The Drosophila DUSP Puckered is phosphorylated by JNK and p38 in response to arsenite-induced oxidative stress

The Drosophila DUSP Puckered is phosphorylated by JNK and p38 in response to arsenite-induced oxidative stress

Biochemical and Biophysical Research Communications 418 (2012) 301–306 Contents lists available at SciVerse ScienceDirect Biochemical and Biophysica...

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Biochemical and Biophysical Research Communications 418 (2012) 301–306

Contents lists available at SciVerse ScienceDirect

Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc

The Drosophila DUSP Puckered is phosphorylated by JNK and p38 in response to arsenite-induced oxidative stress Katerina Karkali, George Panayotou ⇑ Institute of Molecular Oncology, Biomedical Sciences Research Center ‘‘Alexander Fleming’’, 34 Fleming Street, Vari 166 72, Greece

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Article history: Received 21 December 2011 Available online 12 January 2012 Keywords: Puckered JNK p38 Phosphorylation Phosphatase Arsenite

a b s t r a c t Dual-Specificity Phosphatases (DUSPs) are enzymes that remove phosphate groups from both phosphotyrosine and phospho-serine/threonine residues. A subgroup of DUSPs specifically targets Mitogen-Activated Protein Kinases (MAPKs) and has been shown to participate in the regulation of differential cellular responses to the large variety of stimuli conveyed by MAPK-pathways. In Drosophila, Puckered has been identified as a DUSP, exhibiting specificity towards the c-Jun-N-terminal kinase (JNK). Recent studies have signified its role in regulating JNK-dependent processes, including immunity, stress tolerance and longevity. Puckered expression depends on the activation of the JNK pathway whereas it’s degradation is mediated by the ubiquitin–proteasome system. In this study we show that Puckered is phosphorylated by JNK and p38 in response to arsenite-induced oxidative stress and that phosphorylation affects the interaction between Puckered and these MAPKs. In silico analysis of the Puckered amino acid sequence revealed several MAPK consensus phosphorylation motifs. Expression of Puckered in the heterologous system of HEK293 cells and subsequent stimulation with arsenite resulted in reduced mobility of Puckered in SDS–PAGE. Similar results were obtained when Puckered was co-expressed with the constitutively active forms of JNK and p38. This mobility shift was abolished by lambda-phosphatase treatment or by simultaneous inhibition of JNK and p38. Analysis by mass-spectrometry identified Puckered phosphorylation in Ser413, though phosphorylation on this site was found irrespective of stimulation. Finally, phosphorylation of Puckered enhanced its interaction both with JNK and p38. Our results suggest a possible functional role of Puckered phosphorylation by MAPKs. Ó 2012 Elsevier Inc. All rights reserved.

1. Introduction Mitogen-Activated Protein Kinases (MAPKs) are activated by a variety of stimuli to induce distinct cellular responses. Environmental stressors, such as UV radiation, free oxygen radicals and mechanical stress, as well as inflammatory cytokines, are known to preferentially activate the MAPK-subgroup of Stress-Activated Protein Kinases (SAPKs) [1]. In particular, sodium arsenite, the carcinogenic form of trivalent arsenic, has been reported to promote the activation of SAPKs through the production of reactive oxygen species [2]. SAPKs consist of the c-Jun-N-terminal (JNK) kinases and p38 kinases and their activity has been implicated in several physiological and pathological conditions, including inflammation and cancer [1]. Dual Specificity Phosphatases (DUSPs) have emerged as key regulators of MAPK-activity in development, cancer and environmental stress conditions and are promising therapeutic targets for cancer treatment [3]. DUSPs belong to the super-family of Protein Tyrosine Phosphatases and are cysteine-based enzymes ⇑ Corresponding author. Fax: +30 2109653934. E-mail address: [email protected]fleming.gr (G. Panayotou). 0006-291X/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2012.01.015

capable of removing phosphate groups from both phospho-tyrosine and phospho-serine/threonine residues [4]. In mammals, several members of the DUSP-family have been shown to specifically target and inactivate MAPKs. DUSPs are evolutionarily conserved, with homologues identified in budding yeast, Caenorhabditis elegans and Drosophila melanogaster [5]. DUSPs are subjected to tight regulation, both at the transcriptional and post-translational level. Transcriptional induction has been reported for all mammalian class I DUSPs, which are encoded by immediate early genes and are rapidly up-regulated in response to mitogenic and stress stimuli. At the post-translational level, DUSPs are modified by phosphorylation and ubiquitination, while acetylation of DUSP1 was also reported recently [5,6]. Phosphorylation of DUSPs has been shown to affect their stability as well as their ability to bind to target MAPKs [7–9]. Oxidative stress results in activation of JNK and p38 and directly affects DUSP-activity, either by oxidizing their catalytic cysteine or by promoting their degradation [10–13]. Additionally, phosphorylation of DUSP8 by JNK has been reported under arsenite-induced oxidative stress [14]. Six DUSPs have been annotated in the Drosophila genome, of which Puckered is the best characterized. The gene puckered is

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involved in the regulation of the final steps of embryonic dorsal closure, an essential late morphogenetic event [15]. Further, in adult flies, puckered modulates innate immunity, wound healing, stress tolerance and longevity [16–19]. Puckered codes for a JNK-specific phosphatase whose stability has been shown to be regulated by ubiquitination [15,20]. Despite its importance, Puckered remains poorly characterized at the biochemical level. In this study we show that Puckered is post-translationally regulated by phosphorylation. Puckered is basally phosphorylated at Ser413, as identified by mass-spectrometry, upon expression in the heterologous system of HEK293 cells. Arsenite-induced oxidative stress results in enhanced Puckered phosphorylation by the combined activities of JNK and p38. Finally, we provide evidence for the physical interaction of Puckered with JNK and p38. This interaction occurs irrespectively of stimulation for p38, while it is stimulation-dependent for JNK.

described [14,22]. Raw-data were compared against the dmel-alltranslation-r5.33.fasta (flybase) D. melanogaster database. Differentially modified serine/threonine molecular mass values (addition of 79.966 Da) were used to identify phosphorylated peptides.

2. Materials and methods

3.1. Puckered is phosphorylated in response to arsenite-induced oxidative stress

2.1. Plasmids and reagents The plasmids used for transfections were: (1) pCS2-MT-Puckered and pCS2-MT-Puckered-DEAD were a gift of Dr. D.G. McEwen (University of Texas) [21]. (2) pcDNA3.1-flag-MKK6DD was provided by Dr. A. Nebreda (IRB, Barcelona). (3) pCDNA3-flagMKK7B2Jnk1a1 (APF) and pCDNA3-flag-MKK7B2Jnk1a1 were obtained from Addgene. (4) Rc-CMV-flag-JNK1, pCDNA3-flag-p38a and pCDNA3-flag-p38b were a gift of Dr. R.J. Davis (University of Massachusetts). Inhibitors were obtained from Calbiochem and added to the medium at the following final concentrations for 12 h prior to arsenite stimulation: SP600125 (50 lM); SB203580 (10 lM); PD98059 (20 lM). Lambda phosphatase (New England Bioloabs) was applied at 200 units per reaction for 30 min. 2.2. Cell culture and transfection Human Embryonic Kidney cells 293 (HEK293) were cultured in DMEM supplemented with 10% (v/v) fetal bovine serum, 100U/mL penicillin, and 100 lg/mL streptomycin (Gibco) at 37 °C in a humidified 5% CO2 atmosphere. Transient transfections were performed using a standard calcium phosphate protocol. 2.3. Western blotting and immunoprecipitation For western blot analysis proteins were transferred to Protran nitrocellulose membrane (Whatman) and ECL Plus reagent (GE Healthcare) was used for antibody detection. For immunoprecipitations Protein A Sepharose CL-4B (Pharmacia Biotech) was used. The antibodies employed were: c-myc A-14 was used for immunoblotting while c-myc-9E10 was used for immunoprecipitation of myc-Puckered (Santa Cruz); anti-active JNK (Cell Signaling); antiJNK1 (R&D Systems); Pan-actin (Cell Signaling); anti-phosphocJun (KM-1) (Santa Cruz); anti-flag-M2 (Sigma). Goat anti-mouse (Southern Biotech) and goat anti-rabbit (Santa Cruz) were used as secondary antibodies. 2.4. Phosphopeptide mapping Myc-Puckered immunoprecipitates were resolved on SDS–PAGE and the gel stained with silver nitrate. Puckered bands were excised, destained and subjected to in-gel digestion with trypsin. Peptides were fractionated by nano-HPLC and analyzed on an LCQ-Deca mass-spectrometer (Thermo Electron Corporation), as

2.5. Western blot quantification and statistical analysis For western blot quantification, the average band density was measured using ImageJ software (NIH). Data are given as mean of three independent experiments ± SEM. Statistical analysis was performed using GraphPad software (GraphPad Software Inc., La Jolla, CA, USA). In all cases one sample t test was performed and probability values p < 0.05 were regarded as significant. 3. Results

In silico analysis of the Puckered amino acid sequence revealed several potential sites bearing the consensus phosphorylation motif for MAPKs, mapping mainly to the C-terminus of the protein (Supplemental Fig. S1). To experimentally evaluate these predictions, endogenous MAPKs activation was induced by subjecting HEK293 cells, transiently expressing Puckered, to different types of stress. Treatment with arsenite resulted in a dose dependent up-shift of Puckered mobility in SDS–PAGE, the magnitude of the shift correlating with arsenite concentration (Fig. 1A). Treatment with H2O2 or sorbitol only induced a moderate shift in Puckered mobility, while anisomycin had no effect (data not shown). A similar strong effect of arsenite has been reported previously for another JNK-specific DUSP, the mammalian M3/6 (DUSP8) [14]. Therefore this stress-inducing agent was used in subsequent experiments. Arsenite is a potent activator of the JNK and p38 pathways in HEK293 cells, only inducing a moderate activation of ERK (Supplemental Fig. S2). Confirming that the arsenite-induced effect was due to activation of upstream kinases, we found that co-expression of the constitutively active fusion flag-MKK7JNK1a1 with wildtype myc-Puckered also induced a significant up-shift in its mobility, which was not observed when the inactive fusion flagMKK7JNK1a1 (APF) was used. This up-shift was enhanced when a catalytically impaired form of Puckered (myc-Puckered-DEAD) was expressed (Fig. 1B). Weaker effects were obtained for the constitutively active form of MKK6 (flag-MKK6DD) (Fig. 1C), while EGF stimulation of HEK293 cells, resulting in ERK activation, had no effect (Supplemental Fig. S3). Protein phosphorylation is known to affect electrophoretic mobility in SDS–PAGE. To determine if the observed shift of Puckered mobility upon HEK293 cell stimulation was indeed due to phosphorylation, we immunoprecipitated Puckered from cells coexpressing the active or the inactive form of flag-MKK7JNK1a1 and subjected it to lambda-phosphatase treatment (Fig. 1D). In the presence of lambda-phosphatase the induced shift in mycPuckered mobility by the active flag-MKK7JNK1a1 was abolished, confirming that the shift was due to phosphorylation (Fig. 1D, right panel). No alteration in Puckered mobility was observed after incubation with lambda-phosphatase using the inactive form of flagMKK7JNK1a1, that does not induce a mobility shift (Fig. 1D, left panel), suggesting low levels of basal phosphorylation in Puckered. Analysis by mass-spectrometry of immunoprecipitated myc-Puckered from extracts of arsenite treated or untreated HEK293 cells identified the presence of phosphate on serine 9 on the Puckered peptide AISKPNLLpSPTR under both conditions, suggesting that Puckered is basally phosphorylated (Supplemental Fig. S4).

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Fig. 1. Puckered is phosphorylated in response to arsenite and phosphorylation is mediated by JNK and p38 activities. (A) Immunoblotting of extracts from HEK293 cells, transiently expressing myc-Puckered, stimulated with the indicated doses of arsenite for 1 h. Loading control: actin. (B) Immunoblotting of extracts from HEK293 cells coexpressing myc-Puckered or myc-Puckered-DEAD with the active or inactive (APF) form of flag-MKK7JNK1a1 fusion. Loading control: actin. (C) Immunoblotting of extracts from HEK293 cells co-expressing myc-Puckered or myc-Puckered-DEAD with the constitutively active form of MKK6DD (flag-MKK6DD). Empty pcDNA3 vector was cotransfected with myc-Puckered constructs as control for MKK6DD activity. Loading control: actin. (D) Western blot of immunoprecipitated Puckered from cells co-expressing the active or inactive (APF) form of MKK7JNK1a1 fusion after treatment with lambda phosphatase.

However, it was not possible to quantitate the relative abundance of this phophorylation site in stimulated vs. unstimulated cells, nor indeed to identify additional sites that would contribute to the mobility shift. 3.2. Puckered is phosphorylated by both JNK and p38 In order to further confirm that SAPK activation is responsible for Puckered phosphorylation in response to arsenite, we used the competitive ATP-site inhibitors SP600125 and SB203580 to block the activity of JNK and p38, respectively. HEK293 cells transiently expressing myc-Puckered or myc-Puckered-DEAD were simultaneously exposed to both inhibitors and subsequently stimulated with arsenite for 90 min. The transcription factor c-Jun is a common downstream target of JNK and p38 and its phosphorylation levels served as read-out of the inhibitors’ effect on the activity of these kinases. The combined SAPK inhibition resulted in substantial reduction in phospho-c-Jun levels and in an almost complete loss of myc-Puckered and myc-Puckered-DEAD phosphorylation upon arsenite treatment, evident by the abolition of the mobility up-shift (Fig. 2). Use of either inhibitor alone was not sufficient to prevent c-Jun phosphorylation nor Puckered mobility shift to a significant effect (data not shown). In contrast, the MEK1 inhibitor PD98059, while effectively preventing ERK activation, had no effect on Puckered phosphorylation in response to arsenite (Supplemental Fig. S5). These results are in support of the data presented in Fig. 1B and C, obtained upon co-expression of Puckered with the constitutively active forms of JNK and p38 and strongly argue that Puckered is phosphorylated by both SAPKs in response to stress. 3.3. Puckered attenuates arsenite-induced JNK activation Most published data on the JNK-specific phosphatase activity of Puckered have been primarily based on genetic evidence in

Fig. 2. Puckered is phosphorylated by JNK and p38 in response to arsenite. Immunoblotting of extracts from HEK293 cells transiently expressing myc-Puckered or myc-Puckered-DEAD. Cells were simultaneously exposed to the JNK-specific SP600125 inhibitor and the p38-specific SB203580 inhibitor, as described in Section 2. Cells were then stimulated with 200 lM arsenite for 90 min. Phospho-c-Jun levels were used to monitor the extent of JNK and p38 inhibition, while actin served as loading control.

Drosophila [15]. In order to confirm in a more direct manner the phosphatase activity of Puckered and investigate a possible correlation with its phosphorylation, we used our heterologous expression system and performed a time-course stimulation of HEK293 cells expressing catalytically active or inactive Puckered, with 200 lM arsenite. A time-dependent up-shift in myc-Puckered (Fig. 3A) and myc-Puckered-DEAD (Fig. 3B) mobility was observed, reaching its highest level at 60 min of treatment. In the same

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extracts, phospho-JNK levels were quantified in the presence of myc-Puckered (Fig. 3C) and myc-Puckered-DEAD (Fig. 3D) and were compared to the phospho-JNK levels in control cells (HEK293 cells transfected with pcDNA3 vector) at each of the time points tested. In control cells, as in cells expressing myc-Puckered or myc-Puckered-DEAD, rapid phosphorylation of JNK was detected, peaking at 60 min of treatment. However, the presence of catalytically active myc-Puckered resulted in a reduction of JNK phosphorylation (Fig. 3C), which was statistically significant up to 45 min of treatment, and therefore inversely correlated with the shift in myc-Puckered mobility (Fig. 3A). Expression of mycPuckered-DEAD, as expected, had no impact on phospho-JNK levels at any of the time points (Fig. 3D). In contrast, no substantial difference in phospho-p38 levels was observed between control and Puckered expressing cells, confirming Puckered specificity for JNK (data not shown). 3.4. Arsenite enhances the interaction of Puckered with JNK and p38 The phosphorylation of Puckered by JNK and p38 prompted us to test the direct interaction between Puckered and these MAPKs by performing co-immunoprecipitation experiments. Cell extracts from HEK293 cells co-expressing myc-Puckered with flag-tagged forms of either JNK1 or p38a were prepared before and after arsenite stimulation. The expression levels of each of these proteins were determined and the activation of JNK and p38, as a result of arsenite stimulation was confirmed by western blot analysis of the extracts (Fig. 4A and C). In the absence of arsenite, an antibody against the myc epitope precipitated Puckered but no detectable JNK1. After arsenite treatment however, an interaction between myc-Puckered and flag-JNK1 was observed, even though the amount of precipitated Puckered was reduced (Fig. 4B, top). In

the inverse experiment, myc-Puckered in flag-JNK1 immunoprecipitates was below the limit of detection (Fig. 4B, bottom). An interaction between myc-Puckered and flag-p38a was also observed and was enhanced by arsenite treatment (Fig. 4D, top). In this case, the reciprocal immunoprecipitation with anti-flag antibody also showed this interaction (Fig. 4D, bottom). Similar results were obtained for the interaction of Puckered with a different p38 isoform, p38b (data not shown). From the above experiments we conclude that Puckered interacts with JNK and p38 and the interactions are enhanced by arsenite treatment. 4. Discussion Extensive studies have established Puckered as the key regulator of JNK activity in Drosophila. Puckered has been implicated in several developmental processes and its function has been shown to affect immunity as well as the organism’s ability to respond to stress [16,21]. Despite these advances, our knowledge on the regulation of this enzyme remains limited. Puckered’s expression has been directly linked to the activation of the JNK pathway and it has been found up-regulated under oxidative and UV-induced stress conditions [19,21]. In mammals oxidative stress leads to the activation of JNK and p38, among other responses [10,11]. Similarly, arsenite stimulation of Drosophila S2 cells results in a ROS-dependent activation of all MAPKs, while JNK activity in flies is associated with oxidative stress tolerance and has been shown to increase the life span of the organism [18,19,23]. Part of the cellular defense mechanisms activated to attenuate oxidative stress is the transcriptional activation of DUSPs [11]. Further, the activity of these proteins can be post-translationally modified by the same type of stimulus. Oxidative stress increases thiol

Fig. 3. Catalytically active myc-Puckered reduces phospho-JNK levels in HEK293 cells upon stimulation with arsenite. Immunoblotting of extracts from HEK293 cells transiently expressing myc-Puckered (A) or myc-Puckered-DEAD (B), after stimulation with 200 lM arsenite for the indicated time points. Loading control: actin. Immunoblots against phospho-JNK and total JNK1 in extracts from control (transfected with empty pcDNA3 vector) and myc-Puckered (C) or myc-Puckered-DEAD (D) expressing HEK293 cells after treatment with arsenite. Comparison of JNK activation levels in control versus myc-Puckered expressing cells is presented in histograms after western blot quantification. Asterisks denote statistically significant differences (p value < 0.05).

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Fig. 4. Arsenite-treatment promotes Puckered interaction with JNK and p38. (A) Immunoblotting of extracts from HEK293 cells co-expressing myc-Puckered with flag-JNK1 or expressing flag-JNK1 alone. Cells were either treated with 200 lM arsenite for 90 min or left untreated, as indicated. Phospho-JNK detection was used as a reporter of oxidative stress, while actin served as loading control. (B) anti-myc and anti-flag immunoblots of myc-Puckered (IP:myc) and flag-JNK1 (IP:flag) immunoprecipitates performed from the extracts presented in A. (C) Immunoblots of extracts from arsenite treated or untreated HEK293 cells expressing myc-Puckered together with flag-p38a, or flag-p38a alone. The detection of phospho-p38 verified oxidative stress conditions and actin was used as loading control. (D) anti-myc and anti-flag immunoblots of mycPuckered (IP:myc) and flag-p38a (IP:flag) immunoprecipitates performed from the extracts analyzed in C.

modifications on DUSPs and induces oxidation of their catalytic cysteine residue, rendering them inactive [12]. Moreover, signaling events evoked in response to oxidative stress have been shown to indirectly affect DUSPs-function by promoting their degradation [13]. Recently it was reported that DUSP8 is also modified by phosphorylation, under oxidative stress conditions [14]. Here we show that the Drosophila DUSP, Puckered, is phosphorylated in response to arsenite-based oxidative stress. It is likely that Puckered is phosphorylated on multiple sites, since the shift in its mobility occurs gradually after 30 min of arsenite stimulation, reaching a plateau between 60 and 90 min of treatment (Fig. 3A). Moreover, our data argue that Puckered phosphorylation in response to arsenite depends on the activation of JNK and p38. Puckered harbors several putative MAPK phosphorylation sites and we determined that Puckered is phosphorylated at Ser413 (Supplemental Figs. S1 and S4). The phosphorylation of Ser413 was detected irrespective of any stimulus, suggesting that Puckered is phosphorylated under resting conditions, although treatment with lambda-phosphatase failed to alter basal Puckered mobility (Fig. 1D, left panel). It is possible that one-dimensional

SDS–PAGE analysis cannot resolve un-phosphorylated and singlephosphorylated forms of Puckered. The kinase(s) responsible for Puckered phosphorylation in the absence of stimulus remains elusive. One possibility is that basal activation of JNK and p38, maintained by the presence of serum in the culture medium, would be sufficient. In this scenario, Puckered in un-stimulated S2R+ cells has been shown to interact with p38b in a Drosophila protein interactions map (DpiM) generated by large-scale co-affinity purification and tandem mass spectrometry [24]. Puckered’s basal phosphorylation might thus depend on p38 activity in our system. Phosphorylation has also been reported to modify DUSPs binding to their substrate MAPKs [9]. Puckered’s function requires physical interaction with JNK although this was detected only in the presence of stimulus. Docking on the JIP scaffold proteins has been shown to facilitate JNK activation by bringing it in proximity to its upstream kinases [25]. In addition, interaction of DUSPs with JIPs has been reported for DUSP8 and DUSP16 [14,26]. Therefore it is possible that, upon arsenite stimulation, Puckered is recruited to these protein complexes stabilizing its interaction with JNK. On the other hand, the interaction of Puckered with p38a occurs

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irrespectively of stimulus, although it is enhanced after treatment (Fig. 4D). Similar results were obtained for p38b and this observation is in accordance with the interaction data of the DPiM map described above. Interestingly, no JNK–Puckered interaction was reported in the DPiM favoring the idea that this interaction is enhanced under stress conditions [24]. There is no evidence that phosphorylation directly affects the catalytic activity of DUSPs. The presence of active Puckered results in a significant reduction in phospho-JNK levels (Fig. 3C), which is evident up to 60 min after stimulation, when Puckered is maximally phosphorylated (Fig. 3A). This correlation could suggest a role for phosphorylation in reducing Puckered activity, but it would also be consistent with an accumulation of catalytic cysteine oxidation under the strong oxidative stress. It would also be interesting to investigate further the precise role of p38 in regulating Puckered activity towards JNK, since under basal conditions, we found that Puckered interacts with phospho-p38. It is likely however that Puckered’s phosphorylation, driven by the combined action of both kinases and resulting in sustained activation of JNK, is an attenuating regulatory event. Acknowledgments We thank Enrique Martin-Blanco and Angel Nebreda for helpful comments and reagents; Makis Skoulakis, Marina Cotsiki and Wolf Oehrl for critical reading of the manuscript and Emmanuel Fragoulis and Diamantis Sideris for their scientific support; Martina Samiotaki for the MS-analysis; Roger Davis and Donald McEwen for their gift of reagents. This work was supported by the European Commission FP7 program INFLA-CARE (EC contract number 223151). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.bbrc.2012.01.015. References [1] J.M. Kyriakis, J. Avruch, Mammalian mitogen-activated protein kinase signal transduction pathways activated by stress and inflammation, Physiol. Rev. 81 (2001) 807–869. [2] M.S. Iordanov, B.E. Magun, Different mechanisms of c-Jun NH(2)-terminal kinase-1 (JNK1) activation by ultraviolet-B radiation and by oxidative stressors, J. Biol. Chem. 274 (1999) 25801–25806. [3] C. Nunes-Xavier, C. Roma-Mateo, P. Rios, C. Tarrega, R. Cejudo-Marin, L. Tabernero, R. Pulido, Dual-specificity MAP kinase phosphatases as targets of cancer treatment, Anticancer Agents Med. Chem. 11 (2011) 109–132. [4] K.I. Patterson, T. Brummer, P.M. O’Brien, R.J. Daly, Dual-specificity phosphatases: critical regulators with diverse cellular targets, Biochem. J. 418 (2009) 475–489. [5] O. Bermudez, G. Pages, C. Gimond, The dual-specificity MAP kinase phosphatases: critical roles in development and cancer, Am. J. Physiol. Cell Physiol. 299 (2010) 189–202. [6] H. Chi, R.A. Flavell, Acetylation of MKP-1 and the control of inflammation. Sci. Signal 1 (2008) pe44.

[7] J.M. Brondello, J. Pouyssegur, F.R. McKenzie, Reduced MAP kinase phosphatase1 degradation after p42/p44MAPK-dependent phosphorylation, Science 286 (1999) 2514–2517. [8] Y.W. Lin, J.L. Yang, Cooperation of ERK and SCFSkp2 for MKP-1 destruction provides a positive feedback regulation of proliferating signaling, J. Biol. Chem. 281 (2006) 915–926. [9] R.J. Dickinson, L. Delavaine, R. Cejudo-Marin, G. Stewart, C.J. Staples, M.P. Didmon, A.G. Trinidad, A. Alonso, R. Pulido, S.M. Keyse, Phosphorylation of the kinase interaction motif in mitogen-activated protein (MAP) kinase phosphatase-4 mediates cross-talk between protein kinase A and MAP kinase signaling pathways, J. Biol. Chem. 286 (2011) 38018–38026. [10] B. Racz, K. Hanto, A. Tapodi, I. Solti, N. Kalman, P. Jakus, K. Kovacs, B. Debreceni, F. Gallyas Jr., B. Sumegi, Regulation of MKP-1 expression and MAPK activation by PARP-1 in oxidative stress: a new mechanism for the cytoplasmic effect of PARP-1 activation, Free Radic. Biol. Med. 49 (2010) 1978–1988. [11] C.H. Teng, W.N. Huang, T.C. Meng, Several dual specificity phosphatases coordinate to control the magnitude and duration of JNK activation in signaling response to oxidative stress, J. Biol. Chem. 282 (2007) 28395–28407. [12] H. Kamata, S. Honda, S. Maeda, L. Chang, H. Hirata, M. Karin, Reactive oxygen species promote TNFalpha-induced death and sustained JNK activation by inhibiting MAP kinase phosphatases, Cell 120 (2005) 649–661. [13] D.W. Chan, V.W. Liu, G.S. Tsao, K.M. Yao, T. Furukawa, K.K. Chan, H.Y. Ngan, Loss of MKP3 mediated by oxidative stress enhances tumorigenicity and chemoresistance of ovarian cancer cells, Carcinogenesis 29 (2008) 1742– 1750. [14] M. Cotsiki, W. Oehrl, M. Samiotaki, A. Theodosiou, G. Panayotou, Phosphorylation of the M3/6 dual-specificity phosphatase enhances the activation of JNK by arsenite, Cell Signal 24 (2012) 664–676. [15] E. Martin-Blanco, A. Gampel, J. Ring, K. Virdee, N. Kirov, A.M. Tolkovsky, A. Martinez-Arias, Puckered encodes a phosphatase that mediates a feedback loop regulating JNK activity during dorsal closure in Drosophila, Genes Dev. 12 (1998) 557–570. [16] M. Boutros, H. Agaisse, N. Perrimon, Sequential activation of signaling pathways during innate immune responses in Drosophila, Dev. Cell 3 (2002) 711–722. [17] M. Ramet, R. Lanot, D. Zachary, P. Manfruelli, JNK signaling pathway is required for efficient wound healing in Drosophila, Dev. Biol. 241 (2002) 145– 156. [18] J. Karpac, J. Hull-Thompson, M. Falleur, H. Jasper, JNK signaling in insulinproducing cells is required for adaptive responses to stress in Drosophila, Aging Cell 8 (2009) 288–295. [19] M.C. Wang, D. Bohmann, H. Jasper, JNK signaling confers tolerance to oxidative stress and extends lifespan in Drosophila, Dev. Cell 5 (2003) 811–816. [20] J. Liu, M. Ghanim, L. Xue, C.D. Brown, I. Iossifov, C. Angeletti, S. Hua, N. Negre, M. Ludwig, T. Stricker, H.A. Al-Ahmadie, M. Tretiakova, R.L. Camp, M. PereraAlberto, D.L. Rimm, T. Xu, A. Rzhetsky, K.P. White, Analysis of Drosophila segmentation network identifies a JNK pathway factor overexpressed in kidney cancer, Science 323 (2009) 1218–1222. [21] D.G. McEwen, M. Peifer, Puckered, a Drosophila MAPK phosphatase, ensures cell viability by antagonizing JNK-induced apoptosis, Development 132 (2005) 3935–3946. [22] Z. Gkiafi, G. Panayotou, Comparative proteomic analysis implicates COMMD proteins as Epstein-Barr virus targets in the BL41 Burkitt’s lymphoma cell line, J. Proteome Res. 10 (2011) 2959–2968. [23] O.P. Ryabinina, E. Subbian, M.S. Iordanov, D-MEKK1, the Drosophila orthologue of mammalian MEKK4/MTK1, and Hemipterous/D-MKK7 mediate the activation of D-JNK by cadmium and arsenite in Schneider cells, BMC Cell Biol. 7 (2006) 7. [24] K.G. Guruharsha, J.F. Rual, B. Zhai, J. Mintseris, P. Vaidya, N. Vaidya, C. Beekman, C. Wong, D.Y. Rhee, O. Cenaj, E. McKillip, S. Shah, M. Stapleton, K.H. Wan, C. Yu, B. Parsa, J.W. Carlson, X. Chen, B. Kapadia, K. Vijayraghavan, S.P. Gygi, S.E. Celniker, R.A. Obar, S. Artavanis-Tsakonas, A protein complex network of Drosophila melanogaster, Cell 147 (2011) 690–703. [25] D.N. Dhanasekaran, K. Kashef, C.M. Lee, H. Xu, E.P. Reddy, Scaffold proteins of MAP-kinase modules, Oncogene 26 (2007) 3185–3202. [26] E.A. Willoughby, G.R. Perkins, M.K. Collins, A.J. Whitmarsh, The JNK-interacting protein-1 scaffold protein targets MAPK phosphatase-7 to dephosphorylate JNK, J. Biol. Chem. 278 (2003) 10731–10736.