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ERK1/2 Achieves Sustained Activation by Stimulating MAPK Phosphatase-1 Degradation via the Ubiquitin-Proteasome Pathway

2003; Elsevier BV; Volume: 278; Issue: 24 Linguagem: Inglês

10.1074/jbc.m301854200

ISSN

1083-351X

Autores

Yun‐Wei Lin, Show‐Mei Chuang, Jia‐Ling Yang,

Tópico(s)

Protein Kinase Regulation and GTPase Signaling

Resumo

Sustained extracellular signal-regulated kinase 1/2 (ERK1/2) activation does not always correlate with its upstream Ras-Raf-mitogen-activated protein kinase kinase 1/2 (MKK1/2) signal cascade in cancer cells, and the mechanism remains elusive. Here we report a novel mechanism by which sustained ERK1/2 activation is established. We demonstrate that Pb(II), a carcinogenic metal, persistently induces ERK1/2 activity in CL3 human lung cancer cells and that Ras-Raf-MKK1/2 signaling cannot fully account for such activation. It is intriguing that Pb(II) treatment reduces mitogen-activated protein kinase phosphatase 1 (MKP-1) protein levels in time- and dose-dependent manners, which correlates with sustained ERK1/2 activation, and that Pb(II) also induces mRNA and de novo protein synthesis of MKP-1. In Pb(II)-treated cells, MKP-1 is polyubiquitinated, and proteasome inhibitors markedly alleviate the ubiquitination and degradation of MKP-1. Inhibiting the Pb(II)-induced ERK1/2 activation by PD98059 greatly suppresses MKP-1 ubiquitination and degradation. It is remarkable that constitutive activation of MKK1/2 triggers endogenous MKP-1 ubiquitination and degradation in various mammalian cell lines. Furthermore, expression of functional MKP-1 decreases ERK1/2 activation and the c-Fos protein level and enhances cytotoxicity under Pb(II) exposure. Taken together, these results demonstrate that activated ERK1/2 can trigger MKP-1 degradation via the ubiquitin-proteasome pathway, thus facilitating long-term activation of ERK1/2 against cytotoxicity. Sustained extracellular signal-regulated kinase 1/2 (ERK1/2) activation does not always correlate with its upstream Ras-Raf-mitogen-activated protein kinase kinase 1/2 (MKK1/2) signal cascade in cancer cells, and the mechanism remains elusive. Here we report a novel mechanism by which sustained ERK1/2 activation is established. We demonstrate that Pb(II), a carcinogenic metal, persistently induces ERK1/2 activity in CL3 human lung cancer cells and that Ras-Raf-MKK1/2 signaling cannot fully account for such activation. It is intriguing that Pb(II) treatment reduces mitogen-activated protein kinase phosphatase 1 (MKP-1) protein levels in time- and dose-dependent manners, which correlates with sustained ERK1/2 activation, and that Pb(II) also induces mRNA and de novo protein synthesis of MKP-1. In Pb(II)-treated cells, MKP-1 is polyubiquitinated, and proteasome inhibitors markedly alleviate the ubiquitination and degradation of MKP-1. Inhibiting the Pb(II)-induced ERK1/2 activation by PD98059 greatly suppresses MKP-1 ubiquitination and degradation. It is remarkable that constitutive activation of MKK1/2 triggers endogenous MKP-1 ubiquitination and degradation in various mammalian cell lines. Furthermore, expression of functional MKP-1 decreases ERK1/2 activation and the c-Fos protein level and enhances cytotoxicity under Pb(II) exposure. Taken together, these results demonstrate that activated ERK1/2 can trigger MKP-1 degradation via the ubiquitin-proteasome pathway, thus facilitating long-term activation of ERK1/2 against cytotoxicity. Members of the family of mitogen-activated protein kinase (MAPK) 1The abbreviations used are: MAPK, mitogen-activated protein kinase; MKK, MAPK kinase; MKKK, MKK kinase; ERK, extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinase; MKP, MAPK phosphatase; Pb(II), lead acetate; DEF, docking site for ERK, FXFP; ALLN, N-acetyl-Leu-Leu-norleucinal; CHO, Chinese hamster ovary; PBS, phosphate-buffered saline; MBP, myelin basic protein. proteins are vital intracellular signaling components that become phosphorylated and activated in response to a wide diversity of extracellular stimuli, including growth factors, cytokines, and environmental stresses (reviewed in Refs. 1Chang L. Karin M. Nature. 2001; 410: 37-40Crossref PubMed Scopus (4420) Google Scholar, 2Kolch W. Biochem. J. 2000; 351: 289-305Crossref PubMed Scopus (1224) Google Scholar, 3Schaeffer H.J. Weber M.J. Mol. Cell. Biol. 1999; 19: 2435-2444Crossref PubMed Scopus (1407) Google Scholar, 4Lewis T.S. Shapiro P.S. Ahn N.G. Adv. Cancer Res. 1998; 74: 49-139Crossref PubMed Google Scholar, 5Whitmarsh A.J. Davis R.J. J. Mol. Med. 1996; 74: 589-607Crossref PubMed Scopus (1397) Google Scholar). MAPKs are activated through a three-kinase module composed of a MAPK, a MAPK kinase (MKK), and a MKK kinase (MKKK). These MAPK modules are connected to cell surface receptors and activated via interaction with a family of small GTPases and MKKK kinases. Activated MAPKs phosphorylate many substrates, including cytoskeletal proteins, other kinases, phosphatases, enzymes, and transcription factors, thereby orchestrating several cellular alterations including proliferation, differentiation, survival, and apoptosis. The duration and strength of MAPK activation also affects these biological outcomes. Three major MAPK subfamilies have been extensively studied, i.e. the extracellular signal-regulated kinases (ERK1/2), the c-Jun N-terminal kinases (JNKs), and the p38 kinases. Activation of a particular MAPK signal must be controlled with high specificity and efficiency to achieve precise physiological regulation. The recent discovery of specific docking sites among the members of the MAPK cascades provide a mechanism that explains how specific and efficient signaling is established. For instance, a cluster of positively charged amino acids followed by an LXL motif called the D domain (or the kinase interaction motif, KIM) has been identified in MKKs, MAPK phosphatases (MKPs), and several MAPK substrates (6Tanoue T. Adachi M. Moriguchi T. Nishida E. Nat. Cell Biol. 2000; 2: 110-116Crossref PubMed Scopus (691) Google Scholar, 7Tanoue T. Nishida E. Pharmacol. Ther. 2002; 93: 193-202Crossref PubMed Scopus (112) Google Scholar, 8Sharrocks A.D. Yang S.H. Galanis A. Trends Biochem. Sci. 2000; 25: 448-453Abstract Full Text Full Text PDF PubMed Scopus (389) Google Scholar). The D domain binds specifically to an acidic domain (common docking domain) within a docking groove of MAPKs (6Tanoue T. Adachi M. Moriguchi T. Nishida E. Nat. Cell Biol. 2000; 2: 110-116Crossref PubMed Scopus (691) Google Scholar, 7Tanoue T. Nishida E. Pharmacol. Ther. 2002; 93: 193-202Crossref PubMed Scopus (112) Google Scholar, 8Sharrocks A.D. Yang S.H. Galanis A. Trends Biochem. Sci. 2000; 25: 448-453Abstract Full Text Full Text PDF PubMed Scopus (389) Google Scholar). Another docking site found in many ERK substrates is called the DEF motif (docking site for ERK, FXFP) (8Sharrocks A.D. Yang S.H. Galanis A. Trends Biochem. Sci. 2000; 25: 448-453Abstract Full Text Full Text PDF PubMed Scopus (389) Google Scholar, 9Jacobs D. Glossip D. Xing H. Muslin A.J. Kornfeld K. Genes Dev. 1999; 13: 163-175Crossref PubMed Scopus (443) Google Scholar). These docking interactions facilitate phosphorylation of substrates by MAPKs on specific Ser or Thr residues followed by a Pro residue ((S/T)P sites). The small GTPase, MKKK, and MKK in the ERK pathway are known to be Ras, Raf, and MKK1/2, respectively (1Chang L. Karin M. Nature. 2001; 410: 37-40Crossref PubMed Scopus (4420) Google Scholar, 2Kolch W. Biochem. J. 2000; 351: 289-305Crossref PubMed Scopus (1224) Google Scholar, 3Schaeffer H.J. Weber M.J. Mol. Cell. Biol. 1999; 19: 2435-2444Crossref PubMed Scopus (1407) Google Scholar, 4Lewis T.S. Shapiro P.S. Ahn N.G. Adv. Cancer Res. 1998; 74: 49-139Crossref PubMed Google Scholar, 5Whitmarsh A.J. Davis R.J. J. Mol. Med. 1996; 74: 589-607Crossref PubMed Scopus (1397) Google Scholar). Activation of ERK1/2 requires a dual-phosphorylation by MKK1/2 on the Thr and Tyr residues of TEY sites within the activation loop, whereas dephosphorylation of these residues by MKPs terminates such activation (10Sun H. Charles C.H. Lau L.F. Tonks N.K. Cell. 1993; 75: 487-493Abstract Full Text PDF PubMed Scopus (1028) Google Scholar, 11Alessi D.R. Smythe C. Keyse S.M. Oncogene. 1993; 8: 2015-2020PubMed Google Scholar, 12Keyse S.M. Curr. Opin. Cell Biol. 2000; 12: 186-192Crossref PubMed Scopus (712) Google Scholar, 13Tonks N.K. Neel B.G. Curr. Opin. Cell Biol. 2001; 13: 182-195Crossref PubMed Scopus (464) Google Scholar). Among these MKPs, MKP-1, MKP-2, and MKP-3 are prototypes whose functional associations with MAPK signals have been well characterized in several aspects. The MKP-3 is localized predominantly in the cytosol, whereas the MKP-1 and MKP-2 are localized primarily in the nuclear compartment. mkp-1 and mkp-2 are immediate early genes rapidly induced by many of the stimuli that activate MAPKs, whereas mkp-3 is not an immediate early gene. The phosphatase activity of MKPs is markedly elevated by specific interaction with MAPKs. For example, the association of ERK2 with MKP-3 is highly specific, and the complex formation results in a dramatic enhancement of MKP-3 phosphatase activity to down-regulate ERK2 activity in the cytosol (14Camps M. Nichols A. Gillieron C. Antonsson B. Muda M. Chabert C. Boschert U. Arkinstall S. Science. 1998; 280: 1262-1265Crossref PubMed Scopus (438) Google Scholar). MKP-1 selectively associates with ERK1/2, JNK1, and p38α, which results in the catalytic activation of MKP-1 and the subsequent inactivation of MAPKs in the nucleus (15Slack D.N. Seternes O.M. Gabrielsen M. Keyse S.M. J. Biol. Chem. 2001; 276: 16491-16500Abstract Full Text Full Text PDF PubMed Scopus (233) Google Scholar). Similarly, the catalytic activity of MKP-2 is greatly elevated upon binding with ERK1/2 and JNK1 (16Chen P. Hutter D. Yang X. Gorospe M. Davis R.J. Liu Y. J. Biol. Chem. 2001; 276: 29440-29449Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). These findings indicate that MKPs feedback inhibit their activating MAPKs precisely. On the other hand, MKP-1 has been reported to be a labile protein whose stabilization can be enhanced by proteasome inhibitors or glucocorticoids (17Brondello J.M. Pouyssegur J. McKenzie F.R. Science. 1999; 286: 2514-2517Crossref PubMed Scopus (365) Google Scholar, 18Orlowski R.Z. Small G.W. Shi Y.Y. J. Biol. Chem. 2002; 277: 27864-27871Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar, 19Kassel O. Sancono A. Kratzschmar J. Kreft B. Stassen M. Cato A.C. EMBO J. 2001; 20: 7108-7116Crossref PubMed Scopus (407) Google Scholar). Furthermore, ERK1/2 is required for the phosphorylation of MKP-1 on the two extreme C-terminal Ser residues, which enhances MKP-1 stabilization without affecting its phosphatase activity (17Brondello J.M. Pouyssegur J. McKenzie F.R. Science. 1999; 286: 2514-2517Crossref PubMed Scopus (365) Google Scholar). This suggests that ERK1/2 may negatively control its own activity by triggering the stabilization of MKP-1. Sustained ERK1/2 activity is strongly associated with many types of cancers occurring in pancreas, colon, lung, ovary, prostate, and kidney. This may be attributable to elevated Ras-Raf-MKK1/2 signaling (20Hoshino R. Chatani Y. Yamori T. Tsuruo T. Oka H. Yoshida O. Shimada Y. Ari-i S. Wada H. Fujimoto J. Kohno M. Oncogene. 1999; 18: 813-822Crossref PubMed Scopus (614) Google Scholar, 21Sebolt-Leopold J.S. Dudley D.T. Herrera R. Van Becelaere K. Wiland A. Gowan R.C. Tecle H. Barrett S.D. Bridges A. Przybranowski S. Leopold W.R. Saltiel A.R. Nat. Med. 1999; 5: 810-816Crossref PubMed Scopus (899) Google Scholar, 22Gioeli D. Mandell J.W. Petroni G.R. Frierson Jr., H.F. Weber M.J. Cancer Res. 1999; 59: 279-284PubMed Google Scholar). Nonetheless, constitutive activation of ERK1/2 has been reported to be independent of MKK1/2 and Raf-1 (23Grammer T.C. Blenis J. Oncogene. 1997; 14: 1635-1642Crossref PubMed Scopus (177) Google Scholar, 24Barry O.P. Mullan B. Sheehan D. Kazanietz M.G. Shanahan F. Collins J.K. O'Sullivan G.C. J. Biol. Chem. 2001; 276: 15537-15546Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar), and the mechanism by which sustained ERK1/2 activation occurs remains elusive. We recently reported that Pb(II) persistently stimulates ERK1/2 without activating JNK, p38, and ERK5 in CL3 human lung cancer cells and normal human fibroblasts, and sustained ERK1/2 signaling is essential for cellular nucleotide excision repair synthesis, anti-cytotoxicity, and anti-mutagenesis (25Lin Y.-W. Chuang S.-M. Yang J.-L. Carcinogenesis. 2003; 24: 53-61Crossref PubMed Scopus (44) Google Scholar). In this study, we investigate the roles of activators and regulators in controlling the duration and strength of ERK1/2 activation using Pb(II) as a stimulus. Our results show for the first time that ERK1/2 signaling can trigger MKP-1 degradation via the ubiquitinproteasome pathway, thereby accomplishing sustained kinase activation. We further demonstrate that Pb(II) triggers not only MKP-1 degradation but also MKP-1 expression at transcriptional and translational levels. The results presented here indicate that ERK1/2 is capable of performing dual and opposing roles in controlling its own activity through up- and down-regulation of MKP-1, thereby determining the duration and strength of kinase activation. Materials—Lead acetate was purchased from Merck. N-acetyl-Leu-Leu-norleucinal (ALLN), MG132, and PD98059 were obtained from Calbiochem-Novabiochem, cycloheximide came from Sigma, and cell culture media were from Invitrogen. Lead acetate was dissolved in MilliQ-purified water (Millipore, Bedford, MA), and all the other compounds were dissolved in Me2SO. Plasmids containing a full-length wild-type MKP-1 (pSG5-MKP-1-Myc) or a phosphatase-inactive mutant (Cys258 to Ser) of MKP-1 (pSG5-CS-MKP-1-Myc; MKP-1CS) (26Bennett A.M. Tonks N.K. Science. 1997; 278: 1288-1291Crossref PubMed Scopus (305) Google Scholar), were kindly provided by Dr. N. K. Tonks (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY). Plasmids containing a constitutively active form of MKK1 (ΔN3/S218E/S222D; MKK1-CA) (27Mansour S.J. Candia J.M. Gloor K.K. Ahn N.G. Cell Growth & Differ. 1996; 7: 243-250PubMed Google Scholar) or MKK2 (ΔN4/S222E/S226D; MKK2-CA) (28Mansour S.J. Candia J.M. Matsuura J.E. Manning M.C. Ahn N.G. Biochemistry. 1996; 35: 15529-15536Crossref PubMed Scopus (120) Google Scholar) were gifts from Dr. N. G. Ahn (University of Colorado, Boulder). Dominant negative mutants RasN17 and Raf301 were described previously (29Feig L.A. Cooper G.M. Mol. Cell. Biol. 1988; 8: 3235-3243Crossref PubMed Scopus (679) Google Scholar, 30Kolch W. Heidecker G. Lloyd P. Rapp U.R. Nature. 1991; 349: 426-428Crossref PubMed Scopus (355) Google Scholar). The antibodies specific against phospho-ERK1/2(Thr202/Tyr204) (catalog number 9101) and phospho-MKK1/2(Ser217/Ser221) (catalog number 9121) were from Cell Signaling (Beverly, MA). The rabbit polyclonal antibodies against ERK2 (C-14 or K-23), MKK1 (12-B), MKP-1 (V-15), ubiquitin (P4D1), c-Fos (sc-52), and α-tubulin (TU-02) were from Santa Cruz Biotechnology (Santa Cruz, CA). The monoclonal antibody against Myc (MSA-110) was from StressGen Biotechnologies (Victoria, British Columbia, Canada). Cell Culture—The CL3 cell line established from a non-small cell lung carcinoma tumor and the Chinese hamster ovary cell line CHO-K1 was cultured in RPMI 1640 and F12/Dulbecco's modified Eagle's medium complete media, respectively. The human diploid fibroblast line HFW and the human embryonic kidney cell line H293 were cultured in Dulbecco's modified Eagle's medium complete media. The complete media were supplemented with sodium bicarbonate (2.2%, w/v), l-glutamine (0.03%, w/v), penicillin (100 units/ml), streptomycin (100 μg/ml), and fetal calf serum (10%). CL3 and CHO-K1 cells were maintained at 37 °C in a humidified incubator containing 5% CO2 in air, whereas H293 and HFW cells were cultured in a 10% CO2 incubator. Treatment—Cells in exponential growth were plated before serum starvation for 16–18 h. Serum-starved cells were then exposed to lead acetate for 15 min to 24 h in serum-free media. In experiments to determine the effect of de novo protein synthesis, cells were exposed to Pb(II) for 12 h, and cycloheximide (10 μg/ml) was added during the final 1–3 h. To determine the effect of MKK1/2 on MKP-1 and ERK1/2 levels, PD98059 (25 μm) was added 1 h before the addition of Pb(II) for 24 h. To determine the effect of proteasome-mediated proteolysis, ALLN (10 μm) and MG132 (25 μm) were added in the final 3 h during treatment of cells with Pb(II) for 24 h. Transfection—Cells (5 × 105) were plated in a 60-mm Petri dish 1 day before transfection. Plasmids were carried by liposome and transferred into CL3 cells using GenePORTER™2 (Gene Therapy Systems, San Diego, CA). After 24 and 48 h, the cells were exposed to Pb(II) for 24 h and 30 min in serum-free media, respectively. The cells were then subjected to whole cell extract preparation and a colony-forming ability assay. Colony-forming Ability Assay—Immediately after treatment, cells were washed with phosphate-buffered saline (PBS) and trypsinized for determination of cell numbers. The cells were plated at a density of 100–200 cells per 60-mm Petri dish in triplicate for each treatment. The cells were then cultured for 7–14 days, and cell colonies were stained with 1% crystal violet solution (in 30% ethanol). Western Blot Analysis—After treatment, cells were rinsed twice with cold PBS and lysed in a whole cell extract buffer (20 mm HEPES at pH 7.6, 75 mm NaCl, 2.5 mm MgCl2, 0.1 mm EDTA, 0.1% Triton X-100, 0.1 mm Na3VO4, 50 mm NaF, 1 μg/ml leupeptin, 1 μg/ml aprotinin, 1 μg/ml pepstatin and 1 mm 4-(2-aminoethyl)benzenesulfonyl fluoride). The cell lysate was rotated at 4 °C for 30 min and then centrifuged at 10,000 rpm for 15 min, after which the precipitates were discarded. The BCA protein assay kit (Pierce) was employed to determine protein concentrations using bovine serum albumin as a standard. Equal amounts of proteins from each set of experiments were subjected to Western blot analysis as described (31Chuang S.-M. Wang I.-C. Yang J.-L. Carcinogenesis. 2000; 21: 1423-1432Crossref PubMed Google Scholar). Antibodies were stripped from polyvinylidene difluoride membranes using a solution containing 2% SDS, 62.5 mm Tris-HCl, pH 6.8, and 0.7% (w/w) β-mercaptoethanol at 50 °C for 15 min before re-probing with another primary antibody. Relative protein blot intensities were determined using a computing densitometer equipped with the ImageQuant analysis program (Amersham Biosciences). Immunoprecipitation—After treatment, cells were washed twice with ice-cold PBS and harvested at 4 °C in an immunoprecipitation lysis buffer (50 mm HEPES, pH 7.5, 150 mm NaCl, 2 mm EDTA, 0.5% Nonidet P-40, 10% glycerol, 1 mm NaF, 1 mm Na3VO4, 1 mm dithiothreitol, 1 mm 4-(2-aminoethyl)benzenesulfonyl fluoride, 1 μg/ml leupeptin, 1 μg/ml aprotinin and 1 μg/ml pepstatin). Equal amounts of proteins were immunoprecipitated using anti-ERK2, anti-MKP-1, and anti-ubiquitin antibodies and collected with protein A-Sepharose beads at 4 °C for 16 h. The immunoprecipitate was then washed three times in a cold lysis buffer and subjected to Western blot analysis and an ERK kinase assay. ERK Kinase Assay—The anti-ERK2 immunoprecipitate was washed in a kinase reaction buffer (20 mm HEPES, pH 7.6, 20 mm MgCl2, 2 mm dithiothreitol, 0.1 mm Na3VO4 and 1 mm NaF). The kinase assay was carried out in a total volume of 30 μl of a kinase reaction buffer containing 20 μm ATP, 1 μCi of [γ-32P]ATP (6000 Ci/mmol), and 0.5 μg of myelin basic protein (MBP) at 30 °C for 30 min. Phosphorylated MBP was resolved on 12% SDS-polyacrylamide gels followed by autoradiography. Northern Blot Analysis—Total RNA (30 μg), isolated via a guanidium isothiocyanate/phenol/chloroform extraction procedure, was subjected to electrophoresis in a 1% agarose-2.2 m formaldehyde gel, transferred to nylon membranes, and hybridized to 32P-labeled DNA probes that were prepared using T4 kinase end-labeling. The synthetic human mkp-1 probe sequence used was 5′-AGGGGCGAGCAAAAAGAAACC-3′. Hybridization was performed at 42 °Cfor3hina solution containing 50% formamide, 6× SSC (1× SSC contains 180 mm NaCl and 10 mm sodium citrate, pH 8.0), 5× Denhardt's reagent, 0.5% SDS, and 100 μg/ml denatured salmon sperm DNA. Membranes were washed with 2× SSC/0.1% SDS for 5 min at room temperature and 0.1× SSC/0.1% SDS for 5 min at 42 °C for 5 min and then exposed to x-ray film. Ras-Raf-MKK1/2 Signaling Is More Important for the Early than the Persistent ERK1/2 Activation by Pb(II)—CL3 cells were exposed to lead acetate (30 μm) in serum free medium for 15 min to 24 h, and the activation of MKK1/2 and ERK1/2 was determined by Western blot analysis using antibodies specific to phospho-MKK1/2 and phospho-ERK1/2. Fig. 1A shows that Pb(II) increased the phospho-ERK1/2 protein levels in CL3 cells in a time-dependent manner, e.g. exposure for 15 min and 24 h yielded increases of 1.7- and 4.4-fold, respectively, over untreated controls. Pb(II) also increased phospho-MKK1/2 protein levels in CL3 cells with the maximum level of induced phospho-MKK1/2 (2.0-fold) observed at an exposure time of 15 min (Fig. 1A). Reprobing these blots with anti-ERK2 and anti-MKK1 antibodies showed similar endogenous ERK2 and MKK1 protein levels in each cell extract, respectively (Fig. 1A). These results indicate that Pb(II) elicits different kinetics for the activation of ERK1/2 and their upstream MKK1/2 signals. To investigate whether Pb(II) elicits MKK1/2-ERK1/2 activation via the Ras-Raf pathway, CL3 cells were transfected with plasmids containing dominant negative forms of Ras (RasN17) or Raf (Raf301). The cells were then allowed expression for 48 and 24 h before exposure to 100 μm Pb(II) for 30 min and 24 h, respectively. ERK in the whole cell extract was immunoprecipitated with an anti-ERK2 antibody, and its kinase activity was determined using MBP as a substrate. As shown in Fig. 1B, the expression of RasN17 or Raf301 completely blocked the induction of ERK kinase activity in cells exposed to Pb(II) for 30 min. However, only partial suppression of the Pb(II)-elicited ERK kinase activity was observed when these RasN17- or Raf301-transfected cells were exposed to Pb(II) for 24 h (Fig. 1C). These results imply that Ras-Raf-MKK1/2 signaling is essential for early activation of ERK1/2 but cannot fully account for the persistent activation of ERK1/2 elicited by Pb(II). Persistent ERK1/2 Activation by Pb(II) Is Accompanied by Decreased MKP-1 Protein Levels—The activation of ERK1/2 kinase activity is mainly a kinetic control between their activating kinases and inactivating phosphatases. The dual specific phosphatase MKP-1 is known to down-regulate ERK2 activity in the nucleus (10Sun H. Charles C.H. Lau L.F. Tonks N.K. Cell. 1993; 75: 487-493Abstract Full Text PDF PubMed Scopus (1028) Google Scholar, 11Alessi D.R. Smythe C. Keyse S.M. Oncogene. 1993; 8: 2015-2020PubMed Google Scholar, 12Keyse S.M. Curr. Opin. Cell Biol. 2000; 12: 186-192Crossref PubMed Scopus (712) Google Scholar, 13Tonks N.K. Neel B.G. Curr. Opin. Cell Biol. 2001; 13: 182-195Crossref PubMed Scopus (464) Google Scholar). To determine whether constitutive activation of ERK1/2 by Pb(II) is associated with mechanisms involved in down-regulation of MKP-1, we examined MKP-1 protein levels in CL3 cells exposed to 100 μm Pb(II) for 1–24 h. As shown in Fig. 2A, MKP-1 protein levels were slightly elevated during Pb(II) exposure for 1–6 h, whereas MKP-1 levels substantially decreased after longer exposure times (12–24 h). This time-dependent increase and decrease in MKP-1 protein levels was also observed in HFW human diploid fibroblasts (data not shown). Exposing CL3 cells to various doses of Pb(II) for 24 h also led to down-regulation of MKP-1 in a dose-dependent manner (Fig. 2B). Moreover, the reduced MKP-1 levels had not recovered 8 h after removal of the Pb(II) (Fig. 2C). Fig. 2, B and C also show that lowered MKP-1 levels were accompanied by increased ERK1/2 activities under Pb(II) exposure. These results indicate that down-regulation of MKP-1 protein levels by Pb(II) is strongly associated with the persistent activation of ERK1/2. MKP-1 Lowering by Pb(II) Is Not Associated with Down-regulation of Its mRNA or de Novo Protein Synthesis—The long-term effect of Pb(II) in down-regulating MKP-1 suggests a decrease in mRNA expression/protein synthesis or protein stability. To assess whether Pb(II) affects the expression of mkp-1, mRNA was isolated and identified by Northern blotting. Fig. 3A shows that mkp-1 mRNA levels constantly increased during exposure to 100 μm Pb(II), indicating that reduced MKP-1 protein levels are not due to suppression at the transcriptional level. To explore whether Pb(II) exposure affects MKP-1 at the translational level, cycloheximide (10 μg/ml), an inhibitor of de novo protein synthesis, was added for the final 3 h of cell exposure to Pb(II) (100 μm) for 12 h. Cycloheximide did not lower MKP-1 protein levels in untreated cells (Fig. 3B), indicating that the endogenous MKP-1 protein is rather stable. However, cycloheximide co-treatment further decreased MKP-1 levels in cells treated with Pb(II) (Fig. 3B), indicating that new synthesis of MKP-1 does occur with Pb(II) exposure. These results suggest that down-regulation of MKP-1 protein levels is not due to decreases in its mRNA or protein synthesis. MKP-1 Lowering by Pb(II) Is Mediated through Ubiquitin-directed Proteolysis—The 26 S proteasome machinery is known to be the major regulator of protein turnover in eukaryotic cells by which many labile proteins are targeted for degradation mediated through the covalent addition of polyubiquitin chains to lysine residues (32Hershko A. Ciechanover A. Annu. Rev. Biochem. 1998; 67: 425-479Crossref PubMed Scopus (6959) Google Scholar, 33Kornitzer D. Ciechanover A. J. Cell. Physiol. 2000; 182: 1-11Crossref PubMed Scopus (234) Google Scholar, 34Weissman A.M. Nat. Rev. Mol. Cell Biol. 2001; 2: 169-178Crossref PubMed Scopus (1262) Google Scholar). To investigate the involvement of proteasome in the Pb(II) lowering of MKP-1 proteins, ALLN, a common 26 S proteasome inhibitor, was added at a concentration of 10 μm for the final 3 h of the treatment of CL3 cells with 100 μm Pb(II) for 24 h. As shown in Fig. 4A, ALLN restored the Pb(II)-decreased MKP-1 proteins to their untreated level. MG132, another proteasome inhibitor, also prevented the degradation of the MKP-1 protein caused by Pb(II) treatment (Fig. 4A). To determine whether MKP-1 proteins are modified by ubiquitination before degradation, cells were co-exposed to Pb(II) and ALLN as described above, and the MKP-1 protein in whole cell extract was immunoprecipitated and then subjected to immunoblot analysis using an anti-ubiquitin antibody. As shown in Fig. 4B, the endogenous MKP-1 was recognized by the antibody against ubiquitin, indicating that MKP-1 was modified by ubiquitin. Similar results were observed in immunoblot analysis using an anti-MKP-1 antibody to detect ubiquitinated proteins derived from anti-ubiquitin immunoprecipitates in Pb(II)-exposed cells (Fig. 4B). Taken together, these results indicate that Pb(II) triggers MKP-1 protein degradation via the ubiquitin-directed 26 S proteasome pathway in CL3 cells. Ubiquitin-directed Proteolysis of MKP-1 Is Dependent on ERK1/2 Signaling—To determine whether Pb(II)-elicited ERK1/2 signaling is involved in controlling MKP-1 degradation, cells were pretreated with the MKK1/2 inhibitor PD98059 (25 μm) for 1 h before exposure to 100 μm Pb(II) for 24 h. The whole cell extract was isolated for determination of MKP-1 levels by Western blot analysis. As shown in Fig. 5A, PD98059 completely blocked Pb(II)-elicited ERK1/2 activation. Interestingly, PD98059 co-treatment restored the MKP-1 protein lowered by Pb(II) to a level higher than that in untreated cells (Fig. 5A). Moreover, PD98059 prevented significant polyubiquitination of MKP-1 in Pb(II)-treated CL3 cells (Fig. 5B). The role of ERK1/2 signaling in triggering proteolysis of the MKP-1 protein was further examined by transient transfection of CL3 cells with a plasmid carrying MKK1-CA, a constitutively active form of MKK1. As shown in Fig. 6, the expression of MKK1-CA significantly increased MKP-1 ubiquitination and decreased the levels of this protein in CL3 cells. Ubiquitination of the endogenous MKP-1 protein by forced expression of MKK1-CA also occurred in H293 and CHO-K1 cells (Fig. 6B), and forced expression of MKK2-CA also induced MKP-1 ubiquitination and degradation in CL3, H293, and CHO-K1 cells (Fig. 6B, and data not shown). These results indicate that activation of MKK1/2-ERK1/2 signaling that elicits MKP-1 protein ubiquitination and degradation may be a widespread phenomenon. Exogenous MKP-1 Phosphatase Activity Decreases ERK Phosphorylation and c-Fos Protein Level and Enhanced Cytotoxicity upon Pb(II) Exposure—To explore whether the phosphatase activity of MKP-1 is essential for dephosphorylation of ERK1/2 in CL3 cells, plasmids containing either wild-type MKP-1 or the catalytically inactive mutant MKP-1CS were transfected into cells, allowed expression for 24 h, and then exposed to 100 μm Pb(II) for another 24 h. As Fig. 7 shows, forced expression of wild-type MKP-1 decreased the phospho-ERK1/2 levels induced by Pb(II), whereas the MKP-1CS did not have such an effect. On the other hand, phospho-ERK1/2 levels in the untreated control cells were slightly reduced and enhanced by the expression of MKP-1 and MKP-1CS, respectively (Fig. 7). Conversely, the over-expression of either wild-type MKP-1 or MKP-1CS had no effect on MKK1/2 activation by Pb(II) (Fig. 7A). These results indicate that supplementing exogenous MKP-1 can dephosphorylate ERK1/2 in CL3 cells. In an earlier study we demonstrated that Pb(II)-elicited ERK1/2 activity is required for the induction of c-Fos protein levels and the prevention of cytotoxicity (25Lin Y.-W. Chuang S.-M. Yang J.-L. Carcinogenesis. 2003; 24: 53-61Crossref PubMed Scopus (44) Google Scholar). To investigate whether exogenous expr

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