Báo cáo khoa học: Role of extracellular signal regulated kinases 1 and 2 in neuronal survival docx

MINIREVIEW
Role of extracellular signal regulated kinases 1 and 2 in neuronal
survival
Michal Hetman
1,2,3
and Agata Gozdz
1
1
Kentucky Spinal Cord Research Injury Center and Departments of Neurological Surgery and
2
Pharmacology and Toxicology,
University of Louisville, KY, USA;
3
Nencki Institute, Warsaw, Poland
Extracellular signal regulated kinases 1 and 2 (ERK1/2)
regulate cellular responses to a variety of extracellular
stimuli. In the nervous system, ERK1/2 is critical for neur-
onal differentiation, plasticity and may also modulate
neuronal survival. In this minireview, we present evidence
that supports prosurvival activity of ERK1/2 in neurons.
Several reports suggest that ERK1/2 mediates neuropro-
tective activity of extracellular factors, including neurotro-
phins. In addition, ERK1/2 is activated by neuronal injury.
In damaged cells, ERK1/2 activation may act as a defensive
mechanism that helps to compensate for the deleterious
effects of a damaging insult. The emerging mechanisms of
ERK1/2-mediated neuroprotection may involve transcrip-
tional regulation and/or direct inhibition of cell death
machinery.
Keywords: apoptosis; MAP-kinase; neurons; neuropro-
tection; programmed cell death; signal transduction;

survival.
Signaling pathways and neuronal survival
Death of neurons occurs during development of the central
nervous system (CNS). Therefore, restriction of neuronal
death by survival signaling is critical for the proper
formation of the CNS [1]. In CNS pathologies including
stroke, Alzheimer’s disease or CNS trauma, nerve cell death
occurs as a result of cell injury [1]. Consequently, survival
signaling cascades may provide targets for neuroprotective
therapies against these conditions. Neuronal survival sign-
aling involves several pathways including phoshpatidylo-
inositol-3-kinase (PI3K), extracellular signal regulated
kinase 1/2 (ERK1/2) or extracellular signal regulated kin-
ase 5 (ERK5) [2]. Interestingly, while PI3K seems to be
the principal transducer for prosurvival factors involved
in trophic support, several reports suggest preferential
ERK1/2 involvement in protection against damage-induced
cell death. Therefore, manipulations of the latter pathway
may be useful for neuroprotective interventions in diseases.
ERK1/2 signaling
ERK1,alsoknownasp44mitogenactivatedproteinkinase
(MAP kinase), and ERK2 (p42 MAP kinase) are closely
related protein kinases of the MAP kinase family [3]. ERK1/2
is regulated by a cascade of phosphorylations including
a dual phosphorylation at Thr/Tyr residues of the ERK1/2
activation domain that is carried out by MAP kinase kinase
1/2 (MKK1/2) [3]. These dual phosphorylation events
activate ERK1/2 [3]. MKK1/2 is, in turn, activated by
phosphorylation catalyzed by the Raf family of protein
kinases. Raf activators include small GTPases, Ras or Rap3.

ERK1/2 activity is decreased by either phosphotyrosine
phosphatases or dual specificity (Ser/Thr + Tyr) phospha-
tases known as MAP kinase phosphatases (MKPs) [3].
ERK1/2 is considered a Ôproline directed kinaseÕ as it
phosphorylates Ser or Thr residues followed by a proline.
ERK1/2 targets include several transcription factors, sign-
aling mediators, cytoskeletal proteins and protein kinases
[3]. ERK1/2 may also engage in the regulation of cellular
processes via protein–protein interactions rather than
through its kinase activity. For instance, kinase dead mutant
forms of ERK2 may activate MKPs or DNA topoiso-
merase II [3]. It is believed that ERK1/2 interactions with its
activators and/or substrates are enhanced by scaffolding
proteins such as MP-1 [3]. Also, ERK1/2 signaling may be
regulated by subcellular localization [3] and crosstalk to
other signaling mediators including Ca
2+
,cAMPorPI3K
[2,4]. Biological processes involving ERK1/2 include stimu-
lation of cell proliferation and survival, neoplastic transfor-
mation, neuronal differentiation and plasticity [2,3].
Tools to study ERK1/2 signaling
Several approaches have been employed to study the
biological role of ERK1/2 in mammalian cells including
Correspondence to Michal Hetman, Kentucky Spinal Cord
Research Injury Center, University of Louisville, 511 S. Floyd St.,
MDR616, Louisville, KY 40292, USA.
Fax: + 1 502 852 5148, Tel.: + 1 502 852 3619,
E-mail:
Abbreviations: BDNF, brain derived neurotrophic factor; CNS, cen-

tral nervous system; CREB, cAMP response element binding protein;
ERK, extracellular signal regulated kinase; HSV2, herpes simplex
virus type 2; MAP, mitogen activated protein; MKK1/2, MAP kinase
kinase 1/2; MKP, MAP kinase phosphatase; NGF, nerve growth
factor; NMDA, N-methyl-
D
-aspartate; NMDAR, NMDA receptor;
P13K, phoshpatidyloinositol-3-kinase; PARP, poly-(ADP-ribose)-
polymerase; TGFa, transforming growth factor a.
(Received 14 February 2004, accepted 18 March 2004)
Eur. J. Biochem. 271, 2050–2055 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04133.x
overexpression of mutated elements of the pathway, use of
pharmacological inhibitors and genetic ablation in knock-
out mice [3]. Perhaps the most popular tools to manipulate
ERK1/2 signaling are the dominant-negative and the
constitutive-active mutants of MKK1 as well as several
pharmacological inhibitors of MKK1/2. It is important to
realize the limitations of these tools. First, it is possible that
MKK1/2 may also have other targets than ERK1/2 [5].
Secondly, specificity of commonly used MKK1/2 drug
inhibitors is not absolute. For instance, PD98059 and
U0126 may inhibit signaling by ERK5 [6]. PD98059 was
also shown to directly inhibit cyclooxygenases [7] and block
Ca
2+
influx into isolated synaptosomes [8]. The interpret-
ation of the data obtained through experimental modula-
tion of ERK1/2 signaling may be hampered by the fact that
inhibition of one element of the ERK1/2 signaling network
may modulate other signaling units. For instance, selective

inhibition of MKK1/2 increases ERK5 activity [6]. There-
fore, the optimal design of experiments addressing ERK1/2
function could include application of several inhibitors and
dominant-negative mutants that act at various levels of the
ERK1/2 cascade as well as testing the effects of these agents
on other signaling circuitries that may crosstalk to ERK1/2.
ERK1/2 as a transducer of extrinsic survival
signals
The first reported experiments suggesting that ERK1/2
may transduce anti-apoptotic signaling in neurons were
performed in neuronally differentiated PC12 cells [9].
Because nerve growth factor (NGF) activated ERK1/2
in this system, and NGF withdrawal-induced apoptosis
was prevented by overexpression of constitutively active
mutants of MKK1, Xia et al. proposed that trophic
signaling by NGF was mediated through the ERK1/2
pathway [9]. Additional studies showed that ERK1/2
activation may also block cell death induced by trophic
deprivation of retinal ganglion cells, cerebellar granule
neurons or spiral ganglion neurons. In rat retinal ganglion
cells or cerebellar granule neurons, protective ERK1/2 was
activated by brain derived neurotrophic factor (BDNF)
[10–12], or by cAMP signaling triggered by PACAP or
forskolin [10,13]. In rat spiral ganglion neurons, ERK1/2
mediated the protective effect of the neuromodulator
substance P [14]. However, several other studies using
both pharmacological inhibitors and various mutants of
the ERK1/2 pathway suggested that ERK1/2 may not be
the major mediator of neuroprotection afforded by neuro-
trophins, IGF-1 or membrane depolarization in trophic-

deprived neurons (reviewed in [2]). Importantly, as the
studies indicating a lack of ERK1/2 involvement in
antiapoptotic signaling used a variety of neuronal cells,
the role of ERK1/2 in protection against trophic with-
drawal may appear only in some contexts. In contrast,
PI3K/Akt signaling seems to be the principal mediator of
both the anti-apoptotic effects of basal trophic support and
neuroprotective action of agents that suppress trophic
deprivation-induced cell death [2].
Although the contribution of ERK1/2 to support neur-
onal survival under ÔbasalÕ culture conditions may be
minimal, there is accumulating evidence that ERK1/2 may
mediate the neuroprotective activity of such factors that
neuroprotect against damaging insults. For instance,
ERK1/2 activation by BDNF was shown to protect
cultured rat cortical neurons against apoptosis induced by
DNA damage [15,16]. In these studies, DNA damage was
induced by genotoxic anticancer drugs, camptothecin or
cisplatin (CPDD). Interestingly, cisplatin is highly neuro-
toxic, which limits its use against CNS tumors [17]. BDNF
protection against neuronal apoptosis induced by either of
these compounds was inhibited with MKK1/2 blockers,
PD98059 or SL327 (a blood–brain barrier permeable
compound similar to U0126). Because neurons that over-
expressed a constitutive-active form of MKK1 were
protected against camptothecin or CPDD, it seems that
ERK1/2 activation is both necessary and sufficient for the
anti-apoptotic action of BDNF [15,16]. Similarly, Anderson
& Tolkovsky showed that in cultured sympathetic neurons
exposed to another DNA-damaging agent, cytosine arabi-

noside, PD98059 inhibited NGF-mediated protection,
implicating ERK1/2 involvement [18].
There are several reports suggesting that ERK1/2 may
serve as a transducer for agents that protect from other
forms of neuronal injury including excitotoxicity, calcium
overload, oxidative injury, hypoxia or neurotoxic viruses.
ERK1/2 is required for neuroprotection by estrogen
against glutamate excitotoxicity [19]. Also, in hippocam-
pal slice cultures that were challenged by an excitotoxic
insult with N-methyl-
D
-aspartate (NMDA), the protective
effects of nicotine were ERK1/2-dependent [20]. In
another study, transforming growth factor a (TGFa)
was shown to protect neurons from NMDA-induced
death by activating astrocytic ERK1/2. In agreement
with the anti-excitotoxic effects of ERK1/2, cortical
neuron death by ionomycin-induced Ca
2+
overload was
attenuated by BDNF, at least in part, via ERK1/2
activation [21].
ERK1/2 also seems to contribute to the neuroprotective
effects of cAMP. For instance, corticotrophin releasing
hormone protected hippocampal slices from excitotoxicity
by cAMP-mediated activation of ERK1/2 [22]. Also,
prostaglandin E1, a signaling mediator know to activate
the cAMP pathway, protected rat spinal neurons against
nitric oxide, acting partially through ERK1/2 [23]. Similarly,
the cAMP inducer forskolin increased the survival of

cultured dopaminergic neurons by reducing the sponta-
neous oxidative toxicity, via stimulation of the ERK1/2
pathway [24].
ERK1/2 activation mediates the protective effects of
several factors that enhance neuronal survival in hypoxia/
ischemia models. For instance, Han & Holtzman showed
that in P7 rat pups, intraventrical injection of BDNF
protected against hypoxic/ischemic brain injury through
ERK1/2 but not PI3K [25]. Other examples of neuropro-
tectants that counteract hypoxia by activating ERK1/2
include erythropoietin, fructose-1, 6-biphosphate or
N-acetyl-o-methyldopamine [26–28]. Finally, TGF1b-medi-
ated ERK1/2 activation inhibited staurosporine-induced
cell death of cultured hippocampal neurons and was also
suggested to protect in a rat stroke model [29]. An
interesting example of ERK1/2-mediated neuroprotection
against injury is that induced by the herpes simplex virus
type 2 (HSV2). In this case, virally produced ICP10
protein activated ERK1/2 to inhibit apoptosis of infected
Ó FEBS 2004 Role of ERK1/2 in neuronal survival (Eur. J. Biochem. 271) 2051
hippocampal neurons [30]. Thus, ERK1/2 appears to be
required for several neuroprotectants that support neuronal
survival after injury.
Activation of ERK1/2 as a defense mechanism
in neuronal injury
Neurons respond to cell damage by activation of death
signaling pathways. However, at the same time, cells may
also mobilize defense mechanisms in an attempt to coun-
teract cell death and enable damage repair. Interestingly,
defensive ERK1/2 activation is observed after neuronal

injuries. ERK1/2 activation was observed in cultured
cortical or hippocampal neurons exposed to the DNA
damaging agents, camptothecin, CPDD or etoposide
[15,16,31]. In cultured rat cortical neurons, CPDD-mediated
activation of ERK1/2 reached levels similar to those
observed at the peak of BDNF action [16]. Inhibition
of ERK1/2 by either pharmacological inhibitors or by a
dominant-negative mutant form of MKK1 increased
CPDD-induced apoptosis and further reduced survival of
CPDD-treated neurons [16]. Surprisingly, the protective
ERK1/2 activation turned out to be mediated by NMDA
receptors (NMDARs) [16]. Further experiments indicated
that in CPDD-treated neurons, NMDAR signaling to
ERK1/2 is enhanced by activation of poly-(ADP-ribose)-
polymerase (PARP), an enzyme that mobilizes DNA repair
but may also lead to energetic deprivation and necrosis [16].
Elevated PARP activity was suggested to stimulate
NMDAR signaling by depleting neurons of ATP [32].
Because the moderate increase in PARP activity observed in
CPDD-treated neurons did not deplete cellular ATP, PARP
contribution to the defensive ERK1/2 activation may result
from a mechanism that does not involve disturbed neuronal
energetics [16]. Therefore, it appears that DNA damage by
CPDD may activate a novel neuronal defense circuitry that
engages PARP, NMDAR and ERK1/2. It remains to be
tested if a similar pathway may contribute to the defense
against other genotoxic insults. The role of ERK1/2 in
neuronal survival after exposure to DNA-damaging anti-
cancer agents suggests that neurotoxicity induced during
cancer treatment may be enhanced by drugs affecting

protective ERK1/2 activation including the clinically used
NMDAR antagonist, memantine.
ERK1/2 seems to be an important defense mechanism
against hypoxia/ischemia. Induction of ischemic tolerance
by an episode of mild ischemia can be modeled in cultured
rat cortical neurons by transient oxygen/glucose depriva-
tion [33]. Gonzalez-Zulueta et al. showed that in this
system, protective oxygen/glucose deprivation precondi-
tioning activated NMDARs resulting in increased produc-
tion of NO and subsequent activation of Ras, which
ultimately signaled to ERK1/2 [33]. The inhibition of
ERK1/2 activation in this case completely abolished the
protective effects of preconditioning [33]. Furthermore,
in mouse cortical neurons that were exposed to cytotoxic
hypoxia, ERK1/2 was activated and ERK1/2 pathway
blockers increased hypoxia-induced cell death [34].
ERK1/2 may also play a role in neuronal protection in
status epilepticus. Inhibition of ERK1/2 activation with
SL327 increased mortality in rats with pilocarpine-induced
seizures [35].
Interestingly, in several non-neuronal systems, damaging
stimuli including DNA injury, oxidative stress or death
receptor signaling were reported to mobilize an anti-
apoptotic ERK1/2 activity [36–38]. These data suggest an
interesting possibility that ERK1/2 activation may be a
general defense mechanism that is mobilized to protect
different cell types against various forms of damage.
Protective mechanisms downstream
of ERK1/2
The ERK1/2 pathway affects multiple targets that may

mediate its prosurvival activity. These include transcription
factors that could stimulate production of anti-apoptotic
mediators or inhibit accumulation of killer proteins. In
addition, ERK1/2 may also directly affect several cell death/
cell survival regulators. The protective mechanisms that do
not involve gene expression may be particularly relevant for
the survival of damaged neurons, under conditions where
gene expression machinery is disturbed.
In the nervous system, protective ERK1/2 signaling may
target both gene expression-dependent and gene expression-
independent events (Fig. 1). For instance, Bonni et al.
suggested that activation of a transcription factor, cAMP
response element binding protein (CREB) and/or a direct
inhibition of Bad, a pro-apoptotic member of the bcl-2
family, may mediate the prosurvival activity of ERK1/2 in
trophic-deprived cerebellar granule neurons [12]. In this
study, ERK1/2 appeared to mediate its effects through
direct action on its target kinase, p90Rsk2. Inhibition of
Rsk2 with a dominant-negative mutant blocked ERK1/2-
mediated inhibition of apoptosis. Also, the prosurvival
activity of ERK1/2 was reduced by antagonizing the Rsk
target CREB. In addition, a dominant-negative mutant
form of Rsk2 decreased the anti-apoptotic phosphorylation
of Bad at its Ser112 residue. Finally, Rsk2 activation by
ERK1/2 protected against apoptosis that was induced by an
overexpressed wild type, but not a Ser112 fi Ala mutant
form of Bad. Inhibitory Bad phosphorylation is also
implicated as a mechanism of prosurvival ERK1/2 activity
in neurons exposed to damage. In cultured hippocampal
neurons that were protected against staurosporine-induced

apoptosis by TGFb1, ERK1/2 stimulated survival by
Fig. 1. Neuroprotective mechanisms employed by ERK1/2. Bold italic
indicates the targets whose regulation by ERK1/2 appears to be gene
expression-dependent. ERK1/2-mediated inhibition of Bim may
involve both inhibitory phosphorylation and decreased expression.
Dotted lines indicate prosurvival ERK1/2 targets that were found in
non-neuronal systems and may also be involved in neuroprotection.
See text for more details.
2052 M. Hetman and A. Gozdz (Eur. J. Biochem. 271) Ó FEBS 2004
Rsk-mediated phosphorylation of Bad Ser112 [29]. More-
over, in murine brain, TGFb1 protected against ischemia
while activating ERK1/2 and increasing Bad phosphoryla-
tion at Ser112 [29]. This same ERK/Rsk-mediated phos-
phorylation event also occurred in cultured mouse cortical
neurons that were exposed to a neuroprotective hypoxia
treatment [34].
In neuronally differentiated PC12 cells, the anti-apoptotic
protection with NGF was suggested to be a result of
ERK1/2-mediated inhibition of another pro-apoptotic
member of the bcl-2 family, Bim40. The increase in Bim
expression was implicated as one of the killer mechanisms
activated by NGF withdrawal in this system. If trophic-
deprived cells were rescued with NGF, Bim protein levels
decreased. This effect was mediated by the ERK1/2
pathway [39]. Furthermore, ERK1/2 activation induced
Bim phosphorylation at Ser109 and Thr110 residues that
inhibited apoptosis induced by the overexpressed Bim40.
Therefore, it appears that the ERK1/2 pathway may inhibit
Bim function both by the decrease in Bim expression and by
the direct phosphorylation of Bim.

Neuroprotection by ERK1/2 may also involve a CREB-
mediated increase in the expression of anti-apoptotic
members of the bcl-2 family including bcl-2 and bag-1.
For instance, the prosurvival activity of ERK1/2 in PC12
cells may proceed via CREB-stimulated expression of bcl-2
[40]. Furthermore, in HSV2-infected neurons, apoptosis was
suppressed by an ERK1/2-dependent increase of bag-1
expression [30] that was presumed to be CREB-mediated.
Similarly, overexpression of the endogenous CREB antag-
onist ICER induced apoptosis and decreased bcl-2 levels in
cultured rat cortical neurons [41]. Finally, ERK1/2 may
protect from neuronal death by inhibiting the activity of a
pro-apoptotic kinase GSK3b [42].
Neuroprotective mechanisms controlled by ERK1/2 may
also involve indirect effects in non-neuronal CNS cells
including glia and vascular cells. For instance, Gabriel et al.
have shown that in mixed cortical cultures, neurons were
protected from NMDA-mediated excitotoxicity by TGFa-
induced ERK1/2 activation in astrocytes [43]. In these cells,
ERK1/2 increased production of type 1 inhibitor of tissue
type plasminogen activator that blocked the neurotoxic
activity of a secreted protease, tissue-type plasminogen
activator. Interestingly, Notch3 protects against ischemic
stroke by supporting survival of brain vascular smooth
muscle cells [44]. Notch3 mediated protection is suggested
to occur through an ERK1/2-dependent up-regulation of
c-FLIP that subsequently blocks the pro-apoptotic activa-
tion of caspase 8 [44].
Outside the nervous system, there are several interesting
observations pointing to the possible modes of ERK1/2-

mediated neuroprotection. Allan et al. found that ERK2
can directly phosphorylate caspase 9 at Thr125 [45]. This
phosphorylation event blocked caspase 9-mediated activa-
tion of caspase 3 [45]. Indeed, there are reports suggesting
that ERK1/2 may protect by inhibiting apoptosis down-
stream of cytochrome c release, one of the key steps directly
linked to caspase activation in the mitochondrial death
pathway [46,47].
Another example of an interesting anti-apoptotic
ERK1/2 target is a protein encoded by the Drosophila head
involution defective (Hid) gene. Hid activates Drosophila
caspases and mammalian caspase 8 [48,49]. Activity of this
protein is inhibited through a direct phosphorylation by
ERK1/2 [48]. Intriguingly, caspase 8 activation by death
receptor signaling was blocked through ERK1/2 [38]. This
inhibition was independent of protein synthesis indicating
that a Hid-like ERK1/2 target may exist in mammals [38].
Noteworthy, in cardiomiocytes, ERK1/2 protected against
cell death by the upregulation of cyclooxygenase-2 gene
transcription and the resulting increase in production of
cytoprotective prostaglandins [36].
A functional proteomics approach revealed several
candidate ERK1/2 signaling substrates that may participate
in neuroprotection [50]. For instance, ERK1/2 activation
targeted an anti-apoptotic member of the bcl-2 family,
mcl-1 and also DNA repair enzymes of the nucleotide
excision repair pathway [50]. The significance of these
ERK1/2 signaling substrates for neuronal survival remains
to be elucidated. Lastly, ERK1/2 activation may be
beneficial for functional recovery following CNS injury in

the absence of eliciting any effects on the survival of
damaged neurons. For instance, ERK1/2 may enhance
proliferation of neural stem cells that may replace the
deceased neurons [51]. ERK1/2 may also contribute to
the functional plasticity after traumatic brain injury [52].
In conclusion, dissection of the protective mecha-
nisms activated by ERK1/2 in the nervous system is still
incomplete.
ERK1/2: Killer or savior?
There are several reports summarized in the accompanying
review by Chu et al. that ERK1/2 inhibition protects
neurons in ischemia, traumatic brain injury, epilepsy or
oxidative glutamate toxicity [53]. How does one reconcile
these observations with the proposed protective role of
ERK1/2? Interestingly, several signaling systems have also
been recognized to have seemingly contradictory effects on
cellular survival. For instance, NMDAR can trigger cell
death but may also have protective effects [54]. Likewise,
the tumor suppressor protein p53 can trigger apoptosis but
may also enhance DNA repair and cell survival [55]. The
factors that are proposed to determine the beneficial or
deleterious outcome of these multifunctional transducers
include differences in the activation intensity or duration,
the subcellular localization of signaling molecules, the
signaling context provided by other pathways or the
cellular energetic state. Importantly, the nature and extent
of cellular injury may also change the ultimate results of
the same signaling events. Finally, chronic activation of a
signaling pathway may lead to an increased activity of
inhibitory feedback pathways switching off downstream

signaling that is normally activated by this circuitry. Some
or all of these factors may also affect the ultimate outcome
of ERK1/2 activation in the nervous system. A number of
these issues are further discussed in the accompanying
review by Chu et al. with a particular focus on neuro-
degeneration [53].
Perspectives
In summary, it appears that in many cases ERK1/2
activation is neuroprotective and mediates the effects of
Ó FEBS 2004 Role of ERK1/2 in neuronal survival (Eur. J. Biochem. 271) 2053
several extrinsic survival signals. Furthermore, ERK1/2
activation is found in injured neurons where, at least in
some cases, it has the protective, compensatory role. The
neuroprotective mechanisms controlled by ERK1/2 include
regulation of pro- and anti-apoptotic members of the bcl-2
family. It is probable that other mediators underlying
prosurvival activity of ERK1/2 in neurons will be uncovered
in the future. The factors that determine whether ERK1/2
activation will stimulate or inhibit neuronal survival will
also be an interesting target for research. Identification of
these factors may be critical for the development of useful
strategies for targeting the ERK1/2 cascade to intervene
against neurological diseases.
Acknowledgements
This work was supported by a NIH/NCRR grant, RR15576. The
authors wish to thank Drs Richard Benton, Scott R. Whittemore,
Donald DeFranco and Jane E. Cavanaugh for critical reading of this
paper.
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