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The Dual Specificity Mitogen-activated Protein Kinase Phosphatase-1 and -2 Are Induced by the p42/p44MAPK Cascade

The Dual Specificity Mitogen-activated Protein Kinase Phosphatase-1 and -2 Are Induced by the... THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 272, No. 2, Issue of January 10, pp. 1368–1376, 1997 © 1997 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. The Dual Specificity Mitogen-activated Protein Kinase Phosphatase- MAPK Cascade* 1 and -2 Are Induced by the p42/p44 (Received for publication, September 4, 1996, and in revised form, October 28, 1996) Jean-Marc Brondello, Anne Brunet, Jacques Pouysse´gur, and Fergus R. McKenzie‡ From the Universite´ de Nice, Centre de Biochimie, CNRS UMR 134, Parc Valrose, 06108 Nice Cedex 02, France Mitogen-activated protein (MAP) kinase phospha- phoamino acid specificity. Dual specificity phosphatases capa- tase-1 (MKP-1) and MKP-2 are two members of a recently ble of removing both phosphotyrosine and phosphothreonine described family of dual specificity phosphatases that from protein targets are a relatively recent discovery. However, MAPK are capable of dephosphorylating p42/p44 . Overex- an ever increasing number of such phosphatases exist, includ- 14 15 pression of MKP-1 or MKP-2 inhibits MAP kinase-de- ing Cdc25, which dephosphorylates Thr and Tyr of Cdc2 (4), pendent intracellular signaling events and fibroblast and Cdi1 (or KAP) (5, 6), which dephosphorylates Thr of proliferation. By using specific antibodies that recog- Cdk2. A novel family of dual specificity enzymes harboring the nize endogenous MKP-1 and MKP-2 in CCL39 cells, we canonical motif (I/V)HCXAGXXR(S/T)G, originally identified in show that MKP-1 and MKP-2 are not expressed in qui- the active site of the VH-1 phosphatase (7), and able to dephos- escent cells, but are rapidly induced following serum phorylate, at least in vitro, an archetypal substrate, p42/ addition, with protein detectable as early as 30 min MAPK p44 (8, 9), has recently been identified. These phosphata- (MKP-1) or 60 min (MKP-2). Serum induction of MKP-1 ses have been named MAP kinase phosphatases (MKPs), with and MKP-2 is sustained, with protein detectable up to at least eight known members currently identified: MKP-1 14 h after serum addition. Induction of MKP-1 and, to a (CL100, XCL100, HVH-1, 3CH134, or erp) (10–14); MKP-2 lesser extent, MKP-2 temporally correlates with p42/ MAPK (hVH-2, TYP1) (15, 16); hVH-3 (B23) (17, 18); MKP-3 (rVH-6, p44 inactivation. Pyst1) (19, 20, 23); hVH-5 (21), PAC1 (22); and two incomplete To analyze the contribution of the MAP kinase cas- sequences, MKP-X (19) and Pyst2 (23). cade to MKP-1 and MKP-2 induction, we examined MAPK p42/p44 are highly homologous ternary members of a CCL39 cells transformed with either v-ras or a constitu- group of ubiquitously expressed serine/threonine kinases that, tively active direct upstream activator of MAP kinase, in fibroblasts, are activated in response to all mitogenic stimuli mitogen-activated protein kinase kinase-1 (MKK-1; MKK-1(SD/SD) mutant). In both cell models, MKP-1 and (24). Sustained activation of the MAP kinase cascade is an MKP-2 are constitutively expressed, with MKP-2 being absolute requirement for fibroblasts to exit from the quiescent prevalent. In addition, in CCL39 cells expressing an es- G state and to pass the restriction point in G . Thus, blockade 0 1 tradiol-inducible DRaf-1::ER chimera, activation of Raf of MAP kinase signaling with antisense MAP kinase constructs alone is sufficient to induce MKP-1 and MKP-2. The role or dominant-negative MAP kinase molecules (25) or overex- of the MAP kinase cascade in MKP induction was high- pression of MKP-1 (26) all prevent cell cycle reentry. The MAP lighted by the MKK-1 inhibitor PD 098059, which kinase family is exemplified by three distinct subtypes: p42/ MAPK blunted both the activation of p42/p44 and the in- MAPK MAPK JNK p44 (27), p38 (28), and p46/p54 (29), which are duction of MKP-1 and MKP-2. However, the MAP kinase predominately cytoplasmic proteins in quiescent cells. Upon cascade is not absolutely required for the induction of MAPK JNK stimulation, p42/p44 and p46/p54 translocate to the MKP-1, as this phosphatase, but not MKP-2, was induced nucleus, where they may phosphorylate nuclear transcription to detectable levels by agents that stimulate protein ki- factors and thus regulate gene transcription (30–32). Full en- MAPK nases A and C. Thus, activation of the p42/p44 cas- zymatic activity of MAP kinase requires a dual phosphoryla- cade promotes the induction of MKP-1 and MKP-2, tion on a Thr-X-Tyr motif, which is performed in vivo by specific MAPK which may then attenuate p42/p44 -dependent upstream activators termed MAP kinase kinase (MKK-1/2, events in an inhibitory feedback loop. MAPK MAPK MKK-3/6, and MKK-4 for the p42/p44 , p38 , and p46/ JNK p54 enzymes, respectively) (33–35). Consequently, each MAP kinase family member is a candidate for dual dephospho- A major characteristic of protein phosphorylation is its re- rylation by MKPs. This raises the question of specificity be- versibility. In the cell, a dynamic balance exists between phos- tween cognate kinase-phosphatase partners. However, previ- phorylation and dephosphorylation, resulting from interplay ous studies have shown that MKP-1, MKP-2, MKP-3, and between protein phosphatases and protein kinases. As a con- PAC1 (restricted to hematopoietic cell lines) are all able to sequence, a modification of either component is likely to have MAPK inactivate p42/p44 and that specificity is correspondingly an equally important impact on signal transduction (1–3). low (8, 15, 19, 36). Protein phosphatases are generally classified into either ser- Several MKP family members were identified by virtue of ine/threonine or tyrosine phosphatases, depending on phos- their being encoded by immediate-early genes (10, 37). With the exception of Pyst1 (23), MKPs are not expressed in quies- * This work was supported by CNRS Grant UMR134, by l’Association pour la Recherche Contre le Cancer, by La Ligue Nationale contre le Cancer, and by a Roussel Uclaf doctoral fellowship (to J.-M. B.). The costs of publication of this article were defrayed in part by the payment The abbreviations used are: MAP, mitogen-activated protein; MKP, of page charges. This article must therefore be hereby marked “adver- MAP kinase phosphatase; MKK, MAP kinase kinase; GST, glutathione tisement” in accordance with 18 U.S.C. Section 1734 solely to indicate S-transferase; DMEM, Dulbecco’s modified Eagle’s medium; HA, he- this fact. magglutinin; PBS, phosphate-buffered saline; PAGE, polyacrylamide ‡ To whom correspondence should be addressed. Tel.: 33-4-92-07-64- gel electrophoresis; FCS, fetal calf serum; IL-1b, interleukin-1b; PMA, 27; Fax: 33-4-92-07-64-32. phorbol 12-myristate 13-acetate; CRE, cAMP-responsive element. 1368 This paper is available on line at http://www-jbc.stanford.edu/jbc/ This is an Open Access article under the CC BY license. Induction of MKP-1 and MKP-2 by MAP Kinase 1369 Expression of MAP Kinase and MAP Kinase Phosphatase Family cent cells and may be induced upon stimulation with agonists Members in CCL39 Cells that include mitogens, oxidative stress, heat shock, and UV CCL39 cells were seeded at a density of 0.8 3 10 cells/dish (60-cm irradiation (9). Specificity of MKPs for the various MAP kinase plate) (day 0) and transfected the following day (day 1) by the classical members may therefore depend on their presence during a calcium phosphate coprecipitation technique with 2 mg of the relevant particular cellular event. An understanding of the mechanisms HA or Flag epitope-tagged kinase construct associated either with 25 mg controlling MKP induction may explain how MAP kinase sig- of empty vector or with 25 mg of phosphatase-expressing vector. The naling pathways are regulated. cells were then serum-starved overnight (day 2), and on day 3, cells We report that two dual specificity phosphatases (MKP-1 were stimulated by serum or anisomycin for 30 min. Kinase assays were MAPK MAPK performed as described below. For HA-p44 and HA-p38 , im- and MKP-2) may be induced in resting CCL39 fibroblasts fol- munoprecipitation was performed with monoclonal anti-HA antibodies. lowing serum exposure. We have attempted to correlate their JNK For Flag-p46 , immunoprecipitation was performed with monoclonal induction with the activity status of signal transduction path- anti-Flag antibodies. In experiments to determine the effect of MKP-1 ways involving different MAP kinase family members. To this and MKP-2 on CCL39 cell growth, CCL39 cells were seeded at a density end, we investigated the level of MKP-1 and MKP-2 in cells of 3 3 10 cells/well (six-well plate) and transfected with plasmid 1 1 expressing either constitutively active or inducible forms of encoding the Na /H antiporter NHE-3 (2 mg) either with pcDNAneo alone or with pcDNAneo encoding MKP-1 or MKP-2 (20 mg). Cells were each element of the Ras/Raf-1/MKK module. Our results sug- MAPK selected by the established proton-killing technique (25) performed over gest that activation of the p42/p44 cascade is sufficient to a 2-week period, after which colonies were stained with Giemsa blue promote the expression of MKP-1 and MKP-2. We propose that (10% in PBS) and counted. MAPK p42/p44 down-regulation when cells progress through G is dependent on a feedback loop that involves MKP family Northern Blot Analysis members. CCL39 cells were seeded in 10-cm plates and, when confluent, se- rum-starved for 24 h. Cells were then stimulated with the appropriate EXPERIMENTAL PROCEDURES agonist and, following a suitable time period, washed twice with cold Materials PBS and lysed with Bioprobe RNA preparation (acid phenol and gua- dininium chloride). RNA was prepared by phenol/chloroform extraction [g- P]ATP and the enhanced chemiluminescence (ECL) immunode- and sodium acetate precipitation. Following RNA separation (30 mgof tection system were obtained from Amersham Corp.; antiserum E1B, MAPK total RNA) on formaldehyde-agarose gels, RNA was transferred to which specifically recognizes p42 and p44 on Western blots, and MAPK nitrocellulose and probed by the technique of Church and Gilbert (44). antiserum Kelly, which specifically immunoprecipitates p42/p44 The MKP-1 probe was obtained following purification from a StuI activity, were as previously detailed (38). Antisera specifically immu- MAPK digestion (780 base pairs) of pcDNAneo/CL100. The MKP-2 probe was noprecipitating p44 were a kind gift of Dr. Sylvain Meloche (39). purified as a StuI/SacII digest (1055 base pairs) of pcDNAneo/hVH-2. Anti-cyclin D1 antisera were a kind gift of Dr. Ve´ronique Baldin. The GST-Jun-(1–79) and GST-ATF-2-(1–149) expression vectors were kind Western Blot Analysis gifts of Dr. M. Karin and Dr. R. Davis, respectively, and were as JNK described (40). The Flag-p46 expression vector was a kind gift of Dr. Cells were washed twice with cold PBS and lysed in Triton X-100 MAPK B. De´rijard (35). The specific p38 inhibitor SB 203580 was supplied lysis buffer (50 mM Tris-HCl, pH 7.5, 100 mM NaCl, 50 mM NaF, 5 mM by SmithKline Beecham. The specific MKK-1/2 inhibitor PD 098059 EDTA, 40 mM b-glycerophosphate, 200 mM sodium orthovanadate, 10 was purchased from New England Biolabs Inc. Bovine myelin basic M phenylmethylsulfonyl fluoride, 1 mg/ml leupeptin, 1 mM pepstatin A, protein and bovine serum albumin were purchased from Sigma. Triton and 1% Triton X-100) for 15 min at 4 °C. Insoluble material was re- X-100 and Nonidet P-40 were from Pierce. moved by centrifugation at 12,000 3 g for 2 min at 4 °C. Proteins from cell lysates (100 mg) were separated by 10% SDS-PAGE and electro- Cells and Culture Conditions phoretically transferred to Hybond-C Extra membranes (Amersham Corp.) in 25 mM Tris-HCl and 0.19 M glycine. Membranes were blocked The established Chinese hamster lung fibroblast line CCL39 (Amer- in Tris-buffered saline (25 mM Tris-HCl, pH 7.5, and 137 mM NaCl) ican Type Culture Collection) and its derivatives (clones Ras5C and containing 5% nonfat dry milk. The blots were then incubated with MKK-1(SD/SD)) were cultivated in Dulbecco’s modified Eagle’s medium antiserum Alb-1 (1:300) in blocking solution for2hat room tempera- (DMEM; Life Technologies, Inc.) containing 7.5% fetal calf serum, 50 ture. After washing in Tris-buffered saline and 0.1% Triton X-100, blots units/ml penicillin, and 50 mg/ml streptomycin sulfate. CCL39-derived were incubated with horseradish peroxidase-conjugated goat anti-rab- DRaf-1::ER-expressing cells were cultivated in DMEM without phenol bit IgG (1:3000) in blocking solution for 1 h and revealed with ECL. red in the presence of 7.5% fetal calf serum and G418 (400 mg/ml) (42). MAPK Where indicated, the activity status of p42/p44 was determined by HEK 293 cells were cultivated in DMEM containing 7.5% decomple- a mobility shift assay in which, following cell lysis, proteins were mented fetal calf serum. Cells were growth-arrested by serum starva- separated by SDS-PAGE (12.5% acrylamide, 0.0625% bisacrylamide) tion for 16–24 h. and Western blotting was performed with antiserum E1B, which pref- MAPK erentially recognizes p42 . DNA Constructs and Expression in HEK 293 Cells The EcoRI fragment of the full-length cDNA of CL100 (human Immune Complex Kinase Assays MKP-1) previously introduced into the pECE vector (Stratagene) was MAPK p42/p44 —CCL39 cells or their derivatives were seeded in 12- digested by SmaI/XbaI and cloned into the pcDNAneoI mammalian well plates and rendered quiescent at confluence by serum starvation expression vector (EcoRV and XbaI sites). The EcoRI/EcoRI fragment of for 24 h. Cells were stimulated in DMEM with 10% serum (CCL39) or the full-length cDNA of hVH-2 (human MKP-2) in the pBS vector was in phenol red-free DMEM with either estradiol (1 mM) or serum (5%) digested by BamHI/XhoI and cloned into the pcDNAneoI expression (DRaf-1::ER) at 37 °C for the times indicated, prior to being washed vector (BamHI and XhoI sites) (Invitrogen). HEK 293 cells were seeded with ice-cold PBS and lysed with Triton X-100 lysis buffer as described at a density of 0.5 3 10 cells/well (six-well plate) and transfected the for Western blot analysis. Proteins from lysates (200 mg) were incu- following day by the classical calcium phosphate coprecipitation tech- MAPK bated with specific anti-p44 antibodies preadsorbed to protein nique with 10 mg of the corresponding construct (43). 48 h after trans- A-Sepharose-coated beads for2hat4°C. Immune complexes were fection, cells were lysed for Western blot analysis with Laemmli sample washed three times with Triton X-100 lysis buffer and twice with kinase buffer (45). buffer (20 mM HEPES, pH 7.4, 20 mM MgCl ,1mM dithiothreitol, and MAPK 10 mM p-nitrophenyl phosphate). p42/p44 activity was assayed by Preparation of Polyclonal Antiserum against MKP-1 resuspending the final pellet in 40 ml of kinase buffer containing 50 mM A synthetic peptide corresponding to the last 12 C-terminal amino [g- P]ATP (5000 cpm/pmol) and 0.25 mg/ml myelin basic protein. The acids of 3CH134 protein (mouse MKP-1) conjugated to keyhole limpet reaction was incubated for 10 min at 30 °C and stopped by addition of hemocyanin was used for rabbit immunization. This antibody is re- Laemmli sample buffer (45). JNK ferred to here as Alb-1. The specificity of Alb-1 was tested by Western p46/p54 —Cells were deprived of serum for 18 h in DMEM and blotting using cell lysates from HEK 293 cells transfected with the stimulated with FCS (10%), IL-1b (10 ng/ml), sorbitol (300 mM), or pcDNAneo/MKP-1 construct, the pcDNAneo/MKP-2 construct, or an sodium arsenite (200 mM). Cells were washed and lysed with the same MAPK empty vector. protocol as that described for p42/p44 . Cleared lysates (500 mgof 1370 Induction of MKP-1 and MKP-2 by MAP Kinase FIG.1. MKP-1 and MKP-2 are expressed by CCL39 cells. A, CCL39 cells were seeded in 10-cm plates and, when confluent, rendered quiescent. Cells were then stimulated or not with FCS (10%) for 2 h prior to RNA isolation and Northern blotting with specific probes against MKP-1 and MKP-2 as described under “Experimental Procedures.” RNA loading was identical as shown by the 18 S ribosomal subunit signal. B (left panel), HEK 293 cells were transiently transfected as described under “Experimental Procedures” with vector alone (pcDNAneo) or with vector encoding MKP-1 or MKP-2. Cells were lysed, and proteins were separated by 10% SDS-PAGE and Western-blotted with anti-MKP-1/2 antiserum Alb-1 as described under “Experimental Procedures.” B (right panel), CCL39 cells in 10-cm plates were rendered quiescent at confluence and then stimulated or not with FCS (10%) for 3.5 h. Cells were lysed, and proteins were separated by 10% SDS-PAGE and Western-blotted with anti-MKP-1/2 antiserum in the presence or absence of the synthetic peptide employed for rabbit immunization (10 mg/ml) as indicated. C, quiescent CCL39 cells in 12-well plates were stimulated with FCS for 2 h. After lysis, proteins (75 mg) were separated by 10% SDS-PAGE and Western- blotted with anti-MKP-1/2 antiserum. Kb, kilobase pairs; NS, not stimulated. protein) were incubated with glutathione-Sepharose coupled with 6 mg Alb-1 with apparent molecular masses of 40 and 42 kDa, in of GST-Jun-(1–79) for 3 h. Complexes were washed three times with close agreement with the calculated molecular mass from the 150 mM Tris, pH 7.5, and 1% Nonidet P-40; once with PBS and 0.5 M primary sequence of MKP-1 and MKP-2, respectively (Fig. 1B). LiCl; and twice with kinase buffer prior to the kinase reaction. MAPK MAPK We next performed Western blotting on cell lysates derived p38 —Following immunoprecipitation of p38 from cell ly- sates (400 mg of protein), immune complexes were treated exactly as from serum-deprived or serum-stimulated CCL39 cells. Follow- MAPK described for p42/p44 , with myelin basic protein being substituted ing serum stimulation (3.5 h) of quiescent CCL39 cells, Alb-1 in the kinase assay for GST-ATF-2 (5 mg). antisera recognized two major bands with the expected molec- In each case, samples were heated at 95 °C for 2 min and separated ular mass of MKP-1 and MKP-2 (Fig. 1B). In addition, these by 10% SDS-PAGE. Gels were exposed, and the relevant bands were two bands comigrated with MKP-1 and MKP-2 transiently quantified using a Fuji phosphorimager. expressed in HEK 293 cells. When immunoblotting was per- Protein Determinations formed in the presence of the specific peptide used for rabbit Protein determinations were evaluated using the bicinchoninic acid immunization, but not with an unrelated peptide sequence, the protein assay kit (Pierce) with bovine serum albumin as standard. two bands with apparent molecular masses of 40 and 42 kDa Data Reproducibility were no longer detectable. Additionally, serum-mediated in- duction of MKP-1 and MKP-2 was dose-dependent in nature All experiments were performed at least three times. Where indi- cated, data shown are representative of at least two additional experi- (Fig. 1C). These data strongly suggest that our anti-MKP-1/2 ments that gave qualitatively identical results. antiserum is capable of specifically detecting the hamster ho- mologues of MKP-1 and MKP-2 in CCL39 cells. RESULTS MKP-1 and MKP-2 Are Rapidly but Differentially Induced in Characterization of Rabbit Polyclonal Antiserum Directed MAPK Response to Serum: Correlation with p42/p44 Inactiva- against MKP-1 and MKP-2 Proteins—Previous studies that tion Time Course—To analyze the induction of MKP-1 and have examined the expression of MKP family members have MKP-2 in CCL39 cells, we performed Western blotting on cell principally employed Northern blot analysis. In serum-stimu- lysates from serum-stimulated cells (Fig. 2A). In quiescent lated (2 h), but not quiescent, CCL39 cells, two RNA species CCL39 cells, both MKP-1 and MKP-2 are undetectable. Upon that migrate with the size expected of MKP-1 and MKP-2 (;2.4 addition of serum, MKP-1 and MKP-2 are induced, with MKP-1 kilobase pairs) were detected using probes that were designed present as early as 1 h after addition of serum. Comparison of to specifically identify either MKP-1 or MKP-2 mRNA species, a range of experiments shows that MKP-1 is often detectable as respectively (Fig. 1A). A more detailed analysis revealed that early as 30 min after addition of serum (data not shown). In the two mRNA species that were identified with MKP-1 and contrast, MKP-2 is induced later, with detectable protein pres- MKP-2 probes were induced by serum with a time course ent at 2–3 h after serum addition. Each phosphatase is present similar to that previously noted for MKP-1 and MKP-2 (data for at least 14 h after serum addition. After 10–12 h of serum not shown). addition, the CCL39 cell populations had started to enter S To specifically identify MKP-1 and MKP-2 proteins, we phase, correlating with the expression of cyclin D1. Thus, raised rabbit polyclonal antiserum directed against a synthetic MKP-1 and MKP-2 are induced and present during G -G peptide corresponding to the last 12 C-terminal amino acids of 0 1 transition and S phase entry in CCL39 cells. As we have pre- mouse MKP-1 (3CH134 protein) (37). This sequence has only MAPK viously reported (38), serum stimulation of p44 is rapid, one amino acid change from human MKP-1 (Lys to Gln) (10) MAPK being maximal after 5 min. p44 then declines slowly, until and one amino acid change in the same position in MKP-2 (Lys 1–2 h following serum addition, where there is a significant loss to His in both rat and human sequences) (15, 46). In contrast, of activity (Fig. 2B, lower panel) (47). This loss of activity the C-terminal regions of the other MKPs bear little or no correlates temporally with the induction and presence of primary structure similarity. Thus, we expected that our anti- MKP-1 protein and, to a lesser extent, with the production of MKP antisera would be capable of recognizing both MKP-1 and MKP-2, but not other MKP family members. In Western blots MKP-2 (Fig. 2, upper panel). As most members of the MKP of HEK 293 cells transiently transfected with expression vec- family are encoded by immediate-early genes, addition of pro- tors encoding MKP-1 or MKP-2, but not with empty vector, two tein synthesis inhibitors to quiescent CCL39 cells should block single major bands were identified by anti-MKP-1/2 antiserum their induction. To examine the contribution of MKP-1 and Induction of MKP-1 and MKP-2 by MAP Kinase 1371 MAPK FIG.2. Time course of FCS-stimulated MKP-1 and MKP-2 induction, p44 activity, and effect of cycloheximide in CCL39 cells. A, quiescent CCL39 cells in 12-well plates were stimulated for the times indicated with FCS (20%). Cells were lysed, and proteins (100 mg) were separated by 10% SDS-PAGE and Western-blotted with both anti-MKP-1/2 and anti-cyclin D1 (cyc D1) antisera as described in the legend to Fig. 1. B, quiescent CCL39 cells in 12-well plates were stimulated for the times indicated with FCS (20%). Cells were then lysed; and proteins (100 mg) MAPK were separated by 10% SDS-PAGE and Western-blotted with anti-MKP-1/2 antisera (upper panel), or p44 activity was determined as described under “Experimental Procedures” (lower panel). The autoradiograph is shown. C, quiescent CCL39 cells in 12-well plates were stimulated for the times indicated with FCS (20%) in the presence (E) or absence (f) of cycloheximide (CHX;30 mg/ml), which was added 15 min MAPK prior to serum addition. Cells were then lysed, and p44 activity was measured as described under “Experimental Procedures.” At least three experiments were performed that gave qualitatively identical results. NS, not stimulated; MBP, myelin basic protein. MAPK MKP-2 to the inactivation of p42/p44 , we examined the MKP-2 attenuates the activation in vivo of each of the three MAPK MAPK JNK time course of MAP kinase activity in the presence or absence kinases examined: p44 , p38 , and p46 . of cycloheximide (Fig. 2C). As detailed in Fig. 2B, serum-stim- We have previously shown that deregulated expression of MAPK ulated p42/p44 activity is maximal at 5 min and then MKP-1 in CCL39 fibroblasts prevents cell cycle reentry (26). As MAPK declines such that ;30% of the maximal activity elicited by MKP-2 is also able to inhibit p42/p44 in vivo (Fig. 3A), we serum (5-min time point) is present 4 h after addition of serum. expected that MKP-2 would also exert a strong antiprolifera- However, in the presence of cycloheximide, the long-term inac- tive effect. To examine this hypothesis, we transfected CCL39 MAPK tivation of p42/p44 is reduced such that 54% of the max- cells with either MKP-1 or MKP-2 and determined the colony- imal activity elicited by serum (5-min time point) is present 4 h forming ability of transfected cells (Fig. 3B). In the presence of after addition of serum. Additional experiments revealed that both MKP-1 and MKP-2, colony formation and hence cell divi- cycloheximide inhibited the serum-mediated induction of both sion are blocked by up to 80%, demonstrating that both MKP-1 MKP-1 and MKP-2 in a dose-dependent manner that corre- and MKP-2 inhibit the proliferation of CCL39 cells. lated with the extent of protein synthesis inhibition (data not Inactivation of Stress Kinases Does Not Require MKP-1 or shown). Thus, although a considerable fraction of the p42/ MKP-2—The transient expression studies described above MAPK p44 inhibitory activity is independent of cycloheximide (Fig. 3) show that overexpression of MKP-1 or MKP-2 can MAPK JNK treatment, immediate-early genes and probably MKP-1 and inhibit p38 and p46 activity. However, this does not MAPK MKP-2 play a role in setting the level of p42/p44 activity unequivocally demonstrate that endogenous MKP-1 and in CCL39 cells. MKP-2 are required for inactivation of either kinase. Hence, we MAPK JNK JNK MKP-1 and MKP-2 Block the Activation of p44 , p46 , examined the time course of activation of p46/p54 in re- MAPK and p38 and Inhibit CCL39 Cell Proliferation—To explore sponse to a range of agonists, together with the possible induc- JNK the substrate specificity of two MKP family members, MKP-1 tion of MKP-1 or MKP-2 (Fig. 4). Activation of p46/p54 by and MKP-2 expressed by CCL39 cells, we performed cotrans- osmotic shock (sorbitol), sodium arsenite (equivalent to heat fection assays with each phosphatase together with epitope- shock (48)), or IL-1b was slow in comparison with agonist- MAPK MAPK JNK MAPK tagged p44 , p38 ,orp46 (Fig. 3A). In response to mediated activation of p42/p44 (Fig. 2), peaking at ;30 MAPK serum, HA-p44 may be activated and can phosphorylate min after addition of either sorbitol or IL-1b. In the case of its substrate, myelin basic protein. However, following cotrans- sodium arsenite and sorbitol, this activity was sustained, last- MAPK fection with either MKP-1 or MKP-2, p44 is no longer ing for at least3hinthe continual presence of agonist. In JNK activable. In a similar manner, in response to specific stress contrast, stimulation of p46/p54 by IL-1b was transient, MAPK stimulus, such as anisomycin, both HA-p38 and Flag- having returned to basal values within 2–3 h after addition of JNK p46 are able to phosphorylate their respective substrates in agonist. None of the agonists tested were able to induce MKP-1 the absence, but not in the presence, of either MKP-1 or or MKP-2 to detectable levels, in contrast to control cells, which JNK MKP-2. Thus, the transient expression of both MKP-1 and were stimulated with serum. Thus, inactivation of p46/p54 1372 Induction of MKP-1 and MKP-2 by MAP Kinase MAPK JNK MAPK FIG.3. Ectopic expression of MKP-1 and MKP-2 blocks the activation of p44 , p46 , and p38 and inhibits CCL39 cell MAPK JNK MAPK proliferation. A, CCL39 cells were transiently transfected with each of HA-p44 , Flag-p46 , and HA-p38 alone or together with vector or with vector encoding MKP-1 or MKP-2 as described under “Experimental Procedures.” Cells were deprived of serum overnight and then stimulated with either FCS (20%) or anisomycin (Aniso; 50 ng/ml) for 30 min. Cells were lysed, and kinase activities were determined using the 1 1 relevant substrate. A representative autoradiograph is shown. B, CCL39 cells were transfected with plasmid encoding the Na /H antiporter NHE-3 (2 mg) either with pcDNAneo alone or with pcDNAneo encoding MKP-1 or MKP-2 (20 mg). Cells were selected by the established proton-killing technique (25) performed over a 2-week period, after which colonies were stained with Giemsa blue (0.1%) and counted. The control (100%) value represents 184 colonies. Data are means 6 range of duplicate determinations taken from two separate experiments. MBP, myelin basic protein. JNK FIG.4. Inactivation of p46/p54 does not require MKP-1 or MKP-2. CCL39 cells were seeded in 12-well plates and rendered quiescent when confluent. Cells were then stimulated with IL-1b (10 ng/ml), sorbitol (300 mM), sodium arsenite (200 mM), or FCS (20%) for the times indicated JNK (upper panels). Cells were lysed, and p46/p54 activity was determined as described under “Experimental Procedures.” In a parallel experiment (lower panels), cells were lysed, and proteins (100 mg) were separated by 10% SDS-PAGE and Western-blotted with anti-MKP-1/2 antiserum as described in the legend to Fig. 1. Data are from a single experiment that was performed three times. proceeds in the absence of detectable MKP-1 and MKP-2. activate MAP kinase also provoke the induction of MKP-1 and MAPK Serum Induction of MKP-1 and MKP-2 Does Not Require MKP-2, we hypothesized that p42/p44 might regulate the MAPK MAPK MAPK p38 Activity—p38 is weakly, although significantly induction of its own inhibitor. Activation of p42/p44 re- activated by serum in CCL39 cells (data not shown) and could quires the sequential activation of Ras, Raf-1, and MKK-1/2, MAPK therefore play a role in MKP induction. Pretreatment of CCL39 with p42/p44 being the last step in the kinase cascade. To MAPK cells with the specific p38 inhibitor SB 203580 (49, 50) examine the contribution of the MAP kinase cascade to the MAPK completely blocks the activation of p38 (51). However, induction of MKP-1 and MKP-2, we analyzed CCL39 cells following inhibitor pretreatment, the ability of FCS both to expressing constitutively active members of this pathway. In MAPK MAPK stimulate p42/p44 and to induce MKP-1 and MKP-2 was CCL39 cells expressing v-ras, p42/p44 is constitutively MAPK unimpaired (Fig. 5). Hence, activation of the p38 signaling active (Ref. 52 and data not shown), and MKP-2 is constitu- pathway is not a requirement for the serum-mediated induc- tively expressed, with MKP-1 present at a much lower level, tion of MKP-1 and MKP-2. but still detectable. Addition of serum results in the induction MKP-1 and MKP-2 Are Constitutively Expressed in CCL39 of MKP-1, with no appreciable change in MKP-2 (Fig. 6). In MAPK Cells Expressing v-ras or MKK-1(SD/SD)—As both MKP-1 and addition to the p42/p44 cascade, Ras is also known to MAPK MKP-2 are able to inactivate p42/p44 and agents that control signaling pathways such as those linked to phosphati- Induction of MKP-1 and MKP-2 by MAP Kinase 1373 FIG.5. Induction of MKP-1 and MKP-2 does not require FIG.7. Activation of the Raf-1/MKK/MAP kinase module is suf- MAPK p38 activity. CCL39 cells were seeded in 12-well plates and ficient to induce MKP-1 and MKP-2. CCL39 cells transfected with rendered quiescent when confluent. Cells were then pretreated or not and expressing an estrogen-inducible Raf-1 construct (DRaf-1::ER cells) MAPK with the specific p38 inhibitor SB 203580 for 18 h prior to addition were seeded in 12-well plates and rendered quiescent when confluent. or not of FCS (10%) for a further 3 h. Cells were then lysed; and Cells were then stimulated with 5% FCS, 1 mM estradiol (Est), or the MAPK p42/p44 activity was determined following immunoprecipitation as two together for 3 or 5 h. Cells were lysed, and proteins (100 mg) were described under “Experimental Procedures” (upper panel), or proteins separated by 10% SDS-PAGE and Western-blotted with anti-MKP-1/2 (100 mg) were separated by 10% SDS-PAGE and Western-blotted with antiserum as described in the legend to Fig. 1. Data are from a single anti-MKP-1/2 antiserum as described in the legend to Fig. 1 (lower experiment that was performed two times. panel). mented in the presence of serum. Hence, activation of Raf-1, MAPK MKK-1, and, by consequence, p42/p44 is sufficient to in- duce expression of MKP-1 and MKP-2. Serum-mediated Induction of MKP-1 and MKP-2 Requires MKK-1/2 Activity—The experiments above (Fig. 7) show that FIG.6. MKP-1 and MKP-2 are constitutively expressed in activation of Raf-1 is sufficient to induce MKP-1 and MKP-2. To CCL39 cells expressing v-ras or MKK-1(SD/SD). Parental CCL39 MAPK determine whether activation of p42/p44 is necessary for cells or CCL39 cells expressing and transformed by either v-ras or a constitutively active MKK-1 mutant (MKK-1(SD/SD)) were seeded in the serum-mediated induction of MKP-1 and MKP-2, we em- 12-well plates and serum-deprived when quiescent. Cells were then ployed a specific inhibitor of MKK-1/2, PD 098059 (56, 57). In stimulated with FCS (20%) for the times (in minutes) indicated or not the presence of PD 098059 (10 mM), the ability of serum to (NS) prior to cell lysis. Proteins (100 mg) were separated by 10% SDS- MAPK PAGE and Western-blotted with anti-MKP-1/2 antiserum as described stimulate p42 is inhibited by 70%, and the induction of in the legend to Fig. 1. A Western blot is shown that is representative MKP-1 and MKP-2 is significantly, although not completely of four such blots performed. attenuated (Fig. 8). Higher concentrations of PD 098059 are MAPK able to completely block the activation of p42/p44 and the dylinositol 3-kinase or Rac/Rho proteins (53). Hence, to specif- induction of MKP-1 and MKP-2, but also inhibit protein syn- MAPK ically activate only p42/p44 , we analyzed CCL39 cells thesis. However, this inhibitor, when used at a concentration transformed by a mutated, constitutively active MKK (MKK- MAPK of 10 mM, allows us to conclude that p42/p44 are required 1(SD/SD)) (clone SS3 (54)). As with CCL39 cells transformed by for full serum-mediated induction of MKP proteins in CCL39 MAPK v-ras, p42/p44 is constitutively active (54), MKP-2 is con- cells. stitutively expressed, and MKP-1 is barely detectable. Addition MKP-1 and MKP-2 Are Differentially Induced by Protein of serum provokes the induction of MKP-1 in SS3 cells, with Kinase C- and cAMP-elevating Agents—Addition of the tumor- MKP-2 levels not modified to a significant degree (Fig. 6). promoting agent PMA to fibroblasts leads to the stimulation of MAPK Therefore, it appears that sustained p42/p44 activity is MAPK MAPK protein kinase C and p42/p44 , but not p38 and p46/ sufficient to promote the expression of both MKP-1 and MKP-2, JNK p54 . When CCL39 cells are treated with PMA, MKP-1, but with MKP-2 being preferentially induced. not MKP-2, is transiently induced, with maximal induction Activation of Raf-1 Is Sufficient to Induce MKP-1 and MKP- evident 1 h after addition of agonist (Fig. 9A, upper panel). To 2—The transformation of fibroblasts by the ectopic expression determine whether protein kinase C pathways are also associ- of oncogenic proteins may result in the secretion of growth ated with the serum-dependent induction of MKP-1/2, we pre- factors or hormones, which may then stimulate the cell popu- treated quiescent cells with a specific inhibitor of protein ki- lation in an autocrine/paracrine manner. Indeed, conditioned nase C enzymes, GF 109203X (58), and followed the serum- serum-free medium from v-ras-transformed, but not wild-type, mediated induction of MKP-1 and MKP-2. In addition to CCL39 cells can modify signal transduction pathways when attenuating the ability of PMA to induce MKP-1 (Fig. 9B), the added back to wild-type CCL39 cells (data not shown). Hence, serum-mediated induction of MKP-1 and MKP-2 is also re- the constitutive expression of MKP-1/2 in v-ras- or MKK-1(SD/ duced by pretreatment with GF 109203X. In addition to an SD)-transformed CCL39 cells may not be due solely to the MAPK AP-1 element present in the MKP-1 promoter, two cyclic AMP- constitutive activation of the p42/p44 cascade, but may responsive elements (CRE) are evident. Sustained activation of involve additional signaling pathways. To rigorously assess the MAPK protein kinase A in CCL39 cells leads to the induction of role of the p42/p44 cascade alone in the regulation of MKP MKP-1, with MKP-2 undetectable over a 3-h time course. Max- induction, we used CCL39 cells expressing an inducible mem- imal induction of MKP-1 is evident after1hof agonist addition. ber of the MAP kinase signaling cascade, DRaf-1::ER (42, 55). The level of induction of MKP-1 is significantly lower than that This chimeric protein has been shown to be conditionally acti- elicited by serum (Fig. 9A, lower panel). As activation of protein vable by exposure to the estrogen analogue estradiol and to be MAPK MKK-1 MAPK kinase A does not stimulate the p42/p44 pathway in able to specifically activate p45 and p42/p44 with no CCL39 cells, this suggests that although full induction of interference with stress MAP kinase pathways (55). In MAPK MKP-1 and MKP-2 requires p42/p44 activation, p42/ DRaf-1::ER cells, estradiol addition potently activates p42/ MAPK MAPK p44 activity is not an absolute requirement. p44 (Ref. 42 and data not shown) and promotes MKP-1 and MKP-2 expression (Fig. 7), whereas in parental cells, es- MAPK tradiol alone has no effect on p42/p44 activities and MKP- 1/2 expression (data not shown). The ability of the DRaf-1::ER F. R. McKenzie, J. C. Chambard, J.-M. Brondello, and J. Pouysse´- construct to induce MKP-1 and more markedly MKP-2 is aug- gur, manuscript in preparation. 1374 Induction of MKP-1 and MKP-2 by MAP Kinase FIG.8. Serum-mediated induction of MKP-1 and MKP-2 re- MAPK quires p42/p44 activity. CCL39 cells were seeded in 12-well plates and rendered quiescent when confluent. Cells were then stimu- lated or not with FCS (10%) in the presence or absence of the MKK-1 inhibitor PD 098059 (10 mM), which was added 30 min prior to FCS MAPK addition. Cells were lysed; and p42 activity was determined by a mobility shift assay (upper panel), or proteins (100 mg) were separated by 10% SDS-PAGE and Western-blotted with anti-MKP-1/2 antiserum as described in the legend to Fig. 1 (lower panel). Western blots repre- sentative of three performed that gave qualitatively identical results are shown. DISCUSSION MAPK Dual phosphorylated p42/p44 is an excellent substrate for the MKP family of dual specificity phosphatases in vitro (9), FIG.9. Induction of MKP-1 in CCL39 cells by activators of and all MKP family members tested have been shown to inac- MAPK protein kinase C and protein kinase A. A, quiescent CCL39 cells in tivate p42/p44 in vivo. At least eight MKPs are known to 12-well plates were stimulated with either PMA (100 ng/ml) (upper exist in mammalian systems (see the Introduction), which, panel) or with 8-bromo-cAMP (8Br-cAMP;1m M)(lower panel) for the together with the identification of additional MAP kinase fam- times indicated. B, quiescent CCL39 cells in 12-well plates were stim- ulated with FCS (10%) or PMA (100 ng/ml) in the presence or absence ily members, reflects signaling complexity (59). of the protein kinase C inhibitor GF 109203X (GFX;30 mM). In each MKPs, with the exception of Pyst1 (23), are the product of case, cells were lysed, and proteins (100 mg) were separated by 10% immediate-early genes and, based on Northern analysis, share SDS-PAGE and Western-blotted with anti-MKP-1/2 antiserum as de- overlapping tissue distributions (17, 46). Purified MKPs are scribed in the legend to Fig. 1. Western blots representative of two such performed are shown. constitutively active (15, 60, 61). Although this does not rule out the possibility that post-translational regulation of MKP JNK activity occurs, it suggests that the principal point of MKP and p54 , MKP-2 is more discriminating and will dephos- MAPK JNK MAPK regulation is at the level of transcription. Hence, the specificity phorylate p42 and p54 , but not p38 . This discrep- of interaction between MAP kinase and MKP family members ancy may arise from differences in the expression levels of each may depend on the specific induction of one or more MKP. A phosphatase. detailed analysis of the factors required for induction of MKPs To define specificity between endogenous MAP kinases and would increase our understanding of their physiological role. MKPs, we examined the ability of a range of agonists known to MAPK We show that MKP-1 and MKP-2 are expressed in the well stimulate either the mitogenic p42/p44 pathway or the established CCL39 fibroblast cell line (Fig. 1). Both phosphata- stress kinase pathways to induce MKP-1 or MKP-2. In appar- ses are induced by serum, albeit with a different time course of ent contradiction to previous reports based on Northern blot induction, suggesting that the mechanisms involved in their analysis (63), we found that none of the stress agents tested, induction may not be identical (Fig. 2). Induction of MKP-1 IL-1b, osmotic shock, or sodium arsenite, were capable of in- and, to a lesser extent, MKP-2 correlates with an attenuation of ducing MKP-1 or MKP-2 to detectable levels. However, with MAPK MAPK p44 activity. It is therefore possible that p44 is spe- the exception of IL-1b, all of these agents are potent inhibitors cifically targeted by MKP-1 in CCL39 cells. However, it should of protein synthesis in CCL39 cells. Hence, it is not surprising be noted that in contrast to NIH3T3 fibroblasts (8) and Rat-1 that we were unable to detect protein expression of two imme- cells (data not shown), where there is very little inactivation of diate-early genes. In addition, protein synthesis inhibition has MAPK p42/p44 in the presence of cycloheximide, in CCL39 cells, been shown to up-regulate immediate-early gene mRNA induc- MAPK ;50% of the p42/p44 inactivating activity is insensitive to tion, most probably through an increase in mRNA stability cycloheximide and hence does not involve the majority of MKP (64). Thus, an analysis of MKP mRNA induction is response to family members. Overexpression of either MKP-1 or MKP-2 stress agents may be difficult to interpret. Stimulation of MAPK completely blocks the activity of p44 and two members of CCL39 stress pathways with the above agonists, in particular MAPK JNK the stress kinase family, p38 and p46 . Thus, both IL-1b, resulted in only a transient activation of both p46/ JNK MAPK MKP-1 and MKP-2 are potent inhibitors of CCL39 cell cycle p54 and p38 (data not shown). As we were unable to reentry (Fig. 3). We cannot conclude that this block is entirely detect induction of MKP-1 or MKP-2 in response to any stress MAPK due to p42/p44 inhibition or is a result of inhibition of agonists tested, we conclude that neither phosphatase is in- MAPK p42/p44 , stress kinases, and some of the more recently volved in the inactivation of stress kinases following addition of identified MAP kinase family members. However, it is impor- these agonists alone (Fig. 4). Unfortunately, it is not possible to tant to note that overexpression of MKP-1 does not result in a block production of MKP family members with protein synthe- complete loss of substrate specificity as we have previously sis inhibitors and then follow a time course of stress kinase S6K shown that activation of p70 occurs normally in cells that activity in response to agonist stimulation, as an inhibition of overexpress MKP-1 (42). Our results are not in total agreement protein synthesis by itself activates stress kinases (Ref. 65 and MAPK with those reported by Chu et al. (62), who have demonstrated data not shown). The inhibition of p38 failed to modify by a similar transient transfection technique of several cell serum-stimulated induction of MKP-1 and MKP-2, demon- MAPK MAPK types that while MKP-1 dephosphorylates p42 , p38 , strating that activation of this stress kinase is not a require- Induction of MKP-1 and MKP-2 by MAP Kinase 1375 ment for MKP-1 and MKP-2 induction. Whether stress kinase Finally, we have shown that MKP-1 and MKP-2 are princi- pally induced in CCL39 cells by the MAP kinase cascade, but inactivation in response to IL-1b stimulation requires the ex- pression of other members of the MKP phosphatase family maximal induction involves the interplay of at least one addi- tional signaling pathway. However, we have been unable to remains to be determined. address whether the endogenous level of expression of MKP-1 Stimulation of CCL39 cells results in the activation of a MAPK or MKP-2 is sufficient to dephosphorylate p42/p44 and range of signaling pathways, one or more of which lead to the MAPK whether p42/p44 is preferentially dephosphorylated by induction of MKP-1 and MKP-2. To fully analyze the contribu- endogenous MKP-1 or MKP-2. In an attempt to address the tion of the MAP kinase cascade to the induction of MKP-1 and latter question, we are adopting an antisense strategy to spe- MKP-2, we employed three different approaches: (i) CCL39 cifically target and prevent the induction of endogenous MKP-1 cells transformed by constitutively active components of the and MKP-2. Current research in this laboratory is aimed at MAP kinase pathway; (ii) CCL39 cells expressing an inducible developing cell models in which MKPs are maximally inducible Raf-1 construct, DRaf-1::ER; and finally, (iii) a chemical inhib- MAPK in the absence of activation of the MAP kinase pathway, which itor of MKK-1, the direct upstream activator of p42/p44 . may allow us to answer the former question. We thus identified the activation of MKK-1 as being sufficient to strongly induce MKP-2, with MKP-1 induced more weakly. A Acknowledgments—We thank Drs. S. Keyse and K. 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The Dual Specificity Mitogen-activated Protein Kinase Phosphatase-1 and -2 Are Induced by the p42/p44MAPK Cascade

Brondello, Jean-Marc; Brunet, Anne; Pouysségur, Jacques; McKenzie, Fergus R.
Journal of Biological Chemistry – Jan 1, 1997
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THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 272, No. 2, Issue of January 10, pp. 1368–1376, 1997 © 1997 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. The Dual Specificity Mitogen-activated Protein Kinase Phosphatase- MAPK Cascade* 1 and -2 Are Induced by the p42/p44 (Received for publication, September 4, 1996, and in revised form, October 28, 1996) Jean-Marc Brondello, Anne Brunet, Jacques Pouysse´gur, and Fergus R. McKenzie‡ From the Universite´ de Nice, Centre de Biochimie, CNRS UMR 134, Parc Valrose, 06108 Nice Cedex 02, France Mitogen-activated protein (MAP) kinase phospha- phoamino acid specificity. Dual specificity phosphatases capa- tase-1 (MKP-1) and MKP-2 are two members of a recently ble of removing both phosphotyrosine and phosphothreonine described family of dual specificity phosphatases that from protein targets are a relatively recent discovery. However, MAPK are capable of dephosphorylating p42/p44 . Overex- an ever increasing number of such phosphatases exist, includ- 14 15 pression of MKP-1 or MKP-2 inhibits MAP kinase-de- ing Cdc25, which dephosphorylates Thr and Tyr of Cdc2 (4), pendent intracellular signaling events and fibroblast and Cdi1 (or KAP) (5, 6), which dephosphorylates Thr of proliferation. By using specific antibodies that recog- Cdk2. A novel family of dual specificity enzymes harboring the nize endogenous MKP-1 and MKP-2 in CCL39 cells, we canonical motif (I/V)HCXAGXXR(S/T)G, originally identified in show that MKP-1 and MKP-2 are not expressed in qui- the active site of the VH-1 phosphatase (7), and able to dephos- escent cells, but are rapidly induced following serum phorylate, at least in vitro, an archetypal substrate, p42/ addition, with protein detectable as early as 30 min MAPK p44 (8, 9), has recently been identified. These phosphata- (MKP-1) or 60 min (MKP-2). Serum induction of MKP-1 ses have been named MAP kinase phosphatases (MKPs), with and MKP-2 is sustained, with protein detectable up to at least eight known members currently identified: MKP-1 14 h after serum addition. Induction of MKP-1 and, to a (CL100, XCL100, HVH-1, 3CH134, or erp) (10–14); MKP-2 lesser extent, MKP-2 temporally correlates with p42/ MAPK (hVH-2, TYP1) (15, 16); hVH-3 (B23) (17, 18); MKP-3 (rVH-6, p44 inactivation. Pyst1) (19, 20, 23); hVH-5 (21), PAC1 (22); and two incomplete To analyze the contribution of the MAP kinase cas- sequences, MKP-X (19) and Pyst2 (23). cade to MKP-1 and MKP-2 induction, we examined MAPK p42/p44 are highly homologous ternary members of a CCL39 cells transformed with either v-ras or a constitu- group of ubiquitously expressed serine/threonine kinases that, tively active direct upstream activator of MAP kinase, in fibroblasts, are activated in response to all mitogenic stimuli mitogen-activated protein kinase kinase-1 (MKK-1; MKK-1(SD/SD) mutant). In both cell models, MKP-1 and (24). Sustained activation of the MAP kinase cascade is an MKP-2 are constitutively expressed, with MKP-2 being absolute requirement for fibroblasts to exit from the quiescent prevalent. In addition, in CCL39 cells expressing an es- G state and to pass the restriction point in G . Thus, blockade 0 1 tradiol-inducible DRaf-1::ER chimera, activation of Raf of MAP kinase signaling with antisense MAP kinase constructs alone is sufficient to induce MKP-1 and MKP-2. The role or dominant-negative MAP kinase molecules (25) or overex- of the MAP kinase cascade in MKP induction was high- pression of MKP-1 (26) all prevent cell cycle reentry. The MAP lighted by the MKK-1 inhibitor PD 098059, which kinase family is exemplified by three distinct subtypes: p42/ MAPK blunted both the activation of p42/p44 and the in- MAPK MAPK JNK p44 (27), p38 (28), and p46/p54 (29), which are duction of MKP-1 and MKP-2. However, the MAP kinase predominately cytoplasmic proteins in quiescent cells. Upon cascade is not absolutely required for the induction of MAPK JNK stimulation, p42/p44 and p46/p54 translocate to the MKP-1, as this phosphatase, but not MKP-2, was induced nucleus, where they may phosphorylate nuclear transcription to detectable levels by agents that stimulate protein ki- factors and thus regulate gene transcription (30–32). Full en- MAPK nases A and C. Thus, activation of the p42/p44 cas- zymatic activity of MAP kinase requires a dual phosphoryla- cade promotes the induction of MKP-1 and MKP-2, tion on a Thr-X-Tyr motif, which is performed in vivo by specific MAPK which may then attenuate p42/p44 -dependent upstream activators termed MAP kinase kinase (MKK-1/2, events in an inhibitory feedback loop. MAPK MAPK MKK-3/6, and MKK-4 for the p42/p44 , p38 , and p46/ JNK p54 enzymes, respectively) (33–35). Consequently, each MAP kinase family member is a candidate for dual dephospho- A major characteristic of protein phosphorylation is its re- rylation by MKPs. This raises the question of specificity be- versibility. In the cell, a dynamic balance exists between phos- tween cognate kinase-phosphatase partners. However, previ- phorylation and dephosphorylation, resulting from interplay ous studies have shown that MKP-1, MKP-2, MKP-3, and between protein phosphatases and protein kinases. As a con- PAC1 (restricted to hematopoietic cell lines) are all able to sequence, a modification of either component is likely to have MAPK inactivate p42/p44 and that specificity is correspondingly an equally important impact on signal transduction (1–3). low (8, 15, 19, 36). Protein phosphatases are generally classified into either ser- Several MKP family members were identified by virtue of ine/threonine or tyrosine phosphatases, depending on phos- their being encoded by immediate-early genes (10, 37). With the exception of Pyst1 (23), MKPs are not expressed in quies- * This work was supported by CNRS Grant UMR134, by l’Association pour la Recherche Contre le Cancer, by La Ligue Nationale contre le Cancer, and by a Roussel Uclaf doctoral fellowship (to J.-M. B.). The costs of publication of this article were defrayed in part by the payment The abbreviations used are: MAP, mitogen-activated protein; MKP, of page charges. This article must therefore be hereby marked “adver- MAP kinase phosphatase; MKK, MAP kinase kinase; GST, glutathione tisement” in accordance with 18 U.S.C. Section 1734 solely to indicate S-transferase; DMEM, Dulbecco’s modified Eagle’s medium; HA, he- this fact. magglutinin; PBS, phosphate-buffered saline; PAGE, polyacrylamide ‡ To whom correspondence should be addressed. Tel.: 33-4-92-07-64- gel electrophoresis; FCS, fetal calf serum; IL-1b, interleukin-1b; PMA, 27; Fax: 33-4-92-07-64-32. phorbol 12-myristate 13-acetate; CRE, cAMP-responsive element. 1368 This paper is available on line at http://www-jbc.stanford.edu/jbc/ This is an Open Access article under the CC BY license. Induction of MKP-1 and MKP-2 by MAP Kinase 1369 Expression of MAP Kinase and MAP Kinase Phosphatase Family cent cells and may be induced upon stimulation with agonists Members in CCL39 Cells that include mitogens, oxidative stress, heat shock, and UV CCL39 cells were seeded at a density of 0.8 3 10 cells/dish (60-cm irradiation (9). Specificity of MKPs for the various MAP kinase plate) (day 0) and transfected the following day (day 1) by the classical members may therefore depend on their presence during a calcium phosphate coprecipitation technique with 2 mg of the relevant particular cellular event. An understanding of the mechanisms HA or Flag epitope-tagged kinase construct associated either with 25 mg controlling MKP induction may explain how MAP kinase sig- of empty vector or with 25 mg of phosphatase-expressing vector. The naling pathways are regulated. cells were then serum-starved overnight (day 2), and on day 3, cells We report that two dual specificity phosphatases (MKP-1 were stimulated by serum or anisomycin for 30 min. Kinase assays were MAPK MAPK performed as described below. For HA-p44 and HA-p38 , im- and MKP-2) may be induced in resting CCL39 fibroblasts fol- munoprecipitation was performed with monoclonal anti-HA antibodies. lowing serum exposure. We have attempted to correlate their JNK For Flag-p46 , immunoprecipitation was performed with monoclonal induction with the activity status of signal transduction path- anti-Flag antibodies. In experiments to determine the effect of MKP-1 ways involving different MAP kinase family members. To this and MKP-2 on CCL39 cell growth, CCL39 cells were seeded at a density end, we investigated the level of MKP-1 and MKP-2 in cells of 3 3 10 cells/well (six-well plate) and transfected with plasmid 1 1 expressing either constitutively active or inducible forms of encoding the Na /H antiporter NHE-3 (2 mg) either with pcDNAneo alone or with pcDNAneo encoding MKP-1 or MKP-2 (20 mg). Cells were each element of the Ras/Raf-1/MKK module. Our results sug- MAPK selected by the established proton-killing technique (25) performed over gest that activation of the p42/p44 cascade is sufficient to a 2-week period, after which colonies were stained with Giemsa blue promote the expression of MKP-1 and MKP-2. We propose that (10% in PBS) and counted. MAPK p42/p44 down-regulation when cells progress through G is dependent on a feedback loop that involves MKP family Northern Blot Analysis members. CCL39 cells were seeded in 10-cm plates and, when confluent, se- rum-starved for 24 h. Cells were then stimulated with the appropriate EXPERIMENTAL PROCEDURES agonist and, following a suitable time period, washed twice with cold Materials PBS and lysed with Bioprobe RNA preparation (acid phenol and gua- dininium chloride). RNA was prepared by phenol/chloroform extraction [g- P]ATP and the enhanced chemiluminescence (ECL) immunode- and sodium acetate precipitation. Following RNA separation (30 mgof tection system were obtained from Amersham Corp.; antiserum E1B, MAPK total RNA) on formaldehyde-agarose gels, RNA was transferred to which specifically recognizes p42 and p44 on Western blots, and MAPK nitrocellulose and probed by the technique of Church and Gilbert (44). antiserum Kelly, which specifically immunoprecipitates p42/p44 The MKP-1 probe was obtained following purification from a StuI activity, were as previously detailed (38). Antisera specifically immu- MAPK digestion (780 base pairs) of pcDNAneo/CL100. The MKP-2 probe was noprecipitating p44 were a kind gift of Dr. Sylvain Meloche (39). purified as a StuI/SacII digest (1055 base pairs) of pcDNAneo/hVH-2. Anti-cyclin D1 antisera were a kind gift of Dr. Ve´ronique Baldin. The GST-Jun-(1–79) and GST-ATF-2-(1–149) expression vectors were kind Western Blot Analysis gifts of Dr. M. Karin and Dr. R. Davis, respectively, and were as JNK described (40). The Flag-p46 expression vector was a kind gift of Dr. Cells were washed twice with cold PBS and lysed in Triton X-100 MAPK B. De´rijard (35). The specific p38 inhibitor SB 203580 was supplied lysis buffer (50 mM Tris-HCl, pH 7.5, 100 mM NaCl, 50 mM NaF, 5 mM by SmithKline Beecham. The specific MKK-1/2 inhibitor PD 098059 EDTA, 40 mM b-glycerophosphate, 200 mM sodium orthovanadate, 10 was purchased from New England Biolabs Inc. Bovine myelin basic M phenylmethylsulfonyl fluoride, 1 mg/ml leupeptin, 1 mM pepstatin A, protein and bovine serum albumin were purchased from Sigma. Triton and 1% Triton X-100) for 15 min at 4 °C. Insoluble material was re- X-100 and Nonidet P-40 were from Pierce. moved by centrifugation at 12,000 3 g for 2 min at 4 °C. Proteins from cell lysates (100 mg) were separated by 10% SDS-PAGE and electro- Cells and Culture Conditions phoretically transferred to Hybond-C Extra membranes (Amersham Corp.) in 25 mM Tris-HCl and 0.19 M glycine. Membranes were blocked The established Chinese hamster lung fibroblast line CCL39 (Amer- in Tris-buffered saline (25 mM Tris-HCl, pH 7.5, and 137 mM NaCl) ican Type Culture Collection) and its derivatives (clones Ras5C and containing 5% nonfat dry milk. The blots were then incubated with MKK-1(SD/SD)) were cultivated in Dulbecco’s modified Eagle’s medium antiserum Alb-1 (1:300) in blocking solution for2hat room tempera- (DMEM; Life Technologies, Inc.) containing 7.5% fetal calf serum, 50 ture. After washing in Tris-buffered saline and 0.1% Triton X-100, blots units/ml penicillin, and 50 mg/ml streptomycin sulfate. CCL39-derived were incubated with horseradish peroxidase-conjugated goat anti-rab- DRaf-1::ER-expressing cells were cultivated in DMEM without phenol bit IgG (1:3000) in blocking solution for 1 h and revealed with ECL. red in the presence of 7.5% fetal calf serum and G418 (400 mg/ml) (42). MAPK Where indicated, the activity status of p42/p44 was determined by HEK 293 cells were cultivated in DMEM containing 7.5% decomple- a mobility shift assay in which, following cell lysis, proteins were mented fetal calf serum. Cells were growth-arrested by serum starva- separated by SDS-PAGE (12.5% acrylamide, 0.0625% bisacrylamide) tion for 16–24 h. and Western blotting was performed with antiserum E1B, which pref- MAPK erentially recognizes p42 . DNA Constructs and Expression in HEK 293 Cells The EcoRI fragment of the full-length cDNA of CL100 (human Immune Complex Kinase Assays MKP-1) previously introduced into the pECE vector (Stratagene) was MAPK p42/p44 —CCL39 cells or their derivatives were seeded in 12- digested by SmaI/XbaI and cloned into the pcDNAneoI mammalian well plates and rendered quiescent at confluence by serum starvation expression vector (EcoRV and XbaI sites). The EcoRI/EcoRI fragment of for 24 h. Cells were stimulated in DMEM with 10% serum (CCL39) or the full-length cDNA of hVH-2 (human MKP-2) in the pBS vector was in phenol red-free DMEM with either estradiol (1 mM) or serum (5%) digested by BamHI/XhoI and cloned into the pcDNAneoI expression (DRaf-1::ER) at 37 °C for the times indicated, prior to being washed vector (BamHI and XhoI sites) (Invitrogen). HEK 293 cells were seeded with ice-cold PBS and lysed with Triton X-100 lysis buffer as described at a density of 0.5 3 10 cells/well (six-well plate) and transfected the for Western blot analysis. Proteins from lysates (200 mg) were incu- following day by the classical calcium phosphate coprecipitation tech- MAPK bated with specific anti-p44 antibodies preadsorbed to protein nique with 10 mg of the corresponding construct (43). 48 h after trans- A-Sepharose-coated beads for2hat4°C. Immune complexes were fection, cells were lysed for Western blot analysis with Laemmli sample washed three times with Triton X-100 lysis buffer and twice with kinase buffer (45). buffer (20 mM HEPES, pH 7.4, 20 mM MgCl ,1mM dithiothreitol, and MAPK 10 mM p-nitrophenyl phosphate). p42/p44 activity was assayed by Preparation of Polyclonal Antiserum against MKP-1 resuspending the final pellet in 40 ml of kinase buffer containing 50 mM A synthetic peptide corresponding to the last 12 C-terminal amino [g- P]ATP (5000 cpm/pmol) and 0.25 mg/ml myelin basic protein. The acids of 3CH134 protein (mouse MKP-1) conjugated to keyhole limpet reaction was incubated for 10 min at 30 °C and stopped by addition of hemocyanin was used for rabbit immunization. This antibody is re- Laemmli sample buffer (45). JNK ferred to here as Alb-1. The specificity of Alb-1 was tested by Western p46/p54 —Cells were deprived of serum for 18 h in DMEM and blotting using cell lysates from HEK 293 cells transfected with the stimulated with FCS (10%), IL-1b (10 ng/ml), sorbitol (300 mM), or pcDNAneo/MKP-1 construct, the pcDNAneo/MKP-2 construct, or an sodium arsenite (200 mM). Cells were washed and lysed with the same MAPK empty vector. protocol as that described for p42/p44 . Cleared lysates (500 mgof 1370 Induction of MKP-1 and MKP-2 by MAP Kinase FIG.1. MKP-1 and MKP-2 are expressed by CCL39 cells. A, CCL39 cells were seeded in 10-cm plates and, when confluent, rendered quiescent. Cells were then stimulated or not with FCS (10%) for 2 h prior to RNA isolation and Northern blotting with specific probes against MKP-1 and MKP-2 as described under “Experimental Procedures.” RNA loading was identical as shown by the 18 S ribosomal subunit signal. B (left panel), HEK 293 cells were transiently transfected as described under “Experimental Procedures” with vector alone (pcDNAneo) or with vector encoding MKP-1 or MKP-2. Cells were lysed, and proteins were separated by 10% SDS-PAGE and Western-blotted with anti-MKP-1/2 antiserum Alb-1 as described under “Experimental Procedures.” B (right panel), CCL39 cells in 10-cm plates were rendered quiescent at confluence and then stimulated or not with FCS (10%) for 3.5 h. Cells were lysed, and proteins were separated by 10% SDS-PAGE and Western-blotted with anti-MKP-1/2 antiserum in the presence or absence of the synthetic peptide employed for rabbit immunization (10 mg/ml) as indicated. C, quiescent CCL39 cells in 12-well plates were stimulated with FCS for 2 h. After lysis, proteins (75 mg) were separated by 10% SDS-PAGE and Western- blotted with anti-MKP-1/2 antiserum. Kb, kilobase pairs; NS, not stimulated. protein) were incubated with glutathione-Sepharose coupled with 6 mg Alb-1 with apparent molecular masses of 40 and 42 kDa, in of GST-Jun-(1–79) for 3 h. Complexes were washed three times with close agreement with the calculated molecular mass from the 150 mM Tris, pH 7.5, and 1% Nonidet P-40; once with PBS and 0.5 M primary sequence of MKP-1 and MKP-2, respectively (Fig. 1B). LiCl; and twice with kinase buffer prior to the kinase reaction. MAPK MAPK We next performed Western blotting on cell lysates derived p38 —Following immunoprecipitation of p38 from cell ly- sates (400 mg of protein), immune complexes were treated exactly as from serum-deprived or serum-stimulated CCL39 cells. Follow- MAPK described for p42/p44 , with myelin basic protein being substituted ing serum stimulation (3.5 h) of quiescent CCL39 cells, Alb-1 in the kinase assay for GST-ATF-2 (5 mg). antisera recognized two major bands with the expected molec- In each case, samples were heated at 95 °C for 2 min and separated ular mass of MKP-1 and MKP-2 (Fig. 1B). In addition, these by 10% SDS-PAGE. Gels were exposed, and the relevant bands were two bands comigrated with MKP-1 and MKP-2 transiently quantified using a Fuji phosphorimager. expressed in HEK 293 cells. When immunoblotting was per- Protein Determinations formed in the presence of the specific peptide used for rabbit Protein determinations were evaluated using the bicinchoninic acid immunization, but not with an unrelated peptide sequence, the protein assay kit (Pierce) with bovine serum albumin as standard. two bands with apparent molecular masses of 40 and 42 kDa Data Reproducibility were no longer detectable. Additionally, serum-mediated in- duction of MKP-1 and MKP-2 was dose-dependent in nature All experiments were performed at least three times. Where indi- cated, data shown are representative of at least two additional experi- (Fig. 1C). These data strongly suggest that our anti-MKP-1/2 ments that gave qualitatively identical results. antiserum is capable of specifically detecting the hamster ho- mologues of MKP-1 and MKP-2 in CCL39 cells. RESULTS MKP-1 and MKP-2 Are Rapidly but Differentially Induced in Characterization of Rabbit Polyclonal Antiserum Directed MAPK Response to Serum: Correlation with p42/p44 Inactiva- against MKP-1 and MKP-2 Proteins—Previous studies that tion Time Course—To analyze the induction of MKP-1 and have examined the expression of MKP family members have MKP-2 in CCL39 cells, we performed Western blotting on cell principally employed Northern blot analysis. In serum-stimu- lysates from serum-stimulated cells (Fig. 2A). In quiescent lated (2 h), but not quiescent, CCL39 cells, two RNA species CCL39 cells, both MKP-1 and MKP-2 are undetectable. Upon that migrate with the size expected of MKP-1 and MKP-2 (;2.4 addition of serum, MKP-1 and MKP-2 are induced, with MKP-1 kilobase pairs) were detected using probes that were designed present as early as 1 h after addition of serum. Comparison of to specifically identify either MKP-1 or MKP-2 mRNA species, a range of experiments shows that MKP-1 is often detectable as respectively (Fig. 1A). A more detailed analysis revealed that early as 30 min after addition of serum (data not shown). In the two mRNA species that were identified with MKP-1 and contrast, MKP-2 is induced later, with detectable protein pres- MKP-2 probes were induced by serum with a time course ent at 2–3 h after serum addition. Each phosphatase is present similar to that previously noted for MKP-1 and MKP-2 (data for at least 14 h after serum addition. After 10–12 h of serum not shown). addition, the CCL39 cell populations had started to enter S To specifically identify MKP-1 and MKP-2 proteins, we phase, correlating with the expression of cyclin D1. Thus, raised rabbit polyclonal antiserum directed against a synthetic MKP-1 and MKP-2 are induced and present during G -G peptide corresponding to the last 12 C-terminal amino acids of 0 1 transition and S phase entry in CCL39 cells. As we have pre- mouse MKP-1 (3CH134 protein) (37). This sequence has only MAPK viously reported (38), serum stimulation of p44 is rapid, one amino acid change from human MKP-1 (Lys to Gln) (10) MAPK being maximal after 5 min. p44 then declines slowly, until and one amino acid change in the same position in MKP-2 (Lys 1–2 h following serum addition, where there is a significant loss to His in both rat and human sequences) (15, 46). In contrast, of activity (Fig. 2B, lower panel) (47). This loss of activity the C-terminal regions of the other MKPs bear little or no correlates temporally with the induction and presence of primary structure similarity. Thus, we expected that our anti- MKP-1 protein and, to a lesser extent, with the production of MKP antisera would be capable of recognizing both MKP-1 and MKP-2, but not other MKP family members. In Western blots MKP-2 (Fig. 2, upper panel). As most members of the MKP of HEK 293 cells transiently transfected with expression vec- family are encoded by immediate-early genes, addition of pro- tors encoding MKP-1 or MKP-2, but not with empty vector, two tein synthesis inhibitors to quiescent CCL39 cells should block single major bands were identified by anti-MKP-1/2 antiserum their induction. To examine the contribution of MKP-1 and Induction of MKP-1 and MKP-2 by MAP Kinase 1371 MAPK FIG.2. Time course of FCS-stimulated MKP-1 and MKP-2 induction, p44 activity, and effect of cycloheximide in CCL39 cells. A, quiescent CCL39 cells in 12-well plates were stimulated for the times indicated with FCS (20%). Cells were lysed, and proteins (100 mg) were separated by 10% SDS-PAGE and Western-blotted with both anti-MKP-1/2 and anti-cyclin D1 (cyc D1) antisera as described in the legend to Fig. 1. B, quiescent CCL39 cells in 12-well plates were stimulated for the times indicated with FCS (20%). Cells were then lysed; and proteins (100 mg) MAPK were separated by 10% SDS-PAGE and Western-blotted with anti-MKP-1/2 antisera (upper panel), or p44 activity was determined as described under “Experimental Procedures” (lower panel). The autoradiograph is shown. C, quiescent CCL39 cells in 12-well plates were stimulated for the times indicated with FCS (20%) in the presence (E) or absence (f) of cycloheximide (CHX;30 mg/ml), which was added 15 min MAPK prior to serum addition. Cells were then lysed, and p44 activity was measured as described under “Experimental Procedures.” At least three experiments were performed that gave qualitatively identical results. NS, not stimulated; MBP, myelin basic protein. MAPK MKP-2 to the inactivation of p42/p44 , we examined the MKP-2 attenuates the activation in vivo of each of the three MAPK MAPK JNK time course of MAP kinase activity in the presence or absence kinases examined: p44 , p38 , and p46 . of cycloheximide (Fig. 2C). As detailed in Fig. 2B, serum-stim- We have previously shown that deregulated expression of MAPK ulated p42/p44 activity is maximal at 5 min and then MKP-1 in CCL39 fibroblasts prevents cell cycle reentry (26). As MAPK declines such that ;30% of the maximal activity elicited by MKP-2 is also able to inhibit p42/p44 in vivo (Fig. 3A), we serum (5-min time point) is present 4 h after addition of serum. expected that MKP-2 would also exert a strong antiprolifera- However, in the presence of cycloheximide, the long-term inac- tive effect. To examine this hypothesis, we transfected CCL39 MAPK tivation of p42/p44 is reduced such that 54% of the max- cells with either MKP-1 or MKP-2 and determined the colony- imal activity elicited by serum (5-min time point) is present 4 h forming ability of transfected cells (Fig. 3B). In the presence of after addition of serum. Additional experiments revealed that both MKP-1 and MKP-2, colony formation and hence cell divi- cycloheximide inhibited the serum-mediated induction of both sion are blocked by up to 80%, demonstrating that both MKP-1 MKP-1 and MKP-2 in a dose-dependent manner that corre- and MKP-2 inhibit the proliferation of CCL39 cells. lated with the extent of protein synthesis inhibition (data not Inactivation of Stress Kinases Does Not Require MKP-1 or shown). Thus, although a considerable fraction of the p42/ MKP-2—The transient expression studies described above MAPK p44 inhibitory activity is independent of cycloheximide (Fig. 3) show that overexpression of MKP-1 or MKP-2 can MAPK JNK treatment, immediate-early genes and probably MKP-1 and inhibit p38 and p46 activity. However, this does not MAPK MKP-2 play a role in setting the level of p42/p44 activity unequivocally demonstrate that endogenous MKP-1 and in CCL39 cells. MKP-2 are required for inactivation of either kinase. Hence, we MAPK JNK JNK MKP-1 and MKP-2 Block the Activation of p44 , p46 , examined the time course of activation of p46/p54 in re- MAPK and p38 and Inhibit CCL39 Cell Proliferation—To explore sponse to a range of agonists, together with the possible induc- JNK the substrate specificity of two MKP family members, MKP-1 tion of MKP-1 or MKP-2 (Fig. 4). Activation of p46/p54 by and MKP-2 expressed by CCL39 cells, we performed cotrans- osmotic shock (sorbitol), sodium arsenite (equivalent to heat fection assays with each phosphatase together with epitope- shock (48)), or IL-1b was slow in comparison with agonist- MAPK MAPK JNK MAPK tagged p44 , p38 ,orp46 (Fig. 3A). In response to mediated activation of p42/p44 (Fig. 2), peaking at ;30 MAPK serum, HA-p44 may be activated and can phosphorylate min after addition of either sorbitol or IL-1b. In the case of its substrate, myelin basic protein. However, following cotrans- sodium arsenite and sorbitol, this activity was sustained, last- MAPK fection with either MKP-1 or MKP-2, p44 is no longer ing for at least3hinthe continual presence of agonist. In JNK activable. In a similar manner, in response to specific stress contrast, stimulation of p46/p54 by IL-1b was transient, MAPK stimulus, such as anisomycin, both HA-p38 and Flag- having returned to basal values within 2–3 h after addition of JNK p46 are able to phosphorylate their respective substrates in agonist. None of the agonists tested were able to induce MKP-1 the absence, but not in the presence, of either MKP-1 or or MKP-2 to detectable levels, in contrast to control cells, which JNK MKP-2. Thus, the transient expression of both MKP-1 and were stimulated with serum. Thus, inactivation of p46/p54 1372 Induction of MKP-1 and MKP-2 by MAP Kinase MAPK JNK MAPK FIG.3. Ectopic expression of MKP-1 and MKP-2 blocks the activation of p44 , p46 , and p38 and inhibits CCL39 cell MAPK JNK MAPK proliferation. A, CCL39 cells were transiently transfected with each of HA-p44 , Flag-p46 , and HA-p38 alone or together with vector or with vector encoding MKP-1 or MKP-2 as described under “Experimental Procedures.” Cells were deprived of serum overnight and then stimulated with either FCS (20%) or anisomycin (Aniso; 50 ng/ml) for 30 min. Cells were lysed, and kinase activities were determined using the 1 1 relevant substrate. A representative autoradiograph is shown. B, CCL39 cells were transfected with plasmid encoding the Na /H antiporter NHE-3 (2 mg) either with pcDNAneo alone or with pcDNAneo encoding MKP-1 or MKP-2 (20 mg). Cells were selected by the established proton-killing technique (25) performed over a 2-week period, after which colonies were stained with Giemsa blue (0.1%) and counted. The control (100%) value represents 184 colonies. Data are means 6 range of duplicate determinations taken from two separate experiments. MBP, myelin basic protein. JNK FIG.4. Inactivation of p46/p54 does not require MKP-1 or MKP-2. CCL39 cells were seeded in 12-well plates and rendered quiescent when confluent. Cells were then stimulated with IL-1b (10 ng/ml), sorbitol (300 mM), sodium arsenite (200 mM), or FCS (20%) for the times indicated JNK (upper panels). Cells were lysed, and p46/p54 activity was determined as described under “Experimental Procedures.” In a parallel experiment (lower panels), cells were lysed, and proteins (100 mg) were separated by 10% SDS-PAGE and Western-blotted with anti-MKP-1/2 antiserum as described in the legend to Fig. 1. Data are from a single experiment that was performed three times. proceeds in the absence of detectable MKP-1 and MKP-2. activate MAP kinase also provoke the induction of MKP-1 and MAPK Serum Induction of MKP-1 and MKP-2 Does Not Require MKP-2, we hypothesized that p42/p44 might regulate the MAPK MAPK MAPK p38 Activity—p38 is weakly, although significantly induction of its own inhibitor. Activation of p42/p44 re- activated by serum in CCL39 cells (data not shown) and could quires the sequential activation of Ras, Raf-1, and MKK-1/2, MAPK therefore play a role in MKP induction. Pretreatment of CCL39 with p42/p44 being the last step in the kinase cascade. To MAPK cells with the specific p38 inhibitor SB 203580 (49, 50) examine the contribution of the MAP kinase cascade to the MAPK completely blocks the activation of p38 (51). However, induction of MKP-1 and MKP-2, we analyzed CCL39 cells following inhibitor pretreatment, the ability of FCS both to expressing constitutively active members of this pathway. In MAPK MAPK stimulate p42/p44 and to induce MKP-1 and MKP-2 was CCL39 cells expressing v-ras, p42/p44 is constitutively MAPK unimpaired (Fig. 5). Hence, activation of the p38 signaling active (Ref. 52 and data not shown), and MKP-2 is constitu- pathway is not a requirement for the serum-mediated induc- tively expressed, with MKP-1 present at a much lower level, tion of MKP-1 and MKP-2. but still detectable. Addition of serum results in the induction MKP-1 and MKP-2 Are Constitutively Expressed in CCL39 of MKP-1, with no appreciable change in MKP-2 (Fig. 6). In MAPK Cells Expressing v-ras or MKK-1(SD/SD)—As both MKP-1 and addition to the p42/p44 cascade, Ras is also known to MAPK MKP-2 are able to inactivate p42/p44 and agents that control signaling pathways such as those linked to phosphati- Induction of MKP-1 and MKP-2 by MAP Kinase 1373 FIG.5. Induction of MKP-1 and MKP-2 does not require FIG.7. Activation of the Raf-1/MKK/MAP kinase module is suf- MAPK p38 activity. CCL39 cells were seeded in 12-well plates and ficient to induce MKP-1 and MKP-2. CCL39 cells transfected with rendered quiescent when confluent. Cells were then pretreated or not and expressing an estrogen-inducible Raf-1 construct (DRaf-1::ER cells) MAPK with the specific p38 inhibitor SB 203580 for 18 h prior to addition were seeded in 12-well plates and rendered quiescent when confluent. or not of FCS (10%) for a further 3 h. Cells were then lysed; and Cells were then stimulated with 5% FCS, 1 mM estradiol (Est), or the MAPK p42/p44 activity was determined following immunoprecipitation as two together for 3 or 5 h. Cells were lysed, and proteins (100 mg) were described under “Experimental Procedures” (upper panel), or proteins separated by 10% SDS-PAGE and Western-blotted with anti-MKP-1/2 (100 mg) were separated by 10% SDS-PAGE and Western-blotted with antiserum as described in the legend to Fig. 1. Data are from a single anti-MKP-1/2 antiserum as described in the legend to Fig. 1 (lower experiment that was performed two times. panel). mented in the presence of serum. Hence, activation of Raf-1, MAPK MKK-1, and, by consequence, p42/p44 is sufficient to in- duce expression of MKP-1 and MKP-2. Serum-mediated Induction of MKP-1 and MKP-2 Requires MKK-1/2 Activity—The experiments above (Fig. 7) show that FIG.6. MKP-1 and MKP-2 are constitutively expressed in activation of Raf-1 is sufficient to induce MKP-1 and MKP-2. To CCL39 cells expressing v-ras or MKK-1(SD/SD). Parental CCL39 MAPK determine whether activation of p42/p44 is necessary for cells or CCL39 cells expressing and transformed by either v-ras or a constitutively active MKK-1 mutant (MKK-1(SD/SD)) were seeded in the serum-mediated induction of MKP-1 and MKP-2, we em- 12-well plates and serum-deprived when quiescent. Cells were then ployed a specific inhibitor of MKK-1/2, PD 098059 (56, 57). In stimulated with FCS (20%) for the times (in minutes) indicated or not the presence of PD 098059 (10 mM), the ability of serum to (NS) prior to cell lysis. Proteins (100 mg) were separated by 10% SDS- MAPK PAGE and Western-blotted with anti-MKP-1/2 antiserum as described stimulate p42 is inhibited by 70%, and the induction of in the legend to Fig. 1. A Western blot is shown that is representative MKP-1 and MKP-2 is significantly, although not completely of four such blots performed. attenuated (Fig. 8). Higher concentrations of PD 098059 are MAPK able to completely block the activation of p42/p44 and the dylinositol 3-kinase or Rac/Rho proteins (53). Hence, to specif- induction of MKP-1 and MKP-2, but also inhibit protein syn- MAPK ically activate only p42/p44 , we analyzed CCL39 cells thesis. However, this inhibitor, when used at a concentration transformed by a mutated, constitutively active MKK (MKK- MAPK of 10 mM, allows us to conclude that p42/p44 are required 1(SD/SD)) (clone SS3 (54)). As with CCL39 cells transformed by for full serum-mediated induction of MKP proteins in CCL39 MAPK v-ras, p42/p44 is constitutively active (54), MKP-2 is con- cells. stitutively expressed, and MKP-1 is barely detectable. Addition MKP-1 and MKP-2 Are Differentially Induced by Protein of serum provokes the induction of MKP-1 in SS3 cells, with Kinase C- and cAMP-elevating Agents—Addition of the tumor- MKP-2 levels not modified to a significant degree (Fig. 6). promoting agent PMA to fibroblasts leads to the stimulation of MAPK Therefore, it appears that sustained p42/p44 activity is MAPK MAPK protein kinase C and p42/p44 , but not p38 and p46/ sufficient to promote the expression of both MKP-1 and MKP-2, JNK p54 . When CCL39 cells are treated with PMA, MKP-1, but with MKP-2 being preferentially induced. not MKP-2, is transiently induced, with maximal induction Activation of Raf-1 Is Sufficient to Induce MKP-1 and MKP- evident 1 h after addition of agonist (Fig. 9A, upper panel). To 2—The transformation of fibroblasts by the ectopic expression determine whether protein kinase C pathways are also associ- of oncogenic proteins may result in the secretion of growth ated with the serum-dependent induction of MKP-1/2, we pre- factors or hormones, which may then stimulate the cell popu- treated quiescent cells with a specific inhibitor of protein ki- lation in an autocrine/paracrine manner. Indeed, conditioned nase C enzymes, GF 109203X (58), and followed the serum- serum-free medium from v-ras-transformed, but not wild-type, mediated induction of MKP-1 and MKP-2. In addition to CCL39 cells can modify signal transduction pathways when attenuating the ability of PMA to induce MKP-1 (Fig. 9B), the added back to wild-type CCL39 cells (data not shown). Hence, serum-mediated induction of MKP-1 and MKP-2 is also re- the constitutive expression of MKP-1/2 in v-ras- or MKK-1(SD/ duced by pretreatment with GF 109203X. In addition to an SD)-transformed CCL39 cells may not be due solely to the MAPK AP-1 element present in the MKP-1 promoter, two cyclic AMP- constitutive activation of the p42/p44 cascade, but may responsive elements (CRE) are evident. Sustained activation of involve additional signaling pathways. To rigorously assess the MAPK protein kinase A in CCL39 cells leads to the induction of role of the p42/p44 cascade alone in the regulation of MKP MKP-1, with MKP-2 undetectable over a 3-h time course. Max- induction, we used CCL39 cells expressing an inducible mem- imal induction of MKP-1 is evident after1hof agonist addition. ber of the MAP kinase signaling cascade, DRaf-1::ER (42, 55). The level of induction of MKP-1 is significantly lower than that This chimeric protein has been shown to be conditionally acti- elicited by serum (Fig. 9A, lower panel). As activation of protein vable by exposure to the estrogen analogue estradiol and to be MAPK MKK-1 MAPK kinase A does not stimulate the p42/p44 pathway in able to specifically activate p45 and p42/p44 with no CCL39 cells, this suggests that although full induction of interference with stress MAP kinase pathways (55). In MAPK MKP-1 and MKP-2 requires p42/p44 activation, p42/ DRaf-1::ER cells, estradiol addition potently activates p42/ MAPK MAPK p44 activity is not an absolute requirement. p44 (Ref. 42 and data not shown) and promotes MKP-1 and MKP-2 expression (Fig. 7), whereas in parental cells, es- MAPK tradiol alone has no effect on p42/p44 activities and MKP- 1/2 expression (data not shown). The ability of the DRaf-1::ER F. R. McKenzie, J. C. Chambard, J.-M. Brondello, and J. Pouysse´- construct to induce MKP-1 and more markedly MKP-2 is aug- gur, manuscript in preparation. 1374 Induction of MKP-1 and MKP-2 by MAP Kinase FIG.8. Serum-mediated induction of MKP-1 and MKP-2 re- MAPK quires p42/p44 activity. CCL39 cells were seeded in 12-well plates and rendered quiescent when confluent. Cells were then stimu- lated or not with FCS (10%) in the presence or absence of the MKK-1 inhibitor PD 098059 (10 mM), which was added 30 min prior to FCS MAPK addition. Cells were lysed; and p42 activity was determined by a mobility shift assay (upper panel), or proteins (100 mg) were separated by 10% SDS-PAGE and Western-blotted with anti-MKP-1/2 antiserum as described in the legend to Fig. 1 (lower panel). Western blots repre- sentative of three performed that gave qualitatively identical results are shown. DISCUSSION MAPK Dual phosphorylated p42/p44 is an excellent substrate for the MKP family of dual specificity phosphatases in vitro (9), FIG.9. Induction of MKP-1 in CCL39 cells by activators of and all MKP family members tested have been shown to inac- MAPK protein kinase C and protein kinase A. A, quiescent CCL39 cells in tivate p42/p44 in vivo. At least eight MKPs are known to 12-well plates were stimulated with either PMA (100 ng/ml) (upper exist in mammalian systems (see the Introduction), which, panel) or with 8-bromo-cAMP (8Br-cAMP;1m M)(lower panel) for the together with the identification of additional MAP kinase fam- times indicated. B, quiescent CCL39 cells in 12-well plates were stim- ulated with FCS (10%) or PMA (100 ng/ml) in the presence or absence ily members, reflects signaling complexity (59). of the protein kinase C inhibitor GF 109203X (GFX;30 mM). In each MKPs, with the exception of Pyst1 (23), are the product of case, cells were lysed, and proteins (100 mg) were separated by 10% immediate-early genes and, based on Northern analysis, share SDS-PAGE and Western-blotted with anti-MKP-1/2 antiserum as de- overlapping tissue distributions (17, 46). Purified MKPs are scribed in the legend to Fig. 1. Western blots representative of two such performed are shown. constitutively active (15, 60, 61). Although this does not rule out the possibility that post-translational regulation of MKP JNK activity occurs, it suggests that the principal point of MKP and p54 , MKP-2 is more discriminating and will dephos- MAPK JNK MAPK regulation is at the level of transcription. Hence, the specificity phorylate p42 and p54 , but not p38 . This discrep- of interaction between MAP kinase and MKP family members ancy may arise from differences in the expression levels of each may depend on the specific induction of one or more MKP. A phosphatase. detailed analysis of the factors required for induction of MKPs To define specificity between endogenous MAP kinases and would increase our understanding of their physiological role. MKPs, we examined the ability of a range of agonists known to MAPK We show that MKP-1 and MKP-2 are expressed in the well stimulate either the mitogenic p42/p44 pathway or the established CCL39 fibroblast cell line (Fig. 1). Both phosphata- stress kinase pathways to induce MKP-1 or MKP-2. In appar- ses are induced by serum, albeit with a different time course of ent contradiction to previous reports based on Northern blot induction, suggesting that the mechanisms involved in their analysis (63), we found that none of the stress agents tested, induction may not be identical (Fig. 2). Induction of MKP-1 IL-1b, osmotic shock, or sodium arsenite, were capable of in- and, to a lesser extent, MKP-2 correlates with an attenuation of ducing MKP-1 or MKP-2 to detectable levels. However, with MAPK MAPK p44 activity. It is therefore possible that p44 is spe- the exception of IL-1b, all of these agents are potent inhibitors cifically targeted by MKP-1 in CCL39 cells. However, it should of protein synthesis in CCL39 cells. Hence, it is not surprising be noted that in contrast to NIH3T3 fibroblasts (8) and Rat-1 that we were unable to detect protein expression of two imme- cells (data not shown), where there is very little inactivation of diate-early genes. In addition, protein synthesis inhibition has MAPK p42/p44 in the presence of cycloheximide, in CCL39 cells, been shown to up-regulate immediate-early gene mRNA induc- MAPK ;50% of the p42/p44 inactivating activity is insensitive to tion, most probably through an increase in mRNA stability cycloheximide and hence does not involve the majority of MKP (64). Thus, an analysis of MKP mRNA induction is response to family members. Overexpression of either MKP-1 or MKP-2 stress agents may be difficult to interpret. Stimulation of MAPK completely blocks the activity of p44 and two members of CCL39 stress pathways with the above agonists, in particular MAPK JNK the stress kinase family, p38 and p46 . Thus, both IL-1b, resulted in only a transient activation of both p46/ JNK MAPK MKP-1 and MKP-2 are potent inhibitors of CCL39 cell cycle p54 and p38 (data not shown). As we were unable to reentry (Fig. 3). We cannot conclude that this block is entirely detect induction of MKP-1 or MKP-2 in response to any stress MAPK due to p42/p44 inhibition or is a result of inhibition of agonists tested, we conclude that neither phosphatase is in- MAPK p42/p44 , stress kinases, and some of the more recently volved in the inactivation of stress kinases following addition of identified MAP kinase family members. However, it is impor- these agonists alone (Fig. 4). Unfortunately, it is not possible to tant to note that overexpression of MKP-1 does not result in a block production of MKP family members with protein synthe- complete loss of substrate specificity as we have previously sis inhibitors and then follow a time course of stress kinase S6K shown that activation of p70 occurs normally in cells that activity in response to agonist stimulation, as an inhibition of overexpress MKP-1 (42). Our results are not in total agreement protein synthesis by itself activates stress kinases (Ref. 65 and MAPK with those reported by Chu et al. (62), who have demonstrated data not shown). The inhibition of p38 failed to modify by a similar transient transfection technique of several cell serum-stimulated induction of MKP-1 and MKP-2, demon- MAPK MAPK types that while MKP-1 dephosphorylates p42 , p38 , strating that activation of this stress kinase is not a require- Induction of MKP-1 and MKP-2 by MAP Kinase 1375 ment for MKP-1 and MKP-2 induction. Whether stress kinase Finally, we have shown that MKP-1 and MKP-2 are princi- pally induced in CCL39 cells by the MAP kinase cascade, but inactivation in response to IL-1b stimulation requires the ex- pression of other members of the MKP phosphatase family maximal induction involves the interplay of at least one addi- tional signaling pathway. However, we have been unable to remains to be determined. address whether the endogenous level of expression of MKP-1 Stimulation of CCL39 cells results in the activation of a MAPK or MKP-2 is sufficient to dephosphorylate p42/p44 and range of signaling pathways, one or more of which lead to the MAPK whether p42/p44 is preferentially dephosphorylated by induction of MKP-1 and MKP-2. To fully analyze the contribu- endogenous MKP-1 or MKP-2. In an attempt to address the tion of the MAP kinase cascade to the induction of MKP-1 and latter question, we are adopting an antisense strategy to spe- MKP-2, we employed three different approaches: (i) CCL39 cifically target and prevent the induction of endogenous MKP-1 cells transformed by constitutively active components of the and MKP-2. Current research in this laboratory is aimed at MAP kinase pathway; (ii) CCL39 cells expressing an inducible developing cell models in which MKPs are maximally inducible Raf-1 construct, DRaf-1::ER; and finally, (iii) a chemical inhib- MAPK in the absence of activation of the MAP kinase pathway, which itor of MKK-1, the direct upstream activator of p42/p44 . may allow us to answer the former question. We thus identified the activation of MKK-1 as being sufficient to strongly induce MKP-2, with MKP-1 induced more weakly. A Acknowledgments—We thank Drs. S. Keyse and K. Guan for CL100 similar pattern of induction was obtained in cells transformed cDNA and hVH-2 cDNA, respectively; Dr. V. Dulic for critical discus- by v-ras (Fig. 6). These experiments suggest that activation of sions and comments on the manuscript; Dr. M. Bouaboula for providing protein kinase C inhibitors; D. Grall for technical assistance; and M. the MAP kinase pathway is more tightly linked to the induction Valetti for manuscript preparation. of MKP-2 than of MKP-1. However, one should be careful in REFERENCES analyzing signal transduction pathways in transformed cell 1. Dixon, J. E. (1995) Ann. N. Y. Acad. Sci. 766, 18–22 lines. Hence, in untransformed CCL39 cells expressing 2. Cohen, P. (1989) Annu. Rev. Biochem. 58, 453–508 DRaf-1::ER (Fig. 7), we show that activation of the Raf/MKK/ 3. Charbonneau, H., and Tonks, N. K. (1992) Annu. Rev. Cell Biol. 8, 463–493 MAP kinase module is sufficient for the induction of MKP-1 4. Gautier, J., Solomon, M. J., Booher, R. N., Bazan, J. 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Published: Jan 1, 1997

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