Constitutive Induction of p-Erk1/2 Accompanied by ReducedActivities of Protein Phosphatases 1 and 2A and MKP3 Due toReactive Oxygen Species during Cellular Senescence*

Received for publication, November 18, 2002, and in revised form, June 27, 2003Published, JBC Papers in Press, July 2, 2003, DOI 10.1074/jbc.M211739200

Hong Seok Kim, Myeong-Cheol Song, In Hae Kwak, Tae Jun Park, and In Kyoung Lim‡

From the Department of Biochemistry and Molecular Biology, Ajou University School of Medicine, Suwon 442-721, Korea

The mechanism of senescence-associated cytoplasmicinduction of p-Erk1/2 (SA-p-Erk1/2) proteins in humandiploid fibroblasts was investigated. p-Erk1/2 proteinswere efficiently dephosphorylated in vitro by proteinphosphatases 1 and 2A (PP1/2A) and MAPK phosphatase3 (MKP3). Specific activity of PP1/2A and MKP3 activitysignificantly decreased during cellular senescence,whereas their protein expression levels did not. To in-vestigate possible mechanism of phosphatase inactiva-tion, we measured reactive oxygen species (ROS) gener-ation by fluorescence-activated cell sorting analysis andfound it was much higher in mid-old cells than theyoung cells. Treating the young cells once with 1 mM

H2O2 remarkably induced p-Erk1/2 expression; how-ever, it was transient unless repeatedly treated until 72h. Multiple treatment of the cells with 0.2 mM H2O2 sig-nificantly duplicated inactivation of PP1/2A; however,thiol-specific reagents could reverse the PP1/2A activi-ties, suggesting the oxidation of cysteine molecule inPP1/2A by the increased ROS. When the cells were pre-treated with 10 mM N-acetyl-L-cysteine for 1 h, Erk1/2activation was completely blocked. To elucidate whichcysteine residue and/or metal ion in PP1/2A was modi-fied by H2O2, electrospray ionization-tandem mass spec-trometry analyses were performed with purified PP1C-�and found Cys62-SO3H and Cys105-SO3H, implicating thetertiary structure perturbation. H2O2 inhibited purifiedPP1C-� activity by both oxidation of Cys residues andmetal ion(s), evidenced by dithiothreitol and ascorbate-restoration assay. In summary, SA-p-Erk1/2 was mostlikely due to the oxidation of PP1/2A, which resultedfrom the continuous exposure of the cells to vastamounts of ROS generated during cellular senescenceby oxidation of Cys62 and Cys105 in PP1C-� and metalion(s).

Decreased growth rate, limited cell division, flat and largecell shapes, and tight binding of the cells to culture dished (1,2) are well known characteristics of cells entering into replica-tive senescence (3). Primary mouse embryo fibroblasts exposedto oncogenic Ras overexpression undergo premature senes-cence in response to constitutive MAPK1 kinase/MAPK mito-

genic signaling (4, 5), whereas established variants lacking p53or p19ARF are efficiently transformed (6). Ha-Ras mutants thatinteract preferentially with specific Ras effector proteins areknown as V12S35, V12C40, and V12G37 (7, 8); V12S35 binds pref-erentially to Raf-1 and activates MAPK without any effect onmembrane ruffling (7), and the V12C40 associates with phos-phatidylinositol 3-kinase (Akt kinase) and promotes membraneruffling and cell survival independent of Raf-1 (8, 9), but theV12G37 binds to Ral-GDS without binding to Raf-1 or phos-phatidylinositol 3-kinase (7). A distinctive feature of the cellu-lar senescence induced by overexpression of the Ha-ras mu-tants as well as the replicative senescence in normal humandiploid fibroblasts (HDF) is the markedly increased phospho-rylation of extracellular signal-regulated protein kinase (p-Erk1/2) without nuclear translocation (10). However, its bio-chemical mechanism has not yet been clarified.

MAPK activity is tightly regulated by phosphorylation anddephosphorylation. The activation of the MAPK activity re-quires the dual phosphorylation of the Ser/Thr and Tyr resi-dues in the TXY kinase activation motif (11–13), and deactiva-tion occurs through the action of either Ser/Thr proteinphosphatase (14), protein-tyrosine phosphatase (PTP) (14, 15),or dual specificity phosphatases (16, 17). The dual phospha-tases are capable of catalyzing the removal of the phosphorylgroup from Tyr(P) as well as Ser(P)/Thr(P) residues. The dualspecificity phosphatases that specifically dephosphorylate andinactivate the p-Erk1/2 are called MAPK phosphatases(MKPs), and at least 9 mammalian MKPs have been identifiedso far (18, 19). It has been reported that MKP3 (20), alsotermed Pyst1 (21) or VH6 (22), is predominantly localized inthe cytoplasm, is highly specific for Erk1/2 inactivation, and isnot inducible by either growth factor or stress (20–23). Anelegant series of studies have shown that the N-terminal do-main of MKP3 can physically associate with Erk1/2 (24), andthis association can activate phosphatase activity of MKP3present in the C-terminal domain (25). Moreover, the C-termi-nal domain of MKP3 is significantly homologous to VHR, one ofthe MKPs (26), and Erk1/2 proteins are efficient substrates forVHR. MKP3 dual specificity phosphatase is not related tothe Ser/Thr protein phosphatases but belongs to the PTPsuperfamily.

* This work was supported by Grant R05-2002-000-00054-0 fromKorea Science & Engineering Foundation (to I. K. L.). The costs ofpublication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked “advertisement”in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

‡ To whom correspondence should be addressed. Tel.: 82-31-219-5051; Fax: 82-31-219-5059; E-mail: iklim@ajou.ac.kr.

1 The abbreviations used are: MAPK, mitogen-activated protein ki-nase; PP1, phosphatase 1; and PP2A phosphatase 2A; p-Erk1/2, extra-cellular signal-regulated protein kinase 1/2; SA-p-Erk1/2, senescence-

associated persistent induction of p-Erk1/2; HDF, human diploidfibroblasts; PTP, protein-tyrosine phosphatase; MKPs, MAPK phos-phatases; VHR, vaccinia H1-related; rVHR, recombinant VHR; ROS,reactive oxygen species; PBS, phosphate-buffered saline; DTT, dithio-threitol; PMSF, phenylmethylsulfonyl fluoride; pNPP, p-nitrophenylphosphate; IP, immunoprecipitation; FACS, fluorescence-activated cellsorting; H2-DCFDA, 2�,7�-dichlorodihydrofluorescein diacetate; NAC,N-acetyl-L-cysteine; ESI, electrospray ionization; MS, mass spectrome-try; MS/MS, tandem mass spectroscopy; EGF, epidermal growth factor;NBD-Cl, 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole; TOF, time-of-flight;bis-Tris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol.

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 278, No. 39, Issue of September 26, pp. 37497–37510, 2003© 2003 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.

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There is increasing evidence that relatively few Ser- or Thr-specific protein phosphatases with pleiotropic action partici-pate in cellular regulation. Four major classes of protein phos-phatase, type 1 (PP1), type 2A (PP2A), type 2B (PP2B orcalcineurin), and type 2C (PP2C), have been identified in mam-malian cells (27, 28). PP1 occurs in three isoforms (�, �/�, and�1) and contains a catalytic subunit (PP1C), regulatory subunit(inhibitor 2), a G subunit, and an M subunit. PP1C binds to twocytosolic thermostable proteins, termed inhibitor-1 (I-1) andinhibitor-2 (I-2), which completely abolish its activity at nano-molar concentrations (29). The I-1 tightly binds to PP1 both invitro and in vivo and requires phosphorylation by cAMP-de-pendent protein kinase to be inhibitory of a number of phos-phoproteins (30–32), whereas phosphorylation of I-2 at Thr72

by glycogen synthase kinase leads to activation of the enzyme(33, 34). PP2A is a heterotrimeric enzyme consisting of a 36-kDa catalytic subunit (C), 65-kDa structural subunit (A), and avariable regulatory subunit (B). PP2A is unaffected by theabove inhibitors but highly effectively inhibited by low concen-trations of okadaic acid (27, 35, 36). PP2A is active toward mostsubstrates in the absence of divalent cation, whereas PP2B andPP2C have absolute requirements for Ca2� and Mg2�, respec-tively (27).

There are numerous reports that cellular redox status playsan important role in the mechanisms to regulate the function ofgrowth factors and tyrosine phosphorylation-dependent signaltransduction pathways (37–42). Furthermore, ROS such asH2O2 have been shown to be involved in growth factor signalingpathway (43, 44), perhaps as a second messenger. Therefore,PTP appears to be a logical target of H2O2, leading to transientand reversible inactivation of phosphatase activity by oxidizingcatalytic cysteine residue to sulfenic acid (45). It has beenreported that low levels of H2O2 (200 �M) robustly activateErk1/2 (26, 46–49) but only slightly activate p38MAPK and c-JunN-terminal kinase/stress-activated protein kinase in COS1 cellsby inactivating endogenous VHR to dephosphorylate Erk.

Very recently, a paper (50) with great relevance to our workhas suggested that the PP1 is a molecular constraint on learn-ing and memory and a potential mediator of cognitive declineduring aging. It has been shown with tet-O-inhibitor-1 trans-genic mice model that the transgene was expressed in thehippocampus and cortex of the mice brain. Because these tis-sues have been implicated in the memory process, the aboveobservation offers a great credence to the physiological rele-vance of the PP1-dependent mechanism of forgetting, particu-larly in aging-related memory decline (51). Therefore, in orderto investigate the mechanism of SA-p-Erk1/2, enzymatic activ-ities of PP1/2A and MKP3 together with their protein expres-sion levels were evaluated during cellular senescence. Further-more, the difference of ROS generation between the young andold cells and the effects of ROS on PP1/2A and MKP3 were alsoelucidated using primary culture of the young, mid-old, and oldHDF cells.

EXPERIMENTAL PROCEDURES

HDF Cell Cultures—The primary culture of normal HDF was iso-lated from the foreskin of a 4-year-old boy by the method describedpreviously (10) and Ha-ras double mutants (V12S35, V12G37, and V12C40)overexpressing HDF cell lines were also prepared in our laboratory byretrovirus infection (10). HDF cells were maintained in Dulbecco’smodified Eagle’s medium (Invitrogen, catalog number 31600-034) sup-plemented with 10% fetal bovine serum at 37 °C in 5% CO2 incubator.Ha-ras-infected HDF cells were cultured in the media containing hy-gromycin B (100 �g/ml) during the entire experiment. Confluent cellswere transferred to a new dish, and the numbers of population dou-blings as well as the doubling time were continuously counted. Young,mid-old, and old cells used in the present experiments were defined asthe HDF with a doubling time of around 24 h, 10–12 days, and over 2weeks, respectively.

p-Erk1/2 Immunocytochemistry—HDF cells were cultured on a coverglass and fixed with 4% paraformaldehyde in PBS for 20 min at roomtemperature. After washing the monolayer with PBS, cells were per-meabilized with 0.15% Triton X-100 in PBS for 5 min and then blockedwith 2% bovine serum albumin in PBS for 1 h at room temperature.Cells were incubated overnight at 4 °C with anti-p-Erk1/2 antibody(catalog number 9101, New England Biolabs, Beverly, MA), diluted(1:200) in 2% bovine serum albumin solution, followed by washing withPBS, and fluorescein isothiocyanate-labeled anti-rabbit IgG (1:200,Jackson ImmunoResearch, West Grove, PA) was applied for 1 h at roomtemperature. After washing with PBS, the cover glass was mountedwith Mowiol mounting medium (Hoechst Celanese, Charlotte, NC) con-taining antifade agent 1,4-diazabicyclo[2,2,2]octane (Aldrich).

Immunoblot Analyses of Mono- and Di-phosphorylated Erk1/2 Pro-teins—Young and old HDF cells were solubilized with RIPA buffer (50mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% Nonidet P-40, 0.1% SDS, 0.5%deoxycholic acid, 50 mM sodium fluoride, 1 mM sodium vanadate, 1 mM

PMSF, 1 �g/ml leupeptin), and 40 �g of cell lysates were resolved on10% SDS-PAGE in 25 mM Tris-glycine buffer. After transferring proteinbands to polyvinylidene difluoride membrane (catalog number 162-0177, Bio-Rad) at 150 mA for 1 h and 40 min, anti-p-Erk1/2 antibodywas applied overnight at 4 °C. For the loading control, anti-�-tubulin(Oncogene catalog number CP06, Boston), anti-Erk1/2 (Cell Signalingcatalog number 9102, Beverly, MA), or anti-actin (Sigma, catalog num-ber A-5060) antibodies were used. ECL kit was employed to visualizeprotein expression levels. In order to elucidate whether the anti-p-Erk1/2 antibody used in our experiments detects both monophosphory-lated and dual phosphorylated Erk1/2 proteins or not, purified PP1(Sigma, catalog number P7937) and PP2A (Calbiochem, catalog num-ber 539508) enzymes were applied to the activated Erk1 (p-Erk1,Stratagene, catalog number 206110) and cell lysates, respectively,and then repeated immunoblot analyses were performed using anti-Thr(P) (Zymed Laboratories Inc., South San Francisco, catalog num-ber 13-9200), anti-Tyr(P) antibodies (Upstate Biotechnology, Inc.,catalog number 05-321) as well as anti-p-Erk1/2 and anti-Erk1/2antibodies.

When investigating the fraction levels of p-Erk1/2 regulated by PP2Ain vivo, young and old HDF cells, which were maintained in the serum-free media for 24 h, were treated with 40 nM okadaic acid, a specificinhibitor for PP2A, and the cells were harvested every 4 h until 12 h forimmunoblot analysis with anti-p-Erk1/2 antibody.

Alkaline Phosphatase Assay—pNPP tablet set (N-1891, SigmaFASTTM) purchased from Sigma was used for alkaline phosphataseassay. Each pNPP and Tris Buffer tablets were dissolved in 5 ml ofdistilled water and used as substrate. Three hundred and fifty �l of areaction mixture containing 200 �l of the substrate and 150 �l of celllysate (100 �g), which was prepared in 1% Nonidet P-40 in PBS, 1 mM

PMSF, and 1 �g/ml leupeptin, was incubated in the dark for 2 h at roomtemperature, and the reaction was stopped by cooling down in ice.Absorbance at 405 nm was read, and phosphatase activity was calcu-lated from the standard curve prepared with p-nitrophenol.

Protein Phosphatases 1 and 2A Assay—To determine the activity ofPP1/2A, cells were washed twice with phosphate-free medium and lysedwith a buffer solution, containing 100 mM Tris-HCl (pH 7.4), 1 mM

PMSF, 1 �g/ml leupeptin, by sonication (Sonic Dismembrator 550,Fisher) twice at level 2 for 10 s. The whole cell lysates were centrifugedat 11,000 � g for 10 min, and the supernatant was used for the enzymeassay. To separate cytoplasmic and nuclear fractions, whole cell lysateswere prepared with STM (250 mM sucrose, 20 mM Tris-HCl, pH 7.5, 10mM Mg2�) buffer using a Dounce homogenizer, and the lysate wascentrifuged at 800 � g for 10 min. The supernatant was denoted as thecytoplasmic fraction, and the pellet was resuspended in STM bufferafter rinsing the pellet twice. Nuclear fraction was prepared by soni-cating the above pellet and centrifuging at 11,000 � g for 10 min, andthe supernatant was used as a nuclear fraction. Malachite Green Assaykit (Upstate Biotechnology, Lake Placid, NY) was mixed with Thr(P)peptide (KRpTIRR (catalog number 12-219) purchased from UpstateBiotechnology, Inc., as a substrate. An assay mixture (25 �l) containing250 �M substrate and 2 �g of the above subcellular fractions wasincubated at 30 °C for 10 min, and the reaction was stopped with 100 �lof malachite green phosphate detection solution. The assay mixture wasleft for another 15 min at room temperature for color development. Inorder to eliminate phosphate contamination arising from the cell ex-tracts and the reagents, an assay mixture without the substrate orreagent was run for each measurement. Absorbance at 630 nm was readusing microplate reader (Benchmark, Bio-Rad). Phosphate released bythe enzyme reaction was determined from the standard curve preparedwith 0.1 mM KH2PO4 solution.

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Restoration of Protein Phosphatases 1 and 2A Activity by Thiol-specific Reagents—To investigate the role of ROS played in a signifi-cantly reduced PP1/2A activity during cellular senescence, young HDFcells were pretreated once with 1 mM H2O2; since the PP1/2A activity inthe mid-old cells had been reduced, most likely due to accumulatedH2O2, these cells were not pretreated with H2O2. The cells were washedwith phosphate-free medium within 10 min, and the cell lysates werethen prepared according to the method described above. To examinewhether the H2O2 oxidized the active site cysteine residue in PP1/2A ornot, DTT (1 mM), �-mercaptoethanol (0.1%), or NAC (10 mM) wasindividually added to the young and mid-old cell lysates before thePP1/2A assay. All of the experiments with young and mid-old HDF cellswere repeated 3 times. All the thiol reagents were purchased fromSigma.

Preparation of MKP3 Antibody—Recombinant MKP3 protein wasexpressed in the BL21 cells using pGEX4T3-GST-MKP3 cDNA (origi-nally isolated by Muda et al. (20) and kindly donated by Dr. MontserratCamps (Serono Pharmaceutical Research Institute, Geneva, Switzer-land)). MKP3 protein (500 �g) mixed with an equal volume of completeFreund’s adjuvant (F-5881, Sigma) was injected subcutaneously intoback, footpad, and thigh of New Zealand White rabbits. In 3 weeks, the

first booster injection was done with MKP3 protein (500 �g) mixed withequal volume of incomplete Freund’s adjuvant (F-5506, Sigma), and therabbits were sacrificed 1 week after the second booster injected simi-larly to the first. Whole serum was then obtained, and IgG was purifiedby incubating the serum with protein A-agarose beads (Invitrogen,catalog number 15920-010).

MKP3 Immunoprecipitation-Phosphatase (IP-phosphatase) Assay—IP with anti-MKP3 IgG was performed with young and old HDF celllysates (300 �g) in a solution containing 20 mM Tris-HCl (pH 7.0), 1%Triton X-100, 0.25 M sucrose, 1 mM EDTA, 1 mM EGTA, 0.1% �-mer-captoethanol, 1 mM PMSF, and 1 �g/ml leupeptin by the standardmethod. MKP3-IP-phosphatase assay mixture (200 �l) containing theabove MKP3 immunoprecipitates and 50 mM pNPP in 50 mM succinatebuffer (pH 7.0) was incubated at 30 °C for 10 h, and the phosphatereleased was measured with enzyme-linked immunosorbent assayreader (Emax, Molecular Devices, Sunnyvale, CA) at 405 nm. As acontrol blank, heat-inactivated cell lysate and reagent were also run foreach assay. When comparing the amount of p-Erk1/2 proteins seques-tered by MKP3 in young and old HDF cells, whole cell lysates, MKP3immunoprecipitates, and the IP supernatants after MKP3-IP were sep-arated on 10% SDS-PAGE, and immunoblot analysis was performedagainst p-Erk1/2 antibody.

Measurement of ROS by FACS Analysis—H2O2 generation duringcellular senescence was measured by FACS analysis using 50 �M 2�,7�-dichlorodihydrofluorescein diacetate (H2-DCFDA, D-399, MolecularProbes, Eugene, OR) in cell culture system. Young and mid-old HDFcells (old cells were too large to analyze by FACS) were plated in a60-mm culture dish with 25 and 70% confluence, respectively. The nextday, media were changed, and the cells were incubated in completemedium for another 24 h. The cells were then pretreated with 10 mM

NAC (Sigma, catalog number A-7250) for 1 h, and H2-DCFDA wasadded 10 min prior to cell harvest. ROS generation was determinedwith FACScan (BD Biosciences) by measuring the fluorescence of DCFarising from the oxidation of H2-DCFDA.

ESI-MS/MS Analysis—Nano-ESI on a Q-TOF2 mass spectrometer(Micromass, Manchester, UK) was used for sequence analysis of Cys62

and Cys105 containing peptides after in-gel trypsin digestion (overnightat 37 °C). Data were collected for positive ions at m/z 400–2000. Forsequence analysis of peptide, once the parent ions were identified, themass spectrometer was set up to obtain collision-induced dissociation

FIG. 1. Marked induction of p-Erk1/2 proteins and failure of its nuclear translocation in old HDF cells by EGF treatment. Young(A) and old (D) cells were homogenized in RIPA buffer, and 40 �g of the lysates were resolved on 10% SDS-PAGE and then hybridized withanti-p-Erk1/2 and �-tubulin antibodies, used as a loading control. Note the higher level of p-Erk1/2 in the old cells as compared with the young cells.Young (B and C) and old (E and F) cells were treated with (C and F) or without (B and E) EGF (10 ng/ml) for 15 min, and immunocytochemistrywith p-Erk1/2 antibody was performed to reveal the basal level of p-Erk1/2 between the young and old cells as well as its response to EGFtreatment. Note the differences of the basal levels and the cell size between the young and old cells. Arrowheads indicate p-Erk1/2 in nuclei, whichare under mitosis in response to EGF treatment only in the young cells. Magnification, �100.

FIG. 2. Markedly increased p-Erk1/2 proteins are also in theprematurely senescent HDF cells. Overexpression of Ha-ras doublemutants, such as V12C40, V12G37, and V12S35, induced premature senes-cence in HDF cells. Cells lysates were prepared in RIPA buffer bysonication, and immunoblot analyses were performed with the celllysates (40 �g/lane). The rest of the experimental procedures were thesame as described under Fig. 1. Y and O indicate young and old cells,respectively.

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MS/MS spectra of the parent ions. The quadrupole analyzer was used toselect parent ions for fragmentation in the hexapole collision cell. Thecollision gas was argon at a pressure of 6–7 � 10�5 mbars, and thecollision energy was 20–30 V. A potential of 1 kV was applied tothe precoated borosilicate nanoelectrospray needles in the ion sourcecombined with a nitrogen back pressure of 0–5 pounds/square inch toproduce a stable flow rate (10–30 nl/min). A new needle was used foreach analysis to eliminate the risk of cross-contamination betweendifferent peptide digests. Product ions were analyzed using an orthog-

onal time-of-flight (TOF) analyzer, fitted with a reflector, a micro-channel plate detector, and a time-to-digital converter. The data wereprocessed using a Mass Lynx Windows NT PC system.

RESULTS

Constitutive Phosphorylation of Erk1/2 Proteins during Cel-lular Senescence—By employing young HDF cells with a dou-bling time of about 24 h and the old HDF cells with doubling

FIG. 3. SA-p-Erk1/2 cells are competent substrates for alkaline phosphatase, protein phosphatases 1/2A, protein-tyrosine phos-phatase, and dual-phosphatase rVHR. V12S35-induced prematurely senescent HDF cells were homogenized by sonication in lysis buffercontaining 1% Nonidet P-40 in PBS, 1 mM PMSF, and 1 �g/ml leupeptin. A, alkaline phosphatase. Forty �g of the cell lysate were incubated with1 and 5 units of alkaline phosphatase at 30 °C for 10 min. B, protein phosphatase 1C-�. Sixty �l of reaction mixture containing 40 �g of the youngand 20 �g of the old cell lysates were incubated with 10 units of PP1C-� at 30 °C for 1 h. C, PTPase. The old cells were homogenized in the buffercontaining 50 mM bis-Tris, 100 mM sodium acetate, 1 mM DTT, 0.01% bovine serum albumin, and 1% NP-40 (pH 7.0). The reaction mixture (60 �l)contained 20 units of PTP-� and 40 �g of the cell lysate and was incubated at 30 °C up to 60 min. rVHR (0.2 �g) was added to the assay mixtureas a positive control. D, rVHR. The young and old cells were homogenized in the lysis buffer, which was used above for the PTPase. The assaymixture (60 �l) contained 40 �g of the cell lysate and 0.2 �g of rVHR and was incubated at 30 °C for 1 h. The reaction mixtures were resolved on10% SDS-PAGE and transferred to polyvinylidene difluoride membrane for immunoblot analysis with anti-p-Erk1/2 antibody. Expressions ofErk1/2 and �-tubulin were used for loading control.

FIG. 4. Anti-p-Erk1/2 antibody cannot detect SA-p-Erk1/2 after PP1 and PP2A reaction. A, to verify how well the anti p-Erk1/2 antibodywas effective against monophosphorylated Erk1/2 proteins either on threonine or tyrosine residues, 0.1 �g of activated MAPK (p-Erk1, catalognumber 206110, Stratagene, La Jolla, CA) was incubated with either 20 or 40 units (20U or 40U) of PP1C-� at 30 °C for 1 h, and thenphosphorylation status of the protein was determined by repeated immunoblot analyses with anti-Thr(P), Tyr(P), p-Erk1/2, and Erk1/2 antibodies.PP1C-� efficiently dephosphorylated phosphothreonine but not phosphotyrosine residues. Also, it was confirmed that anti-p-Erk1/2 antibody couldsensitively unravel the monophosphorylated Erk/12 proteins on immunoblot analysis. B, the activity of the purified PP2A1 was examined with 40�g of old HDF cell lysates in the reaction mixture of 50 mM Tris-HCl (pH 7.4), 2 mM EGTA, 0.1% �-mercaptoethanol, 1 mM PMSF, and 2 �g/mlleupeptin at 30 °C for 1 h. Immunoblot analyses were done with anti p-Erk1/2 and Erk1/2 antibodies. PP2A also specifically dephosphorylatedSA-p-Erk1/2 proteins that increased in the old cells.

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time of over 2 weeks, p-Erk1/2 levels were measured by immu-noblot analysis. Expression of p-Erk1/2 was significantlyhigher in the old cells (Fig. 1D) as compared with the youngcells (Fig. 1A). Moreover, immunocytochemical analysis re-vealed no response to EGF treatment in the old cells, signifi-cant induction of p-Erk1/2 level in the young cells (Fig. 1, B andC), and no change in the old cells (Fig. 1, E and F). It shouldalso be noted that the basal level of p-Erk1/2 was markedlydifferent between these cells (compare Fig. 1, B versus E). Toinvestigate whether the marked induction of p-Erk1/2 was

limited only to replicative senescence HDF cells, Ha-ras doublemutant-induced premature senescence cells were also em-ployed, and immunoblot analysis was carried out. As shown inFig. 2, the constitutive induction of p-Erk1/2 was apparent inboth the replicative senescence (HDF control) and oncogenicRas-induced premature senescence cells (V12C40, V12G37, andV12S35). Therefore, we denote this phenomenon as ‘‘senescence-associated persistent induction of p-Erk1/2’’ or ‘‘SA-p-Erk1/2.’’

Which Phosphatase Can Possibly Be Involved in Dephospho-rylation of p-Erk1/2 Proteins?—To discern phosphatases possi-

FIG. 5. Decreased protein phosphatases 1 and 2A activities, but not their protein expression levels, during cellular senescence. A,significant reduction of alkaline phosphatase activity in the mid-old HDF cells. Phosphatase activity was assayed according to the methoddescribed under ‘‘Experimental Procedures.’’ Note statistically significant reduction of the activity in the mid-old and old cells. B, significantdecrease of PP1/2A activities in the mid-old and old cells as compared with the young cells. C, nuclear (Nu) and cytoplasmic (Cyt) distribution ofPP1/2A activities in the young and old HDF cells. Nuclear and cytoplasmic fractions were prepared from the young and old cells, and PP1/2Aactivities were measured as described under ‘‘Experimental Procedures.’’ To ascertain the purity of the nuclear fractions used for PP1/2A assay,�-tubulin immunoblot analysis was performed as a cytoplasmic marker. D, protein expression of PP1 isozyme in the young and old HDF cells.Immunoblot analyses with anti-PP1C-� and PP1C-� antibodies were performed with the whole cell lysates of the young and old HDF cells. Therest of the procedures were the same as described under ‘‘Experimental Procedures.’’ E, inhibition of PP2A activity by okadaic acid increasedp-Erk1/2 levels in both young and old cells. The young and old cells were plated, and serum-free medium was added 24 h later, and the cells werethen cultured for another 24 h. Okadaic acid (40 nM) was added to the culture medium, and the cells were harvested at 0, 4, 8, and 12 h after thetreatment. Cells lysates were immunologically analyzed as described under ‘‘Experimental Procedures.’’

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bly involved in the induction of SA-p-Erk1/2 in the old cells,whole homogenates (40 �g) of the young and old HDF cellswere incubated with either a recombinant alkaline phospha-tase (M0290S, New England Biolabs, Beverly, MA), proteinphosphatase 1C-�, PTP-� (14–350, Upstate Biotechnology,Inc., Lake Placid, NY), or rVHR (generously donated by Dr.J. M. Denu at Oregon Health Sciences University), and themixtures were analyzed by immunoblot with anti-p-Erk1/2 an-tibody. Five units of alkaline phosphatase (Fig. 3A), 10 units ofPP1C-� (Fig. 3B), 20 units of PTP-� (Fig. 3C), and 0.2 ng ofrVHR (Fig. 3D) completely removed phosphate from thep-Erk1/2 of the young cells after 30 min of incubation. On theother hand, both PP1C-� (10 units) and rVHR (0.2 ng) onlypartially removed the phosphate in the old cells under identicalconditions as above (Fig. 3, B and D). These data indicate thatSA-p-Erk1/2 might not only be regulated by MKP such asrVHR, but also by PP1 and PTP as well as the nonspecificalkaline phosphatase. In order to verify how well the anti-p-Erk1/2 antibody works against monophosphorylated Erk1/2proteins either on threonine or tyrosine residue, 0.1 �g ofactivated MAPK (p-Erk1) was incubated with both 20 and 40units of PP1C-� at 30 °C for 1 h, and the phosphorylationstatus of the protein was then determined by repeated immu-noblot analyses with anti-Thr(P), Tyr(P), p-Erk1/2, and Erk1/2antibodies. As shown in Fig. 4A, PP1C-� efficiently dephospho-rylated Thr(P) but not Tyr(P) residue, confirming that anti-p-Erk1/2 antibody hybridized only with the -pTXpY- dual phos-phorylated Erk1/2 proteins. Additionally, PP2A1 activity onp-Erk1/2 proteins was also examined by incubating the purifiedPP2A1 (catalog number 539508, Calbiochem) with old cell ly-sates in 50 mM Tris-HCl (pH 7.4) containing 2 mM EGTA, 0.1%�-mercaptoethanol, 1 mM PMSF, and 2 �g/ml leupeptin at30 °C for 1 h. When the reaction mixture was analyzed byimmunoblot with anti-p-Erk1/2 and Erk1/2 antibodies, PP2A1was also found to be able to be dephosphorylated by p-Erk1/2(Fig. 4B). Therefore, these results suggest that not only thepurified PP1 but also the PP2A enzymes could efficiently reg-ulate the level of SA-p-Erk1/2, which was highly increasedduring cellular senescence.

Are the Activities of Phosphatases Significantly Changedduring Cellular Senescence?—To investigate whether the ac-tivities of phosphatases were altered during cellular senes-cence or not, alkaline phosphatase and PP1/2A were assayedwith the homogenates of the young, mid-old, and old HDF cells.As illustrated in Fig. 5, alkaline phosphatase activity was

2.61 � 0.05, 1.43 � 0.06, and 1.45 � 0.14 pmol of phosphate/min/�g of protein (Fig. 5A), and PP1/2A activities were 28.78 �8.05, 12.50 � 1.59, and 16.03 � 3.33 pmol of phosphate/min/�gof protein in the young, mid-old, and old cells (Fig. 5B), respec-tively. Both phosphatases activities were already significantlydepressed in the mid-old cells as compared with the youngcells.

Because we are herein primarily concerned with the traffick-ing of Erk1/2 between nucleus and cytoplasm and their phos-phorylation status, we determined the changes of protein phos-phatase activities in these two subcellular fractions duringcellular senescence. Thus, nuclear and cytoplasmic fractionswere prepared from the young and old cells, and possible con-tamination of nuclear fraction with cytoplasm was first deter-mined by �-tubulin expression. As shown in the right panel ofFig. 5C, the nuclear preparation was free of �-tubulin compo-nent. With these preparations, both the nuclear and cytoplas-mic PP1/2A activities were found to be decreased more in theold than in the young cells (left panel). The possibility that thedecrease of PP1/2A activity in the old cells was due to lessexpression of catalytic PP1 subunit was investigated by immu-noblot analysis with anti-PP1C-� and anti-PP1C-� antibodies(Fig. 5D); however, no difference between the young and oldcells was found.

Next, in order to evaluate the specific effect of PP2A on theSA-p-Erk1/2 in vivo, the young and old HDF cells were treatedwith 40 nM okadaic acid, a specific PP2A inhibitor (27, 35, 36).Thus, the cells were harvested at 4, 8, and 12 h after thetreatment, and cell lysates were analyzed by immunoblot anal-ysis. As shown in Fig. 5E, inhibition of PP2A with okadaic acidsignificantly increased phosphorylation levels of Erk1/2 pro-teins in both the young and old cells, strongly indicating thatthe change of PP1 and PP2A activities had significant effectson the SA-p-Erk1/2 in both young and old cells. In order tocompare the PP1/2A proteins expression and their activities inthe young and old cells, total PP1/2A activity, specific activity(pmol/min/�g), and total amount of protein in 16,500 cells weremeasured. As shown in Table I, the old cells had 4 times moreprotein and 2.5 times higher total PP1/2A activity as well asmuch larger cell size than the young cells. Therefore, the spe-cific activity in the old cells was only 60% of the young cells,indicating that specific activity of PP1/2A was significantlydecreased during cellular senescence.

Is MKP3 Responsible for Cytoplasmic Retention of p-Erk1/2Proteins?—Because p-Erk1/2 has been shown to strongly bindto the N-terminal domain of MKP3, allosterically activatesMKP3 C-terminal phosphatase domain (52), and is exclusivelylocated in the cytoplasm of sympathetic neurons (20), the pos-sibility of MKP3 as a potential candidate to sequester SA-p-Erk1/2 in the cytoplasm was first investigated by determiningthe amounts of p-Erk1/2 bound to MKP3 in the IP-precipitate,the IP-supernatant, and the whole homogenates by immuno-blot against p-Erk1/2 antibody. As seen in Fig. 6A, a largeamount of p-Erk1/2 protein was bound to MKP3 in the old cells,whereas there was hardly any p-Erk1/2 bound to MKP3 in theyoung cells. Next, we measured the MKP3 protein expressionin both the young and old cells with IP-immunoblot analysis(Fig. 6B). However, no significant change was observed duringHDF senescence. On the other hand, when its activity wasmeasured by IP-phosphatase assay, the activity in the old cells(21.2 � 4.2 pmol/min/mg protein) was significantly lower thanin the young cells (34.5 � 2.7 pmol/min/mg protein) (Fig. 6C).

What Is the Mechanism of Reduction of PP1/2A and MKP3Activities during Cellular Senescence?—In order to investigatefurther the mechanism underlying the significant reduction ofPP1/2A and MKP3 activities and elevated p-Erk1/2 level dur-

TABLE IComparison of protein phosphatase 1 and 2A expressed in young

and old HDF cells

* Numbers in parentheses indicate relative values between the youngand old cells. The old cells used in these experiments could divide oncein over 2 weeks.

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ing cellular senescence, ROS generation was measured duringcellular senescence by FACS analysis after incubating both theyoung and mid-old HDF cells with H2-DCFDA. As shown inFig. 7A, the mid-old cells contained about 20 times more ROSthan the young cells, and the ROS generation by both couldefficiently be inhibited by pretreatment with 10 mM NAC.When young HDF cells were treated once with 1 mM H2O2,Erk1/2 phosphorylation was markedly increased at 10 min,however, the increase was greatly attenuated in 40 min. At thesame time, when the cells were pretreated for 1 h with 10 mM

NAC, phosphorylation of Erk1/2 protein was efficiently inhib-ited (Fig. 7B). To discern whether H2O2 was responsible for thereduction of PP1/2A activities or not, the enzyme activitieswere measured after treatment of the young cells once with 200�M H2O2. As seen in Fig. 7C, the enzyme activities were tran-siently inhibited in 10 min, but started to recover 40 min laterand finally returned to the control level in 8 h. To furtherinvestigate the regulation of p-Erk1/2 level and activity ofPP1/2A by H2O2 treatment, we measured the changes of theabove two parameters by immunoblot analysis and the activityassay, respectively. As shown in Fig. 7D, when the young cells

were repeatedly treated with 200 �M H2O2 at every 8 h, PP1/2Aactivities remained significantly inhibited after 72 h; controlHDF young cells had specific activity of 28.31 � 0.97 pmol/min/�g protein, and it changed to 24.63 � 2.35, 30.50 � 3.81,29.06 � 2.08, 25.38 � 1.54, and 23.63 � 2.68 pmol/min/�gprotein at 5 min, 24, 48, 72, and 80 h, respectively. Concomi-tant with the changes of PP1/2A activities, phosphorylation ofErk1/2 proteins was simultaneously affected by H2O2 treat-ment. These data indicate that repeated damage of the youngcells by H2O2 exposure persistently inhibited PP1/2A activities,resulting in the accompanying induction of p-Erk1/2 levels.

To elucidate whether decrease of PP1/2A activities in themid-old cells was due to the oxidation of cysteine residues inthe enzyme molecules or not, thiol-specific reducing agentssuch as DTT, �-mercaptoethanol, and NAC were added to thelysates of the young HDF cells, which had been pretreated withH2O2 for 10 min. As shown in the left panel of Fig. 8, the controlPP1/2A activity was decreased from 27.96 � 0.76 to 24.04 �0.63 pmol/min/�g protein (p � 0.005) by the treatment withH2O2. However, the inhibition was reversed to 33.38 � 0.66,29.79 � 1.26, and 27.63 � 1.32 pmol/min/�g protein by the

FIG. 6. Sequestration of p-Erk1/2 by MKP3 in the old HDF cells. A, to investigate whether the p-Erk1/2 was bound to MKP3 or not andalso to compare the bound p-Erk1/2 between the young and old cells, 300 �g of each cell lysate in immunoprecipitation buffer (1% Triton X-100,250 mM sucrose, 20 mM Tris-HCl (pH 7.0), 1 mM EGTA, 1 mM EDTA, 0.1% �-mercaptoethanol, 1 mM PMSF, 2 �g/ml leupeptin) were incubated with1 �g of anti-MKP3 antibody. The immunoprecipitate was incubated with 50% protein G-agarose beads at 4 °C overnight and then washed 3 timeswith the same buffer. The IP-supernatant and the washing solution were collected, and they were concentrated by Centricon-10 (catalog number4206, Millipore, Bedford, MA). The whole cell lysates equivalent to the amount of the supernatants were also concentrated with Centricon. Thewhole cell lysate, IP-supernatant, and the MKP3-IP were resolved on 10% SDS-PAGE and then immunoblotted with anti-p-Erk1/2 antibody. Lanes1–3 and lanes 4–6 indicate the whole cell lysates, IP-supernatants, and MKP3-precipitate of the young and old cells, respectively. Note the p-Erk1/2bound to MKP3 only in the old cells but not in the young cells. B, MKP3 protein expression in the young (1st lane) and old (2nd lane) cells, with40 �g of whole cell lysate applied per each lane. �-Tubulin bands represent loading control between the young and old cells. C, MKP3-IPphosphatase activity. Five hundred �g of the young and old cell lysates were applied for IP with anti-MKP3 antibody and then washed 3 times withIP-buffer. Phosphatase assay was carried out with 50 mM pNPP in 50 mM succinate buffer (pH 7.0). Assays were repeated 3 times with duplicatesin each case, and the results of 34.5 � 2.7 and 21.2 � 4.2 pmol/min/mg protein indicate the mean � S.D. in the young and old cells, respectively.Upper panel reveals the MKP3 IP-immunoblot findings used for the MKP3 IP-phosphatase assays.

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FIG. 7. Reactive oxygen species generated during cellular senescence-regulated PP1/2A activities and p-Erk1/2 levels. A, markeddifference of ROS level between young and mid-old HDF cells, measured by FACS analysis. The young and mid-old cells were seeded into culturedishes up to 25 and 70%, respectively. After 24 h, the monolayer was re-fed with complete medium and then treated with H2DCF-DA 10 min prior

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addition of DTT, �-mercaptoethanol, and NAC, respectively.All three reagents significantly reactivated PP1/2A activity ascompared with that of the H2O2 alone treatment (�, p � 0.002,and #, p � 0.02). However, only DTT, but not �-mercaptoetha-nol and NAC, significantly increased PP1/2A activity of theyoung cells more than the control (p � 0.005). Interestingly,when the assay was carried out with mid-old cell lysates, whosePP1/2A activity had been already reduced, most likely due toaccumulated H2O2 during senescence, �-mercaptoethanolcould significantly increase PP1/2A activity as compared withthe control (22.04 � 0.80 versus 18.71 � 0.76 pmol/min/�gprotein, p � 0.005, right panel in Fig. 8), thus strongly suggest-ing that the high level of ROS found in senescent cells oxidizedthiol residues, which are important for phosphatase activity.To investigate whether elimination of ROS generated duringcellular senescence could revert the morphological changescharacteristic to senescent cells or not, the mid-old cells weretreated with NAC twice a day for 2 and 4 days, and theirmorphology was examined. As seen in Fig. 9, the flat and largecell characteristic to the old indeed reverted to the morphologycharacteristic to young cells: small, slender, and cylindricalfibroblast. In order to investigate whether the morphologicalchanges of the young-like cells were accompanied by any bio-chemical changes specific to cellular senescence, the expressionof senescence-associated �-galactosidase in the cells was meas-ured. As shown in Table II, treatment of the cells with NAC for

2 days was sufficient to significantly reduce senescence-associ-ated �-galactosidase expression in the mid-old cells.

Which Cysteine Residue and/or Metal Ion in PP1C-� Is Oxi-dized by H2O2?—To confirm oxidation of Cys-SH by H2O2 invitro, 0.5 mM 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl)was incubated with purified PP1C-� (Sigma) based on Denuand Tanner (45). As shown in Fig. 10A, incubation of PP1C-�with 2.5 mM H2O2 for 10 min clearly oxidized Cys-SH to Cys-SOH, indicated by NBD-sulfoxide modification and the changeof maximum absorbance from 420 to 347 nm. To elucidate thenet effect of oxidation(s) on their activities, thiol-reducingagents and metal-reducing ascorbate were separately added toPP1C-� after incubation with 2.5 mM H2O2 for 10 min at 30 °C.Activity of PP1C-� could be restored not only by Cys-reducingagents but also by ascorbate (Fig. 10B). To determine the mostvulnerable site of oxidation in cysteine of the purified PP1C-�,MS/MS analysis was performed using nano-electrospray ioni-zation on a Q-TOF2 mass spectrometry. As shown in Fig. 11, Aand C, two oxidized cysteine-containing peptides were detectedin the m/z range of 740–860; Cys62-containing peptide encom-

TABLE IISignificant change of SA-�-galactosidase expression by repeated

treatments of mid-old HDF cells with N-acetyl-L-cysteineCells were treated with 10 mM NAC every 12 h for 4 days. At least

3000 cells were counted for each group in duplicate, and the sameexperiments were repeated more than twice.

Treatment SA-�-galactosidase expression

% total cells

Control 4.05 � 0.33NAC (2 days) 1.13 � 0.20a

NAC (4 days) 1.73 � 0.47a

a p � 0.05 versus control.

FIG. 8. Restoration of protein phosphatase 1 and 2A activitiesby thiol-specific reducing agents in mid-old HDF cells. In thecase of the young HDF cells, the cells were pretreated with 1 mM H2O2.However, the mid-old cells were not pretreated, because the PP1/2Aactivity was already reduced, most likely due to accumulated H2O2during senescence. The cell lysates were prepared within 10 min in thephosphate-free condition. By using the phosphopeptide as a substrate,PP1/2A activity was measured by the described method with the control(C) and H2O2-treated cell lysates. The assay was also performed eitherwith DTT (1 mM), �-mercaptoethanol (2ME, 0.1%), or NAC (10 mM)addition to the cell lysates of the H2O2-treated cell lysates. Recovery ofthe senescent-induced PP1/2A activity was also evaluated with themid-old cell lysates by addition of 0.1% �-mercaptoethanol to the controlcell lysate. All the samples were triplicated, and the assays were per-formed more than three times.

FIG. 9. Conversion of mid-old HDF cells to young-like cells byrepeated NAC treatment. Mid-old cells were treated with NAC (10mM) twice per day for 4 days. As revealed in these figures, morphologyof the mid-old cells (top right) clearly changed to young cell-like ones(bottom 2 dishes) after 2 days.

to cell harvest. The cells were pretreated with NAC 1 h prior to harvest and treated with H2DCF-DA for 10 min before the harvest. Changes offluorescence by generated ROS were measured by FACS analysis. Note 19-fold (4.25 versus 79.82) higher ROS in the mid-old cells than the youngcells. B, treatment of HDF young cells with 1 mM H2O2 significantly increased Erk1/2 phosphorylation, but it was efficiently blocked by NACpretreatment. Control HDF cells (lane 1) were treated with 1 mM H2O2 for 10 (lane 2) and for 40 min (lane 4), pretreated for 1 h with 10 mM NAC,and then treated with H2O2 for 10 (lane 3) and for 40 min (lane 5). The cells were harvested, and p-Erk1/2 expression level was measured byimmunoblot analysis. C, to investigate whether ROS regulated PP1/2A activity or not, HDF young cells were treated once with 200 �M H2O2. Theenzyme activity was transiently inhibited in 10 min; however, it was recovered in 40 min and then returned to control level in 8 h. D, simultaneousregulation of PP1/2A activities and Erk1/2 phosphorylation of young HDF cells by repetitive treatment with H2O2. Young HDF cells were treatedwith 200 �M H2O2 every 8 h and then harvested after 0 min (lane 1), 5 min (lane 2), 24 h (lane 3), 48 h (lane 4), 72 h (lane 5), and 80 h. The celllysates were used for p-Erk1/2 immunoblot analysis (upper panel) and PP1/2A activity assay (lower panel). Note the reciprocal changes of p-Erk1/2(increase) and the PP1/2A activities (decrease) at 5 min. The reciprocal change was consistent by repeated treatments for 72 h.

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passing 61ICGDIHGQYYDLLR74 (calculated mass, 1712.8) andCys105-containing peptide encompassing 99QSLETLCLL-LAYK111 (calculated mass, 1543.8). The results are consistentwith the difference between oxidation of Cys62 to Cys62-SO3H(Fig. 11A) and Cys105 to Cys105-SO3H containing peptide (Fig.11B), entailing the addition of 48 mass units. In contrast, otherdominant oxidation states of Cys-containing peptide were notdetected in m/z 400–4000, except for another Cys62 containingpeptide (Glu44–Arg74, calculated mass of 3598.9, data notshown). When H2O2 (25 mM)-oxidized PP1C-� digested over-night with trypsin in-gel, Cys-SO3H-containing peptide becamethe major oxidation product, whereas Cys-SOH- and Cys-SO2H-containing peptides were not observed. To demonstratethe formation of Cys-SO3H, the parent ions were subjected tocollision-induced dissociation MS/MS spectrum. The aminoacid sequence of the dominant ion (M � 2H)2� derived frompeptide 61–74 and 99–111 (m/z 857.4 and 771.9, respectively)were determined from the masses of the fragments arisingfrom collision-induced dissociation of the peptide, and it wasproved to be as Cys-SO3H. As shown in Fig. 11C, the majordaughter ions from y12 to y13 corresponded to peptide bondbetween Cys62-SO3H and Gly63, and the mass difference be-tween y6 and y7 (151.0 Da) clearly indicated presence of Cys105-SO3H (Fig. 11D).

DISCUSSION

Replicative senescence of human fibroblasts has frequentlybeen used as an aging model in vitro (53). In the present study,we have shown that a large amount of p-Erk1/2 proteins, de-noted as SA-p-Erk1/2, were found in the cytoplasm of bothsenescent and prematurely senescent HDF cells induced byHa-ras double mutants (Figs. 1 and 2) and that the SA-p-Erk1/2s were competent substrates for PP1/2A, PTP, andMKP-rVHR in vitro as well as in vivo (Fig. 3). During theprocess of cellular senescence, PP1/2A (Fig. 5B), and MKP3(Fig. 6C), activities were significantly reduced; however, theirprotein expression levels were not affected (Figs. 5D and 6B).The decrease seemed to coincide temporarily with the agingprocess, because the phosphatase activities were already re-

duced in the mid-old HDF cells (Fig. 5, A and B). Furthermore,a large amount of SA-p-Erk1/2 proteins was bound and seques-tered by MKP3 in the cytoplasm of old cells (Fig. 6A).

Erk1/2 is one of MAPK isoforms together with c-Jun N-terminal kinase/stress-activated protein kinase and p38MAPK,and the upstream components of Erk1/2 include c-Ras, c-Raf1,and MAPK kinase (54, 55). When cells are stimulated withvarious ligands such as growth factors, hormones, neurotrans-mitters, or tumor promoters, Erk1/2 is activated through dual-phosphorylation at the -pTEpY- motif. Subsequently, p-Erk1/2translocates into the nucleus and phosphorylates Elk-1,thereby acting as a transcription factor for cell proliferation(56).

The phosphorylation state of any given protein in vivo de-pends on the balance between the activities of phosphatasesand kinases, and there is increasing evidence that relativelyfew Ser- or Thr-specific protein phosphatases with pleiotropicaction participate in cellular regulation. Therefore, the findingthat a significant decrease of PP1 activities in both the cyto-plasm and nuclear fractions of the senescent cells without anychange in their protein expression (Fig. 5D) resulted in theinduction of p-Erk1/2 levels that might possibly be explained bythe existence and induction of phosphatase inhibitors such asinhibitor-1 or an unidentified inhibitor during cellular senes-cence. Therefore, in order to verify the presence or increase ofa putative inhibitor in the old cells, we carried out two ap-proaches as follows: detection of the known inhibitor 1 expres-sion in the young and old cells by immunoblot analysis, and theother to measure the PP1/2A activity after mixing the youngand old cell lysates. When analyzed by immunoblot analysis,the protein expression levels among the two were basically thesame (data not shown). Also when increasing amounts of theold cell lysates were added to a fixed amount of young cells andthe PP1/2A activity was measured, the sums of the activitymeasured individually were the same as those in the mixture,indicating the absence or no increase of an inhibitor in the oldcell lysates (data not shown). The above two experiments ver-ified the fact that the decreased PP1/2A activity in the old cells

FIG. 10. Spectroscopic analysis of PP1C-� oxidation and restoration of the phosphatase activity. Based on the sulfoxide adductformation of sulfenic acid with NBD-Cl and the spectrum change from 420 to 347 nm (45), 1.33 �M PP1C-� (Sigma) was treated with 2.5 mM H2O2for 10 min at 30 °C and then further treated with 0.5 mM NBD-Cl at 25 °C for 2 h. Dialysis and concentration of the mixture up to 400 �l was donewith Centriplus YM-10 (Millipore, MA), and the final product was analyzed spectrophotometrically by 250–600 nm scanning (Ultrospec 4300 pro,Biochrom Ltd., Cambridge, UK). Maximum absorbance at 420 nm indicates Cys-S-NBD (dotted, control), whereas the peak at 347 nm indicatesCys-SO-NBD (solid line) by H2O2 oxidation (A). To investigate the net effect of the oxidation(s) on their activities, purified PP1C-� (1.66 �g/ml) wasincubated with 2.5 mM H2O2 for 10 min at 30 °C, and then the residual H2O2 was removed by 100 units of catalase. In 10 min, phosphatase activitywas measured with pNPP as a substrate and the various reducing agents, 7 mM DTT, 14 mM glutathione (GSH), 14 mM �-mercaptoethanol (2-ME),and 15 mM ascorbate (Asc). Activity of PP1C-� could be restored by both Cys-reducing agents and metal-reducing ascorbate (B).

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FIG. 11. Mass spectrometry analysis of oxidation state of PP1C-�. Control and H2O2-oxidized PP1C-� (each 5 �g/sample) were separatelydigested with trypsin (0.1 �g) in-gel at 37 °C for overnight and desalted using C18 nano-columns. For analysis by MS/MS, peptides were elutedwith 1.5 �l of 50% methanol, 49% H2O, 1% formic acid solution directly into a precoated borosilicate nanoelectrospray needle. A and B spectra showthe oxidized Cys62-containing peptide and Cys105-containing peptide, respectively. C and D are tandem mass spectra of a peptide with m/z 857.4and 771.9, respectively. The C-O3 denotes Cys-SO3H.

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was not due to increase of a putative inhibitor in the old cells.To investigate further the mechanistic evidence of the re-

duced PP1/2A activity during cellular senescence, thiol-specificreducing agents were added to the cell lysates whose cells hadbeen pretreated with H2O2. As seen in Fig. 8, the reducingagents could significantly restore the H2O2-inhibited PP1/2Aactivities in the young HDF cells and increase the PP1/2Aactivity in mid-old cells. These data strongly indicated that theinhibition of phosphatase activity might have been due to theoxidation of reactive site cysteine residue(s) by ROS duringcellular senescence. It should be noted that the catalytic do-mains of PP1, PP2A, and calcineurin are highly homologous,i.e. 49% identity between PP1 and PP2A and 40% identitybetween PP2A and calcineurin. However, their substrate spec-ificities and interactions with regulatory molecules are verydifferent. The crystal structure of their catalytic domains re-vealed similarly folded catalytic cores, each of which containstwo catalytic metals (57, 58). There is very little informationavailable about the effects of oxidants on the activity of PP1/2A.However, Sommer et al. (59) recently revealed that the activi-ties of PP1/2A in SK-N-SH cell lysates were reversibly inhib-ited by H2O2. ROS-inhibited PP1 activity could be reversed bytreating SK-N-SH cells either with thiol-reducing agent DTT ormetal-reductant ascorbate. Our results together with the aboveobservation led us to suggest that the mechanism of inhibitionof PP1/2A in HDF cells was due to direct attack of H2O2 oncysteine residue(s) of PP1/2A but not due to a regulating factoractivity.

As shown in Fig. 3 and Fig. 4, SA-p-Erk1/2 served as asubstrate for PP1/2A. Because the inhibition constants (Ki) ofPP1 and PP2A by okadaic acid are 150 nM and 32 pM, respec-tively (60), induction of p-Erk1/2 in the young and old HDFcells 4 h after the treatment with 40 nM okadaic acid (Fig. 5E,lanes 2–4 and 6–8) strongly indicated that the preferentialinactivation of PP2A during cellular senescence could also besignificantly responsible for SA-p-Erk1/2 proteins.

The reason why a huge induction of p-Erk1/2 by a singletreatment with 10 mM H2O2 was greatly attenuated in 40 minmight be found in the rapid induction of MAPK through EGFreceptor (61, 62), and the significant but transient reduction ofPP1 and PP2A in HDF cells, which were significantly inhibitedin 10 min but recovered by 40 min and reached the control levelin 8 h (Fig. 7C). It is important to note that when the cells weretreated with H2O2 every 8 h for more than 72 h, activities ofPP1/2A were persistently reduced, and the level of p-Erk1/2was constitutively high (Fig. 7D), resulting in SA-p-Erk1/2.These findings implicate that cells in vivo might be continu-ously exposed to the higher level of ROS during the senescenceprocess, thus resulting in the inhibition of PP1/2A and theconstitutive increase of p-Erk1/2. This was supported by the 20times more ROS measured in the mid-old cells than the youngcells (Fig. 7A). When FACS analysis was employed to measureROS generation, data acquisition from the old cells was impos-sible to obtain population-gated, because the size was too largeand because of the fragility of the old cells, thus necessitatingthe use of the mid-old cells for the experiment. Even though thecells were stimulated with 1 mM H2O2 and markedly inducedp-Erk1/2 levels, the huge induction could be completely pre-vented by NAC pretreatment (Fig. 7B). Furthermore, treat-ment of the young cells only once with a lower concentration ofH2O2 (200 �M) significantly inhibited phosphatase activityalong with the concomitant ‘‘increase’’ of p-Erk1/2 proteins 5min after the treatment. However, when treated repeatedlywith H2O2 at every 8 h, the PP1/2A activities remained signif-icantly inhibited even after 72 h (Fig. 7D). The levels ofp-Erk1/2 in the young cells were reciprocally regulated with the

PP1/2A activities by the same treatment. Moreover, treatmentof the mid-old cells with antioxidant NAC induced morpholog-ical changes of the senescent to the young cells (Fig. 9), con-comitant with the reduction of senescence-associated �-galac-tosidase expression (Table II). H2O2 could directly oxidizePP1C-�, as proved by NBD-Cl modification of Cys-SOH species(Fig. 10A). However, H2O2 reduced PP1 activity not only bycysteine oxidation but also by oxidation of metal ions, whichwere located in the active site of the PP1C-� (63). This wasevidenced by ascorbate (15 mM) recovery of H2O2-inhibited PP1activity as much as DTT (Fig. 10B), indicating the oxidation ofmetal ions in the active site during cellular senescence. Theconcentration of ROS was 20 times higher in the mid-old cellsthan the young HDF; therefore, when the cysteine residueoxidized by H2O2 was determined by ESI-MS/MS analysis, 25mM H2O2 was applied instead of 2.5 mM. H2O2 completelyoxidized Cys62 and Cys105 of PP1C-� to sulfonic acid withoutsulfenic and sulfinic acids (Fig. 11, C and D); however, theeffect of air oxidation could not be ruled out during the in-geltrypsin digestion at 37 °C overnight. Sulfinic acid is sensitive toair, as has been pointed out (64). Moreover, oxidation of Cys62

might be very important for reducing the PP1/2A activity dur-ing cellular senescence, because of its conservation between thetwo enzymes. The bulky Cys-SO3H mediated disruption of ter-tiary structure might be expected based on the stereo view ofPP1C-� (Fig. 12).

These data strongly indicate that H2O2 generated duringcellular senescence directly regulated both PP1/2A activitiesand p-Erk1/2 level through oxidation of cysteine residues ofPP1/2A, further suggesting that H2O2 might be the major cul-prit of the significantly reduced PP1/2A activity in the senes-cent HDF cells. Considering that the inhibition of PP1 activityin brain cortex of the inhibitor-1 transgenic mice was signifi-cantly different depending on the training schedule and thatthe long term memory was dependent on the PP1 activity in theexperimental animal, which mimics the physiological aging inanimal and human being (50), the significant inhibition ofPP1/2A activities and accompanying phosphorylation of Erk1/2proteins in the mid-old and old HDF cells in this study imposea great credence upon the physiological importance of PP1/2Aduring cellular senescence.

It has been reported recently (65) that aging compromisesperoxisomal targeting signal 1 (PTS1) protein import, with thecritical antioxidant enzyme catalase; the number and appear-ance of peroxisomes are altered in old cells accompanied by theaccumulation of Pex5p on their membranes. Concomitantly, old

FIG. 12. Stereo view of the catalytic site of PP1C-a. Metal ionsare drawn as pink spheres. Space-filling residues are showing Cys62-SO3H and Cys105-SO3H, respectively. The structure of PP1C-� reportedby Goldberg et al. (57) was modified by Insight II. Hydrophobic residuessurrounding the oxidized cysteines are shown.

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cells produced increasing amounts of the toxic metabolite hy-drogen peroxide, and increased load of ROS may further exac-erbate the effects of aging. It is now generally accepted thatPex5p, the receptor for most peroxisomal matrix proteins, cy-cles between the cytosol and the peroxisomal compartment,and insertion of Pex5p into the peroxisomal membrane is cargoprotein-dependent (66). Pex5p binding to Pex13p, one of theimport machinery, is essential for import of catalase withPTS1-like signal KANL (67). Therefore, failure of catalase im-port into peroxisomal matrix might plausibly explain the 20times higher H2O2 level in the mid-old cells than that in theyoung cells (Fig. 7A).

MKP3 can physically associate with Erk1/2 (24), and thisassociation can consequently activate the phosphatase activityof MKP3 (25) through conformational change of the latter. Ithas been proposed that MKP3 can exist in two distinct confor-mations, the open and closed (52). In the open conformation,the enzyme is catalytically incompetent, because the generalacid (Asp262) is positioned away from the active site (His/Val)-Cys293-X5-Arg299-(Ser/Thr), and the side chains adopted by theactive site Cys293 and Arg299 residues are not optimal for ca-talysis. However, the binding of Erk2 to the N-terminal domainof MKP3 facilitates the repositioning of active site residues andaccelerates the general acid loop closure, accompanied by theactivation of MKP3 (52). The N-terminal domain of MKP3 andErk2 revealed a high affinity for binding interaction (68). Fur-thermore, the p-Erk2 is a highly specific substrate for MKP3with kcat/Km of 3.8 � 106/M/s that is over 6 orders of magnitudehigher than Erk2-derived phosphopeptide encompassing theTEY motif (69). Excess H2O2 found in the mid-old HDF cells(Fig. 7A) was most likely responsible for inactivation of MKP3during cellular senescence. Because Cys293 constitutes partof the triad for the reactive site and H2O2 has been shown tooxidize Cys to sulfenic acid (45), it was highly possible thatH2O2 oxidized the Cys293 residue in MKP3 during senes-cence, resulting in the disruption of formation of the activeclosed conformation. Consequently, although MKP3 bound alarge amount of p-Erk1/2 in the old cells (Fig. 6A), it failed todephosphorylate p-Erk1/2 and held the proteins incytoplasm.

In summary, SA-p-Erk1/2 proteins accumulated in the cyto-plasm of old HDF cells were due to the reduced PP1/2A activ-ities and inactivation of cytoplasmic MKP3, resulting in reten-tion of p-Erk1/2 in the cytoplasm and prevention of theirtranslocation to the nucleus. Failure of nuclear translocation ofp-Erk1/2 might result in stoppage or slow down of mitosis in oldcells. These observations are most plausibly explained by oxi-dation of the conserved Cys62 in PP1/2A and the moleculesaround the active site of MKP3 by ROS, which was immenselygenerated during the senescence process.

Acknowledgments—We thank Prof. Woon Ki Paik at Ajou Universityfor encouragement and for reviewing the manuscript. We also thankProf. Sang Chul Park at Seoul National University for helpful discus-sions. The Aging and Apoptosis Research Center was the recipient ofGrant R11-2002-0012 from the Korea Science & EngineeringFoundation.

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Hong Seok Kim, Myeong-Cheol Song, In Hae Kwak, Tae Jun Park and In Kyoung LimSenescence

Phosphatases 1 and 2A and MKP3 Due to Reactive Oxygen Species during Cellular Constitutive Induction of p-Erk1/2 Accompanied by Reduced Activities of Protein

doi: 10.1074/jbc.M211739200 originally published online July 2, 20032003, 278:37497-37510.J. Biol. Chem. 

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