©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Role of Mitogen-activated Protein Kinase Phosphatase during the Cellular Response to Genotoxic Stress
INHIBITION OF c-Jun N-TERMINAL KINASE ACTIVITY AND AP-1-DEPENDENT GENE ACTIVATION (*)

Yusen Liu , Myriam Gorospe , Chunlin Yang (1), Nikki J. Holbrook(§)(¶)

From the (1) Section on Gene Expression and Aging and Laboratory of Biological Chemistry, Gerontology Research Center, NIA, National Institutes of Health, Baltimore, Maryland 21224

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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.


INTRODUCTION

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)() 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) .

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.


MATERIALS AND METHODS

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.

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-p42antiserum (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)


RESULTS AND DISCUSSION

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.

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).

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.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Gene Expression and Aging Section, NIA, 4940 Eastern Ave., Baltimore, MD 21224. Tel.: 410-558-8197; Fax: 410-558-8335.

The abbreviations used are: UVC, short wavelength ultraviolet light; MMS, methyl methanesulfonate; MAP kinase, mitogen-activated protein kinase; MEK, MAP kinase kinase; ERK, extracellular signal-regulated kinases; ATF2, activator of transcription factor 2; AP-1, activator protein 1; JNK, c-Jun N-terminal kinase; MKP-1, MAP kinase phosphatase 1; HA, hemagglutinin; LUC, luciferase; CAT, chloramphenicol acetyltransferase; TPA, 12- O-tetradecanoylphorbol-13-acetate; GST, glutathione S-transferase.

Y. Liu, M. Gorospe, and N. J. Holbrook, unpublished findings.


ACKNOWLEDGEMENTS

We thank J. Luethy-Martindale for technical assistance and M. Lee for helpful discussions.


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.