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• W2155132585 abstract "Oncogenic ras activates multiple signaling pathways to enforce cell proliferation in tumor cells. The ERK1/2 mitogen-activated protein kinase pathway is required for the transforming effects of ras, and its activation is often sufficient to convey mitogenic stimulation. However, in some settings oncogenic ras triggers a permanent cell cycle arrest with features of cellular senescence. How the Ras/ERK1/2 pathway activates different cellular programs is not well understood. Here we show that ERK1/2 localize predominantly in the cytoplasm during ras-induced senescence. This cytoplasmic localization seems to be dependent on an active nuclear export mechanism and can be rescued by the viral oncoprotein E1A. Consistent with this hypothesis, we showed that E1A dramatically down-regulated the expression of the ERK1/2 nuclear export factor PEA-15. Also, RNA interference against PEA-15 restored the nuclear localization of phospho-ERK1/2 in Ras-expressing primary murine embryo fibroblasts and stimulated their escape from senescence. Because senescence prevents the transforming effect of oncogenic ras, our results suggest a tumor suppressor function for PEA-15 that operates by means of controlling the localization of phospho-ERK1/2. Oncogenic ras activates multiple signaling pathways to enforce cell proliferation in tumor cells. The ERK1/2 mitogen-activated protein kinase pathway is required for the transforming effects of ras, and its activation is often sufficient to convey mitogenic stimulation. However, in some settings oncogenic ras triggers a permanent cell cycle arrest with features of cellular senescence. How the Ras/ERK1/2 pathway activates different cellular programs is not well understood. Here we show that ERK1/2 localize predominantly in the cytoplasm during ras-induced senescence. This cytoplasmic localization seems to be dependent on an active nuclear export mechanism and can be rescued by the viral oncoprotein E1A. Consistent with this hypothesis, we showed that E1A dramatically down-regulated the expression of the ERK1/2 nuclear export factor PEA-15. Also, RNA interference against PEA-15 restored the nuclear localization of phospho-ERK1/2 in Ras-expressing primary murine embryo fibroblasts and stimulated their escape from senescence. Because senescence prevents the transforming effect of oncogenic ras, our results suggest a tumor suppressor function for PEA-15 that operates by means of controlling the localization of phospho-ERK1/2. Normal organisms keep stable cell numbers by regulating the process of cell proliferation and cell death. Cancer cells bypass these controls because of multiple genetic and epigenetic alterations that block the action of cell proliferation inhibitors or mimic growth factor activation (1Hanahan D. Weinberg R.A. Cell. 2000; 100: 57-70Abstract Full Text Full Text PDF PubMed Scopus (22406) Google Scholar). Fortunately, several fail-safe mechanisms have evolved to prevent the expansion of cells threatened by oncogenic stimuli. For example, the myc oncogene induces abnormal cell proliferation but promotes cell death as well (1Hanahan D. Weinberg R.A. Cell. 2000; 100: 57-70Abstract Full Text Full Text PDF PubMed Scopus (22406) Google Scholar, 2Evan G.I. Vousden K.H. Nature. 2001; 411: 342-348Crossref PubMed Scopus (2711) Google Scholar). Also, oncogenic ras leads to a permanent cell cycle arrest with features of cellular senescence (3Serrano M. Lin A.W. McCurrach M.E. Beach D. Lowe S.W. Cell. 1997; 88: 593-602Abstract Full Text Full Text PDF PubMed Scopus (3965) Google Scholar). Tumor formation is therefore very much stimulated by the inactivation of key mediators of the apoptosis and senescence programs such as p53 and the Rb 1The abbreviations used are: Rb, retinoblastoma protein; BrdUrd, bromodeoxyuridine; ERK, extracellular signal-regulated kinase; ERK2*, ERK2-L73P, S151D; MAP, mitogen-activated protein; MEK, MAP kinase/ERK kinase; GST, glutathione S-transferase; MEF, mouse embryo fibroblast; PBS, phosphate-buffered saline; PML, promyelocytic leukemia; RNAi, RNA interference; shRNA, short hairpin RNA; SA-β-gal, senescence-associated β-galactosidase. tumor suppressor proteins (1Hanahan D. Weinberg R.A. Cell. 2000; 100: 57-70Abstract Full Text Full Text PDF PubMed Scopus (22406) Google Scholar, 2Evan G.I. Vousden K.H. Nature. 2001; 411: 342-348Crossref PubMed Scopus (2711) Google Scholar). The induction of a senescent cell cycle arrest by oncogenic ras in normal fibroblasts contrasts with the well known mitogenic effects of Ras and the MAP kinase pathway in multiple cell lines and transgenic animals (4Johnson G.L. Lapadat R. Science. 2002; 298: 1911-1912Crossref PubMed Scopus (3511) Google Scholar). Endogenous Ras proteins are required for cell proliferation (5Stacey D.W. Tsai M.H. Yu C.L. Smith J.K. Cold Spring Harbor Symp. Quant. Biol. 1988; 53: 871-881Crossref PubMed Google Scholar), and Ras activation is part of the response to growth factors during normal development (6Kayne P.S. Sternberg P.W. Curr. Opin. Genet. Dev. 1995; 5: 38-43Crossref PubMed Scopus (60) Google Scholar). In addition, constitutively active oncogenic alleles of ras are often found in human tumors (7Barbacid M. Annu. Rev. Biochem. 1987; 56: 779-827Crossref PubMed Scopus (3778) Google Scholar) where they convey mitogenic signals. To explain this apparent paradox, it was proposed that normal cells interpret aberrant mitogenic signaling by activating a p53- and Rb-dependent senescence program that is absent in most tumor cell lines (3Serrano M. Lin A.W. McCurrach M.E. Beach D. Lowe S.W. Cell. 1997; 88: 593-602Abstract Full Text Full Text PDF PubMed Scopus (3965) Google Scholar). Another explanation is that the ERK1/2 MAP kinase pathway activates different cellular programs depending on the strength and duration of its stimulation. For example, sustained activation of the ERK1/2 MAP kinase pathway by constitutively active Ras or nerve growth factor triggers growth arrest and differentiation in the PC12 pheochromocytoma cell line (8Bar-Sagi D. Feramisco J.R. Cell. 1985; 42: 841-848Abstract Full Text PDF PubMed Scopus (569) Google Scholar). In contrast, a transient activation of ERK1/2 by epidermal growth factor induces cell proliferation (8Bar-Sagi D. Feramisco J.R. Cell. 1985; 42: 841-848Abstract Full Text PDF PubMed Scopus (569) Google Scholar). To date, we do not know whether induction of senescence in normal fibroblasts by constitutive Ras activation involves sustained and strong ERK1/2 activity. As a matter of fact, it is equally possible that these cells activate feedback mechanisms to counteract constitutive Ras activation, blocking the mitogenic activity of the ERK1/2 MAP kinase cascade. Here we report that constitutive Ras expression in IMR90 fibroblasts induced a senescent cell cycle arrest accompanied by a sustained phosphorylation of ERK1/2. Shortly after the introduction of oncogenic ras, ERK1/2 were phosphorylated and translocated to the nucleus. Upon establishment of the senescent phenotype, ERKs remained phosphorylated but relocalized to the cytoplasm. Blocking the senescent response to Ras by the viral oncoprotein E1A restored the nuclear localization of ERK1/2 normally observed in growing cells. The ERK nuclear export factor PEA-15 seems to be required for this relocalization of ERK and, in agreement, PEA-15 levels were reduced by E1A. Also, RNAi-mediated inactivation of PEA-15 in mouse fibroblasts rescued them from Ras-induced senescence. Hence, limiting ERK nuclear localization by PEA-15 keeps the aberrant mitogenic activity of oncogenic ras and the ERK1/2 pathway under control. Cells and Retroviruses—Normal human diploid fibroblast IMR90 cells (American Type Culture Collection) were cultured in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% fetal bovine serum (Hyclone, Logan, Utah) and 1% penicillin G/streptomycin sulfate (Invitrogen). Primary mouse embryo fibroblasts (MEFs) were obtained from 13.5-day-old embryos as described (9Spector D.L. Goldman R.D. Leinwand L.A. Cells: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1998: 8.4-8.8Google Scholar). The retroviruses pBabe, pBabeRas, pLPC, pLPCE1A, pWZL, and pWZLRas were described previously (10Ferbeyre G. de Stanchina E. Querido E. Baptiste N. Prives C. Lowe S.W. Genes Dev. 2000; 14: 2015-2027Crossref PubMed Google Scholar). pBabeERK was constructed as follows. ERK2-L73P, S151D (abbreviated ERK2*) was obtained from pCMVERK2* (11Emrick M.A. Hoofnagle A.N. Miller A.S. Ten Eyck L.F. Ahn N.G. J. Biol. Chem. 2001; 276: 46469-46479Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar) by XbaI and partial EcoRI digestion, blunted with Klenow, and ligated into the SnaBI site of pBabe. Retroviral-mediated gene transfer was performed as described previously (10Ferbeyre G. de Stanchina E. Querido E. Baptiste N. Prives C. Lowe S.W. Genes Dev. 2000; 14: 2015-2027Crossref PubMed Google Scholar). RNA Hairpins Trigger RNA Interference—To construct the gene cassettes that express RNA hairpins (shRNAs) from the U6 polymerase III promoter, we used shagging-PCR as described (cshl.org/public/SCIENCE/hannon.html). shRNAs (primers are available upon request) were designed against nucleotide locations 1–29 and 295–324 of the mouse PEA-15 mRNA sequence (GenBank™ accession number NM_011063) with the computer-assisted RNAi Oligo Retriever program (cshl.org/public/SCIENCE/hannon.html). The common upstream primer U6-BglII (5′-TCTGCCAGATCTGATTTAGGTGACACTATAG3-′), complementary to a region upstream of the U6 promoter on the pGEMU6 plasmid (N. Hernandez, Cold Spring Harbor Laboratory), was used to amplify a PCR cassette containing the U6 promoter, a target-specific hairpin sequence, the Pol-III terminator, and two terminally placed restriction sites (BglII and EcoRI) for convenient directional cloning. The resulting 400-bp PCR product was cloned between the BglII and EcoRI sites of the retroviral vector pMSCVpuro (Clontech). Cell Proliferation and Senescence Determination—To determine cell proliferation rates we used a BrdUrd incorporation assay as described (10Ferbeyre G. de Stanchina E. Querido E. Baptiste N. Prives C. Lowe S.W. Genes Dev. 2000; 14: 2015-2027Crossref PubMed Google Scholar). Senescence was scored by determining the percentage of the population exhibiting a senescence-associated β-galactosidase (SA-β-gal) activity (12Dimri G.P. Lee X. Basile G. Acosta M. Scott G. Roskelley C. Medrano E.E. Linskens M. Rubelj I. Pereira-Smith O. Peacocke M. Campisi J. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 9363-9367Crossref PubMed Scopus (5738) Google Scholar). Senescence Rescue Assay—Primary MEFs were co-infected with the retroviral vector pBabe or its derivative expressing oncogenic ras and with pMSCV or its derivatives expressing anti-PEA-15 RNA hairpins. Two days after double selection (2 μg/μl puromycin for 3 days and 75 μg/μl hygromycin for 5 days), all populations expressing oncogenic ras entered senescence, whereas the control cells remained growing for one additional week (two passages) and then entered into senescence induced by culture shock (13Sherr C.J. DePinho R.A. Cell. 2000; 102: 407-410Abstract Full Text Full Text PDF PubMed Scopus (658) Google Scholar). We kept all of these cells for another 2 weeks in subconfluent cultures, changing media every 3 days. We used morphological criteria and the SA-β-gal assay to evaluate whether the cells remained senescent or resumed proliferation. Protein Expression—To prepare total cell protein, cells were collected by trypsinization, washed with PBS, lysed in 100 μl of SDS sample buffer (60 mm Tris-HCl pH 6.8, 10% glycerol, 2% SDS, and 5% 2-mercaptoethanol), and boiled for 5 min. For Western blots, 20 μg of total cell protein were separated on SDS-polyacrylamide gels and transferred to Immobilon-P membranes (Millipore). The primary antibodies used were anti-pRb (G3–245, 1:250; BD Biosciences), anti-p53 (CM5, 1:2000; Novocastra), anti-mouse PML (36-1-104, 1:5000; a gift from S. Lowe), anti-phosphoserine 259 Raf, anti-phospho-MEK1/2, anti-phospho ERK1/2, anti-phospho-p90Rsk, anti-phospho-Elk1 (Phospho-ERK1/2 Pathway sampler kit, all used 1:500; Cell Signaling), anti-total ERK (K-23. 1:1000; Santa Cruz Biotechnology), anti-PEA-15 (1:1000, kindly provided by Dr J. Ramos, Rutgers University), and anti-α-tubulin (B-5–1-2, 1:5000; Sigma). Signals were revealed after incubation with anti-mouse or anti-rabbit secondary antibodies coupled to peroxidase (Amersham Biosciences) by using enhanced chemiluminescence (ECL, Amersham Biosciences) or Lumi-Light Plus (Roche Applied Science). ERK1/2 kinase assays were performed according to an immunoprecipitation/kinase assay (Cell Signaling Technologies Inc.) using 5 μg of GST-Elk1 as substrate. Phosphorylated GST-Elk1 was detected by Western blot using the anti-phospho-Elk1 (Ser-383) antibody (Cell Signaling). Fluorescence Microscopy—For fluorescence microscopy, 2 × 104 control IMR90 cells or Ras-senescent IMR90 cells were plated on coverslips placed on 12-well plates (Costar, Corning Inc.). Twenty-four hours after plating, the cells were fixed with 4% paraformaldehyde for 15 min at room temperature. Subsequently, the cells were washed in PBS and permeabilized using ice-cold 0.2% Triton X-100 in PBS/bovine serum albumin solution for 5 min. Then the cells were washed three times with PBS/bovine serum albumin and incubated for 1 h at room temperature with anti-phospho-ERK1/2 (9106, 1:300; Cell Signaling); anti-p53 (CM5, 1:50, Novocastra), or anti-mouse PML (36-1-104, 1:200) as primary antibodies. Next, the cells were washed and incubated for 1 h with Oregon-Green-conjugated secondary antibody (1:1000; Molecular Probes, Eugene, OR) or (Alexa 568 anti-mouse, 1:2000; Molecular Probes). Finally, the cells were washed three times with PBS, incubated in 300 nm 4′,6-diamidino-2-phenylindole for 10 min, and mounted on microscope slides. Images were obtained using MetaMorph software (Universal Imaging Corp.) and processed using Adobe Photoshop 6.0. For studying ERK localization in MEFs, cells were first serum-starved for 48 h and later fixed at different times after adding serum. The staining and image processing was as described above. Oncogenic ras Induces a Constitutive Stimulation of the MAP Kinase Pathway—It is known that both proliferation and senescence induced by oncogenic ras (RasV12) require signaling through the ERK1/2 MAP kinases (14Lin A.W. Barradas M. Stone J.C. van Aelst L. Serrano M. Lowe S.W. Genes Dev. 1998; 12: 2997-3007Crossref PubMed Google Scholar, 15Zhu J. Woods D. McMahon M. Bishop J.M. Genes Dev. 1998; 12: 3008-3019Crossref PubMed Scopus (766) Google Scholar). However, MAP kinase signaling is the subject of a negative feedback regulation by the ERK1/2-dependent induction of specific phosphatases that dephosphorylate ERK1/2 (16Brondello J.M. Brunet A. Pouyssegur J. McKenzie F.R. J. Biol. Chem. 1997; 272: 1368-1376Abstract Full Text Full Text PDF PubMed Scopus (318) Google Scholar, 17Sun H. Charles C.H. Lau L.F. Tonks N.K. Cell. 1993; 75: 487-493Abstract Full Text PDF PubMed Scopus (1027) Google Scholar). In particular, it has been reported that the levels of MKP-2 are increased after the induction of cellular senescence by serial passage (replicative senescence) (18Torres C. Francis M.K. Lorenzini A. Tresini M. Cristofalo V.J. Exp. Cell Res. 2003; 290: 195-206Crossref PubMed Scopus (38) Google Scholar). Consequently, it is possible that cell cycle exit in response to oncogenic ras in primary fibroblasts is due to these feedback signals mediated by protein phosphatases. To test this idea, we infected human diploid fibroblast IMR90 cells with a retrovirus expressing oncogenic ras and a vector control. As shown previously (3Serrano M. Lin A.W. McCurrach M.E. Beach D. Lowe S.W. Cell. 1997; 88: 593-602Abstract Full Text Full Text PDF PubMed Scopus (3965) Google Scholar, 10Ferbeyre G. de Stanchina E. Querido E. Baptiste N. Prives C. Lowe S.W. Genes Dev. 2000; 14: 2015-2027Crossref PubMed Google Scholar, 14Lin A.W. Barradas M. Stone J.C. van Aelst L. Serrano M. Lowe S.W. Genes Dev. 1998; 12: 2997-3007Crossref PubMed Google Scholar, 19Ferbeyre G. de Stanchina E. Lin A.W. Querido E. McCurrach M.E. Hannon G.J. Lowe S.W. Mol. Cell. Biol. 2002; 22: 3497-3508Crossref PubMed Scopus (248) Google Scholar), Ras-expressing cells entered a permanent cell cycle arrest with features of cellular senescence (Fig 1, A and B). To verify whether ERK1/2 remained active during Ras-induced senescence or whether their activity is down-regulated by dephosphorylation, we performed a series of immunoblots with antibodies recognizing phosphorylated forms of ERK1/2, their upstream regulators (Raf and MEK1/2), and two of their substrates (Elk-1 and p90Rsk). The results of such an analysis are presented in Fig. 1C. In Ras-expressing cells, all members of the ERK1/2 pathway downstream of Ras displayed a pattern of phosphorylation consistent with constitutive signaling. Interestingly, the downstream target of Ras, Raf, was dephosphorylated at Ser-259 in cells expressing oncogenic ras. Phosphorylation of Raf at this residue is considered to inhibit its activity (20Dhillon A.S. Pollock C. Steen H. Shaw P.E. Mischak H. Kolch W. Mol. Cell. Biol. 2002; 22: 3237-3246Crossref PubMed Scopus (190) Google Scholar). Whether oncogenic ras mediates this dephosphorylation by activating a phosphatase or inhibiting a protein kinase requires further study. We concluded that the MAP kinases ERK1/2 remained active and phosphorylated both at the beginning of the senescent arrest induced by Ras (4 days after infection) and when the senescent arrest was well established (10 days after infection). Clearly, exit from the cell cycle in response to oncogenic ras is not the result of inactivation of the ERK1/2 pathway by dephosphorylation. Constitutive Expression of Activated ERK Is Not Sufficient to Induce Senescence—Senescence can also be induced in primary fibroblasts by activated alleles of Raf and MEK, which, like Ras, activate ERK1/2 (14Lin A.W. Barradas M. Stone J.C. van Aelst L. Serrano M. Lowe S.W. Genes Dev. 1998; 12: 2997-3007Crossref PubMed Google Scholar, 15Zhu J. Woods D. McMahon M. Bishop J.M. Genes Dev. 1998; 12: 3008-3019Crossref PubMed Scopus (766) Google Scholar). Because we showed that all the members of the Ras-ERK1/2 signal transduction cascade (Ras, Raf, MEK, and ERK1/2) are constitutively active during Ras-induced senescence, we decided to investigate whether an activated ERK2 allele also induces senescence. To do this experiment we used the constitutively active ERK2* described by Ahn and colleagues (11Emrick M.A. Hoofnagle A.N. Miller A.S. Ten Eyck L.F. Ahn N.G. J. Biol. Chem. 2001; 276: 46469-46479Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). This allele bears mutations that enhance the rate of autophosphorylation of ERK2 at Thr-183 and Tyr-185, which are the sites phosphorylated by the ERK activators MEK1/2. More importantly, expression of this constitutively active ERK2 allele in human embryonic kidney cells (293) led to phosphorylation of the ERK substrates Elk1 and Rsk as efficiently as does a constitutively active MEK1 allele (11Emrick M.A. Hoofnagle A.N. Miller A.S. Ten Eyck L.F. Ahn N.G. J. Biol. Chem. 2001; 276: 46469-46479Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). Surprisingly, activated ERK2* did not induce senescence (Fig 1, A and B) despite activating Rsk to a similar extent as Ras did (Fig. 1D). We also performed immunoprecipitation/kinase assays, using an excess of GST-Elk1 as a substrate. In this experiment, phospho-ERK, immunoprecipitated from ERK2*-expressing cells was as active as the phospho-ERK1/2 immunoprecipitated from control cells growing in serum or RasV12-expressing cells (Fig. 1E). Together, our results are consistent with those reported before about this ERK2* allele (11Emrick M.A. Hoofnagle A.N. Miller A.S. Ten Eyck L.F. Ahn N.G. J. Biol. Chem. 2001; 276: 46469-46479Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). However, we can not entirely eliminate the possibility that the two mutations present in ERK2* impair its interactions with key substrates that mediate cell cycle arrest. Alternatively, ERK2* and RasV12-activated ERK1/2 may differ in some other functions of the ERK1/2 MAP kinase pathway. Phosphorylated ERKs Localized Predominantly to the Cytoplasm during Ras-induced Senescence—To further investigate ERK physiology during senescence and explain why activated Ras, Raf, or MEK but not ERK induced senescence, we performed localization studies by indirect immunofluorescence using antibodies specific for phosphorylated ERK1/2. We found that phosphorylated ERK1/2 (phospho-ERK) localized both in the in the nucleus and in the cytoplasm early after introducing oncogenic ras in primary fibroblasts, when cells are highly proliferative (Fig. 2). The same pattern of staining was observed for control cells bearing an empty vector. This situation is the result of the steady state equilibrium of ERK shuttling between the nucleus and the cytoplasm (21Brunet A. Roux D. Lenormand P. Dowd S. Keyse S. Pouyssegur J. EMBO J. 1999; 18: 664-674Crossref PubMed Scopus (517) Google Scholar). However, by the time cells entered senescence (6 days after the introduction of Ras), a significant number of cells (∼60%) displayed phospho-ERK mainly in the cytoplasm, apparently excluding the nucleus and often forming a perinuclear halo (Fig. 2). In contrast, the constitutively active ERK2*, which failed to induce senescence, was found predominantly localized in the nucleus (Fig. 2). In addition, blocking Ras-induced senescence with the adenoviral oncoprotein E1A was accompanied by a dramatic enrichment of phospho-ERK1/2 in the nucleus. A similar pattern of staining was also observed in cells that were serum-starved and then serum-stimulated (Fig. 2). Thus, senescence strongly correlated with the nuclear exclusion of phospho-ERK1/2. We then treated Ras-senescent cells with the CRM1 (chromosome region maintenance 1) inhibitor leptomycin B, which blocks nuclear export. We reasoned that this drug should relocalize ERK to the nucleus in Ras-senescent cells if an active export mechanism was responsible for the observed cytoplasmic ERK localization. The results, shown in Fig. 3, indicate that leptomycin B induced the relocalization of phospho-ERK1/2 to the nucleus of Ras-senescent cells. The data are consistent with a model in which phospho-ERK1/2 freely enter the nucleus followed by an active export to the cytoplasm during Ras-induced senescence. However, it is also plausible that an ERK1/2-anchoring protein required nuclear export to localize in the cytoplasm and was therefore able to retain ERK1/2 in this cellular compartment. E1A Inhibits Ras-induced Senescence in Human Cells and Blocks the Expression of the ERK Export Factor PEA-15—Ras-induced senescence in human cells can be efficiently bypassed by the oncoprotein E1A from the adenovirus (3Serrano M. Lin A.W. McCurrach M.E. Beach D. Lowe S.W. Cell. 1997; 88: 593-602Abstract Full Text Full Text PDF PubMed Scopus (3965) Google Scholar, 10Ferbeyre G. de Stanchina E. Querido E. Baptiste N. Prives C. Lowe S.W. Genes Dev. 2000; 14: 2015-2027Crossref PubMed Google Scholar). E1A has multiple activities, including inhibition of the Rb and p53 tumor suppressors (22Lill N.L. Grossman S.R. Ginsberg D. DeCaprio J. Livingston D.M. Nature. 1997; 387: 823-827Crossref PubMed Scopus (596) Google Scholar, 23Whyte P. Buchkovich K.J. Horowitz J.M. Friend S.H. Raybuck M. Weinberg R.A. Harlow E. Nature. 1988; 334: 124-129Crossref PubMed Scopus (1038) Google Scholar, 24Whyte P. Williamson N.M. Harlow E. Cell. 1989; 56: 67-75Abstract Full Text PDF PubMed Scopus (542) Google Scholar). However, inhibition of Rb and p53, using the oncoproteins E6 and E7 from human papillomavirus, did not rescue the cell cycle arrest induced by oncogenic ras in primary human fibroblasts (25Mallette F.A. Goumard S. Gaumont-Leclerc M.F. Moiseeva O. Ferbeyre G. Oncogene. 2004; 23: 91-99Crossref PubMed Scopus (76) Google Scholar), nor did it prevent ERK localization in the cytoplasm (not shown). Obviously, additional E1A targets are responsible for maintaining Ras-induced cell cycle arrest and ERK localization in the cytoplasm. One attractive hypothesis is that E1A affects proteins responsible for cytoplasmic localization of ERK1/2. Hence, E1A can be used as a tool to discover the mechanism responsible for cytoplasmic ERK localization in senescent cells. We asked whether E1A targets factors known to mediate ERK nuclear export. An obvious candidate was PEA-15, recently characterized as an ERK nuclear export factor (26Formstecher E. Ramos J.W. Fauquet M. Calderwood D.A. Hsieh J.C. Canton B. Nguyen X.T. Barnier J.V. Camonis J. Ginsberg M.H. Chneiweiss H. Dev. Cell. 2001; 1: 239-250Abstract Full Text Full Text PDF PubMed Scopus (258) Google Scholar). PEA-15 binds ERK1/2 in the nucleus and uses CRM1-mediated nuclear export to localize them to the cytoplasm. Accordingly, PEA-15 null cells displayed increased cell proliferation and constitutive localization of ERK in the nucleus (26Formstecher E. Ramos J.W. Fauquet M. Calderwood D.A. Hsieh J.C. Canton B. Nguyen X.T. Barnier J.V. Camonis J. Ginsberg M.H. Chneiweiss H. Dev. Cell. 2001; 1: 239-250Abstract Full Text Full Text PDF PubMed Scopus (258) Google Scholar). Interestingly, E1A inhibited Ras-induced senescence (Fig 4, A and B) and dramatically reduced the expression of PEA-15 in human fibroblasts (Fig 4C). This finding suggested that PEA-15 could control ERK localization during Ras-induced senescence. RNAi-mediated Inactivation of PEA-15, an ERK1/2 Nuclear Export Factor, Rescued Primary Murine Embryo Fibroblasts from Ras-induced Senescence—To confirm a role for PEA-15 in cytoplasmic ERK localization during Ras-induced senescence, we blocked its expression using RNAi. To induce RNAi, we designed two retroviral vectors that express RNA hairpins with complementary sequences for mouse PEA-15 (Fig. 5A). Cells expressing one of these hairpins showed reduction of PEA-15 expression 2 days after infection (Fig. 5B). More importantly, although these cells entered into Ras-induced senescence, many of them escaped senescence, forming foci of highly proliferative cells that can be established as permanent, immortalized cultures. This ability to escape senescence induced by PEA-15 RNAi was scored in four independent experiments using four different MEF isolates. In contrast, cells with the non-functional hairpin or cells with an empty vector used as a control rarely did so (Fig. 5C). In MEFs, Ras-induced senescence depends on p53 functions and the induction of p53 targets genes such as PML (3Serrano M. Lin A.W. McCurrach M.E. Beach D. Lowe S.W. Cell. 1997; 88: 593-602Abstract Full Text Full Text PDF PubMed Scopus (3965) Google Scholar, 27de Stanchina E. Querido E. Narita M. Davuluri R.V. Pandolfi P.P. Ferbeyre G. Lowe S.W. Mol. Cell. 2004; 13: 523-535Abstract Full Text Full Text PDF PubMed Scopus (263) Google Scholar). We used immunofluorescence and immunoblots to evaluate the status of p53 and PML at day 10 after infection. By this time, all cells expressing oncogenic ras were arrested and senescent, whereas cells expressing oncogenic ras and the anti-PEA-15 hairpin were divided into two populations, one of senescent cells and one of small cells that escaped senescence. Cells with the vector controls or with the anti-PEA-15 hairpin alone were also in senescence due to culture shock (13Sherr C.J. DePinho R.A. Cell. 2000; 102: 407-410Abstract Full Text Full Text PDF PubMed Scopus (658) Google Scholar). All cell populations expressed p53 and PML immunoreactive material, but the strongest signal was found in cells expressing oncogenic ras alone. Blocking PEA-15 by RNA interference reduced p53 and PML levels in Ras-expressing cells to the levels seen in cells with vector alone (Fig. 5, D and E). This reduction was observed in all cells of the population (senescent and non-senescent), and, therefore, it was not a consequence of escaping senescence. Thus, escape from senescence of cells expressing an anti-PEA-15 RNA hairpin was not due to mutations that result in a loss of p53 expression. Altogether, our data suggest that PEA-15 is required to connect signals from oncogenic ras to p53 and maintain the state of senescence. To investigate whether blocking PEA-15 by RNAi would increase the nuclear localization of ERK1/2, we studied ERK1/2 localization in cells expressing oncogenic ras or oncogenic ras plus the anti-PEA-15 hairpin. For this experiment we used cells at day 10 after infection. To limit the effects that the cell cycle and cell proliferation may have on ERK localization, we serum-starved the cells for 48 h, added serum for 1 h, and then fixed the cells for immunofluorescence. As shown previously, most cells expressing oncogenic ras alone displayed a cytoplasmic staining for phospho-ERK1/2. This finding is consistent with reports indicating that senescent cells do not respond to serum stimulation (28Meyyappan M. Atadja P.W. Riabowol K.T. Biol. Signals. 1996; 5: 130-138Crossref PubMed Scopus (26) Google Scholar). However, more than half of the cells expressing the anti-PEA-15 hairpin exhibited a predominant nuclear staining of phospho-ERK1/2 (Fig. 6B). Hence, rescuing senescence by blocking PEA-15 expression correlates very well with an increase in the nuclear localization of phopho-ERK1/2. To further characterize how blocking PEA-15 functions affects ERK localization, we developed a kinetic protocol to look for ERK nuclear export in cells expressing oncogenic ras alone or with an anti-PEA-15 RNA hairpin. Again, we used cells at day 10 after infection and treated them with leptomycin B to allow for nuclear accumulation of ERK1/2. Then we studied ERK localization at different times after the withdrawal of leptomycin B. In cells expressing oncogenic ras alone, ERK quickly moved out of the nucleus to accumulate in the cytoplasm. In cells expressing oncogenic ras and the anti-PEA-15 hairpin, ERKs were retained for a longer time in the nucleus. One hour after the withdrawal of leptomycin B, ERK1/2 was found both in the nucleus and in the cytoplasm (Fig. 7). Similar results were obtained with an anti-ERK1/2 antibody that recognizes non-phosphorylated ERK1/2. The main conclusion from this experiment is that PEA-15 plays an important role in controlling the nuclear export of phospho-ERK1/2. However, because ERK1/2 still manage to exit the nucleus in cells with very low levels of PEA-15, other factors might regulate ERK1/2 nuclear localization as well. The ERK1/2 MAP kinase pathway conveys mitogenic signals from growth factor receptors. However, this pathway also mediates a senescent cell cycle arrest in normal fibroblasts expressing oncogenic ras (3Serrano M. Lin A.W. McCurrach M.E. Beach D. Lowe S.W. Cell. 1997; 88: 593-602Abstract Full Text Full Text PDF PubMed Scopus (3965) Google Scholar) or in cells from transgenic mice that age prematurely due to expression of a short isoform of the p53 tumor suppressor protein (29Maier B. Gluba W. Bernier B. Turner T. Mohammad K. Guise T. Sutherland A. Thorner M. Scrable H. Genes Dev. 2004; 18: 306-319Crossref PubMed Scopus (492) Google Scholar). Here we present evidence indicating that the localization of ERK1/2 distinguishes mitogenic signaling from senescence. First, ERK1/2 localize mainly in the cytoplasm of senescent cells. Second, the viral oncoprotein E1A, which blocks Ras-induced senescence and stimulates transformation, prevents the cytoplasmic localization of ERK1/2. Third, E1A inhibits the expression of the ERK1/2 export factor PEA-15. Finally, RNAi-mediated inhibition of PEA-15 stimulated the escape from senescence in mouse fibroblasts expressing oncogenic ras and reduced the levels of p53 induced by oncogenic ras. In quiescent cells, ERK1/2 are normally localized in the cytoplasm. Upon stimulation by growth factors, ERK1/2 are phosphorylated by MEK (MAPKK), which induces their translocation to the nucleus (30Lenormand P. Brondello J.M. Brunet A. Pouyssegur J. J. Cell Biol. 1998; 142: 625-633Crossref PubMed Scopus (193) Google Scholar). It has been proposed that MEK acts as a cytoplasmic anchor for inactive ERK1/2 and that their interaction is disrupted after phosphorylation of ERK1/2 by MEK, allowing the nuclear import of ERK1/2 (31Adachi M. Fukuda M. Nishida E. EMBO J. 1999; 18: 5347-5358Crossref PubMed Scopus (241) Google Scholar). However, we think it is unlikely that MEK anchors ERK1/2 in senescent cells. MEK and ERK1/2 remained constitutively phosphorylated during Ras-induced senescence and, therefore, were unable to form stable complexes in these cells. Cytoplasmic accumulation of phosphorylated ERKs was observed in cells stimulated with angiotensin II and treated with protein kinase C inhibitors (32Seta K. Nanamori M. Modrall J.G. Neubig R.R. Sadoshima J. J. Biol. Chem. 2002; 277: 9268-9277Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar). Interestingly, upon treatment with leptomycin B, ERK1/2 nuclear localization was restored (32Seta K. Nanamori M. Modrall J.G. Neubig R.R. Sadoshima J. J. Biol. Chem. 2002; 277: 9268-9277Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar). These observations are consistent with a model in which ERK1/2 are translocated to the nucleus after MEK-dependent phosphorylation and exported to the cytoplasm via CRM1 by a factor normally inhibited by protein kinase C activity. Intriguingly, PEA-15 is phosphorylated by protein kinase C (26Formstecher E. Ramos J.W. Fauquet M. Calderwood D.A. Hsieh J.C. Canton B. Nguyen X.T. Barnier J.V. Camonis J. Ginsberg M.H. Chneiweiss H. Dev. Cell. 2001; 1: 239-250Abstract Full Text Full Text PDF PubMed Scopus (258) Google Scholar), suggesting that it might be the protein kinase C-sensitive factor that controls ERK localization. Consistent with this idea, we have shown here that PEA-15 is required for ERK-nuclear export and for maintaining Ras-induced senescence. Also, the ability of E1A to block senescence correlated with a dramatic reduction of PEA-15 levels. The factors controlling ERK localization are, for the most part, still unknown. In addition to nuclear export, several studies have indicated that ERKs can be retained in specific compartments by anchoring proteins. For example, the β-arrestins have been implicated in mediating a cytoplasmic sequestration of ERK1/2 (33Tohgo A. Pierce K.L. Choy E.W. Lefkowitz R.J. Luttrell L.M. J. Biol. Chem. 2002; 277: 9429-9436Abstract Full Text Full Text PDF PubMed Scopus (329) Google Scholar), and unidentified nuclear anchoring proteins may sequester ERKs in the nucleus (34Pouyssegur J. Volmat V. Lenormand P. Biochem. Pharmacol. 2002; 64: 755-763Crossref PubMed Scopus (361) Google Scholar). For these reasons, it is not a surprise that knocking out PEA-15 expression in our experiments did not block entirely the process of ERK nuclear export or the ability of primary MEFs to enter oncogenic ras-induced senescence. Genetically, MEFs with low PEA-15 levels behaved similarly to MEFs null for the candidate tumor suppressor Mnt. Mnt is a Max-interacting transcriptional repressor that suppresses Myc-dependent transactivation. Mnt null MEFs, like PEA-15-disabled MEFs, arrested proliferation in response to oncogenic ras but frequently escaped from senescence (35Hurlin P.J. Zhou Z.Q. Toyo-oka K. Ota S. Walker W.L. Hirotsune S. Wynshaw-Boris A. EMBO J. 2003; 22: 4584-4596Crossref PubMed Scopus (74) Google Scholar). Mnt-induced immortalization paralleled with inhibition of the expression of Myc target genes, including CDK4 (35Hurlin P.J. Zhou Z.Q. Toyo-oka K. Ota S. Walker W.L. Hirotsune S. Wynshaw-Boris A. EMBO J. 2003; 22: 4584-4596Crossref PubMed Scopus (74) Google Scholar), confirming the well known role of myc as an immortalizing oncogene (36Land H. Parada L.F. Weinberg R.A. Nature. 1983; 304: 596-602Crossref PubMed Scopus (1963) Google Scholar). Whether PEA-15 or cytosolic ERK1/2 stimulate Mnt functions or block Myc activity requires further study. A defective accumulation of ERK1/2 in the nucleus was reported previously in cells that entered senescence after serial passage (37Lim I.K. Won Hong K. Kwak I.H. Yoon G. Park S.C. Mech. Ageing Dev. 2000; 119: 113-130Crossref PubMed Scopus (50) Google Scholar, 38Tresini M. Lorenzini A. Frisoni L. Allen R.G. Cristofalo V.J. Exp. Cell Res. 2001; 269: 287-300Crossref PubMed Scopus (54) Google Scholar) or the introduction of oncogenic ras (37Lim I.K. Won Hong K. Kwak I.H. Yoon G. Park S.C. Mech. Ageing Dev. 2000; 119: 113-130Crossref PubMed Scopus (50) Google Scholar). In the study described by Park and colleagues, the reduction in nuclear ERK was accompanied by the nuclear accumulation of actin and Racl proteins and a marked induction of RhoA expression (37Lim I.K. Won Hong K. Kwak I.H. Yoon G. Park S.C. Mech. Ageing Dev. 2000; 119: 113-130Crossref PubMed Scopus (50) Google Scholar). To explain their observations, they suggested that senescent cells have a general defect in protein translocation across the nuclear membrane. This argument is in apparent conflict with our model proposing normal nuclear import followed by the active export of ERK1/2 from the nucleus by PEA-15. On the other hand, our results are entirely consistent with the idea proposed by both groups (37Lim I.K. Won Hong K. Kwak I.H. Yoon G. Park S.C. Mech. Ageing Dev. 2000; 119: 113-130Crossref PubMed Scopus (50) Google Scholar, 38Tresini M. Lorenzini A. Frisoni L. Allen R.G. Cristofalo V.J. Exp. Cell Res. 2001; 269: 287-300Crossref PubMed Scopus (54) Google Scholar) that a failure to accumulate ERK in the nucleus explains the lack of response to proliferative stimuli exhibited by senescence cells. It remains to be investigated whether cytoplasmic ERK signaling plays a role in senescence or cell cycle control. Our work has important implications for the understanding of the interplay between factors supporting the transforming effect of oncogenic ras and factors that oppose it. It is well established that tumor suppressor pathways controlled by Rb, p53, and PML block Ras-dependent transformation via the establishment of a senescent cell cycle arrest (3Serrano M. Lin A.W. McCurrach M.E. Beach D. Lowe S.W. Cell. 1997; 88: 593-602Abstract Full Text Full Text PDF PubMed Scopus (3965) Google Scholar, 10Ferbeyre G. de Stanchina E. Querido E. Baptiste N. Prives C. Lowe S.W. Genes Dev. 2000; 14: 2015-2027Crossref PubMed Google Scholar, 39Serrano M. Blasco M.A. Curr. Opin. Cell Biol. 2001; 13: 748-753Crossref PubMed Scopus (346) Google Scholar, 40Weinberg R.A. Cell. 1997; 88: 573-575Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar, 41Pearson M. Carbone R. Sebastiani C. Cioce M. Fagioli M. Saito S. Higashimoto Y. Appella E. Minucci S. Pandolfi P.P. Pelicci P.G. Nature. 2000; 406: 207-210Crossref PubMed Scopus (1136) Google Scholar). It is also well known that cellular transformation involves the suppression of the senescence program by different oncogenic activities (42Hahn W.C. Counter C.M. Lundberg A.S. Beijersbergen R.L. Brooks M.W. Weinberg R.A. Nature. 1999; 400: 464-468Crossref PubMed Scopus (1982) Google Scholar, 43Hahn W.C. Dessain S.K. Brooks M.W. King J.E. Elenbaas B. Sabatini D.M. DeCaprio J.A. Weinberg R.A. Mol. Cell. Biol. 2002; 22: 2111-2123Crossref PubMed Scopus (519) Google Scholar). One particularly powerful anti-senescence oncogene is the adenoviral oncoprotein E1A (3Serrano M. Lin A.W. McCurrach M.E. Beach D. Lowe S.W. Cell. 1997; 88: 593-602Abstract Full Text Full Text PDF PubMed Scopus (3965) Google Scholar, 10Ferbeyre G. de Stanchina E. Querido E. Baptiste N. Prives C. Lowe S.W. Genes Dev. 2000; 14: 2015-2027Crossref PubMed Google Scholar). Unlike other oncoproteins such as papillomavirus E6/E7, E1A is sufficient to rescue normal fibroblasts from Ras-induced arrest (25Mallette F.A. Goumard S. Gaumont-Leclerc M.F. Moiseeva O. Ferbeyre G. Oncogene. 2004; 23: 91-99Crossref PubMed Scopus (76) Google Scholar). E1A, like E6, blocks the transcriptional activity of p53 (22Lill N.L. Grossman S.R. Ginsberg D. DeCaprio J. Livingston D.M. Nature. 1997; 387: 823-827Crossref PubMed Scopus (596) Google Scholar) and, like E7, binds Rb blocking its ability to repress E2F-dependent promoters (23Whyte P. Buchkovich K.J. Horowitz J.M. Friend S.H. Raybuck M. Weinberg R.A. Harlow E. Nature. 1988; 334: 124-129Crossref PubMed Scopus (1038) Google Scholar, 24Whyte P. Williamson N.M. Harlow E. Cell. 1989; 56: 67-75Abstract Full Text PDF PubMed Scopus (542) Google Scholar, 44Fischer R.S. Quinlan M.P. J. Virol. 1998; 72: 2815-2824Crossref PubMed Google Scholar). Clearly, therefore, it would seem that E1A must have other activities conferring its unique ability to block Ras-induced senescence. Our data suggest that this new E1A activity results in down-regulation of the ERK1/2 nuclear export factor PEA-15. Thus, PEA-15 is an E1A-modulated gene that normally provides a fail-safe mechanism to avoid constitutive activation of the MAP kinase pathway. As a consequence, we predict that PEA-15 may have a tumor suppressor activity, particularly, in tumors driven by oncogenic ras. We thank Dr. Joe Ramos, Dr. Athena Lin, Alice Rae, and Frédérick A. Mallette for comments. We are also grateful of Dr. J. Ramos (Rutgers University, New Jersey) and Dr. N. Ahn for reagents." @default.
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