/ml). Kinase activity was measured via phosphorylation of
myelin basic protein.
Figure 2:
Kinetics of JNK activation in response to
UVC and MMS treatment. JNK activity was determined in cell extracts
from HeLa cells treated with UVC (40 J/m) or MMS (100
µg/ml). Kinase activity was measured via phosphorylation of
GST-c-Jun(1-135) substrate.
Deactivation of JNK1 by rMKP-1 Expression in
Vivo
To test whether JNK can serve as a substrate for MKP-1
in vivo, we used a transient cotransfection assay to deliver
plasmids expressing HA-tagged JNK1 along with either the plasmid
expressing rMKP-1 (pSG5-rMKP-1) or an empty pSG5 vector. Transfected
cells were either left untreated or were treated with UVC or MMS.
HA-JNK1 protein was immunoprecipitated from cell extracts using anti-HA
antiserum and the immunocomplex was assayed for its ability to
phosphorylate the GST-c-Jun(1-135) substrate (Fig. 4). HA-JNK1
activity was markedly elevated in the transfected cells following UVC
and MMS treatment (58- and 30-fold, respectively). Cotransfection of
HA-JNK1 with pSG5-rMKP-1 at a ratio of 1:4 resulted in a significant
inhibition (>4-fold decline) of JNK activity following both UVC and
MMS treatment. The inhibitory effect was dose-dependent with respect to
the amount of pSG5-rMKP-1 DNA transfected, and was absent with a
construct expressing antisense rMKP-1 (data not shown). While we did
not examine the ability of rMKP-1 to deactivate JNK1 in vitro,
recent findings by Sun et al. (21) , provided such
evidence, although they found in their system that the phosphatase
displayed 30-fold greater activity for dephosphorylation of MAP kinase
relative to JNK. Such a difference in selectivity does not, however,
preclude a role for MKP-1 in the regulation of JNK activity,
particularly in instances where MKP-1 expression is high and MAP kinase
is not involved.
MKP-1 Expression Inhibits AP-1-dependent Gene
Induction
AP-1 transcription factor activity is regulated by
pathways that include JNK and MAP kinase
(7, 8, 9, 10, 32, 33, 34, 35, 36) .
To examine the effect of constitutive rMKP-1 expression on
AP-1-mediated gene induction, we employed two different reporter
constructs, coll-CAT and jun-LUC. Both of these
constructs have been shown to rely on an AP-1 site for enhanced
expression following UVC treatment
(1, 4, 35, 36, 37) . HeLa cells
were transfected with either construct along with plasmids expressing
rMKP-1 in the sense or antisense orientation. Transfected cells were
subsequently treated with TPA, UVC, or MMS and on the following day
monitored for CAT or luciferase expression. In the absence of either
MKP-1 expression plasmid, coll-CAT activity was enhanced
>100-, 6-, and 6-fold by TPA, UVC, and MMS treatments, respectively.
jun-LUC expression was enhanced 25-, 20-, and >50-fold by
the same treatments. rMKP-1 had little effect on the basal levels of
either coll-CAT or jun-LUC, but markedly inhibited
induction of both in response to all three treatments. In contrast, the
plasmid expressing antisense rMKP-1 had no effect (Fig. 5).
Importantly, rMKP-1 does not act nonspecifically to inhibit all
promoter activation, as similar studies performed by cotransfecting
rMKP-1 with the heat shock protein 70 promoter linked to the CAT
reporter showed no inhibition of heat shock-induced HSP70 promoter
activation (data not shown).
General Discussion
There is good evidence to
support a role for MKP-1 in regulating MAP kinase-dependent gene
activation in response to proliferative stimuli
(20, 21, 32) . Despite its high level of
induction following treatment of cells with DNA damaging agents, a
function for the phosphatase during the cellular response to genotoxic
stress has not been established. Here we have provided evidence for the
involvement of MKP-1 in the regulation of the gene activation in
response to two different genotoxic treatments, UVC and MMS. In the
case of UVC irradiation, both MAP kinase and JNK1 are activated and
both could be subject to deactivation by the phosphatase. However,
given the relative selectivity of MKP-1 for MAP kinase compared to JNK
(21) , it is reasonable to assume that MKP-1 has greater
influence on MAP kinase-mediated gene activation than that mediated via
JNK in response to UVC irradiation. With MMS treatment, however, MAP
kinase shows little activation. This argues that MAP kinase is unlikely
to be the target for the MMS-induced MKP-1 protein. However, induction
of MKP-1 expression following UVC and MMS treatment in HeLa cells was
found to coincide with deactivation of JNK1, suggesting that JNK
activity could be regulated by MKP-1 in this system. Indeed, we have
shown that MKP-1 can inhibit activation of JNK1 in response to both UVC
and MMS treatments in vivo, and have demonstrated that both
UVC and MMS-induced activation of two different AP-1-dependent
promoters is markedly inhibited by constitutive expression of rMKP-1.
Finally, in other recent studies in our laboratory we have found that
cycloheximide treatment of cells during the UVC response (which
prevents MKP-1 protein accumulation) does not prolong MAP kinase
activation, but does extend and potentiate UVC-induced increases in
JNK1 activity.()
Taken together, these findings
strongly support a role for MKP-1 in regulating gene expression during
genotoxic stress and suggest that JNK is a target for the phosphatase.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.