Báo cáo khoa học: ERK and cell death: Mechanisms of ERK-induced cell death – apoptosis, autophagy and senescence doc

MINIREVIEW
ERK and cell death: Mechanisms of ERK-induced cell
death – apoptosis, autophagy and senescence
Sebastien Cagnol
1
and Jean-Claude Chambard
2
1 Department of Anatomy and Cellular Biology, Faculty of Medicine and Health Sciences, University of Sherbrooke, Canada
2 Institute of Developmental Biology and Cancer, University of Nice, France
Ras
⁄
Raf
⁄
ERK, the pathway
ERK2 ⁄ ERK1 (also known as p42 ⁄ p44MAPK, respec-
tively, 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-Jun-
NH
2
-terminal kinases (JNK1 ⁄ 2 ⁄ 3) and the p38 MAP
kinases (p38 a,b,d,c). These enzymes are activated
through a sequential phosphorylation cascade that
amplifies and transduces signals from the cell mem-
brane to the nucleus. Upon receptor activation, mem-
brane-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 phos-
phorylates 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 phos-
phorylation 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
Keywords
apoptosis; autophagy; DUSP; ERK; ROS;
senescence
Correspondence
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:
(Received 18 June 2009, revised 26 August
2009, accepted 9 September 2009)
doi:10.1111/j.1742-4658.2009.07366.x
The Ras ⁄ Raf ⁄ extracellular signal-regulated kinase (ERK) signaling path-
way 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.
Abbreviations
ATA, aurintricarboxylic acid; cPLA2, cytosolic phospholipase A2; DAPK, death-associated protein kinase; DUSP, dual-specificity phosphatase;
EGF, epidermal growth factor; ERK, extracellular signal-regulated kinase; FADD, Fas-associated death domain; GAIP, G-interacting protein;
IGF, insulin-like growth factor; JNK, c-JunNH
2
-terminal kinase; MAPK, mitogen-activated protein kinase; MEK1/2, mitogen protein kinase
kinase 1 and 2 (also known as MAP2K1 and MAP2K2, respectively); MEKCA, constitutively activated forms of MEK; MKP, mitogen-activated
protein kinase phosphatase; MOS, v-mos Moloney murine sarcoma viral oncogene homolog; PARP, poly(ADP-ribose) polymerase; ROS,
reactive oxygen species; TNF, tumor necrosis factor; TRAIL, tumor necrosis factor-related apoptosis-inducing ligand.
2 FEBS Journal 277 (2010) 2–21 ª 2009 The Authors Journal compilation ª 2009 FEBS
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 dephos-
phorylate 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 ⁄ Ra-
f ⁄ ERK signaling pathway stimulation.
The Ras ⁄ Raf ⁄ ERK pathway is frequently deregulat-
ed in tumors as a result of activating mutations in Ras
or B-Raf, observed particularly in malignant
melanoma, pancreas intestine and thyroid tumors (cos-
mic database: />cosmic). 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 sug-
gest 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-autono-
mous mechanism that relies on pathway-controlled
activation of caspases and nucleases leading to the
death of the injured cells without affecting neighbor-

ing cells. The intrinsic pathway of apoptosis regu-
lates the activity of the Bcl-2 family proteins that
control the integrity of the mitochondrial membrane.
The release of proapoptotic factors from the mito-
chondria, 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 pro-
teins such as Fas-associated death domain (FADD)
or TNFRSF1A-associated via death domain (TRADD).
Strong caspase-8 activity may directly activate effec-
tor caspases; it may also require signal amplification
through induction of the intrinsic pathway via cleav-
age 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 nega-
tive 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).

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 irradia-
tion [21]. ERK activity has been particularly impli-
cated 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 res-
veratrol [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], TNFa [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], H
2
O
2
[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], inter-
feron-a [75], cephalosporin [76], the calcium mobilizer
calcimycin [77], epidermal growth factor (EGF) depri-
vation [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 inhibi-
tion of the Ras ⁄ Raf ⁄ ERK pathway (Table 1).
Conversely, constitutive activation of ERK by domi-
nant active Raf1 combined with c-Myc expression [85]
or p53 induction [86], or constitutively active MEK
combined with death-associated protein kinase
S. Cagnol and J C. Chambard ERK and cell death
FEBS Journal 277 (2010) 2–21 ª 2009 The Authors Journal compilation ª 2009 FEBS 3
Table 1. Models of ERK-mediated apoptosis and autophagy. LDH, lactate dehydrogenase; MDC, monodansylcadaverine; MTT, 3-(4,5-dim-
ethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide; ND, not determined; TGHQ, 2,3,5-tris-(glutathione-S-yl)hydroquinone; TPA, 12-O-tetradeca-
noylphorbol-13-acetate.
In vivo ⁄ cellular model
Stimuli inducing
cell death
Duration of
ERK activation
promoting
cell death
Characteristics of
cell death
Evidence
implicating
MEK-ERK in cell
death Reference

Transformed mouse
fibroblast
Etoposide 24 h Caspase-3 PD98059 [12]
NIH 3T3 Etoposide
UV
Doxorubicin
24 h DNA degradation PD98059 [13]
Human keratinocytes
HaCaT
Etoposide 24 h DNA condensation
Caspase-3
PARP cleavage
PD98059
DN ERK
[14]
Human hepatocellular
carcinoma HepG2
and Huh-7
Doxorubicin ND PARP cleavage PD98059 [16]
Human promonocytic
leukemia
TPA
ArsenicCadmium
Doxorubicin
24 h DNA fragmentation
DNA condensation
U0126
PD98059
[17]
Human breast

adenocarcinoma
MCF-7
Doxorubicin 12 h Cell viability U0126 [18]
NIH 3T3, human
immortalized
keratinocytes HaCaT
Doxorubicin 24 h DNA fragmentation
PARP cleavage
PD98059
DN ERK
[19]
Rat immortalized
cardiomyocytes H9c2
Doxorubicin 48 h nuclear DNA fragmentation
Caspase-9, -3
PARP cleavage
U0126 [20]
NIH 3T3 c irradiation 48 h Membrane integrity
Annexin V
PD98059
DN ERK
[21]
Human cervix
adenocarcinoma HeLa
Human lung
carcinoma A549
Cisplatin 20 h DNA condensation
Caspase-3
PARP cleavage
Cytochrome c release

U0126
PD98059
[22]
Human ovarian
adenocarcinoma A2780
Cisplatin ND DNA fragmentation PD98059 [23]
Human osteosarcoma Saos-2 Cisplatin 24 h Cell viability
DNA fragmentation
U0126
PD98059
[24]
Rabbit primary renal
proximal tubular cells
Cisplatin 24 h DNA condensation
Caspase-3
U0126
PD98059
[25]
Mouse immortalized
proximal tubule
cell line (TKPTS)
Cisplatin 72 h Caspase-3 U0126 [26]
Human carcinoma
NCCIT and NTERA
Cisplatin 24 h DNA condensation
Caspase-8, -9, -3
U0126
PD98059
[27]
In vivo mouse kidney Cisplatin injection 72 h DNA fragmentation

Caspase-8, -3
U0126 [28]
Opossum immortalized
kidney cells OK cells
Cisplatin 48 h DNA degradation
Caspase-3
U0126
PD98059
DN MEK
[29]
Human cervix
adenocarcinoma HeLa
Cisplatin 18 h Caspase-9
PARP cleavage
U0126 [30]
Human papillary thyroid carcinoma
BHP 2–7 and BHP 18–21
Resveratrol 24 h nuclear DNA degradation PD98059 [33]
Human prostate
adenocarcinoma PC3
Phenethyl
isothiocyanate
24 h Annexin V
ROS production
PD98059 [35]
ERK and cell death S. Cagnol and J C. Chambard
4 FEBS Journal 277 (2010) 2–21 ª 2009 The Authors Journal compilation ª 2009 FEBS
Table 1. Continued.
In vivo ⁄ cellular model
Stimuli inducing

cell death
Duration of
ERK activation
promoting
cell death
Characteristics of
cell death
Evidence
implicating
MEK-ERK in cell
death Reference
Human melanoma
C8161,WM164 Mel Juso
Betulinic acid 96 h DNA fragmentation
DNA condensation
PARP cleavage
U0126 [36]
Human cervix
adenocarcinoma HeLa
Apigenin 8 h Cell viability U0126
PD98059
[37]
Human melanoma
A375-S2
Oridonin 12 h DNA fragmentation
Cytochrome c release
PD98059 [38]
Human glioblastoma
T98G and U87MG
Miltefosine 12 h Cell viability U0126 [39]

Human cervix
adenocarcinoma HeLa
Shikonin 12 h Caspase-8, -3 PD98059 [40]
Human breast
adenocarcinoma MCF-7
Taxol 24 h DNA fragmentation PD98059 [41]
Normal human
embryonic kidney HEK
TRAIL Constitutive DNA fragmentation
Caspase-8
U0126
PD98059
[42]
Human primary
fibroblast BJ
TRAIL ND cell viability U0126 [43]
Human colorectal
HT29 cells
TRAIL 5 h Membrane integrity
Nuclear condensation
PARP cleavage
PD98059 [44]
Human prostate
cancer LNCaP
TRAIL 4 h Annexin V
Caspase-3
U0126 [45]
Human prostate
tumor DU-145
Quercetin

TRAIL
24 h Membrane integrity PD98059 [34]
Human neuroblastoma
SHEP-1
TRAIL
H
2
O
2
ND Membrane integrity PD98059 [46]
Huaman neuroblastoma
SHEP
FasL 30 min DNA condensation DN MEK1 [50]
Rat primary
Sertoli cells
Human acute
T leukemia Jurkat
Fas (CH11) 5 min Membrane integrity
DNA fragmentation
PD98059 [51]
Diffuse large
B-cell lymphoma
CD40 ligation 3 h Membrane integrity
DNA fragmentation
U0126
PD98059
[52]
Primary human
cholangiocytes
CD40 ligation 24 h DNA condensation

Caspase-3 activity
PD98059 [53]
Pig renal tubular
epithelial cells
LLCÆPK1
Zinc 24 h nuclear Cell viabilityROS
production
U0126 [54]
In vivo ⁄ isolated
rat renal cortical
slices
ZnCl
2
injection 90 min nuclear Cell viability U0126 [55]
Pig renal tubular
epithelial cells
LLCÆPK1
TGHQ 5 h Cell viability PD98059 [56]
Rabbit primary
renal proximal
tubular cells
tert-butylhydroperoxide 8 h Annexin V U0126
PD98059
[58]
Human primary
retinal pigmented
epithelial ARPE19
cells
tert-butylhydroperoxide 6 h Cell viability
Caspase-9

DNA fragmentation
U0126 [59]
S. Cagnol and J C. Chambard ERK and cell death
FEBS Journal 277 (2010) 2–21 ª 2009 The Authors Journal compilation ª 2009 FEBS 5
Table 1. Continued.
In vivo ⁄ cellular model
Stimuli inducing
cell death
Duration of
ERK activation
promoting
cell death
Characteristics of
cell death
Evidence
implicating
MEK-ERK in cell
death Reference
Murine transformed
lung epithelial
MLE12
Hyperoxia 4 h Caspase-9, -3
Cytochrome c release
PD98059 [57]
In vivo mouse lung Hyperoxia 72 h DNA fragmentation
Caspase-3
PD98059 [57]
Primary rat
pulmonary
myofibroblasts

ONOO
)
30 min Cell viability PD98059 [60]
Immortalized
mouse fibroblast L929
H
2
O
2
3 h DNA fragmentation
Annexin V
PD98059
DN ERK
[61]
Mouse immortalized
osteoblast
H
2
O
2
Biphasic 12 h Cell viability
Membrane integrity
PD98059 [62]
Rabbit primary
renal proximal
tubular cells
H
2
O
2

2 h constitutive DNA condensation
Caspase-3
U0126
PD98059
MEKCA
[63]
Rabbit primary
renal proximal
tubular cells
H
2
O
2
2 h Membrane integrity
Annexin V
U0126
PD98059
MEKCA
DN MEK
[64]
Human transformed
bronchial epithelial
cell line BEAS-2B
NO donor 24 h Cell viability PD98059 [65]
Human melanoma NO donor ND PARP cleavage U0126 [67]
HEK293 Cadmium Biphasic 96 h Caspase-8, -3
PARP
U0126 [68]
Human hepatocellular
carcinoma HepG2

Benzo[a]pyrene 48 h Cell viability PD98059 [70]
Rat primary pleural
mesothelial cells
Crocidotite
Asbestos
48 h DNA condensation
ROS production
PD98059 [71]
In vivo Caenorhabditis
elegans
Arsenic ND DNA fragmentation mek-2 (n1989)
mpk-1 (ku1)
[72]
Murin bone marrow-derived
primary osteoclasts and
murine long bone-derived
osteocytes MLO-Y4
Estradiol 24 h Membrane integrity U0126 [73]
Human breast
adenocarcinoma MCF-7
Tamoxifen 1 h Membrane integrity PD98059 [74]
Human myeloma
cell line U266-1984
and RHEK-1
Interferon-a 16 h Annexin V
Caspase-3
U0126 [75]
Isolated rat renal
cortical slices
Cephaloridine 90 min nuclear Cell viability

ROS production
U0126
PD98059
[76]
Immortalized rabbit
lens epithelial cells
N ⁄ N1003A
Calcimycin 10 h Membrane integrity
DNA fragmentation
Annexin V
Cytochrome c release
Caspase-3
U0126
DN Raf
DN ERK
[77]
Primary mouse
kidney proximal
tubular epithelial cells
EGF deprivation 120 h Cell viability U0126
PD98059
[78]
Primary human
bone marrow
stromal cells
Leptin 12 h Cell viability
Cytochrome c release
Caspase-3
U0126
PD98059

[79]
ERK and cell death S. Cagnol and J C. Chambard
6 FEBS Journal 277 (2010) 2–21 ª 2009 The Authors Journal compilation ª 2009 FEBS
Table 1. Continued.
In vivo ⁄ cellular model
Stimuli inducing
cell death
Duration of
ERK activation
promoting
cell death
Characteristics of
cell death
Evidence
implicating
MEK-ERK in cell
death Reference
Human leukemia U937 Bufalin 12 h DNA fragmentation MEK antisense [11]
Pig renal tubular
epithelial cells LLCÆPK1
Escherichia coli
toxin
44 h AnnexinV
PARP cleavage
PD98059 [80]
Mouse monocyte ⁄
macrophage J774.2
Francisella
tularensis
infection

42 h Annexin V
DNA fragmentation
U0126
PD98059
[81]
Human osteosarcoma
cell line HOS and U2OS
Chelerythrine 4 h DNA fragmentation
Caspase-8, -9, -7
PARP cleavage
MEKCA U0126
PD98059
DN Ras
[82]
Mouse embryonary fibroblast RacN17 Cdc42N17 ND Membrane integrity PD98059 [83]
Mouse embryonary fibroblast RacN17 Cdc42N17 24 h Annexin V PD98059
DN DUSP6
[84]
Rat fibroblast Rat1 RAF-CAAX Constitutive DNA condensation RAF-CAAX [85]
Mouse immortalized
fibroblast NIH 3T3
DAPK Constitutive Cell viability MEKCA [87]
Murine erythroleukemia
DP16.1 ⁄ p53ts cells
P53 induction Constitutive Annexin V U0126
Raf CA
[86]
Human breast
adenocarcinoma MCF-7
DRAF1 Constitutive DNA fragmentation

Vacuolization
DRAF1 [88]
Human primary osteoblast DRaf1
HrasV12 T35S
Constitutive Cell viability
Membrane integrity
Annexin V
DNA fragmentation
DRaf1
HrasV12T35S
[89]
HEK293T IGF-I receptor 48 h Membrane integrity
Caspase-3
Vacuolization
U0126
MEK siRNA
[90]
HEK293 DRaf1:ER
Anti-Fas(CH11)
Constitutive Membrane integrity
DNA fragmentation
DNA condensation
Annexin V
Caspase-8, -3
PARP cleavage
Vacuolization
DRaf1:ER
U0126
[91]
Murine fibrosarcoma

cells L929
TNFa Cell viability
LC3-II induction
Beclin induction
U0126
PD98059
[48]
Human breast
adenocarcinoma MCF-7
TNFa 10 h Cell viability
LC3-II induction
U0126
PD98059
[49]
Mouse RAW264.7
macrophages
NO 2 h
constitutive
Cell viability
Membrane integrity
BNIP-3 induction
U0126 [66]
Transformed mouse
mesengial MES-13 cells
Cadmium 3 h Cell viability MTT
LC3-II induction
PD98059 [69]
Human colon
adenocarcinoma HT29
Amino acid

depletion
4 h GAIP phosphorylation
Autophagic LDH
sequestration
PD98059 [114]
Human colon
adenocarcinoma HT29
Amino acid
depletion
ATA
RasV12S35
4h
constitutive
Proteolysis
LDH sequestration
RasV12S35 [115]
Human colon
adenocarcinoma
HCT-15
Soyaponin 3 h MDC incorporation U0126 [116]
S. Cagnol and J C. Chambard ERK and cell death
FEBS Journal 277 (2010) 2–21 ª 2009 The Authors Journal compilation ª 2009 FEBS 7
(DAPK) expression [87], could induce apoptosis with-
out any other stimulus. Moreover, in rare cases, acti-
vation 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
DRaf1:ER induction [91] was sufficient to promote cell
death.
Mechanisms of Ras

⁄
Raf
⁄
ERK-mediated
apoptosis
ERK activity has been associated with classical mark-
ers 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). Depend-
ing on the cell type and the nature of the injury, acti-
vation 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 TNFa [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 acti-
vation. 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 obser-
vations 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 induc-
ing 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 mitochon-
drial 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 upregulat-
Table 1. Continued.
In vivo ⁄ cellular model
Stimuli inducing
cell death
Duration of
ERK activation
promoting
cell death
Characteristics of
cell death
Evidence
implicating
MEK-ERK in cell
death Reference
Mouse 42GPA9
Sertoli cell line
Lindane 24 h LC3 relocalization
Vacuolization
PD98059
U0126
[117]
Primary human lung
fibroblasts WI38
Dihydrocapsaicin 4 h LC3-II induction
Caspase-3, -7
LC3 relocalization

PD98059
ERK siRNA
[118]
Human OVCA-420 PEA-15 48 h Membrane integrity
Acidic vacuoles
ERK siRNA [119]
Primary human
fibroblast IMR90
RasV12 Constitutive LC3-II induction [120]
ERK and cell death S. Cagnol and J C. Chambard
8 FEBS Journal 277 (2010) 2–21 ª 2009 The Authors Journal compilation ª 2009 FEBS
ed 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 proteo-
lytic 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 upregu-
lation 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 phos-
phorylation 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-a
[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 acti-
vation. ERK can directly activate cPLA2 by phosphor-
ylation at serine 505 [100,101]. The use of cPLA2
inhibitor AACOCF3 suggests that cPLA2 was neces-
sary 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 recep-
tor function [47].
In MCF-7 cells and primary osteoblasts, activation
of Raf ⁄ ERK pathway-induced apoptosis was the con-
sequence of cellular detachment from the matrix,
which was in this case due to a decrease in integrin b1
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 vivo
Following tissue injury
ERK activity has been clearly implicated in neuro-
degenerative 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 apop-
tosis and tissue destruction in these models.
During development
Proapoptotic ERK activity has also been reported in
developmental models. During germinal cell develop-
ment, PEA-15, a cytoplasmic death domain-containing
protein that binds and sequesters ERK, is highly
expressed in the cytoplasm of Sertoli cells, spermatogo-
nia and spermatocytes, inducing a cytoplasmic ERK
localization. Interestingly, testis isolated from PEA-15-
deficient mice display an abnormal nuclear accumula-
tion 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
S. Cagnol and J C. Chambard ERK and cell death
FEBS Journal 277 (2010) 2–21 ª 2009 The Authors Journal compilation ª 2009 FEBS 9
and Marthasterias glaciali, v-mos Moloney murine
sarcoma viral oncogene homolog (MOS)-dependent
sustained ERK activity led to protein synthesis-depen-
dent 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, downregula-
tion 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. cas-
pase-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 activa-
tion 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 forma-
tion 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 acti-
vation [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 TNFa treatment [49] have clearly associ-
ated 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 neuro-
degenerative 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 aurintricarb-
oxylic acid (ATA) [115] in human colorectal cancer cell
line HT29, soyasaponins [116] in human colon adeno-
carcinoma HCT-15, lindane [117] in the mouse Sertoli
cell line, dihydrocapsaicin [118] in WI38 lung fibro-
blasts, cadmium in mesengial MES-13 cells [69,113]
and TNFa treatment in MCF-7 [49] and L929 cells
[48]. Interestingly, in human ovarian cancer cells, cyto-

plasmic 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 associ-
ated with the autophagic process, as ERK-mediated
phosphorylation of p53 on serine 392 [48] was involved
in TNFa-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
ERK and cell death S. Cagnol and J C. Chambard
10 FEBS Journal 277 (2010) 2–21 ª 2009 The Authors Journal compilation ª 2009 FEBS
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 pre-
mature oncogene-induced senescence [122]. Although
aberrant activation of the Ras ⁄ Raf ⁄ ERK pathway pro-
motes 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 constitu-
tively 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).
Mechanisms of ERK-induced senescence
Ras ⁄ Raf ⁄ ERK pathway-induced senescence correlates
with increased b-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 fibro-
blasts IMR90, when senescence is provoked by induc-
ible 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 constitu-
tively activated forms of MEK (MEKCA) requires
ERK-induced p38 activation [124]. Interestingly, a
Table 2. Models of ERK-mediated senescence.
In vivo ⁄ cellular model
Induction of
ERK activity Markers of senescence MEK activity Reference
Primary human fibroblastic IMR90 Ras V12
MEKCAQ56P
b-galactosidase activity
p21 p53 and p16 ⁄ INK4A
PD98059 [123]
Primary human fibroblastic BJ cells RasV12
MEKCA
b-galactosidase activity

p16 ⁄ INK4A
U0126 [124]
Primary mouse fibroblastic cells Ras V12
PAK4
b-galactosidase activity
p21, p19 ⁄ ARF and p16 ⁄ INK4A
PD98059 [125]
Mouse immortalized NIH 3T3 fibroblast DRaf1:ER p21 [128]
Primary human fibroblast IMR90 DRaf1:ER b-galactosidase activity
p21 and p16 ⁄ INK4A
PD98059 [129]
Human prostate cancer LNCaP cells DRaf1:ER b-galactosidase activity
p21
[130]
Mouse embryonic fibroblast DRaf1:ER p21, p53 and p19 ⁄ Arf [131]
Primary human fibroblast IMR90 Raf-CAAX
DRaf1:ER
b-galactosidase activity [97]
Primary human melanocytes
Primary human fibroblast BJ
In vivo ⁄ human naevi
B-Raf V600E b-galactosidase activity
p21, p53 and p16 ⁄ INK4A
Heterochromatic foci
[127]
Primary human melanocytes
In vivo ⁄ human Naevi
B-Raf V600E b-galactosidase activity
Heterochromatic foci
p16 ⁄ INK4A

[135]
In vivo ⁄ mouse lung tumor model B-Raf V600E p19 ⁄ ARF Dec1
Heterochromatic foci
[133]
Primary human melanocytes B-Raf V600E
MEKCAQ56P
b-galactosidase activity
p16 ⁄ INK4A,
cH2AX
Heterochromatic foci
[132]
Primary human melanocytes B-Raf V600E
NRasQ61R
b-galactosidase activity
p53 and p16 ⁄ INK4A
[126]
Primary human intestinal epithelial cells MEKCA SS218 ⁄ 222DD b-galactosidase activity
p21 p53 and p16 ⁄ INK4A
[134]
Primary human fibroblast WI38 DUSP4 (MKP-2) b-galactosidase activity [136]
S. Cagnol and J C. Chambard ERK and cell death
FEBS Journal 277 (2010) 2–21 ª 2009 The Authors Journal compilation ª 2009 FEBS 11
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 regula-
tors, 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 senes-
cence 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 expres-
sion is then continuously required for transformation,
as downregulation of c-Myc in tumor-derived mela-
noma 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 inacti-
vate 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 onco-
gene 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 melano-
cyte-derived benign tumors restrained from malignant
progression by engagement of senescence. Naevi fre-
quently 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 b-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 hyper-
plastic lesions undergoing senescence-associated growth
arrest [133]. In this model, p53 invalidation was neces-

sary to promote B-RafE600-mediated transformation
and malignant tumor formation [133].
The hallmarks of ERK-mediated cell
death: sustained and sequestered
activity
ROS 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 activa-
tion 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 continu-
ously required to induce cell death [91]. Despite con-
stitutive 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 phospha-
tases 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 H
2
O
2
, peroxynitrite ONOO
)

or NO (see
Table 1), induce ERK, whereas many stimuli implicat-
ing 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, cat-
alyze the formation of ROS [142]. In addition, ERK
activity could be directly responsible for ROS produc-
tion 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.
ERK and cell death S. Cagnol and J C. Chambard
12 FEBS Journal 277 (2010) 2–21 ª 2009 The Authors Journal compilation ª 2009 FEBS
ROS promote sustained ERK activation
Mechanisms 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 oxida-
tion of residue C118 on Ras, a reaction that potenti-
ates 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)-d
[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 sensi-
tive to oxidation [3,4]. ROS have been shown to inhi-
bit ERK-directed phosphatases, DUSP1 and DUSP6,
by oxidation of their catalytic cysteine residues, C258
and C293, respectively [148,149], as well as ERK tyro-
sine phosphatases PP1 ⁄ 2A by oxidation of their con-
served 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, subcellu-
lar localization of ERK is tightly regulated by scaf-
fold proteins and docking phosphatases that allow

nuclear accumulation of dephosphorylated ERK to
terminate signaling [2]. Thus, in addition to a sus-
tained 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]. Inter-
estingly, some of the compounds that induce nuclear
ERK activity are associated with the production of
ROS [54,55,76], which could promote nuclear accumu-
lation 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. Cytoplas-
mic sequestration of ERK by binding to PEA-15 pro-
motes 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).
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 path-
way could promote apoptosis of meduloblastoma cells
[152] or thymocytes [153,154], through Nur77-depen-
dent 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 inhibi-
tor that does not target MEK5 [155], could help to
distinguish between the effects of MEK1 ⁄ 2 and MEK5
in those cells.
S. Cagnol and J C. Chambard ERK and cell death
FEBS Journal 277 (2010) 2–21 ª 2009 The Authors Journal compilation ª 2009 FEBS 13
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 kinet-
ics 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 prevent-
ing uncontrolled cell proliferation.
The Ras ⁄ Raf ⁄ ERK pathway is among the most com-
monly deregulated pathways identified in tumors, as
indicated by frequently observed activating mutations
in Ras or B-Raf oncogenes. Thus, this pathway is cur-
rently the target of new antitumor strategies, based on
the inhibition of upstream ERK regulators. However,
because ERK activation is implicated in DNA-damag-
ing agent-induced cell death (see Table 1), inhibiting
ERK activity in combination therapy with classical
antitumor compounds, such as cisplatin or doxorubi-

cin, 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 senes-
cence 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 regu-
lation of ERK might be lethal. Moreover, because sus-
tained 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

Fig. 1. 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 phos-
phatases). 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 inhi-
bit 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 character-
ized by a deregulation of subcellular active ERK localization. Sus-
tained 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.
ERK and cell death S. Cagnol and J C. Chambard
14 FEBS Journal 277 (2010) 2–21 ª 2009 The Authors Journal compilation ª 2009 FEBS
manuscript. [Correction added on 30 October 2009
after first online publication: in the preceding sentence
the name ‘Dr Brendan Bell’ was corrected.]
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