Sustained Activation Erk

Physiological induction of ERK signalling depends on upstream activation of RAS by receptor-induced signalling 7, 8. PLX4032 induced ERK signalling in SKBR3 breast.

• You have free access to this content ERK and cell death: Mechanisms of ERK-induced cell death – apoptosis, autophagy and senescence.
• 1. Neurochem Res. 2005 Feb;30 2 :263-70. Role of ERK in hydrogen peroxide-induced cell death of human glioma cells. Lee WC 1, Choi CH, Cha SH, Oh HL, Kim- YK.
• The Ras-ERK pathway. ERK extracellular signal-regulated kinase is a MAPK mitogen-activated protein kinase that functions as the major effector of the Ras oncoprotein.
• Growth factors and mitogens use the Ras/Raf/MEK/ERK signaling cascade to transmit signals from their receptors to regulate gene expression and prevent apoptosis.
• Original Article. Oncogene 2013 32, 564–576; doi:10.1038/onc.2012.88; published online 19 March 2012. Oncogenic KRAS and BRAF activation of the MEK / ERK.

Abstract

Chronic hepatitis C virus HCV infection is a leading cause of liver cirrhosis and hepatocellular carcinoma HCC worldwide. The HCV capside core is a multifunctional protein with regulatory functions that affects transcription and cell growth in vitro and in vivo. Here, we show that both HCV genotype 1a and 3 core proteins activate MEK1 and Erk1/2 MAP kinases and that the costitutive expression of the HCV core results in a high basal activity of Raf1 and MAP/kinase/kinase, as determined by endogenous Raf1 in vitro kinase assay and immunodetection of hyperphosphorylated Erk1 and Erk2 even after a serum starvation. Moreover, the activation of both Erk1/2 and the downstream transcription factor Elk-1 in response to the mitogenic stimulus EGF is significantly prolonged. The sustained response to EGF in cells expressing the HCV core occurs despite a normal induction of the MAP phosphatases MKP regulatory feedback and is likely due to the costitutive activation of Raf-1 activity. The ability of HCV core proteins to directly activate the MAP kinase cascade and to prolong its activity in response to mitogenic stimuli may contribute to the neoplastic transformation of HCV infected liver cells.

Fig. 1. Changes in intracellular Ca 2 concentration, ERK phosphorylation, and H1K activity following fertilization in the Phallusia nigra egg.

S. Cagnol, Department of Anatomy and Cellular Biology, Faculty of Medicine and Health Sciences, University of Sherbrooke, Sherbrooke, Quebec, Canada

Fax: 1 819 564 5320

Tel: 1 819 820 6868 ext. 15715

E-mail: sebcagnol yahoo.frAbstract

The Ras/Raf/extracellular signal-regulated kinase ERK signaling pathway plays a crucial role in almost all cell functions and therefore requires exquisite control of its spatiotemporal activity. Depending on the cell type and stimulus, ERK activity will mediate different antiproliferative events, such as apoptosis, autophagy and senescence in vitro and in vivo. ERK activity can promote either intrinsic or extrinsic apoptotic pathways by induction of mitochondrial cytochrome c release or caspase-8 activation, permanent cell cycle arrest or autophagic vacuolization. These unusual effects require sustained ERK activity in specific subcellular compartments and could depend on the presence of reactive oxygen species. We will summarize the mechanisms involved in Ras/Raf/ERK antiproliferative functions.Provide feedback or get helpYou are viewing our new enhanced HTML article.

If you can t find a tool you re looking for, please click the link at the top of the page to Go to old article view. Alternatively, view our Knowledge Base articles for additional help. Your feedback is important to us, so please let us know if you have comments or ideas for improvement. Enhanced PDFStandard PDF 241.4 KB AbbreviationsATA

aurintricarboxylic acidcPLA2

cytosolic phospholipase A2DAPK

death-associated protein kinaseDUSP

dual-specificity phosphataseEGF

epidermal growth factorERK

extracellular signal-regulated kinaseFADD

Fas-associated death domainGAIP

G-interacting proteinIGF

insulin-like growth factorJNK

c-JunNH2-terminal kinaseMAPK

mitogen-activated protein kinaseMEK1/2

mitogen protein kinase kinase 1 and 2 also known as MAP2K1 and MAP2K2, respectively MEKCA

constitutively activated forms of MEKMKP

mitogen-activated protein kinase phosphataseMOS

v-mos Moloney murine sarcoma viral oncogene homologPARP

poly ADP-ribose polymeraseROS

reactive oxygen speciesTNF

tumor necrosis factorTRAIL

tumor necrosis factor-related apoptosis-inducing ligandRas/Raf/ERK, the pathway

ERK2/ERK1 also known as p42/p44MAPK, respectively, and officially named MAPK 1 and 3 are two isoforms of extracellular signal-regulated kinase ERK that belong to the family of mitogen-activated protein kinases MAPKs, which include ERK5, the c-JunNH2-terminal kinases JNK1/2/3 and the p38 MAP kinases p38 α,β,δ,γ. These enzymes are activated through a sequential phosphorylation cascade that amplifies and transduces signals from the cell membrane to the nucleus. Upon receptor activation, membrane-bound GTP-loaded Ras recruits one of the Raf kinases, A-Raf, B-Raf and C-Raf or Raf1, into a complex where it becomes activated. Then, Raf phosphorylates two serine residues on the kinase mitogen protein kinase kinase 1 and 2 MEK1/2; also known as MAP2K1 and MAP2K2, respectively, which in turn activate ERK1/2 by tandem phosphorylation of threonine and tyrosine residues on the dual-specificity motif T-E-Y. Finally, active ERKs regulate by phosphorylation many cytoplasmic and nuclear targets that perform important biological functions 1.

Depending on the duration, the magnitude and its subcellular localization, ERK activation controls various cell responses, such as proliferation, migration, differentiation and death 2. Protein phosphatases play an important role as negative regulators by controlling the Ras/Raf/ERK signaling pathway at different levels. Phosphotyrosine phosphatases target the tyrosine kinase receptors, whereas phosphoserine/phosphothreonine phosphatases target the adapter protein Shc and MEK1/2. Dual-specificity phosphatases DUSP; also called MAPK phosphatases MKP, are able to dephosphorylate both of the threonine and tyrosine residues within the activation loop of MAPK. Specific DUSPs tightly regulate subcellular ERK activity. DUSP1/MKP-1, DUSP2/PAC-1, DUSP4/MKP-2 and DUSP5 are mainly nuclear, whereas DUSP6/MKP-3, DUSP7/MKP-X and DUSP9/MKP-4 are cytoplasmic. Moreover, the expression of DUSP1, -2, -4 and -6 is increased following ERK activation 3,4, taking part in a negative feedback loop aimed at terminating Ras/Raf/ERK signaling pathway stimulation.

The Ras/Raf/ERK pathway is frequently deregulated in tumors as a result of activating mutations in Ras or B-Raf, observed particularly in malignant melanoma, pancreas intestine and thyroid tumors cosmic database: Many studies associate its oncogenic potential to increased cell survival, mainly by promoting the activity of antiapoptotic proteins, such as Bcl-2, Bcl-XL, Mcl-1, IAP, and repressing proapoptotic proteins, such as Bad and Bim 5.

Paradoxically, a growing number of studies also suggest that in certain conditions, aberrant ERK activation can promote cell death. This review will summarize the different cellular models in which the Ras/Raf/ERK pathway plays an antiproliferative role. The specific pro-death function of ERK activity in neurons 6 and lymphocytes 7 and its role in cadmium toxicity 8 will also be discussed in this minireview series.Ras/Raf/ERK pathway induces apoptosis

Programmed cell death by apoptosis is a cell-autonomous mechanism that relies on pathway-controlled activation of caspases and nucleases leading to the death of the injured cells without affecting neighboring cells. The intrinsic pathway of apoptosis regulates the activity of the Bcl-2 family proteins that control the integrity of the mitochondrial membrane. The release of proapoptotic factors from the mitochondria, such as cytochrome c, into the cytoplasm promotes the activation of initiator caspase-9, which in turn activates effector caspases such as caspase-3 or -7. The extrinsic pathway relies on the activation of death receptors from the tumor necrosis factor TNF receptor family that promote the recruitment and activation of initiator caspase-8 via adaptor proteins such as Fas-associated death domain FADD or TNFRSF1A-associated via death domain TRADD. Strong caspase-8 activity may directly activate effector caspases; it may also require signal amplification through induction of the intrinsic pathway via cleavage of the Bcl-2 family protein Bid 9.

Early reports of a proapoptotic function of the Ras/Raf/ERK pathway appeared in 1996. Depletion of Raf by the benzoquinone ansamycin geldanamycin was shown to protect MCF-7 cells from apoptosis induced by the antitumor compound taxol 10, whereas MEK antisense cDNA expression prevented bufalin-induced apoptosis in U937 leukemic cells 11. A growing number of studies using MEK inhibitors PD98059, U0126 and expression of dominant negative or constitutively active forms of Ras, Raf, MEK or ERK have confirmed the implication of the Ras/Raf/ERK pathway in the induction of apoptosis see Table 1 for details. Table 1.   Models of ERK-mediated apoptosis and autophagy. LDH, lactate dehydrogenase; MDC, monodansylcadaverine; MTT, 3- 4,5-dimethylthiazol-2-yl -2,5-diphenyl-tetrazolium bromide; ND, not determined; TGHQ, 2,3,5-tris- glutathione-S-yl hydroquinone; TPA, 12-O-tetradecanoylphorbol-13-acetate. Transformed mouse fibroblastEtoposide24 hCaspase-3PD98059 12 NIH 3T3Etoposide

UV

Doxorubicin24 hDNA degradationPD98059 13 Human keratinocytes HaCaTEtoposide24 hDNA condensation Caspase-3

PARP cleavagePD98059

DN ERK 14 Human hepatocellular carcinoma HepG2 and Huh-7DoxorubicinNDPARP cleavagePD98059 16 Human promonocytic leukemiaTPA

ArsenicCadmium

Doxorubicin24 hDNA fragmentation

DNA condensationU0126

PD98059 17 Human breast adenocarcinoma MCF-7Doxorubicin12 hCell viabilityU0126 18 NIH 3T3, human immortalized keratinocytes HaCaTDoxorubicin24 hDNA fragmentation

DN ERK 19 Rat immortalized cardiomyocytes H9c2Doxorubicin48 h nuclearDNA fragmentation

Caspase-9, -3

PARP cleavageU0126 20 NIH 3T3γ irradiation48 hMembrane integrity

Annexin VPD98059

DN ERK 21 Human cervix adenocarcinoma HeLa

Human lung carcinoma A549Cisplatin20 hDNA condensation

Caspase-3

PARP cleavage

Cytochrome c releaseU0126

PD98059 22 Human ovarian adenocarcinoma A2780CisplatinNDDNA fragmentationPD98059 23 Human osteosarcoma Saos-2Cisplatin24 hCell viability

DNA fragmentationU0126

PD98059 24 Rabbit primary renal proximal tubular cellsCisplatin24 hDNA condensation

Caspase-3U0126

PD98059 25 Mouse immortalized proximal tubule cell line TKPTS Cisplatin72 hCaspase-3U0126 26 Human carcinoma NCCIT and NTERACisplatin24 hDNA condensation

Caspase-8, -9, -3U0126

PD98059 27 In vivo mouse kidneyCisplatin injection72 hDNA fragmentation

Caspase-8, -3U0126 28 Opossum immortalized kidney cells OK cellsCisplatin48 hDNA degradation

PD98059

DN MEK 29 Human cervix adenocarcinoma HeLaCisplatin18 hCaspase-9

PARP cleavageU0126 30 Human papillary thyroid carcinoma BHP 2–7 and BHP 18–21Resveratrol24 h nuclearDNA degradationPD98059 33 Human prostate adenocarcinoma PC3Phenethyl isothiocyanate24 hAnnexin V

ROS productionPD98059 35 Human melanoma C8161,WM164 Mel JusoBetulinic acid96 hDNA fragmentation

DNA condensation

PARP cleavageU0126 36 Human cervix adenocarcinoma HeLaApigenin8 hCell viabilityU0126

PD98059 37 Human melanoma A375-S2Oridonin12 hDNA fragmentation

Cytochrome c releasePD98059 38 Human glioblastoma T98G and U87MGMiltefosine12 hCell viabilityU0126 39 Human cervix adenocarcinoma HeLaShikonin12 hCaspase-8, -3PD98059 40 Human breast adenocarcinoma MCF-7Taxol24 hDNA fragmentationPD98059 41 Normal human embryonic kidney HEKTRAILConstitutiveDNA fragmentation

Caspase-8U0126

PD98059 42 Human primary fibroblast BJTRAILNDcell viabilityU0126 43 Human colorectal HT29 cellsTRAIL5 hMembrane integrity

Nuclear condensation

PARP cleavagePD98059 44 Human prostate cancer LNCaPTRAIL4 hAnnexin V

Caspase-3U0126 45 Human prostate tumor DU-145Quercetin

TRAIL24 hMembrane integrityPD98059 34 Human neuroblastoma SHEP-1TRAIL

H2O2NDMembrane integrityPD98059 46 Huaman neuroblastoma SHEPFasL30 minDNA condensationDN MEK1 50 Rat primary Sertoli cells

Human acute T leukemia JurkatFas CH11 5 minMembrane integrity

DNA fragmentationPD98059 51 Diffuse large B-cell lymphomaCD40 ligation3 hMembrane integrity

PD98059 52 Primary human cholangiocytesCD40 ligation24 hDNA condensation

Caspase-3 activityPD98059 53 Pig renal tubular epithelial cells LLC PK1Zinc24 h nuclearCell viabilityROS productionU0126 54 In vivo/isolated rat renal cortical slicesZnCl2 injection90 min nuclearCell viabilityU0126 55 Pig renal tubular epithelial cells LLC PK1TGHQ5 hCell viabilityPD98059 56 Rabbit primary renal proximal tubular cellstert-butylhydroperoxide8 hAnnexin VU0126

PD98059 58 Human primary retinal pigmented epithelial ARPE19 cellstert-butylhydroperoxide6 hCell viability

Caspase-9

DNA fragmentationU0126 59 Murine transformed lung epithelial MLE12Hyperoxia4 hCaspase-9, -3

Cytochrome c releasePD98059 57 In vivo mouse lungHyperoxia72 hDNA fragmentation

Caspase-3PD98059 57 Primary rat pulmonary myofibroblastsONOO 30 minCell viabilityPD98059 60 Immortalized mouse fibroblast L929H2O23 hDNA fragmentation

DN ERK 61 Mouse immortalized osteoblastH2O2Biphasic 12 hCell viability

Membrane integrityPD98059 62 Rabbit primary renal proximal tubular cellsH2O22 h constitutiveDNA condensation

MEKCA 63 Rabbit primary renal proximal tubular cellsH2O22 hMembrane integrity

Annexin VU0126

MEKCA

DN MEK 64 Human transformed bronchial epithelial cell line BEAS-2BNO donor24 hCell viabilityPD98059 65 Human melanomaNO donorNDPARP cleavageU0126 67 HEK293CadmiumBiphasic 96 hCaspase-8, -3

PARPU0126 68 Human hepatocellular carcinoma HepG2Benzo a pyrene48 hCell viabilityPD98059 70 Rat primary pleural mesothelial cellsCrocidotite

Asbestos48 hDNA condensation

ROS productionPD98059 71 In vivo Caenorhabditis elegansArsenicNDDNA fragmentationmek-2 n1989

mpk-1 ku1 72 Murin bone marrow-derived primary osteoclasts and murine long bone-derived osteocytes MLO-Y4Estradiol24 hMembrane integrityU0126 73 Human breast adenocarcinoma MCF-7Tamoxifen1 hMembrane integrityPD98059 74 Human myeloma cell line U266-1984 and RHEK-1Interferon-α16 hAnnexin V

Caspase-3U0126 75 Isolated rat renal cortical slicesCephaloridine90 min nuclearCell viability

ROS productionU0126

PD98059 76 Immortalized rabbit lens epithelial cells N/N1003ACalcimycin10 hMembrane integrity

DNA fragmentation

Annexin V

Cytochrome c release

DN Raf

DN ERK 77 Primary mouse kidney proximal tubular epithelial cellsEGF deprivation120 hCell viabilityU0126

PD98059 78 Primary human bone marrow stromal cellsLeptin12 hCell viability

PD98059 79 Human leukemia U937Bufalin12 hDNA fragmentationMEK antisense 11 Pig renal tubular epithelial cells LLC PK1Escherichia coli toxin44 hAnnexinV

PARP cleavagePD98059 80 Mouse monocyte/macrophage J774.2Francisella tularensis infection42 hAnnexin V

PD98059 81 Human osteosarcoma cell line HOS and U2OSChelerythrine4 hDNA fragmentation

Caspase-8, -9, -7

PARP cleavageMEKCA U0126

DN Ras 82 Mouse embryonary fibroblastRacN17 Cdc42N17NDMembrane integrityPD98059 83 Mouse embryonary fibroblastRacN17 Cdc42N1724 hAnnexin VPD98059

DN DUSP6 84 Rat fibroblast Rat1RAF-CAAXConstitutiveDNA condensationRAF-CAAX 85 Mouse immortalized fibroblast NIH 3T3DAPKConstitutiveCell viabilityMEKCA 87 Murine erythroleukemia

 DP16.1/p53ts cellsP53 inductionConstitutiveAnnexin VU0126

Raf CA 86 Human breast adenocarcinoma MCF-7ΔRAF1ConstitutiveDNA fragmentation

VacuolizationΔRAF1 88 Human primary osteoblastΔRaf1

HrasV12 T35SConstitutiveCell viability

Membrane integrity

DNA fragmentationΔRaf1

HrasV12T35S 89 HEK293TIGF-I receptor48 hMembrane integrity Caspase-3

VacuolizationU0126

MEK siRNA 90 HEK293ΔRaf1:ER

Anti-Fas CH11 ConstitutiveMembrane integrity DNA fragmentation

Caspase-8, -3

VacuolizationΔRaf1:ER

U0126 91 Murine fibrosarcoma cells L929TNFα Cell viability

LC3-II induction

Beclin inductionU0126

PD98059 48 Human breast adenocarcinoma MCF-7TNFα10 hCell viability

LC3-II inductionU0126

PD98059 49 Mouse RAW264.7 macrophagesNO2 h

constitutiveCell viability

BNIP-3 inductionU0126 66 Transformed mouse

 mesengial MES-13 cellsCadmium3 hCell viability MTT

LC3-II inductionPD98059

69 Human colon adenocarcinoma HT29Amino acid depletion4 hGAIP phosphorylation

Autophagic LDH sequestrationPD98059 114 Human colon adenocarcinoma HT29Amino acid depletion

ATA

RasV12S354 h

constitutiveProteolysis

LDH sequestrationRasV12S35 115 Human colon adenocarcinoma

HCT-15Soyaponin3 hMDC incorporationU0126 116 Mouse 42GPA9 Sertoli cell lineLindane24 hLC3 relocalization

VacuolizationPD98059

U0126 117 Primary human lung fibroblasts WI38Dihydrocapsaicin4 hLC3-II induction

Caspase-3, -7

LC3 relocalizationPD98059

ERK siRNA 118 Human OVCA-420PEA-1548 hMembrane integrity

Acidic vacuolesERK siRNA 119 Primary human fibroblast IMR90RasV12ConstitutiveLC3-II induction  120

The proapoptotic function of the Ras/Raf/ERK pathway is well documented for apoptosis induced by DNA-damaging agents, such as etoposide 12–15, doxorubicin 13,16–20, UV 13 and gamma irradiation 21. ERK activity has been particularly implicated in cisplatin-mediated apoptosis in renal cells 15,22–32 Table 1.

ERK activity has also been involved in cell death induced by various antitumor compounds, such as resveratrol 33, quercetin 34, phenethyl isothiocyanate 35, betulinic acid 36, apigenin 37, oridonin 38, miltefosine 39, shikonin 40 or taxol 10,41 Table 1.

Most of these drugs induce the intrinsic apoptotic pathway. However, ERK activity has also been involved in activation of the extrinsic pathway by death receptor ligands such as TNF-related apoptosis-inducing ligand TRAIL 34,42–46, TNFα 47–49, Fas 50,51 or CD40 ligand 52,53. Cell death induced by other death pathways that occur in response to zinc 54,55, oxidation 56–59, especially in response to ONOO 60, H2O2 61–64 or NO treatment 65–67, toxic compounds such as cadmium, 17,68,69, benzo a pyrene 70, asbestos 71 or arsenic 17,72, also require ERK activity. Many death stimuli, such as estradiol 73 or its antagonist tamoxifen 74, interferon-α 75, cephalosporin 76, the calcium mobilizer calcimycin 77, epidermal growth factor EGF deprivation 78, leptin 79, bufalin 11, bacterial infection 80,81, chelerythrine 82 and the dominant negative form of Rac and Cdc42 83,84, are sensitive to inhibition of the Ras/Raf/ERK pathway Table 1.

Conversely, constitutive activation of ERK by dominant active Raf1 combined with c-Myc expression 85 or p53 induction 86, or constitutively active MEK combined with death-associated protein kinase DAPK expression 87, could induce apoptosis without any other stimulus. Moreover, in rare cases, activation of the Raf/ERK pathway alone mediated by Raf1 88,89, RasV12S35 89, insulin-like growth factor type I IGF-I receptor 90 expression or by ΔRaf1:ER induction 91 was sufficient to promote cell death.Mechanisms of Ras/Raf/ERK-mediated apoptosis

ERK activity has been associated with classical markers of apoptosis execution, such as effector caspase-3 activation, poly ADP-ribose polymerase PARP cleavage, annexin-V staining, DNA condensation or DNA fragmentation see Table 1 for details. Depending on the cell type and the nature of the injury, activation of the Ras/Raf/ERK pathway is associated with the intrinsic apoptotic pathway characterized by the release of cytochrome c from mitochondria 22,38,57,77,79 and activation of initiator caspase-9 20,27,29,57,59,82 or with the extrinsic apoptotic pathway, which relies on the activation of an initiator caspase-8 27,28,40,42,68,82. ERK promotes caspase-8 signaling and activation

The Ras/Raf/ERK pathway potentiates activation of death receptors by increasing the level of death ligands such as TNFα 28 or FasL 51 or death receptors such as Fas 39,89,91, DR4 44,46 or DR5 42,44,46. ERK activity also promotes the induction of FADD, an adaptator of caspase-8 to the death receptors 39,91. Because ERK-mediated caspase-8 activation requires de novo protein synthesis 68,91, it may reflect the activation of transcription factors regulated by ERK, such as c-Fos, which has been associated with the upregulation of DR4 and DR5 44. However, we have shown that ERK-induced caspase-8 activation could be independent of Fas and FADD upregulation, suggesting death receptor-independent modes of caspase-8 activation by ERK 91. FADD bears a death effector domain that mediates caspase-8 activation. A very similar structure is found to bind ERK in vanishin 92 and PEA-15 93, proteins that regulate both ERK and FADD activity 94. These observations suggest that differential interactions between death effector domain-containing proteins that bind either ERK or caspase-8 could mediate death receptor-independent activation of the extrinsic pathway of apoptosis.The control of cytochrome c release by Bcl-2 family proteins

As shown above, ERK activity is associated with DNA-damaging agents and antitumor compound-induced apoptosis, which are often described as inducing the intrinsic pathway of apoptosis. Therefore, several studies have suggested that the Ras/Raf/ERK pathway is involved in this pathway. Indeed, ERK activity has been shown to directly affect mitochondrial function by decreasing mitochondrial respiration 25,58 and mitochondrial membrane potential 58,63,79, which could lead to mitochondria membrane disruption and cytochrome c release 22,38,57,77,79. Interestingly, active ERK has been found to be localized to mitochondrial membranes 25,58,63.

ERK activity could also promote cytochrome c release by modulating Bcl-2 family protein expression. MEK/ERK activity has been associated with the upregulation of proapoptotic members of the Bcl-2 family, such as Bax 20,30,40,62,67,77, p53 upregulated modulator of apoptosis PUMA 20,86 and Bak 75, as well as the downregulation of antiapoptotic members, such as Bcl-2 13,18,20,23,41,45,89 and Bcl-Xl 23,45. In addition, ERK-activated caspase-8 induces the release of cytochrome c through proteolytic activation of the proapoptotic member Bid 45. ERK promotes p53 stability and activity

The regulation of Bcl-2 family proteins has been tightly associated with transcriptional activity of the tumor suppressor gene p53. Apoptosis induced by DNA-damaging agents correlates with p53 upregulation and modulation of Bcl-2 family proteins in an MEK-dependent manner 13,18,20,23,24,33,40,41,48,77,95. ERK-mediated p53 upregulation is associated with p53 phosphorylation on serine 15 20,24,33,70,86,95,96, which stabilizes p53 protein and promotes its accumulation by inhibiting an association with Mdm2 96. This is supported by the ability of ERK to bind p53 18,95 and to phosphorylate p53 on serine 15 in vitro 95,96. Moreover, Mdm2 phosphorylation on serine 166, which is associated with its ubiquitin ligase activity toward p53, is inhibited upon sustained ERK activation 97. ERK activity is implicated in p53 phosphorylation on threonine 55, promoting DNA-binding activity and Bcl-2 downregulation 18.

c-Myc, which is stabilized by ERK through phosphorylation on serine 62, increases the proapoptotic functions of p53 98. Interestingly, when combined with c-Myc overexpression, constitutive activation of ERK is sufficient to induce apoptosis in Rat-1 cells 85 and to potentiate TRAIL-induced apoptosis in primary fibroblasts 43.

The use of p53-deficient cells 35,83, p53 siRNA 70, p53 antisense 33,77, p53 inhibitor pifithrin-α 20,33,41, temperature-sensitive allele of p53 86 or inducible p53 24 showed that ERK-mediated p53 expression is required for apoptosis. However, in other studies, the Ras/Raf/ERK pathway is able to induce apoptosis independently of p53 24,35,36,41.

Together, these data suggest that upregulation of the tumor suppressor p53 may be an important mechanism of Ras/Raf/ERK-induced apoptosis.Other mediators of Ras/Raf/ERK-induced apoptosis

Cytosolic phospholipase A2 cPLA2 is a potential mediator of Ras/Raf/ERK pathway-induced apoptosis through intrinsic as well as extrinsic pathways. The Fas receptor in Sertoli cells 51, B cell receptor BCR in B lymphoma 99 or leptin in adipocytes 79, all promote MEK-dependent cPLA2 induction and activation. ERK can directly activate cPLA2 by phosphorylation at serine 505 100,101. The use of cPLA2 inhibitor AACOCF3 suggests that cPLA2 was necessary for ERK-induced apoptosis by a mechanism that promotes FasL induction 51 or cytochrome c release 79.

Like death receptors and FADD, DAPK contains a death domain. ERK was shown to bind to DAPK and increase its catalytic activity by phosphorylation on serine 735 87. DAPK activation results in apoptosis due to cell detachment 87 or increase in TNF receptor function 47.

In MCF-7 cells and primary osteoblasts, activation of Raf/ERK pathway-induced apoptosis was the consequence of cellular detachment from the matrix, which was in this case due to a decrease in integrin β1 expression 88,89.

DNA-damaging agents have been shown to mediate sustained ERK activation through the protein kinase ataxia telangiectasia mutated 13,102,103. Implication of Ras/Raf/ERK pathway during apoptosis in vivoFollowing tissue injury

ERK activity has been clearly implicated in neurodegenerative diseases and brain injury following ischemia/reperfusion in rodents for a review see 6,104,105. The Ras/Raf/ERK pathway also plays a key role in mouse models of acute renal failure induced by cisplatin 28 or lung injury induced by hyperoxia 57, as treatment with MEK inhibitors prevents apoptosis and tissue destruction in these models.During development

Proapoptotic ERK activity has also been reported in developmental models. During germinal cell development, PEA-15, a cytoplasmic death domain-containing protein that binds and sequesters ERK, is highly expressed in the cytoplasm of Sertoli cells, spermatogonia and spermatocytes, inducing a cytoplasmic ERK localization. Interestingly, testis isolated from PEA-15-deficient mice display an abnormal nuclear accumulation of ERK in germinal cells, which correlates with increased apoptosis 106. In Caenorhabditis elegans, loss-of-function alleles of lin-45 RAF homolog, mek-2 MEK homolog and mpk-1 ERK homolog, have presented genetic evidence for a direct role of the Ras/Raf/ERK pathway in germinal cell apoptosis 72. In unfertilized eggs of starfishes Asterina pectinifera and Marthasterias glaciali, v-mos Moloney murine sarcoma viral oncogene homolog MOS -dependent sustained ERK activity led to protein synthesis-dependent synchronous apoptosis 107–109. Moreover, maintaining ERK activity in fertilized eggs by MOS injection is sufficient to induce apoptosis 107. During metamorphosis in ascidian Ciona intestinalis, sustained nuclear activity of ERK homolog Ci-ERK in the tail is required for the induction of apoptosis caspase-3-like activity and necessary for tail regression 110. Finally, during limb development in chick embryos, ERK activity is inhibited in the mesenchyme by FGF8-induced DUSP6 activity. When activated by the expression of constitutively active MEK1, downregulation of DUSP6 or by the expression of a phosphatase-inactive mutant of DUSP6 C294S, sustained ERK activity induces massive apoptosis and prevents limb development 111. These results strongly indicate that Ras/Raf/ERK pathway-mediated apoptosis is not only associated with in vitro manipulation of cell lines, but also plays a key role in vivo during development and following tissue injury.

The role of the Raf/ERK pathway in the induction of cell death should not be restricted to apoptosis, i.e. caspase-dependent cell death. In some cases, the methods used to assess cell viability, based on cell metabolism or membrane permeabilization measurements see Table 1, cannot distinguish between apoptosis and other forms of cell death, such as necrosis or autophagy.Cytoplasmic vacuolization: lysosomal cell death and autophagy

Autophagy is a genetically regulated program, initially identified as a cell survival mechanism to protect from nutrient deprivation. However, in certain conditions, autophagy results in a form of cell death now described as type II programmed cell death 112.

We and others have shown that constitutive activation of ERK by active Raf 88,91, cadmium 68 or IGF-I receptor 90 induced a form of cell death that correlated with extensive cell rounding and the formation of cytoplasmic macrovacuoles, which pushed the nucleus and the cytoplasm to the side of the dying cell. Although cell death was associated with caspase-8 activation 68,91, this massive vacuolization is unrelated to the classical features of apoptosis. This morphology could be a sign of autophagic programmed cell death, but also of paraptosis, a form of caspase-independent cell death associated with cytoplasmic vacuolization 90. Interestingly, other studies using cadmium 69,113 or TNFα treatment 49 have clearly associated ERK activation with autophagic programmed cell death rather than with apoptosis. This is supported by several studies that have associated ERK activity with neuron autophagic cell death in the course of a neurodegenerative disease 6,104,105. In addition, ERK activity has also been associated with autophagy and autophagic cell death in many non-neuronal cellular models see Table 1 in response to different stresses, such as amino acid depletion 114 and aurintricarboxylic acid ATA 115 in human colorectal cancer cell line HT29, soyasaponins 116 in human colon adenocarcinoma HCT-15, lindane 117 in the mouse Sertoli cell line, dihydrocapsaicin 118 in WI38 lung fibroblasts, cadmium in mesengial MES-13 cells 69,113 and TNFα treatment in MCF-7 49 and L929 cells 48. Interestingly, in human ovarian cancer cells, cytoplasmic sequestration of ERK by PEA-15 has been shown to promote autophagy 119. Moreover, direct ERK activation by overexpression of constitutively active MEK can promote autophagy without any other stimulus 117.

ERK-dependent autophagic activity is associated with classical markers of autophagy, such as induction of LC3 and conversion of LC3-I to LC3-II 48,49,118, induction of beclin 1 48, induction of BNIP-3 66 and activation of G-interacting protein GAIP by phosphorylation on serine 151 114. p53 is also associated with the autophagic process, as ERK-mediated phosphorylation of p53 on serine 392 48 was involved in TNFα-induced autophagy.

The lysosomal compartment plays an important role in autophagy by fusing with autolysosome vacuoles. In NIH3T3 and in human colon carcinoma HCT-116 cells, oncogenic forms of Ras, respectively, v-H-Ras and K-Ras, lead to increased sensitivity to the lysosomal cell death pathway induced by cisplatin or etoposide in an MEK-dependent manner. In these models, constitutive ERK activation leads to a decrease in the levels of lysosome-associated membrane protein-1 and -2 due to the induction and activation of cysteine–cathepsin B 15.

In humans, ERK activity is potentially associated with limb sporadic inclusion body myositis, a disease characterized by cytoplasm vacuolization of muscle fibers. Interestingly, immunostaining of muscle samples from patients revealed a strong ERK accumulation in cytoplasmic vacuoles 120.

Together, these results suggest that the Ras/Raf/ERK pathway can mediate autophagic type II programmed cell death.

ERK-induced cytoplasm vacuolization associated with autophagy has some similarity with senescence-associated vacuoles. Interestingly, autophagy has recently been reported to be required for the efficient establishment of senescence induced by a constitutively active form of Ras or MEK 121. The Ras/Raf/ERK pathway and senescence

Cellular senescence is an irreversible form of cell cycle arrest that prevents proliferation of damaged cells or cells that have surpassed their capacity to proliferate. In response to oncogenic hyperproliferative signals, primary cells undergo cell cycle arrest leading to premature oncogene-induced senescence 122. Although aberrant activation of the Ras/Raf/ERK pathway promotes oncogenic transformation of immortalized cells, it is also tightly associated with senescence of primary cells. In human and rodent primary fibroblastic and melanocytic cells, senescence is triggered by constitutively active forms of Ras 123–126, PAK4 125,127, Raf 97,126,128–133 or MEK 123,124,132,134. This process is often prevented by use of MEK inhibitors See Table 2. Table 2.   Models of ERK-mediated senescence. Primary human fibroblastic IMR90Ras V12

MEKCAQ56Pβ-galactosidase activity

p21 p53 and p16/INK4APD98059 123 Primary human fibroblastic BJ cellsRasV12

MEKCAβ-galactosidase activity

p16/INK4AU0126 124 Primary mouse fibroblastic cellsRas V12

PAK4β-galactosidase activity

p21, p19/ARF and p16/INK4APD98059 125 Mouse immortalized NIH 3T3 fibroblastΔRaf1:ERp21  128 Primary human fibroblast IMR90ΔRaf1:ERβ-galactosidase activity

p21 and p16/INK4APD98059 129 Human prostate cancer LNCaP cellsΔRaf1:ERβ-galactosidase activity

p21  130 Mouse embryonic fibroblastΔRaf1:ERp21, p53 and p19/Arf  131 Primary human fibroblast IMR90Raf-CAAX

ΔRaf1:ERβ-galactosidase activity  97 Primary human melanocytes

Primary human fibroblast BJ

In vivo/human naeviB-Raf V600Eβ-galactosidase activity

p21, p53 and p16/INK4A

Heterochromatic foci  127 Primary human melanocytes

In vivo/human NaeviB-Raf V600Eβ-galactosidase activity

Heterochromatic foci

p16/INK4A  135 In vivo/mouse lung tumor modelB-Raf V600Ep19/ARF Dec1

Heterochromatic foci  133 Primary human melanocytesB-Raf V600E

p16/INK4A, γH2AX

Heterochromatic foci  132 Primary human melanocytesB-Raf V600E

NRasQ61Rβ-galactosidase activity

p53 and p16/INK4A  126 Primary human intestinal epithelial cellsMEKCA SS218/222DDβ-galactosidase activity

p21 p53 and p16/INK4A  134 Primary human fibroblast WI38DUSP4 MKP-2 β-galactosidase activity  136 Mechanisms of ERK-induced senescence

Ras/Raf/ERK pathway-induced senescence correlates with increased β-galactosidase activity and induction of classical senescence-associated genes, such as p16/INK4A, p53, p21 and p14-p19/ARF Table 2, senescence-associated heterochromatic foci 127,132 and DNA damage foci 132. In human primary fibroblasts IMR90, when senescence is provoked by inducible activation of Raf1:ER, it correlates with inhibition of AKT and dephosphorylation of Mdm2, which lead to p53 accumulation and growth arrest 97. In the case of human BJ foreskin primary fibroblasts, senescence induced by ectopic expression of RasV12 or constitutively activated forms of MEK MEKCA requires ERK-induced p38 activation 124. Interestingly, a phenotypic comparison between RasV12-, RasR61- and B-RafE600-induced senescence in human melanocytes suggests that the senescence programs are different: Ras-induced senescence was faster and was associated with massive cytoplasmic vacuolization, whereas B-Raf-expressing cells exhibited a more rounded morphology 126,132. However, human and rodent cells induce different senescence programs. The use of mouse embryonic fibroblasts derived from knock-out mouse models suggested that Ras/Raf/ERK pathway-induced senescence relies on the induction of cell cycle regulators, such as p16/INK4A 123,125, p21 126, p53 123, p19/ARF 125. In human primary fibroblasts, however, knock-down or inhibition of either p16 127 or p53 126 was not sufficient to reverse senescence, suggesting that these genes may have a redundant function controlling human senescence. Oncogene-induced senescence prevents transformation of human primary cells unless overridden by the presence of a cooperating oncogene, such as Myc. Indeed, overexpression of c-Myc in normal human melanocytes suppressed B-Raf- or N-Ras-induced senescence 126. Myc expression is then continuously required for transformation, as downregulation of c-Myc in tumor-derived melanoma cells was shown to induce senescence 126. Senescence and subcellular ERK localization

ERK-induced senescence has been associated with an aberrant control of its spatial activity. Kim et al. 135 found that reactive oxygen species ROS produced during senescence of human primary fibroblasts inactivate the cytosolic ERK phosphatase DUSP6, resulting in cytoplasmic sequestration of active ERK. However, other studies have suggested that senescence could also be the result of inhibition of nuclear ERK activity due to an increase in nuclear DUSP4 activity 136,137. Thus, coordinate gene expression induced by nuclear ERK might be required to prevent the completion of a senescence program induced by increased cytoplasmic ERK activity.Implication of the Ras/Raf/ERK pathway in senescence in vivo

Senescence induced by ectopic expression of an oncogene might reflect an artificially high expression level, as discussed in the study by Tuveson et al. 138 of the oncogene KRasD12. However, several results based on the expression of B-RafE600 at physiological level under the control of its own promoter, support the idea that Ras/Raf/ERK pathway-induced senescence is a physiological cellular response. For instance, in humans, naevi moles can be considered as an in vivo example of B-Raf-driven senescence. Naevi are melanocyte-derived benign tumors restrained from malignant progression by engagement of senescence. Naevi frequently harbor the oncogenic B-RafE600 or NRasR61 mutation 139 which promote senescence of primary melanocytes 126,127,132 and display markers of senescence such as β-galactosidase activity and high p16 expression 127,140. Recently, a mouse model of inducible tumorigenesis in lung epithelium driven by the B-RafE600 oncogene revealed that expression of B-RafE600 alone was not sufficient to promote a severe tumoral phenotype, leading instead to benign hyperplastic lesions undergoing senescence-associated growth arrest 133. In this model, p53 invalidation was necessary to promote B-RafE600-mediated transformation and malignant tumor formation 133. The hallmarks of ERK-mediated cell death: sustained and sequestered activityROS as mediators of ERK-induced cell death

In the majority of the studies related to cell death induced by the Ras/Raf/ERK pathway, ERK activation is unusually prolonged, i.e. ERK is maintained phosphorylated for between 6 and 72 h see Table 1. Moreover, delayed treatments with U0126, a MEK inhibitor, have revealed that ERK activity is continuously required to induce cell death 91. Despite constitutive activation of the pathway by oncogenes, levels of ERK phosphorylation in tumor cells are very variable 141, presumably due to phosphatase-driven feedback mechanisms. Because ERK-specific phosphatases are sensitive to ROS, we speculate that the main cause of sustained ERK activation is the presence of ROS, perhaps reflecting the levels of ROS scavengers in each particular model. The use of different ROS inhibitors demonstrated that ERK activation requires ROS production to induce cell death 14,54,56,57,60,61,65,76,78. Indeed, chemical oxidants, such as H2O2, peroxynitrite ONOO or NO see Table 1, induce ERK, whereas many stimuli implicating ERK in cell death promote the production of ROS 14,35,54,55,71,76,82. Moreover, DNA-damaging agents, such as doxorubicin, cisplatin or etoposide, catalyze the formation of ROS 142. In addition, ERK activity could be directly responsible for ROS production by upregulating inducible NO synthase 80. Thus, ROS-mediated prolonged ERK activation might be the crucial mechanism implicating the functions of the Ras/Raf/ERK pathway in cell death.ROS promote sustained ERK activationMechanisms of ROS-mediated ERK activation upstream of ERK

ROS can stimulate the Ras/Raf/ERK pathway by promoting the activation of tyrosine kinase receptors, such as platelet-defined growth factor receptor or EGF receptor 26,60,61, and adaptor proteins, such as Shc 143. ROS can also increase signaling by direct oxidation of residue C118 on Ras, a reaction that potentiates recruitment and activation of Raf at the plasma membrane 144,145. Other proteins implicated in Raf activation, such as Src 61, protein kinase C PKC -δ 25,29,61 or the cGMP pathway 146, could also be activated by ROS. Moreover, direct oxidation of cysteine residues in the cystein-rich domain of Raf promotes its autoactivation 147. Downstream of Raf, peroxynitrite ONOO can also cause nitration and autophosphorylation of MEK 61. ROS and inhibition of ERK phosphatases

Finally, ERK activity could be prolonged through the inhibition of tyrosine phosphatases and DUSP by ROS. Indeed, enzymatic activity of DUSP and tyrosine phosphatases requires a catalytic cysteine residue sensitive to oxidation 3,4. ROS have been shown to inhibit ERK-directed phosphatases, DUSP1 and DUSP6, by oxidation of their catalytic cysteine residues, C258 and C293, respectively 148,149, as well as ERK tyrosine phosphatases PP1/2A by oxidation of their conserved catalytic residue C62 135. Thus, the control of phosphatases that downregulate ERK activity plays a crucial role in the outcome of Ras/Raf/ERK pathway signaling.

Together, these data indicate that ROS can initiate and sustain ERK activation by different mechanisms. Interestingly, cell death has been associated with a biphasic activation of ERK, which could reflect this dual control of ERK activity by ROS 62,68. The importance of subcellular localization of ERK activity

In most cases, prolonged ERK activation alone, such as in models expressing constitutively active forms of upstream kinases, is not sufficient to promote cell death 15,63,64,66,82,85–87. In normal cells, subcellular localization of ERK is tightly regulated by scaffold proteins and docking phosphatases that allow nuclear accumulation of dephosphorylated ERK to terminate signaling 2. Thus, in addition to a sustained ERK activity, the outcome of ERK-mediated cell death might also rely on an aberrant subcellular localization. Indeed, apoptosis induced by estradiol 73, tamoxifen 74, zinc 54,55 cephaloridine 76, doxorubicin 20, revestratol 33 or dominant negative mutant of Rac or Cdc42 84 correlated with sustained nuclear ERK activity. As mentioned previously, nuclear activation of ERK is also associated with apoptosis during Ciona intestinalis development 106 and in mouse testis deficient for PEA-15 106. Interestingly, some of the compounds that induce nuclear ERK activity are associated with the production of ROS 54,55,76, which could promote nuclear accumulation of active ERK due to inhibition of DUSP. In MDA-MB-231 human breast cancer cells, taxol-induced apoptosis was abrogated by induction of nuclear DUSP1. In this study, DUSP1 induction clearly inhibited both ERK and JNK activity 150. Because nuclear DUSPs especially DUSP1 and -4 also control JNK and p38 phosphorylation 3,4, any modification of DUSP activity or expression could also increase cell death by activation of the stress pathways.

Cytoplasmic sequestration of ERK has also been associated with different forms of cell death. Cytoplasmic sequestration of ERK by binding to PEA-15 promotes autophagy 119, whereas sustained cytoplasmic ERK activity induces senescence in human primary fibroblasts 135–137. Together, these data suggest that sustained activation of ERK in different subcellular compartments is not tolerated and results in different forms of cell death see Fig. 1. Figure 1. Open FigureDownload Powerpoint slide

 The hallmarks of ERK-mediated cell death: sustained and sequestered activity. ERK activity induces the expression of many genes, including its own regulators, the DUSP ERK-specific phosphatases. Thus, ERK activity rapidly reaches a steady state and its death-promoting activity remains at low levels gray line. Any agent that provokes a sustained activation of ERK, such as ROS that inhibit DUSP, induces the progressive accumulation of death-promoting factors up to a level that induces cell death. The activation of ERK arrow might also transiently increase death-promoting activities of other death stimuli, such as chemotherapeutic agents. In addition to DUSP inhibition by ROS, ERK-mediated cell death is characterized by a deregulation of subcellular active ERK localization. Sustained cytoplasmic ERK activity might promote senescence or autophagy, whereas sustained nuclear sequestration of ERK activity might trigger apoptosis. In both conditions, sequestration of ERK depends on subcellular anchors, such as PEA-15, in the cytoplasm.The limits of ERK1/2-mediated cell death studies, the specificity of MEK inhibitors

In many studies, implication of the Ras/Raf/ERK pathway in the induction of cell death is based uniquely on the sole use of MEK1/2 inhibitors PD05059 or U0126 see Table 1. The weakness of these inhibitors is that they inhibit both MEK1/2 and MEK5 151. Interestingly, it has recently been shown that constitutive activation of the MEK5/ERK5 pathway could promote apoptosis of meduloblastoma cells 152 or thymocytes 153,154, through Nur77-dependent mechanisms 153,154. As a consequence, in those types of cell, some of the effect attributed to ERK1/2 might also be caused by the MEK5/ERK5 pathway. The use of PD184352, a more recent MEK1/2 inhibitor that does not target MEK5 155, could help to distinguish between the effects of MEK1/2 and MEK5 in those cells.Conclusions

Together, these data clearly demonstrate that the Ras/Raf/ERK pathway plays a critical role in promoting several forms of cell death in response to numerous stress stimuli both in vitro, with various cellular models, and in vivo. A common hallmark of this response is the sustained activation of ERK, which contrasts with the transient nature of ERK stimulation found in situations where ERK regulates other cell fates. As depicted in Fig. 1, ERK activates its own phosphatases, inducing a feedback loop that, within hours, restores a basal level of ERK activity. At least in the cellular models depicted in Table 1, ERK stimulation induces the expression of gene products with death-promoting activity. We can speculate that the feedback loop decreasing subcellular ERK activity over time prevents these death-promoting factors reaching a threshold concentration that triggers cell death. Consequently, any agent affecting the kinetics of ERK activity in a given cellular compartment such as the ROS that inhibit DUSP in Fig. 1 has a potential to induce cell death. Given the importance of the spatiotemporal regulation of ERK activity for the control of cell division 2, the induction of cell death could be seen as negative feedback mechanism preventing uncontrolled cell proliferation.

The Ras/Raf/ERK pathway is among the most commonly deregulated pathways identified in tumors, as indicated by frequently observed activating mutations in Ras or B-Raf oncogenes. Thus, this pathway is currently the target of new antitumor strategies, based on the inhibition of upstream ERK regulators. However, because ERK activation is implicated in DNA-damaging agent-induced cell death see Table 1, inhibiting ERK activity in combination therapy with classical antitumor compounds, such as cisplatin or doxorubicin, might affect the efficiency of such compounds.

Because prolonged ERK activation has been shown to promote the death of human cancer cell lines from different origins see Table 1, this property of the Ras/Raf/ERK pathway to induce cell death could be used to target cancer cells. However, although tumor cells escape Ras/Raf/ERK pathway-induced senescence by inactivating effectors of senescence, such as p53 or p16/INK4A, mechanisms involved in ERK-induced cell death might also be silenced in tumor cells. Tumor cells with high ERK activity might also have re-modeled the ERK signaling to escape ERK-mediated cell death. Thus, the crucial biochemical events underlying sensitivity or resistance to ERK-mediated cell death remain to be fully understood. We propose the hypothesis that in tumor cells harboring strong ERK activity, the alteration of compensating pathways PI3K/AKT, Wnt, etc would unleash the cell killing ability of ERK. Alternatively, if reagents were able to sequester ERK in a given subcellular compartment, the changes in the spatiotemporal regulation of ERK might be lethal. Moreover, because sustained ERK activity is required to promote cell death, such strategies would only target cancer cells with deregulated ERK activity and not normal cells in which ERK activation is transient.Acknowledgements

S. Cagnol is supported by the Canadian Institutes for Health Research grant CIHR MT-14405. We thank Dr Brendan Bell for careful reading of the manuscript. Correction added on 30 October 2009 after first online publication: in the preceding sentence the name Dr Brendan Bell was corrected. AncillaryArticle InformationDOI10.1111/j.1742-4658.2009.07366.x

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Keywordsapoptosis; autophagy; DUSP; ERK; ROS; senescence

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